USE OF PRIMARY QUAIL EMBRYO FIBROBLAST CELLS FOR PROPAGATION AND ASSAY OF AVIAN VIRUSESMaj may 2015

MALAYSIAN JOURNAL OF VETERINARY RESEARCHVOLUME 6 NO. 1 JANUARY 2015

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EDITORIAL BOARD

ADVISORS

Datuk Dr. Abd. Aziz Bin JamaluddinDirector-General of Veterinary Services, Malaysia

Dr. Abu Hassan Bin Muhammad Ali Director of Research Department of Veterinary Services, Malaysia

CHIEF EDITOR

Dr. Ramlan Bin [email protected]

EDITORS

Dr. P. ChandrawathaniDr. N. MuniandyDr. Jasbir SinghProf. Dr Helan Nair Dr. Cheah Tong SoonDr. S. ShanmugaveluDr. Latiff ah HassanDr. Zaini Mohd ZainDr. D. SabapathyDr. S. VellayanMr. Syed Hussien Syed AbdullahMr. Chang Kum WahMr. Adrien Kumar Raymond

SECRETARIATS

Mdm. Yeoh No [email protected]

Mr. Goon Swee [email protected]

Mdm. Debbra [email protected]

Mr. Mohd Hasril Muhamad [email protected]

Copyright of all pages belongs to the DVS. Authorisation has been granted to the DVS to reproduce these pages.

All DVS publications are protected by international copyright law. Extracts may be copied, reproduced, translated, adapted or published in journals, documents, books, electronic media and any other medium destined for the public, for information, educational or commercial purposes, provided prior written permission has been granted by the DVS.

Performance evaluation of quail closed-house system in Institute of Poultry Technology, Malaysia

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Use of primary quail embryo fi broblast cells for propagation and assay of avian viruses

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Mixed viral infections in village chickens 21

Histomoniasis & incidental fi nding of Schistosoma mansoni in turkey birds

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Ectoparasitic fauna of scavenging chickens (Gallus domesticus) from Penang island, Peninsular Malaysia

33

The follicle characteristic and immature oocyte quality obtained from repeated transvaginal oocyte retrieval in Bos indicus beef cows

43

Multiple factors causing death in Brucella-positive Murrah and Nili-Ravi buff aloes: a case report

53

Antibiotic susceptibility of bacteria isolated from the genital system of cows in Al Hilla, Iraq

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Antimicrobial resistance of Salmonella enterica serovar typhimurium from various meats received in vri

73

Surgical management of unilateral seminoma in a dog

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Experimental Trypanosoma evansi infection in albino mice – a histopathological study

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McMaster method of worm egg count from faecal samples of goats: a comparison of single and double chamber enumeration of worm eggs

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Vectors of veterinary importance in Malaysia: a survey of biting fl ies in relation to trypanosomiasis in Perak

101

CONTENTS


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VOLUME 6 NO. 1 JANUARY 2015 • pages 1-8

PERFORMANCE EVALUATION OF QUAIL CLOSED-HOUSE SYSTEM IN INSTITUTE OF POULTRY TECHNOLOGY, MALAYSIANURUL AINI M.Y.1*, NURSHUHADA S.1 AND IRDAYU H.2

1 Department of Veterinary Services, Wisma Tani, Presint 4, 62630 Putrajaya

2 Institute of Poultry Technology, KM 34, Ramuan China Besar, 78309 Masjid Tanah, Melaka

* Corresponding author: [email protected]

ABSTRACT. Quail closed-house system is a house that support quails’ optimum requirements as far as temperature, relative humidity, ventilation and light are concerned. The Department of Veterinary Services (DVS), Malaysia had developed 4 units of quail closed-house systems at Institute of Poultry Technology, Malacca and the closed-house used in this study named as House A. The objective of this project was to evaluate the performance of the quail closed-house system by comparing the condition of the house under current management condition and after the Standard Operating Procedure determined by DVS was adhered. The house performance was evaluated by observing its ability to achieve an optimum temperature range of 20 and 27°C with a relative humidity between 60 to 80% as perfect surroundings for quails. At the end of the experiments, the results showed a decline in internal temperature at almost 3°C and increasing in a relative humidity of 10% after all the specifications and procedures were followed.

Keywords: closed-house system, quail, internal environmental condition

INTRODUCTION

Institute of Poultry Technology, Malacca is accommodates of 4 units of quail closed-house systems. The closed-house system named as House A which is being studied hold the adult quails with dimension of 45 meters length, 10.8 meters width and 3.7 meters height. The house is equipped with evaporative cooling pad and automatic drop curtain system along the house. The curtains must be ensured in a good condition as any hole and leaking can affect the house ventilation. It is also equipped with 6 units of exhaust fans located at the end of the house. Stale air is removed by the exhaust fans and fresh air bSincrought in through intake openings. A good condition and operation of the exhaust fans and evaporative cooling pad can reduce the internal temperature and at the same time increase the relative humidity in accordance to the specifi cations (Berry and Huhnke, 2003). Proper ventilation in a poultry house is very crucial and must be maintained as it is essential to sustain life apart to remove excess heat, moisture, dust and odors from the building (The Poultry Site, 2012).

RE# MJVR -0030-2014

MALAYSIAN JOURNAL OF VETERINARY RESEARCH VOLUME 6 NO. 1 JANUARY 2015

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House A used in this study is an A-shaped closed-house without ceiling installation as shown in Figure 1. The roof is insulated with thermal refl ective insulation in order to reduce heat inside the house especially during hot weather. However, complaints have been raised that the quail closed-house in Institute of Poultry Technology has recorded high temperature especially at the middle of the house. House A has already been installed with control panel that show the internal temperature of the house but the reading displayed was slightly higher than the specifi cation due to the house operation did not adhere to the guidelines and standards determined by the DVS. Table 1 shows the allowable internal environmental condition as recommended to build a poultry closed-house system (Sulaiman et al., 2005). The current environmental condition affect the growth of quails as well as the effi ciency of production cost and at the same time creates discomfort to the workers since the high temperature will affect the odor and air quality inside the house.

The aim of this study was to evaluate the performance of the quail closed-house system by comparing the operation of House A under current management condition and after all specifi cations and Standard Operating Procedure (SOP) followed as determined by the Department of Veterinary Services.

MATERIALS AND METHODS

The performance of the quail closed-house system was evaluated by locating 4 units of HOBO data loggers near the evaporative cooling pad, at the front, middle and end of the quail’s cage line as shown in Figure 2. The data loggers were placed at the same level of the quail’s cage so that the temperature and relative humidity recorded exactly the same as those accepted by the quails. The internal temperature and relative humidity were recorded at two-hour interval using the data logger.

There were two conditions of experiments conducted named as (a) Experiment 1 and (b) Experiment 2. In Experiment 1, the data were recorded in the current management operation without operating the evaporative cooling pad and the exhaust fans were set in a manual mode in which only one or two fans were operated simultaneously. Experiment 2 was conducted in accordance to the specifi cations and procedures determined by DVS in which the evaporative cooling pad was operated properly while the exhaust fans were set in an automatic mode as mentioned by Sulaiman et al. (2005). In

Table 1: Allowable Internal Environmental Condition of Closed-House System (Sulaiman et al., 2005)

Items Reading

Temperature, T (C) 20 – 27

Relative Humidity, RH (%) 60 – 80

Ammonia, NH3 (ppm) < 20

Hydrogen Sulfi de, H2S (ppm) < 5

Carbon Dioxide, CO2 (%) < 0.3

Carbon Monoxide, CO (%) 0


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Figure 1. Roof without ceiling

Figure 2: Location of HOBO data logger inside the house


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Table 2. Gases reading of House A

DateRecording

Point

Gases ReadingLast

Manure Collection

Oxygen, 02 (%)

Ammonia NH3, (ppm) Hydrogen Sulfi de, H2S

(ppm)Reading 1 Reading 2

5/3/2013

Front 20.9 0 0 0

4/3/2013

Middle 20.9 0 0 0

End 20.9 0 0.1 0

6/3/2013

Front 20.9 0 0 0

Middle 20.9 0 0 0

End 20.9 0 0 0

7/3/2013

Front 20.9 0 0 0

Middle 20.9 0 0 0

End 20.9 0.1 0.1 0

12/3/2013

Front 20.9 0 0.1 0

11/3/2013

Middle 20.9 0 0.1 0

End 20.9 0 0.1 0

13/3/2013

Front 20.9 0 0.6 0

Middle 20.9 0.1 1.1 0

End 20.9 1.1 1.4 0

14/3/2013

Front 20.9 0 0.7 0

Middle 20.9 0 1.1 0

End 20.9 1.0 1.4 0

both experiments, the data were recorded within 14 days.

The results obtained were compared to identify and determine the differences of the internal environmental condition of House A before and after all procedures were followed. In addition, three types of gases namely oxygen, ammonia and hydrogen sulfi de were analysed to monitor the air composition inside the house.

RESULTS AND DISCUSSION

Gases Reading

The gases reading of oxygen, ammonia and hydrogen sulfi de of House A are as presented in Table 2. The readings were taken at three different points during Experiment 1 was conducted. The amount of ammonia and hydrogen sulfi de were recorded between 0-1 ppm and 0 ppm respectively. The results showed that all


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three gases did not exceed the acceptable limit as recommended to operate a closed-house system as shown in Table 1. Hence, no further study was carried out for Experiment 2 in terms of gases reading as the changes are not signifi cant. The amount of ammonia should be controlled to ensure the air quality of the house since high concentrations of ammonia can reduce weight gains and increase susceptibility to disease like irritate to mucous membranes of the respiratory tract and the conjunctivae and corneas of the eyes (World Poultry, 2010). Meanwhile, the oxygen level should be monitored since it is crucial to sustain life and carries out the harmful gases and undesirable odors caused by respiration and waste decomposition (The Poultry Site, 2012).

Temperature and Relative Humidity

In this study, 4 units of HOBO data loggers were installed near the evaporative cooling pad, at the front, middle and end of the quail’s cage line respectively. Figure 3, Figure 4, Figure 5 and Figure 6 show the temperature and relative humidity recorded at four different points for both experiments; before and after Standard Operating Procedure was adhered (Sulaiman et al., 2005). All data were recorded at two-hour intervals.

Based on the results obtained, the internal temperature was slightly dropped after Experiment 2 was conducted. The temperature dropped at almost 3°C while a relative humidity increased about 10% after all procedures were followed.

Figure 3: Temperature and relative humidity near the cooling pad


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Figure 4: Temperature and relative humidity at the front point

Figure 5: Temperature and relative humidity at the middle point


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The automatic operation of evaporative cooling pad and exhaust fans affect the environmental condition inside the house. However, the temperature recorded for both experiments were between 28 and 38°C and it exceeded the temperature range of 20°C and 27°C as recommended by DVS to operate a closed-house system. Meanwhile, the relative humidity recorded a satisfactory rate in a range of 70% and 80% for both experiments.

CONCLUSION

The closed-house system has a lot of advantages such as it can control the house ventilation, reduce internal temperature and give more comfortable to the birds as weak ventilation system can lead to poor

air quality and, resulting in bird health and performance issues. A proper environment must be maintained to increase quail’s performance. Even though the closed-house performance was increased after the Standard Operating Procedures followed, it is not achieve a satisfactory level. Therefore, the quail closed-house system in Institute of Poultry Technology, Malacca needs more improvements in terms of building structure since no signifi cant differences of the temperature and relative humidity were shown after the study was completed.

Figure 6: Temperature and relative humidity at the end point


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REFERENCES

1. Berry, J.G. and Huhnke, R.L. (2003). Hot Weather Management in the Poultry House. Division of Agricultural Sciences and Natural Resources, Oklahoma State University.

2. Sulaiman, A.K, Radim, D., Saonah, M.N., Elva S.M., and Nurizan Y. (2005). Reban Tertutup Ayam Daging. Department of Veterinary Services. First Edition. pp. 1-61.

3. The Poultry Site: Key Factors for Poultry House Ventilation. (2012). (http://www.thepoultrysite.com/articles/2321/key-factors-for-poultry-house-ventilation)

4. World Poultry: Harmful Effects of Ammonia on Birds. (2010). (http://www.worldpoultry.net/Breeders/Health/2010/10/Harmful-effects-of-ammonia-on-birds-WP008071W/)


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ABSTRACT. A primary fi broblast cells from embryos of brown quail Coturnix ypsilophora has been established and partially characterized. The cells were maintained in Modifi ed Eagle’s medium (MEM) supplemented with 10% fetal bovine serum. The cells were able to grow at temperatures between 35°C and 38°C with optimum temperature of 37°C. The growth rate of primary quail fi broblast cells increased as the FBS proportion increased from 5% to 20% at 37°C with optimum growth at the concentrations of 10% or 15% FBS. The cells showed no microbial contamination throughout the period of experiment and the total chromosome number of a diploid cell was 78, according to karyotyping and chromosome analysis.

The susceptibility of quail primary cells for avian viruses was investigated in this study after inoculation with ND and IB viruses. Both viruses showed a satisfactory CPE development and the infectivity were assayed by virus titration (TCID50). This suggests that the quail primary cells can be used for isolation of various avian viruses with further steps of infectivity confi rmation in the future.

INTRODUCTION

Although embryonating eggs can support the growth of a broad range of avian viruses, many fi eld viruses do not readily grow in eggs. Therefore, obtaining a sufficient number of reliable, high-quality eggs to avoid false negatives (sample mishandling) is a considerable limitation in their use. Many attempts have been made to fi nd suitable alternatives to the use of eggs for avian virus isolation and research purposes. However, all the previously studied cell lines for avian viruses were mammalian in origin, and limitations (including the restricted host specifi city of the cell and a possible change in receptor specifi city) arise when using a mammalian cell line for avian virus study. Thus, it would be ideal to use a well-established avian-origin cell line for avian virus study.

Infectious bronchitis virus (IBv) infects the respiratory tract, kidneys and oviduct of poultry of all ages, causing delay in growth, mortality, reduced egg production and poor egg shell quality and in many countries the disease remains one of the main problems affecting existing or

USE OF PRIMARY QUAIL EMBRYO FIBROBLAST CELLS FOR PROPAGATION AND ASSAY OF AVIAN VIRUSESMAYADA F. HASOON1*, MAJED H. MOHAMMED2 AND JALADET M.S. JUBRAEL1

Scientific Research Center, Faculty of Science, Duhok University,Kurdistan Region, Iraq

Pathology Department, College of Veterinary Medicine, Baghdad University, Baghdad, Iraq



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developing poultry industries. Until now, there is no cure for the disease (OIE, 1996). Prevention is to import birds from disease-free fl ocks only or through vaccination. broilers are normally vaccinated at 1 day of age with live attenuated vaccines (Cavanagh & Naqi, 1997). In addition, breeders and egg layers are also vaccinated at approximately 8 week intervals with live attenuated vaccines, and with inactivated vaccines after they start laying eggs (Cook et al., 1999).

NDv causes Newcastle disease (ND), which is an acute highly contagious viral disease of domestic poultry as well as other species of birds regardless of age and sex variation (Alexander, 2003). According to the strain variation of NDV, the rate of morbidity and mortality of poultry in a fl ock due to ND varies from 90-100%, thereby poultry industry all over the world facing serious economic losses every year.

However IBv and NDv infections can also be diagnosed by detection of viral RNA, which make the diagnostic rapid and also dependable (Capua et al., 1999; Cavanagh, 2001), but researchers used to produce the virus by growing in embryonated chicken eggs and this takes long time and requires large area for egg incubation. Thus, cell cultures considered are more suitable and less expensive than eggs and also convenient to inspect microscopically for indication of viral proliferation (Dhinakar & Jones, 1997; de Wit et al., 1997). There is therefore a serious need to improve IB virus isolation

technologies based on quail fi broblast cells (QFC).

In this study, the authors evaluated the characteristics of quail fi broblast cells to investigate their ability to support the growth of avian viruses


The 8-day-old embryos of quail in this research were provided by chicken breeding farm of faculty of Agricultural, Dept. Animal Production, Duhok Univ. Kurdistan Region, Iraq. MEM (Gibco, USA), special grade fetal bovine serum (Biochrom, German), DMSO (Sigma, USA), Hoechst 33258 (Invitrogen, USA) were used in this study. Reference strains of IB (QX strain) and ND (AG68,1968 Iraq. VI. 4a. AF001108) viruses were kindly provided by Al-Kindi Company for Veterinary Vaccines & Drugs, Baghdad, Iraq. This study was performed and accomplished at Animal Biotechnology Lab., Scientifi c Research Center/Faculty of Science/ Duhok Univ.

Cell culture

The quail eggs incubated for 8 days were sterilized using alcohol swabs, and then the embryos were isolated and washed three times with phosphate buffered saline (PBS) supplemented with high level of antibiotic (3% Penicillin-Streptomycin). The embryos were cropped into pieces of 1 mm3 in size and seeded onto the surface of 25 cm2 tissue culture fl ask, and cultured at


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37°C in a humidifi ed atmosphere, 5% CO2

for 1 to 2 hrs. Modifi ed Eagle’s medium (MEM) containing 10% fetal bovine serum (FBS) was added into the fl ask. The medium was refreshed after 1 to 2 days. The cells were harvested at 80% to 90% confl uence using 0.25% trypsin (m/v) solution and were seeded again into culture fl asks at the ratio of 1:2 as passage 1 (Guan et al., 2005; Zhou et al., 2004).

Culture media optimization

Three different media (MEM, RFLP and DMEM) were used to evaluate the growth of quail fibroblast cell (QFC) cultures according to the method of Wolf and Quimby (1976) (Table 1). All media were supplemented with 10% FBS, 1% (100 IU/ml) Antibiotic (Penicillin-Streptomycin), 1% (100 IU/ml) antifungal-Nystatin. A total of 6 wells of tissue culture plate were seeded with new cells at a density of 106

cells/ml at initial P1 passage. Duplicate wells of each treatment were treated with different media. 1 ml of old media was removed and replaced with fresh media every next day during the fi rst week of growth period. The cells were trypsinized

and the effi ciency of each medium was evaluated by the number of viable cells/ml of cell suspension.

Temperature optimization

MEM complete growth media (15% FBS, 1% Penicillin-Streptomycin and 1% Nystatin-antifungal) was used as base medium in this study. Temperature required for growth of quail primary cells was optimized according to the method described by Wolf and Quimby (1976). Cells were cultured in multi-well tissue culture plate with seeding density of 106

cells/ml and incubated at 37°C for 24 hours to allow for attachment. Later, each plate with attached cells was incubated in a selected temperature of 35, 37 and 39°C respectively. Every two alternate days, cells at each temperature were counted to determine the best growth. The numbers of viable cells were expressed as cells per ml.

Serum optimization

The optimal fetal bovine serum (FBS) percentage for the growth of primary

Table 1. Media evaluation study. Three types of commercial media were used to evaluate the growth of quail fi broblast cells

serial number Type of media

1 Basal media of Minimum Essential Medium (MEM Eagle M0268 Sigma®, UK)

2 Roswell Park Memorial Institute (RPMI media-1640 (Sigma-Aldrich®, Germany)

3 DMEM - Dulbecco’s Modifi ed Eagle Medium (Gibco® DMEM)


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quail cells at initial P1 passage was assessed following the technique of Wolf and Quimby (1976). Cells were plated at density of 106 cells/ml in multi-well tissue culture plates and incubated at 37°C for 24 hours to allow cell attachment. MEM complete growth media with different percentage of FBS (5%, 10%, 15% and 20%) were selected for cell growth test. Every two alternate days, wells at each FBS percentage were trypsinized and the cells were stained with trypan blue and counted using haemocytometer chamber. The cell concentration was expressed as cells/ml of culture medium.

Microbial detection

Detection of bacteria and fungi

The cells were cultured in complete MEM media free of antibiotics and observed for the presence of bacteria and fungi at 3 days after subculture according to the method described by Doyle et al., 1990).

Detection of viruses

Hay’s hemadsorption protocol was used to examine the samples for cytopathogenesis using phase contrast microscopy (Hay et al., 1992; Wu et al., 2008).

Karyotyping and chromosome analysis

Chromosome spreads were prepared, fi xed and stained following standard methods (Costa et al., 2005). Cells were harvested

when 80 to 90% confl uent, and subjected to hypotonic treatment and fi xed, then the chromosome numbers were counted from 100 spreads under an oil immersion objective lens upon Giemsa staining. Chromosome distribution was calculated according to the protocol described by Sun et al. (2006).

Virus inoculation

The stocks of IB and ND viruses were originally obtained from Al-Kindi com.for vaccines development in Baghdad. Viruses were isolated from the allantoic fl uid of embryonated chicken eggs. Initially, 0.3 ml of the virus stock was diluted in 30 ml of 10X PBS giving a dilution of 1:100. The diluted viruses were used to inoculate QFC cells. 85-90% confl uent fl asks of cells were used for virus passage in an attempt to adapt the virus to replicate in these cells and show CPE.

Tissue culture infective dose 50 (TCID50)

The infectivity of replicated IBV and ND to QFC cells were determined by calculating 50% end point, as described by Reed & Muench (1938). Ten-fold serial dilution of each virus (NDv and IBv) was prepared in PBS from 10-1 to10-10. A 96 multi-well tissue culture microtiteration plate (Titertek, UK) was used to prepare QFC cells monolayers. 100μl of each virus dilution was added in each well of fi rst row leaving last two wells as negative


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control. The plate was incubated at 37°C for 1 hour to allow adsorption. Then 100μl of prewarmed maintenance medium (MEM+2%FBS) was added in each well and again incubated at 37°C in 5% CO2. The plate was observed daily for CPEs. The highest dilution of virus showing 50% CPEs was considered as end point to calculate TCID50

RESULTS

Morphological observation of quail fi broblast cells

Cells derived from trypsinized tissue of quail embryo muscles were well attached and started to differentiate after one day of being placed on the bottom of tissue culture fl asks (Figure 1A), and then they continued to proliferate and were subcultured when reached 80 to 90% confl uent within about 5 days (Figure 1B & C). The cells displayed typical fi brous and fusiform morphology. less than 50% of the cells shown cuboidal epithelial-like appearance, while the fi broblasts grew rapidly and replaced the epithelial cells gradually after 2 passages, and then a relatively purifi ed fi broblast line was obtained (Figure 1). The viability of quail fi broblasts evaluated through trypan blue exclusion tests was 85%.

Optimum media composition

Comparison between three different media of MEM, DMEM and RFLP revealed the best growth for QFC cells maintained in

Figure 1. Morphology development of QFC .Primary cells grew and differentiated from the trypsinized quail embryo body muscles; (A) 1 d after initial culturing; (B) 3 ds after culturing; (C) 90% confl uent monolayer in about 5 ds after culturing. Unstained cells. 200μm.


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MEM media. QFC cells seeded at initial density of 106 cell /ml and maintained with complete growth MEM media exhibited a high yield of viable cells (7x106 cell /ml) and reached confl uence within 7 days (Table 2). Cells that were maintained with DMEM complete growth media comprised cell viability of 103 cell /ml and reached confl uence after 14 days. Cells that grew in RPMI complete growth media showed low viability of 102 cells /ml and no confl uence was achieved.

Optimum temperature

QFC cells maintained in MEM complete growth media and seeded at initial density of 106 cell /ml exhibited different growth levels at different temperatures of 35°C, 37°C, and 39°C . The cells were able to grow at temperature range from 35 to 39°C. However, maximum growth with high viable cells (6.5 x106 cell /ml) was obtained at 37°C after 7 days post- culture (Figure 2).

Table 2: Culture media used to evaluate the growth potential of primary QFC cells

Medium Cell viability and confl uency No. of days to reach confl uent

MEM +++ 7

DMEM + 14

RPMI +/- -(+++): Excellent growth, <106 viable cells, <80% confl uence(+): Medium growth, 103 viable cell, 20-40% confl uence(+/-): Little growth, 10-102 viable cells (-): No growth, no confl uence

Figure 2. Various incubation temperatures used to evaluate potential growth of QFC cells. Optimum growth was achieved at 37°C while at 35°C, and 39°C the cells showed less growth.


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Optimum serum concentration

QFC cells seeded at initial density of 106 cell /ml and maintained in MEM media supplemented with different levels of serum (5, 10, 15 and 20%), were exhibited viable cell yield of 9x106 cell /ml on day 6th post- seeding at level 20% serum. Cells maintained in media supplemented with 15 and 10% serum showed less in number of viable cells yield (6 x106 and 5.5 x106 cell /ml respectively) at day 6th post- seeding. Only 0.8 x105 cell /ml viable cells obtained when cells maintained in media supplemented with 5% FBS.

Microorganism detection

The medium was clear all the time and no abnormalities were observed under the microscope. As is shown by the cytopathogenic evidence for virus contamination was also negative. The

results indicated that the QFC were free of bacterial, fungal and viral contamination.

Chromosome number

In this experiment 100 representative spreads at metaphase of passage 1 to 3 were observed under the microscope to count the chromosome number of diploid QFC, including the mean proportion of diploid cells which was 78±1% (Figure 3).

Cytopathic effect (CPE)

Infected cells monolayer of QFC was steadily became rounded and broken as the virus proliferates to occupy extra cells in culture. In the fi rst and second passage, the infectivity of both viruses (IB & ND) were sluggish and not very clear as the virus was just begin to adapt on cells. Through the third passage, CPE was rapid and changes have been recognized within

Figure 3. Chromosome analysis of primary quail fi broblast cells showing the chromosome number distribution at passage P2 with model peak at 78


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Figure 4: Cytopathology produced by IBv. infection in quail fi broblast cells 48hrs postinoculation.100μm.

Table 3: Virus titer determined by TCID50

Virus category and passage level Virus titer TCID50/ml

IBv NDv1 4.5 2.5

2 6.5 3.5

3 7.4 4.7

Figure 5: Cytopathology produced by NDv. infection in quail fi broblast cells 72hrs postinoculation. x 100μm.


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two days (in compare with the control). It was characterized by rounding of cells, failure of adhesion, vacuolization in cells,

clustering of infected cells (Figures 4 & 5).

TCID50

The infectivity titre of both viruses was found to increase gradually from the 1st to the 3rd passage. The TCID50 titer was 7.4 for IBv and 4.7 for NDv after 3 passages (Table 3)

DISCUSSION

Quail embryonic fi broblasts (QFC) were selected in this experiment as they offer many advantages, such as high vitality, easy access, and low probability of bacterial and other contamination. QFCs were successfully established from 40 embryo samples by adherent culture. The biological and morphological characteristics may be altered and cells may stop dividing by in vitro culture after many passages, so a minimal number of passages are recommended to protect the cells against degeneration and death (Bai et al., 2011). Morphological observation indicated that the cells developing a fibroblast-like appearance during the primary and the fi rst several passages of trypsinized tissues. Due to their different tolerance to trypsinization, the fi broblasts-like cells detached from the fl asks earlier when digested with trypsin and adhered again quickly after passage, (Rosenberg

etal., 1991). For this reason, a purifi ed fi broblast line could be obtained after 2-3 passages.

Infectious bronchitis (IB) and ND virus, early described in 1930 (Schalk & Hawn, 1931) continues to be main causes of disease in poultry of all ages and types in all parts of the world (Anon, 1991). The disease is occurring in all countries with a concentrated poultry industry, with the occurrence of infection up to 100% in most locations (Ignjatovic & Sapats, 2000).

Among the various media tested were DMEM - Dulbecco’s Modifi ed Eagle Medium, Eagle’s minimum essential medium (Eagle’s MEM), and Roswell Park Memorial Institute (RPMI) medium. MEM media was found to be the most suitable for the attachment and proliferation of QFCs cells. Several researchers have reported the successful used of MEM to support the growth of poultry cells (Lakra et al., 2006; Hameed et al., 2006). However, some primary cell lines required specifi c culture medium designed specifi cally to optimize growth during development of the primary culture (Wang et al., 1995), more than 80% of the cell lines established after 1994 used MEM media.

The growth temperature range used for QFC cells in this study was 30-39°C with optimum growth at 37°C. These results were consistent with other results reported previously (Nicholson et al., 1987; Tong et al., 1997, Kang et al., 2003; Hameed et al., 2006). One of the advantages of cell lines that grow over a wide temperature range is their potential suitability for isolating wide


18

range of pathogenic viruses (Nicholson et al., 1987).

The growth rate of QFC cells increases as the FBS concentration increased from 5% to 20%. However, a 10% concentration of FBS also provided relatively good growth and this is an advantage to be recommended to maintain those cells of QFC at low cost (Ye et al., 2006). In addition, eliminating serum proteins, often improves product quality and/or cell growth. As Barnes and Sato (1980) suggested in their strategies for optimizing cell growth; the goal is sometimes not to maximize cell growth rate, but rather to optimize viability and growth condition, whereas serum reduction is, almost useful in meeting this goal.

The newly developed QFC primary cell line was free from contaminating microbes as detected by tests on special media as described earlier by Hopert et al., (1993).

The proportion of 2n = 78 cells were 98% as detected in 100 cells. Most poultry chromosomes were very small ones, which were easily lost in the preparation process and by the interference of dye, rendering the difficulty to count chromosome number and to observe the morphology. Therefore, time point and duration of colchicine administration and low-osmotic duration should be precisely controlled in experiments and must be tightly controlled within appropriate time (Ashraful, 2012).

The cytopathic effects (CPEs) appeared after 72 hours of infection in third passage and this observation was

also noticed by (Hopkins, 1974), but slightly varied from fi ndings of Mahgoub et al. (2010) where the IBV and ND were adapted to Vero cell line after the third passage. The difference might be due to the cell culture passage level of the virus strains used or variation in sensitivity of cell culture to different strains. The total infectious titer at passage 3 was found to be 107.4 and 104.7 TCID50/ml for IBv and NDv respectively. These fi nding supports the previous report by (Otsuki et al., 1979).

REFERENCES

1. Alexander, D.J. 2003. Newcastle Disease, Other Avian Paramyxoviruses, and Pneumovirus Infections. In: Saif, Y.M. Diseases of Poultry (11th Edition) Blackwell Publishing Company, Iowa State Press. Iowa, USA pp 63

2. Anon, 1991. proceedings of the Second International Symposium on Infectious Bronchitis, Rouischholzhausen Germany Capua, I., Minta, Z., Karpinska, E., Mawditt, K., Britton, P., Cavanagh, D. and Gough, R.E. 1999. Co-circulation of four types of infectious bronchitis virus (793/B, 624/I, B1648 and Massachusetts). Avian Pathology., 28: 587 -593.

3. Ashraful K. M. 2012. Karyotypic analysis of chickens and other birds. Journal of Biotechnology (GARJB) Vol. 1(2) pp. 023-032

4. Barnes, D. and Sato, G.H. 1980. Methods for the growth of cells in serum free medium. Analysis Biochemistry102: 255-270.

5. Bai,C., D. Wang, C. Li, D. Jin, C. Li, W. Guan and Y. Ma. 2011. Establishment and biological characteristics of a Jingning chicken embryonic fi broblast bank. European Journal of Histochemistry; volume 55:e4:19-23.

6. Capua, 1., Minta, Z. Karpinska, E., Mawditt, K., Britton, P., Cavan agh, D. and Gough, R. E 1999. Co- Circulation of four types of infectious bronchitis virus (793/B, 624/1, B 1648 and Massachusetts) Avian Pathology, 28, 587 – 592.

7. Cavanagh, D. and Naqi, S.A. 1997. Infectious bronchitis. In B.W. Calnek, H.J. Barnes, C.W. Bearol, L.R. Mc Daugald, and Y.M. Saif (eds). Disease of Poultry 10th. pp 511-526. Ed. Lawa University Press.


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8. Cavanagh, D., Mawditt, K., Sharma, M., Drury, S.E., Ainsworth, H.L., Britton, P. and Gough, R.E. 2001. Detection of a coronavirus from turkey poults in Europe genetically related to infectious bronchitis virus of chickens. Avian Pathology., 30: 365 -378.

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23. Mahgoub, KM., Bassiouni AA., Manal A, Afi fy., and Nagwa, RS. 2010. The prevalence of infectious bronchitis (IB) outbreaks in some chicken farms. I. Spotlight on the status of IB outbreaks in some chicken fl ocks. Journal American Science., 6(9): 57-70.

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25. Offi ce international des Epizootics (OIE) 1996. Avian infectious bronchitis, chapter 3.6.6. In Manual of standards for diagnostic test and vaccines, 3rd Ed. pp 539- 548. OIE, Paris.

26. Otsuki, K., Noro, K., Yamamoto, H. and Tsubokura, M. 1979. Studies on avian bronchitis virus (IBV). Propagation of IBV in several cultured cells. Archives of Virology., 60: 115 -/122.

27. Reed, L.J. and Muench, H. 1938. A simple method for estimating fi fty percent end points. American Journal Hygiene., 27: 493-497.

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29. Schalk, A.F. and Hawn, M.C. 1931. An apparently new respiratory disease of chicks. J. Am. Veterinary. Medicine. Association., 78: 413-422.

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31. Tong, S.L., Lee, H. and Miao, H.Z. (1997). The establishment and par tial characterization of a continuous fi sh cell line FG-9307 from the gill of fl ounder Paralichthys olivaceus. Aquaculture 156: 327–333.

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35. Ye, H. Q., Chen, S. L., Sha, Z. X. and Xu, M. Y. (2006). Development and characterization of cell lines from heart, liver, spleen and head kidney of sea perch Lateolabrax japonicus. Journal of Fish Biology 69: 115– 126.

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ACKNOWLEDGEMENTS. This research was supported by the faculty of science, scientifi c research center, and the viral references were kindly provided by Al-Kindee Com. in Baghdad for viral vaccine production. The microbial tests were demonstrated at bacteriology Lab/ Faculty of Science, Duhok Univ. Quail embryonated chickens were kindly provided by head Dept. of Animal production, Faculty of Agriculture, Duhok Univ.


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ABSTRACT. A mixed viral infection of Newcastle Disease (ND), Marek’s Disease (MD) and Avian Leukosis (AL) was reported in village chickens. Analysis of the deduced amino acid sequences of the F protein cleavage site of ND virus showed that the isolate was virulent with sequence 112KRRKR116 for the C-terminus of the F2 protein and phenylanine (F) at residue 117, the N-terminus of the F1 protein. Basic Local Alignment Search Tool (Blast) analysis showed the isolate was ND which was 96% similar identity with Indonesia Sukorejo genotype VII, MD 99% similar identity with China very virulent strain and ALV 100% similar identity with Taiwan ALV strain. Due to the free ranging type of management, causes of the diseases in the poultry were uncertain as many factors can contribute to the disease occurrence. However, good hygiene practices can help to improve the farm’s sanitary. Basic biosecurity system can be applied although the success may be limited. Vaccination is another alternative that can be considered to prevent the diseases with the help from certain parties.

Keywords: viral diseases, village chicken, hygiene, biosecurity

INTRODUCTION

In South-east Asian countries including Malaysia, poultry keeping is still practised as a backyard operation among some rural families although commercial poultry has been developing rapidly over the years (Ramlah, 1999). The Malaysian indigenous chicken, the village chicken which is also known as ‘ayam kampung’ is a crossbreed of the Red Jungle Fowl with mixed exotic domestic breeds imported by Europeans, mainly the British (Aini, 1990). Rural families still rear village chicken due to its economic importance. The village chicken production which includes egg and chicken meat serves as a side income for the owner as well as cheap protein source in a family’s diet. Cost of the production is very small and the chickens also play an important role in waste disposal system where the chickens help in converting the kitchen scraps into valuable protein. Chicken’s manure can be used as fertilizer for the fruit trees and the sale of the fruits serves as an additional income (Aini, 1990).

In this case study, a poultry farmer complained of his village chickens showing dullness, inappetance and producing greenish faeces for 2 days. The farmer had 45 local kampong chickens aged 6

MIXED VIRAL INFECTIONS IN VILLAGE CHICKENS LEOW B.L*., FAIZUL FIKRI M.Y., ONG G.H., MUHAMMAD REDZWAN S., BASIRAH M.A., NORAZURA B., MAIZATUL Z., MOHD KHAIRIL A., MOHD. JIHAN R. AND RAMLAN M.Veterinary Research Institute, 59 Jalan Sultan Azlan Shah, 31400 Ipoh, Perak


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months old which were unvaccinated. The morbidity rate was 33.3% and mortality rate was 22.2%. Complete post-mortem and diagnosis was conducted on a dead carcass to elucidate the cause of mortality in the farm.


On post-mortem, ND and MD were suspected. Pooled organ obtained from the post-mortem was homogenised and diluted with TPB prior centrifugation. The supernatant was collected and fi ltered. Virus isolation was attempted for Newcastle Disease virus. It was carried out by inoculating the fi ltrate sample into 9-10 days old embryonated SPF eggs via intra-allantoic route and incubated for three days at 37oC for two passages. The infected allantoic fl uid was harvested and the presence of the virus was identifi ed by Haemagglutination test (HA) and Hemagglutination-Inhibition (HI) test (Manual OIE 2012).

Molecular detection was carried out for the three avian diseases: ND, MD and AL. For ND, the viral RNA was extracted from the infected allantoic fl uid using TRI LS Reagent (Molecular Research centre, Inc). RT-PCR is performed to detect the ND virus by using primer set MV1/B2 (Herczeg et al., 1999) that amplify the partial of matrix and fusion gene. The RNA was also amplifi ed by using primer set of MV1/ NDVIR2 (in- house designed primers) to determine the pathotype of the virus. For both MD and AL, the viral DNA

was extracted directly from the pooled organ by using DNeasy Blood and Tissue kit (Qiagen). The DNA was amplifi ed to detect Unique Long (UL) region of MD virus (MDV) by using primer set MDV UL 19 P5/ MDV UL 19 P6 (Ottiger, 2010) and Pol gene for AL virus (ALV) using primer set H5/AD1 (Maaz et al., 2005). The amplicons generated were cut from the gel and purifi ed using the Qiagen QIAquick gel extraction kit. Purifi ed products were sent for sequencing to identify the isolate. Sequences were assembled and analysed using Lasergene’s SeqMan Pro software. Analysis of protein sequence was done by BioEdit Sequence Alignment Editor. Nucleotide sequence of the isolate was checked and compared with published sequences deposited in the Gene Bank database using a BLAST (Basic Local Alignment Search Tool) search via the National Center of Biotechnology Information (NCBI).

RESULTS

The isolate presented HA titer of 64 and was positive for ND by HI test. A PCR product of 557bp was amplifi ed for common detection of NDV (Figure 1). An amplicon of 495bp was detected by using pathotype determination primer set indicating that the isolate was virulent ND strain (Figure 1). A PCR product of 521bp and approximately 295-326bp was detected for MDV and ALV respectively (Figure 2). Analysis of the deduced amino acid sequences of the F protein cleavage


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Figure 1. Gel photo for molecular detection of the ND isolate. The PCR products were separated on a 1.5% agarose gel stained with SYBR Safe DNA gel stain. Well 1-4 showed the PCR products for common detection of ND whereas PCR products for ND pathotype determination were showed in well 5-8. Well 1: ND Isolate; well 2: positive control (ND virulent strain); well 3: positive control (ND non-virulent strain); well 4: non template control; well 5: ND isolate; well 6: positive control (ND virulent strain); well 7: positive control (ND non-virulent strain); well 8: non template control, M: 100bp DNA ladder.

100 bp

1000 bp

500 bp 495 bp

557 bp

521

~300

1000

100

500

M 1 2 3 4 5 6

Figure 2. Gel photo for molecular detection of the ALV and MDV positive samples. The PCR products were separated on a 1.5% agarose gel stained with SYBR Safe DNA gel stain. Wells 1-3 and 4-6 showed the PCR products for ALV and MDV respectively. Well 1:sample; well 2: positive control; well 3: non template control; well 4: sample; well 5: positive control; well 6: non template control, M: 100 bp DNA ladder.


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site of NDV showed that the isolate was virulent with the amino acid sequence 112KRRKR116 for the C-terminus of the F2 protein and phenylanine (F) at residue 117, the N-terminus of the F1 protein. BLAST analysis showed the isolate was ND which was 96% similar identity with Indonesia Sukorejo genotype VII, MD 99% identity to China very virulent strain and AL 100% identity to Taiwan ALV strain.

DISCUSSION AND CONCLUSION

Many factors can cause mortality in free-range poultry keeping. 80-90% mortality of the poultry in the fi rst year after hatching mostly due to improper management, short of fresh water and supplementary feed supply, predators and diseases. Among these, disease is the main problem in the production of village chicken (Permin et al., 1999). Due to free ranging and unconfi ned type of management, disease control is very diffi cult and is therefore rarely practiced by the farmers (Permin et al., 1999).

In this study, the isolate was confi rmed ND by both virus isolation and molecular detection. Sequence analysis of the F protein cleavage site showed the isolate was virulent strain of ND and BLAST analysis revealed that it was 96% identity to Indonesia Sukorejo genotype VII ND. MDV and ALV were also detected in the samples by molecular detection. BLAST analysis of UL gene for MDV and Pol gene for ALV showed the isolate was 99% identity to China very virulent MD

strain and 100 % identity to Taiwan AL strain. The mixed viral infection reported in this case was in agreement with Aini (1990 and 1999) and Permin et al. (1999) said that the common viral diseases found in village chicken are Newcastle disease, Marek’s Disease, lymphoid leukosis, infectious bronchitis etc.

ND is a highly contagious disease in chicken where outbreaks with mortality up to 100% are common and has been one of the major causes of economic losses in the poultry industry. MD, a lymphoproliferative and neuropathic disease of chickens is caused by highly contagious cell-associated alphaherpesvirus MDV serotype 1 (Tulman et al., 2000). MD causes huge economic losses as yearly economic losses of total $1 billion worldwide have been reported (Tulman et al., 2000). Like MD, ALV is an avian oncogenic virus which can cause variety of neoplastic disease condition in chickens. The infection results in huge economic losses due to reduced productivity and ALV induced tumor (Fadly, 2000). The mixed viral infection consists of ND, MD and AL contributed to the morbidity and mortality of the village chickens with rate of 33.3% and 22.2% respectively.

According to Aini (1999), all countries in South-East Asia reported that among all the diseases, ND is the most important disease that causes the highest economic losses in indigenous chickens. The disease is endemic in village poultry populations in Africa and Asia (Spradbrow, 1993/94). The isolation of ND virulent strain in this


25

study was in agreement with the fi nding of Spradbrow (1993/94) stated that virulent strain of ND was detected when virus isolation was attempted in village poultry. Alexander et al. (2004) reported that the occurrence of ND in village chicken is mostly due to introduction of infected chicken. Domestic birds such as ducks, geese, doves, and guinea fowl can carry the virus and spread the disease. They may or may not manifest clinical syndrome of ND where the development of clinical signs is depends on the strain of ND and species of the bird (Alexander et al., 2004). Role of wild bird in spreading the disease was uncertain although ND virus of different virulence had been detected in many species of wild birds (Alexander 1988). Chicken’s manure which sometimes used by the farmers as fertilizer can become the source of infection as the virus can persist in uncultivated feces for more than six months (G. Arzey, 1989).

Persistence of the viral agents may play a role in the spreading of the disease. MD virus can survived for at least 8 months in infected feather materials and 440 days in the dust in dry state from poultry houses at room temperature (Hlozanek et al., 1977). Like MDV, NDV can survive well in the environment. It may persist at 8°C for months and 37°C for several days (Anonymous, 2003). The virus can also survive on materials such as paper, polythene and cloth for 25-35 days at room temperature (G.Arzey, 1989). As the village chickens like to roam freely to search for food, they may have come into

contact with the infected materials and get infected.

How the village chickens were infected by the three viral diseases in this case is uncertain. Due to the free-ranging type of poultry management, there are too many factors that the village chickens can be infected by the agents as discussed earlier. Factors such as lack of biosecurity measures and good hygiene practices, no vaccination, introduction of the viruses by infected chickens or birds, contaminated poultry keeping area and etc can contribute to the occurrence of the diseases.

Generally, due to the management system in village chicken, disease control is very minimal (Aini, 2000). Therefore, good farm management and strict biosecurity play an important role in controlling poultry disease. Traffi c control which includes both human and vehicles into and within the farm is important as disease can enter the farm by this route. Implementation of strict biosecurity measure may be hard with limited effectiveness in free range fl ocks but some of these practices may be applicable (Aini, 2000). Thus, raising the awareness of the farmers about the concept of f lock diseases, principle of disease control and importance of biosecurity is crucial.

Good hygiene which includes effective cleaning and disinfection is another key component in disease control. Due to the free range poultry management systems, cleaning of the environment may be diffi cult to carry out but limited disinfection can be applied (Aini, 2000).


26

For example, use of lime to disinfect the affected area after a disease outbreak. Lime is always used by farmers due to its low cost (Aini, 2000). Despite the facts that the viruses can persist for a period of time, the viruses is readily to be inactivated by disinfectants. NDV can be inactivated by a wide range of disinfectants including chlorine-based chemicals and lipid solvents (Anonymous, 2003). Organic iodine solution can be used to inactivate the MDV infected dust (Holzanek et al., 1977) and 1% NaOH was effective against MDV infected feather extract (Calnek et al., 1973). As most common route of MD transmission is inhalation of the infected dust by susceptible chicken (Payne et al., 2000), therefore proper cleaning and disinfection of the poultry rearing area is important to eliminate the disease.

Vaccination is an effective way to control and prevent MD and ND. However, it may be diffi cult to carry out vaccination programme in village chickens due to the free ranging and unconfi ned type of management. Therefore, the route of vaccine administration should be appropriate and convenient to the vaccine administer. Educate farmers about the importance of vaccination in preventing the disease and to convince them that the vaccine used is safe, effi cacious, available and affordable is important (Alexander et al., 2004). Farmers, veterinary services personnel, livestock and social scientist, private businessman (including chicken traders) and non-government organisations should involve in the disease control

activities from the beginning (Alexander et al., 2004). Also, government’s role in the disease control cannot be neglected. Vaccine distributors, training the farmers on the administration of the vaccine, cost of the vaccine, government policy are the elements that have to take into account.

There is no specific treatment or vaccines for Avian Leukosis (Payne et al., 2000). ALV can be transmitted through infected hens to the eggs, hence current approach is to eradicate the ALV from primary breeding level to produce an infection free parent breeding stock (Payne et al., 2000). ALV is not highly contagious compared to other viral agents. Transmission can be reduced by strict sanitation. Therefore, hatchery hygiene is particularly important to prevent the introduction of the disease (Payne et al., 2000). ALV is a fragile virus outside the host, with a half-life of few hours at room temperature and it is susceptible to all common disinfectants (Payne et al., 2000). Thus, eradication programme as well as good hygiene is a very effective measure in preventing ALV infection.

CONCLUSION

A mixed viral infection of ND, MD and AL is reported in village chickens. How the chickens were infected by the agents is unknown. There are too many factors that can contribute to the occurrence of the diseases. However, diseases can be prevented or controlled by some measures. Good hygiene practises including


27

cleaning and disinfection is important in disease control. Due to the free ranging management system, a basic biosecurity system can be applied by farmers although the success may be limited. Vaccination the poultry is another effective way to prevent disease. However, many factors and elements have to take into account.

REFERENCES

1. Aini, I. (1990). Indigenous chicken production in South-east Asia. World’s Poultry Science Journal 46: 51-57

2. Aini, I. (1999). Diseases in rural family chickens in South-East Asia. International Network for Family Poultry Development, INFPDE-Conference 37-41

3. Aini I. (2000). Biosecurity in family fl ocks. Proceeding 21st World’s Poultry Congress, Montreal, Canada, CD-ROM.

4. Anonymous. (2003). Persistence of Disease Agents in Carcases and Animal Products, Report for Animal Health Australia by Scott Williams Consulting Pty Ltd, Revised - December 2003, pp30-33

5. Alexander, D.J. (1988). Methods of Spread. In: Newcastle Disease, D.J. Alexander, Ed., Kluwer Academic Publishers, Boston, pp. 256-272

6. Alexander, D. J., Bell, J.G. and Alders R. G. (2004). A technology review: Newcastle Disease, with special emphasis on its effect on village chickens. FAO Animal Production and Health. Chapter 4.

7. Calnek, B. W. and Hitchner, S. B. (1973). Survival and disinfection of Marek’s Disease virus and the effectiveness of fi

8. lters in preventing airborne dissemination. Poultry Science 52:35-43

9. Fadly, A. M. (2000). Isolation and identifi cation of Avian Leukosis viruses: a review. Avian Pathology 29: 529-535

10. G.Arzey (1989). Mechanism of spread of Newcastle Disease. Technical Bulletin 42. NSW Agriculture and Fisheries.

11. Herczeg J., Wehmann E., R. R. Bragg, P. M. Travassos Dias, G. Hadjiev, O. Werner and B. Lomniczi (1999). Two novel genetic groups (VIIb and VIII) responsible for recent Newcastle Disease outbreaks in SouthernAfrica, one (VIIb) of which reached Southern Europe. Arch Virol 144: 2087-2099

12. Hlozanek I., Jurajda V. and Benda V. (1977). Disinfection of Marek’s Disease virus in poultry dust. Avian Pathology 6(3): 241-250.

13. Maaz Majzoub Abdel-Latif and Abdelmelik Ibrahim Khalafalla (2005). Detection by PCR of multiple subgroups of Avian Leukosis Virus (ALV) in broilers in the Sudan. Journal of Animal and Veterinary Advances 4(3): 407-413

14. Ottiger Hans-Peter (2010). Development, standardization and assessment of PCR systems for purity testing of avian viral vaccines. Biologicals 38: 381-388

15. Payne L. N. and Venugopal K. (2000). Neoplastic diseases: Marek’s Disease, avian leucosis and reticuloendotheliosis. Rev. sci. tech. Off. Int. Epiz 19 (2), 544-564

16. Permin, A. and Bisgaard, M. (1999) A general review on some important diseases in free-range chickens. International Network for Family Poultry Development, INFPDE-Conference 163-167

17. Ramlah A. H. (1999). Production aspects of village chicken in South-east Asian region. International Network for Family Poultry Develpoment, INFPDE-Conference 61-76

18. Spradbrow, P.B. (1993-94). Newcastle disease in village chickens. Poultry Science Review 5: 57-96.

19. Tulman E. R., Afonso C. L., Lu Z., Zsak L., Rock D. L. and Kutish G. F. (2000). The Genome of a very virulent Marek’s Disease virus. Journal of Virology, 7980-7988

20. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2012 NB Version adopted May 2012 Chapter 2.3.14 Newcastle Disease

ACKNOWLEDGEMENTS. We would like to thank the Director-General of the Department of Veterinary Services Malaysia for his kind permission to publish the paper. Special thanks to the staff of Avian Virology for their contributions in the study.


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ABSTRACT. This report describes the parasitological and pathological information retrieved from the samples submitted to Regional Veterinary Laboratory, Bukit Tengah, Penang (MVKBT) following an episode of histomoniasis in a small scale poultry farm. Liver and ceca from two dead turkey birds, water (n=5) and freshwater snails (Pomacea sp, n=7) were stored between 4°C to 10°C until analyzed. Soil samples (n=7) were preserved in 5% formalin and kept at room temperature prior to further testing. The soil samples were filtered through laboratory test sieves before the fi ltrates at each level were examined using direct microscopy. Portions of the liver were processed using routine paraffin technique for histopathological examination and found positive for Histomoniasis. Results from direct microscopy of the liver samples revealed Schistosoma mansoni ova, Sarcocystis sporocysts and Fasciola sp. Heterakis sp ova were detected from cecal content. Four out of seven soil samples were positive for Sarcocystis sporocysts, Coccidia oocyst, Strongyles ova and nematod larva. Water and freshwater snail samples were negative for cercaria

of Schistosoma mansoni. This case was concluded as Histomoniasis, Sarcocystosis and helminthiasis with incidental fi nding of Schistosoma mansoni.

Keywords: liver, histomoniasis, turkey, Schistosoma mansoni, Sarcocystis

INTRODUCTION

The Perlis State Veterinary Services was contacted in early January 2014 by a small scale turkey farmer to advise him regarding the mortality of a number of newly purchased turkeys. The turkeys (n=29) were kept in a mixed farming practice farm with various species of animals including village chicken, ducks, geese and twelve cats. All of them shared the same free-ranging compound. The mortality in the turkeys began after three days upon arrival of the birds from Penang. Neither mortality nor morbidity was noted in the other animals in the farm. This paper describes the findings retrieved from the liver, ceca specimens submitted to Regional Veterinary Laboratory Bukit Tengah, Penang (MVKBT) for confirmation of Histomoniasis. In addition, parasitological information of

HISTOMONIASIS & INCIDENTAL FINDING OF Schistosoma mansoni IN TURKEY BIRDSSHAHAZA O.1*, JIN SENG O.2, NORINA L.1, JAMAIYAH I.1, SALMEAH A.R.1, SUHAIMI A.1, NURUL ANA A.B.1, JAMAL-NASIR M.H.1 AND MASWATI M.A.1

1 Regional Veterinary Laboratory Bukit Tengah, 14007 Bukit Mertajam, Pulau Pinang, Malaysia.

2 State Veterinary Services, Perlis, Malaysia.


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the water, soil, and freshwater snails of the affected farm were also documented.


Two adult dead turkey birds were examined; liver and ceca of the birds were sampled for confi rmation of histomoniasis. Water (n=4), soil (n=7) and freshwater snails (n=7) from the farm compound were also sampled. All samples were stored between 4°C to 10°C and sent to MVKBT for direct microscopic examination with exception to the soils samples. The soil samples were preserved in 5% formalin (Albonico et al., 2013) and kept at room temperature until analysed. The soil samples were filtered through 75 μm and 45 μm laboratory test sieves before the fi ltrates at each level were examined using direct microscopy. Portions of the liver were processed using routine paraffi n technique for histopathological examination. Direct microscopy of the samples and photomicrographs were performed using

light microscope (Olympus BX41, Japan) mounted with camera head (Olympus DP 20, Japan).


Grossly, necrotic foci with alternating pattern of pale and hemorrhagic circle were observed on the liver (Figure 1). Histopathologically, numerous trophozoites which were characterized by pale eosinophilic and surrounded by narrow rims of clear spaces were noted in liver parenchyma(Figure 2). Those fi ndings were consistent with histomoniasis or Blackhead Disease. The fi nding of Heterakis sp. (Figure 3), the intermediate host of Histomonas meleagridis in the cecal content of the dead turkeys had supported the diagnosis of histomoniasis. Histomonas meleagridis causes acute caecal and liver damage in Galliformes (Wei et al. 2011). Life cycle of this protozoa is complex, however, Hu et al., 2004 demonstrated that histomoniasis could spread through a

Figure 1. Hepatic histomoniasis. Figure 2. Oval-shaped histomonad (black arrows ) in liver parenchyma. HE, x1000.


31

fl ock of turkey by phenomenon of cloacal drinking. Interestingly, they also found that direct transmission from bird to bird is not possible in chickens (Hu et al., 2006). It is highly suggestive that direct transmission may occur in turkeys which subsequently accelerate the Histomoniasis rate in the turkeys fl ock, thus explained the high mortality witnessed in turkeys whilst; none of the other Galliformes reared in the farm was affected. The situation experienced in this farm was consistent with statement made by McDougald, 2005 in his review paper of Histomoniasis; “…mortality in turkey could reach 100%, whereby in chickens many outbreaks pass unnoticed”. Direct examination of the fl uid from squashed liver specimen revealed, Fasciola sp. and also Sarcocystis sp. sporocysts. In addition, an oval ovum with lateral spine (Figure 4) which closely resembles ova of Schistosoma mansoni was detected. Sarcocystis is a protozoon that causes sarcocystosis in birds and mammals. Many species of mammals can

act as intermediate hosts for sarcocystis including cattle, sheep, and goats. Defi nitive hosts include human, domestic dogs and cats. Geese, domestic chickens can act as intermediate hosts. In Malaysia, Sarcocystis sp. have been reported in wildlife, for instance, civet cat, squirrel and terrapin (Fazly et al., 2013).

Schistosoma or blood fl uke is a snail-transmitted trematodes. Schistosoma japonicum that cause human intestinal schistosomiasis in Asia was documented to be present in certain area of Malaysia (IAMAT, 2012). However, Schistosoma mansoni which also lead to similar disease in Africa, the Americas and the Middle East (Akinwale et al., 2013) had not been discovered in Malaysia. Successful transmission of these parasite between their various intermediate (freshwater snails) and fi nal hosts (human) may also involve free swimming stages (cercaria) and the encystment of larvae on aquatic vegetation (Woodruff and Upatham, 1992). Therefore, further investigation was

Figure 3. Ovum of Heterakis sp. Figure 4. Schistosoma mansoni ovum


32

performed on water, soil and freshwater snails sampled from the farm compound. Four out of seven soil samples were positive for Sarcocystis sp. sporocysts, strongyles ova, nematode larva and coccidia oocysts. Cercaria of the Schistosoma mansoni was not detected from water and snail samples. The snails were identifi ed as Pomacea sp. Based on this investigation, it can be concluded that the fi nding of Schistosoma mansoni in this case was incidental. Nevertheless, enviromental sampling techniques described by Worrell et al. 2011 could be followed to increase the chance for detection of Schistosoma spp in future sampling. Overall, the fi ndings on multiparasitic infection in the farm described, indicates the need for a better fl ock health and farm management system to be put in place

REFERENCES

1. Akinwale, P.O., Tang, T.H., Doblin, A.S. and Cheah, H.L. (2013). Molecular Survey of Freshwater Snail Intermediate Hosts of Schistosoma malayensis in Northern Peninsular Malaysia, Continental J. Biological Sciences, 6(2):33-41..

2. Albonico, M., Rinaldi, L., Sciascia, S., Morgoglione, M.E., Piemonte, M., Maurell i, M.P., Musella , V., Utzinger, J., Ali, S.M., Ame, S.M., Cringoli, G. (2013). Comparison of three copromicroscopic methods to assess albendazole effi cacy against soil-transmitted helminth infections in school-aged children on Pemba Island, Trans R Soc Trop Med Hyg., 107(8):493-501.

3. Fazly Ann Zainalabidin, Nurulaini Raimy,Mohd Shafarin Shamsudin, Niny Fariza Junoh, Zawida Zahari, Mohd Iswadi Ismail and Chandrawathani Panchadcharam. (2013). Histological findings of Sarcocystis spp. in exotic meat. Malaysian Journal of Veterinary Research. 4(2):51-54.

4. Hu, J., Fuller, L. and McDougald, L.R. (2004). Infection of Turkeys with Histomonas meleagridis by the Cloacal Drop Method, Avian Dis., 48(4):746-50.

5. Hu, J., Fuller, L., Armstrong, P.L. and McDougald, L.R. (2006). Histomonas meleagridis in Chickens: Attempted Transmission in the Absence of Vectors, Avian Dis., 50(2):277-9.

6. International Association for Medical Assistance to Travellers (IAMAT). (2012). World Schistosomiasis Risk Chart. www.iamat.org. Accessed on 16 July 2014.

7. McDougald , L.R. (2005). Black head d isease (histomoniasis) in poultry: a critical review, Avian Dis., 49(4):462-76.

8. Wei, L., Jun-yu, P., Fen, L., Hong-yan, S., Ying, D., Jing, H., and Yi, L. (2011). Identifi cation of Histomonas meleagridis by In Vitro Microculture and Polymerase Chain Reaction, Reports in Parasitology, Dove Press Journal, www.dovepress.com. Accessed on 16 July 2014.

9. Woodruff, D.S and Upatham, E.S. (1992). Snail-transmitted disease of medical and veterinary importance in Thailand and the Mekong Valley, J.Med. & Appl. Malacol, 4: 1-12.

10. Worrel, C., Xiao, N., Vidal, J.E., Chen, L., Zhong, Bo. and Remais, J. (2011). Field Detection of Schistosoma japonicum Cercariae in Environmental Water Samples by Quantitative PCR, Applied and Enviromental Microbiology, 77(6): 2192-2195.

ACKNOWLEDGEMENTS. The authors would like to thank all the staffs of Perlis State Department of Veterinary Services, Penang Department of Veterinary Services for their involvement, full support and help during this case investigation. Special thanks to Mohd. Fairuz Noor Din, staff of MVKB T; Olaoluwa P. Akinwale, Director of Research (Neglected Tropical Diseases), Nigerian Institute of Medical Research, Nigeria; and Tang Thean Hock, Infectious Disease Clusters, Advanced Medical & Dental Institute, Universiti Sains Malaysia for their technical assistance.


33


ABSTRACT. A study was conducted on 240 scavenging chickens randomly obtained from various districts from the state of Penang, Peninsular Malaysia. The chickens were closely examined for visible ectoparasites in the laboratory. The ectoparasites were collected using a blunt forceps and stored in universal bottles containing 70% ethanol. Ten species of ectoparasites were noted which consisted of fi ve species of lice, two species of mites, two species of ticks and one species of chigger. The lice identifi ed were Menopon gallinae, Menacanthus pallidulus, Lipeurus caponis, Goniocotes gallinae and Goniodes dissimilis. These lice occurred in the fl uff of the feathers of the body especially the neck, back, abdomen and wings. The mites were Megninia sp. and Pterolichus sp. Examinations of the ears and combs revealed Haemaphysalis sp., the hard tick. Meanwhile, Ornithonyssus sp., the soft tick was found on feathers, whereas chigger, Leptotrombidium sp. was found attached to the skins. The study also revealed that M. gallinae was the most common ectoparasite with 76.7% occurrence, followed by Pterolichus sp. (69.6%), L. caponis (63.3%), M. pallidulus

(41.7%), Leptotrombidium sp. (17.5%), G. gallinae (9.5%), Haemaphysalis sp. (6.7%), Megninia sp. (3.8%) and Ornithonyssus sp. (3.8%). The least common ectoparasite was G. dissimilis occurring in 2.1% of the chickens.

Keywords : ectoparasites, scavenging chickens, Penang, Peninsular Malaysia

INTRODUCTION

Chickens are the most abundant birds in the world which provide protein in the form of meat and eggs. Scavenging chickens or ‘ayam kampung’ meat has a strong fl avour and is juicier than that of commercial chickens. Therefore, they command higher prices than commercial chickens, more so, if they have not been treated with antibiotics, hormones or antihelminthics. Most of the rural villagers still keep chickens in small fl ocks. They are allowed to range free around the house or the backyard. They require little attention and feed mainly on kitchen wastes, broken grains, worms, snails, insects and vegetation. Due to their free-range and scavenging habits, parasitic infections are commonly high. The chickens have an increased opportunity

ECTOPARASITIC FAUNA OF SCAVENGING CHICKENS (Gallus domesticus) FROM PENANG ISLAND, PENINSULAR MALAYSIAWAHAB A. RAHMAN* AND FARAH HAZIQAHSchool of Food Science and Technology, Universiti Malaysia Terengganu, Kuala Terengganu 21030, Malaysia

* Corresponding address: [email protected]


34

to encounter infective eggs, larvae and intermediate hosts of parasites that can cause serious debilitating infections. On the other hand, inadequate hygiene and the physical environment such as rainfall, humidity, and ambient temperature provide optimum conditions to maintain helminth populations. Severe cases of parasitism can cause mortality (Soulsby, 1982). Heavy infestations can result in increased stress to the chickens and subsequently reduced egg production, poor health, anemia and severely affected chickens may die (Shanta et al, 1971).

There had been few studies on ectoparasitic fauna of chickens in Malaysia (Amin-Babjee and Ragavan, 1985; Sani et. al 1986; Amin-Babjee and Lee, 1994; Wu, 1994). However, most of the chickens from these studies were either obtained from markets or confi ned to certain states within Peninsular Malaysia.

This study examined for ectoparasitic fauna of scavenging chickens kept in free-ranged systems, and randomly obtained from the State of Penang, Peninsular Malaysia. The state of nutrition or body condition of the scavenging chickens was recorded. In addition, parasite infection in relation to sex and age were described.


The study was conducted on 240 scavenging chickens randomly obtained from various districts from the State of Penang, Peninsular Malaysia. The chickens had been left scavenging around

the backyards during daytime and confi ned during night. They fed mainly on kitchen wastes, broken grains, worms, snails, insects, vegetation, food remnants and offal. They were generally small in size with body weights ranging from 1.3 kg to 2.4 kg. All chickens were bought directly from the owners. Estimated age of the chickens was provided by owners. The age of chickens was classifi ed into three groups: (i) below 6 months, (ii) 6 months to 12 months and (iii) 12 months to 24 months.

In the laboratory the chickens were closely examined for visible ectoparasites such as ticks on the ears, comb and wattle. The ectoparasites were collected using a blunt forceps and stored in universal bottles containing 70% ethanol.

Ectoparasites such as lice and mites were collected after slaughtering the chickens. The skin and the feathers were pulled off from the whole carcass, soaked in bottles containing 70% ethanol. Feathers and the skin were carefully examined under a dissecting microscope. Ectoparasites found attached were removed with a pair of fi ne forceps and preserved in universal bottles containing 70% ethanol for further identification. The specimens were removed from 70% ethanol and soaked in lactophenol (a clearing agent) for one week at room temperature. Small punctures were made with a fi ne needle at the abdomen of each specimen in order to remove body contents as well as to facilitate entry of lactophenol into the specimen. The specimens were


35

observed under a stereomicroscope. At least 10 specimens of each species were measured to calculate the mean dimension of the adult according to the procedures of Mustaffa Babjee (1977). Ectoparasite species were identifi ed based on keys and illustrations developed by Soulsby (1968) and Mustaffa Babjee (1977). The severity of the infestation was noted and ranked as low, medium or high. These categories were designated depending on the number of ectoparasites on each chicken (low infestation: 1-50, medium infestation: 51-100 and high infestation: more than 100). It can be considered analogous to those used by Harshbarger and Raffensperger

(1961) and Sychra et. al (2008). With the exception of mites, species present were noted (+) but the number of individuals were not counted due to its overwhelming abundance.

RESULTS

Table 1 shows different species of ectoparasites isolated from scavenging chickens in Penang Island. Ten species of ectoparasites were noted which consisted of fi ve species of lice, two species of mites, two species of ticks and one species of chigger. The lice identifi ed were Menopon gallinae, Menacanthus pallidulus,

Table 1. Prevalence of ectoparasites in 240 scavenging chickens.

Ectoparasites

No. of chickensinfested

Prevalence (%)

Mean abundance

± SE

Range of ectoparasites/

chickenPredilection

sites

Lice

Menopon gallinae 184 76.7 67.3 ± 8.244 1-210 feathers

Menacanthus pallidulus

100 41.7 11.0 ± 1.793 1-40 feathers

Lipeurus caponis 152 63.3 20.1 ± 4.394 1-125 feathers

Goniocotes gallinae 23 9.5 1.1 ± 0.466 1-9 feathers

Goniodes dissimilis 5 2.1 5.2 ± 1.338 1-43 feathers

Mites

Megninia sp. 9 3.8 1.5 ± 1.143 1-40 feathers

Pterolichus sp. 167 69.6+

(too numerous)- feathers

Ticks

Haemaphysalis sp. 16 6.7 1.9 ± 1.439 1-48 combs/ears

Ornithonyssus sp. 9 3.8 14.0 ± 7.414 1-210 feathers

Chiggers

Leptotrombidium sp. 42 17.5 23.8 ± 13.630 1-508 skin+ Not counted


36

Lipeurus caponis, Goniocotes gallinae and Goniodes dissimilis. These lice occurred in the fl uff of the feathers of the body especially the neck, back, abdomen and wings. The mites were Megninia sp. and Pterolichus sp. Examinations of the ears and combs revealed Haemaphysalis sp., the hard tick. Meanwhile, Ornithonyssus sp., the soft tick was found on feathers, whereas chigger, Leptotrombidium sp. was found attached to the skins.

The study revealed that M. gallinae was the most common ectoparasite with 76.7% occurrence, followed by Pterolichus sp. (69.6%), L. caponis (63.3%), M. pallidulus (41.7%), Leptotrombidium sp. (17.5%), G. gallinae (9.5%), Haemaphysalis sp. (6.7%), Megninia sp. (3.8%) and Ornithonyssus sp. (3.8%). The least common ectoparasite was G. dissimilis occurring in 2.1% of the chickens.

The highest mean abundance was recorded for M. gallinae with 67.3 (±8.244), followed by Leptotrombidium sp. and L. caponis with 23.8 (±13.630) and 20.1 (±4.394) respectively. Meanwhile, Ornithonyssus sp., M. pallidulus and G. dissimilis were recorded with the mean abundance of 14.0 (±7.414), 11.0 (±1.793) and 5.2 (±1.338) respectively. The lowest mean abundance were recorded for Haemaphysalis sp., Megninia sp. and G. gallinae with 1.9 (±1.439), 1.5 (±1.143) and 1.1 (±0.466) respectively.

At least one species of ectoparasite was found on each infested chicken. It was found that 7% were infected with single infections and 75% with multiple

endoparasites infections. Most of the chickens had between three to fi ve species of ectoparasites. The highest number of chickens was recorded to harbour four species of ectoparasites affecting 47 (20%) chickens, followed by three and fi ve species of ectoparasites with 39 (16%) and 32 (13%) chickens respectively. None of the chickens harbored all ten species of ectoparasites. Only one chicken was heavily infested with nine species of ectoparasites (Figure 1).

Table 2 shows ectoparasite infestations with different groups of arthropods. Seventy-seven (32%) of the chickens were infested with just only lice, whereas 71 (30%) were infested with lice and mites. Twenty two (9%) chickens were infested with lice, mites and chiggers. Only 3 (1%) chickens were found infested with all four groups of arthropods (lice, mites, ticks and chiggers). Most of the chickens were found to be infested with at least one group of arthropod. However, none were found infected with chiggers alone.

As shown in Table 3, 108 chickens were recorded to have high ectoparasite infestation with the prevalence of 45%, followed by low infestation with 83 chickens infested by ectoparasites and the prevalence of 35%. Medium infestation was the least common degree of infestation, affecting 49 chickens and a prevalence of 20%.

Out of 43 chickens belonging to below 6 months of age, 11 (5%) were infested with high, 6 (3%) with medium and 6 (3%) with low infestations (Table


37

Figure 1. Number of ectoparasite species per chicken.

1720

39

47

3228

11

3 105

101520253035404550

One Two Three Four Five Six Seven Eight Nine

Num

ber

of c

hick

ens

Number of ectoparasite species per chicken

Table 2. Ectoparasite infestations with different groups of arthropods.

Arthropod group No. of chickens infested

1) Lice 77 (32%)

2) Mites 5 (2%)

3) Ticks 3 (1%)

4) Chiggers 0

5) Lice and mites 71 (30%)

6) Lice and ticks 2 (0.8%)

7) Lice, mites and ticks 15 (6%)

8) Lice, mites and chiggers 22 (9%)

9) Lice, mites, ticks and chiggers 3 (1%)

Table 3. Degree of ectoparasite infestation.

Ectoparasite infestations

No. of chickens infested

Low 83 (35%)

Medium 49 (20%)

High 108 (45%)

Table 4. Degree of ectoparasite infestations according to age group.

AgeDegree of ectoparasite infestations Total

chickens Low Medium High

< 6 months 26 (11%) 6 (3%) 11 (5%) 43

6 months - 12 months

40 (17%) 25 (10%) 46 (19%) 111

> 12 months 17 (7%) 18 (7.5%) 51 (21%) 86


38

4). In the age group of 6 months to 12 months, 46 (19%) chickens were found with high infestation, followed with low and medium infestations with 30 (13%) and 25 (10%) chickens respectively. High infestation was the most common degree of infestation among chickens above 12 months of age, with 51 (21%) chickens, followed by medium and low infestations with 18 (7.5%) and 16 (7%) chickens respectively. Chickens above 12 months of age recorded the highest mean number of ectoparasite species with 5 species, followed by 6 months to 12 months of age and below 6 months of age with 3 and 2 species respectively (Table 5).

There was a signifi cant difference somewhere among the mean number of ectoparasites species on the dependent variable for the three age groups [F(2, 237)=29, p=0.000]. Post-hoc comparisons using the LSD test indicated that the mean number of ectoparasite species for Group 1 (M=1.98, SD=2.262), Group 2 (M=2.91, SD=1.998) and Group 3 (M=4.63, SD=1.898) was signifi cantly different from

one another. Table 6 shows the mean difference between the three age groups.

Figure 2 shows the prevalence of ectoparasite infestations according to gender. Out of a total of 240 scavenging chickens examined, 198 chickens infested which comprised of 105 (44%) males and 104 (43%) females. However, no infestation were recorded on 42 (13%) chickens.

As shown in Table 7, high infestation was the most common degree of infestation in males and females with 54 (23%) chickens infested respectively. Medium infestation was found in 28 (12%) male and 21 (9%) female chickens. However, low infestation was found in 38 (32%) male and 45 (38%) female chickens.

Table 8 shows the mean number of ectoparasite species according to gender. Male and female chickens have the same mean number of ectoparasite species. The mean number of ectoparasite for both genders was three species.

There was no signifi cant difference in the mean number of ectoparasites species for males (M=3.26, SD=2.167) and

Table 5. Mean number of ectoparasite species at different ages.

AgeMean number

of ectoparasites species

< 6 months 2

6 months - 12 months

3

> 12 months 5

Table 6. Mean difference of ectoparasites at different ages.

(L) Age (J) Age Mean ± SE

< 6 months6 -12 months -0.933 ± 0.362*

> 12 months -2.651 ± 0.376*

6 -12 months< 6 months 0.933 ± 0.362*

> 12 months -1.718 ± 0.289*

> 12 months< 6 months 2.651 ± 0.376*

6 -12 months 1.718 ± 0.289** The mean difference is signifi cant at the 0.05 level


39

females [M=3.46, SD=2.322; t(238)=-0.69, p=0.491]. Meanwhile, the magnitude of the differences in the means was very small (eta squared=0.002).

DISCUSSION

In Malaysia, production of poultry meat and eggs are dominated by commercial broiler sector although most of rural villagers in this country still continue

rearing native chickens under the backyard system (Aini, 1990).

Scavenging chicken or ‘ayam kampung’ is of a dual-purpose type, reared for both its meat and eggs. It has a low egg-laying performance and the eggs are smaller than that of commercial chicken eggs. Generally, ‘ayam kampung’ meat is preferred by most consumers, probably due to the specifi c texture and taste, more so, it is free of drug residues. Nowadays,

Male44%

Female43%

No ectoparasite infesta�ons

13%

Figure 2. Prevalence of chickens infested with ectoparasites according to gender.

Table 7. Degree of ectoparasite infestations according to gender.

Gender

Degree of ectoparasite infestations

TotalLow Medium High

Male 38 (32%) 28 (12%) 54 (23%) 120

Female 45 (38%) 21 (9%) 54 (23%) 120

Table 8: Mean number of ectoparasites species according to gender.

GenderMean number of

ectoparasites species

Male 3

Female 3


40

‘ayam kampung’ has become popular in rural households, where there is an emerging trend of consumer awareness towards organically grown chickens, with customers increasingly willing to pay higher prices for good quality meat.

Most of the rural villagers in Peninsular Malaysia still keep chickens in small fl ocks. The free-range system is the most popular and viable production system practiced, because it requires very little inputs for housing, feeding and health care (Ramlah and Shukor, 1987). The chickens are allowed to range free around the house or the backyards and fi nd their feeds from the surrounding environment that takes the forms of kitchen wastes, worms, snails, insects, food remnants or offal. They enjoy more freedom of movement as compared to chickens reared under the intensive system, where they are crammed and may lack movement.

According to Soulsby (1982), poultry can easily be infected with diseases mainly caused by bacteria, viruses, fungal and parasitic pathogens. However, parasitic infections are often neglected. It often constitutes to low productivity and impairs the health status of chickens. Parasitic infection is commonly high in scavenging chickens due to an increased opportunity to encounter the infective eggs, larvae, and intermediate hosts of parasites. These can cause considerable damage due to anemia, malnutrition, weight loss, lowered egg production as well as mortality in young and weak chickens (Fatunmbi and Adene, 1979; Soulsby, 1982; Pandey et. al 1992).

The majority of the chickens examined were found to be infested with ectoparasites. There were four groups of arthropods (lice, mites, ticks and chiggers) reported in this study with ten species of ectoparasites consisting of fi ve species of lice, two species of mites, two species of ticks and one species of chigger. The fi nding is similar to the study conducted in Selangor with ten species of ectoparasites as reported by Sani et. al (1986). The majority of the chickens examined were infested with at least one species of lice. The lice identifi ed were M. gallinae, M. pallidulus, L. caponis, G. gallinae and G. dissimilis. These lice occur in the fl uff of the feathers of the body especially the neck, back, abdomen and wings. The mites were Megninia sp. and Pterolichus sp. Examinations of the ears and combs revealed Haemaphysalis sp., the hard tick. Meanwhile, Ornithonyssus sp., the soft tick was found on feathers, whereas chigger, Leptotrombidium sp. was found attached to the skins.

Benbrook (1965) reported that the most common ectoparasite of chickens were lice. In the present study, the shaft louse, M. gallinae was recorded as the most common ectoparasite with the highest mean abundance. A similar fi nding has reported by Hagos and Eshetu (2005) in a study conducted on ectoparasites of local scavenging chickens in Central Ethiopia although Amin-Babjee et. al (1997) showed that the tick, Haemaphysalis wellingtoni was the most common ectoparasite observed in Selangor, Malaysia. This louse


41

was found on the shafts than beneath the wings and body surfaces. The typical symptom of M. gallinae infection is feather-loss (Ikpeze et. al 2008).

In this study, Pterolichus sp. was observed for the fi rst time in this country. This feather mite was found attached on the wing feathers of the chickens. Besides, it is very rare to fi nd infestation of chigger (larval trombiculid mites) in domestic chickens. However, this study revealed that chiggers from the genus Leptotrombidium was found attached to the skin of 35 chickens. Although it had been described previously by Nadchatram and Dohany (1974) in a pictorial key of Southeast Asian chiggers, this six-legged larva had not been reported on G. domesticus in Malaysia. However, chiggers, Neoschongastia gallinarum reported by Amin-Babjee et. al (1997) and Eutrombicula hirsti listed in the check-list of domestic chickens by Lee et. al (1991) were absent.

Multiple ectoparasite infestation was recorded in the chickens investigated in which the highest number of chickens was recorded to harbor four species of ectoparasites. The greater diversity of ectoparasites in scavenging chickens was due to the free-range habit where the chickens have high exposure to the environment as well as low input management system. Besides, Mekuria and Gezahegn (2010) also stated that suitable environmental conditions may enhance the propagation and life cycle progression of the diverse ectoparasites species.

Actual enumeration of ectoparasites obtained was used to access the severity of infestations. The overall rate of infestation in chickens examined was high, with more than 100 ectoparasites per chicken. High infestation of ectoparasites was probably because the chickens were left to scavenge and not confi ned, where they have more access to outdoor areas which exposure them to the sources of infestations.

In the present study, there was a signifi cant difference in the mean number of ectoparasite species between young and adult chickens. According to Nnadi and George (2010), adults may have high infections due to their gregariousness to the environment. They scavenge over greater areas as compared to growers or younger individuals. This could partly explain fi ndings that adult recorded the highest mean number of ectoparasite species compared to growers or younger chickens.

On the other hand, this may be associated with long periods of exposure. Besides, the variation in ectoparasite infestations in different ages may also be related to the habitat or environments with poor hygiene. Despite differences in ectoparasite infestations between age groups, this study also examined the ectoparasites infestations in male and female chickens. It showed that there was no difference between the mean number of ectoparasite species and the sexes of chickens. According to Mekuria and Gezahegn (2010), infestations might be


42

by chance and there is no direct relation to animal reproductive system.

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2. Amin-Babjee, S. M. and Lee, C. C. (1994) Parasites of the fi ghting breed of domestic chicken, Gallus gallus domesticus. In: Applied Biology beyond the Year 2000. Third Symposium of Malaysian Society of Applied Biology, M. K. Vidyadaran and S.C. Quah (Eds). Malacca. pp. 16-18.

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17. Sani, R. A., Harisah, M. and Bohari, Y. (1986) Parasites of the indigenous fowl (Gallus gallus domesticus) in Selangor. Kajian Veterinar 18 : 183-189.

18. Shanta, C. S., Song, C. Y. and Wan, S. P. (1971) Helminth parasites of the alimentary tract of broilers in North Malaya. Malaysian Veterinary Journal 5: 1-8.

19. Soulsby, E. J. L. (1968) Helminths, Arthropods and Protozoa of Domesticated Animals (Sixth Edition of Mönnig’s Veterinary Helminthology and Entomology). Baillière, Tindall and Cassell Ltd. pp. 5-683.

20. Soulsby, E. J. L. (1982) Helminths, Arthropods and Protozoa of Domesticated Animals (Seventh Edition of Mönnig’s Veterinary Helminthology and Entomology). Baillière, Tindall and Cassell Ltd. pp. 5-683.

21. Sychra, O., Harmat, P. and Literák, I. (2008) Chewing lice (Phthiraptera) on chickens (Gallus gallus) from small backyard fl ocks in the eastern part of the Czech Republic. Veterinary Parasitology 152: 344-348.

22. Wu, K. Y. (1994) In: A study of tick (Haemaphysalis wellingtoni) and trombiculid mites (Neoschongastia gallinarum) feeding lesions in the domestic chicken, Gallus gallus domesticus. An Animal Industry Project Paper, Faculty of Veterinary Medicine and Animal Science. pp. 20.


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ABSTRACT. The development and availability of follicles is an indicator to predict which of the follicle sizes are suitable to recover the oocytes assisted by means of ultrasonography of transvaginal oocyte retrieval (OPU). Thus, the study was done in order to characterize the follicular recruitment and distribution in response to the repeated removal of follicles, and thus to determine the availability of follicles and immature oocytes harvested repeatedly for two consecutive days of OPU in beef cows. Results indicated that 24-h OPU showed signifi cantly greater numbers of medium and large follicles than small categories (P<0.05). However the 48-hr of OPU does not showed any differences of follicles categories (P>0.05). The mean total number of follicles and immature oocytes recovered were higher (P<0.05) in 24-hr OPU (13.76±1.2 and 7.38 ± 1.7) compared to 48-hr OPU (9.08 ± 1.5 and 3.54 ± 1.00) with the oocyte retrieval rate of 51.22% and 38.17%, respectively. The

morphological classifi cation indicated the 24-hr oocyte retrieval produced 62% of suitable immature oocytes that can be used for in vitro embryo production. In conclusion, the repeated removal of two consecutive days of OPU has averted the development of dominant follicle, and thus, gave an atmosphere to the subordinate follicles to continue growth relatively to an equal proportion of small, medium and large categories of follicles. Due to the reduction of follicle and recovery rate at 48-hr it is suggested that OPU be carried out later than 48 hour so that the follicle has more time to increase the diameter size.

Keywords: follicle development, immature oocytes, transvaginal oocyte retrieval (OPU), follicle stimulating hormone (FSH), oocyte quality, in vitro embryo production,

THE FOLLICLE CHARACTERISTIC AND IMMATURE OOCYTE QUALITY OBTAINED FROM REPEATED TRANSVAGINAL OOCYTE RETRIEVAL IN Bos indicus BEEF COWSAZIZAH A.1*, AHMAD J.2, AHMAD NAZRI A.R.3, IZUAN BAHTIAR A.J.2 AND SAADIAH J.1

1 Strategic Livestock Research Centre, Malaysian Agricultural Research and Development Institute (MARDI),

Persiaran MARDI- UPM, Serdang, Selangor

2 Strategic Livestock Research Centre, Malaysia Agricultural Research and Development Institute (MARDI),

Kluang Research Station, Locked Bag 525, 86009 Kluang, Johor.

3 Ruminant Section, Livestock Commodity Development Division, Department of Veterinary Services, Wisma

Tani, Level 1, Lot 4G2, Precinct 4, Federal Government Administrative Center, 62630 Putrajaya


RE# MJVR -0027-2014


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INTRODUCTION

Ultrasound-guided technology has emerged to impale the small and preovulatory follicles in cattle (Pieterse et al., 1989; Pieterse et al., 1991). This technology offers a new reproductive approach in harvesting oocytes from live cattle (Callesen et al., 1987; Pieterse et al., 1988: 1991; Kruip et al., 1991) which is so called transvaginal ultrasound-guided oocyte retrieval (OPU).

OPU is an alternative procedure to domestic females which are genetically valuable but unable to produce viable embryos through the natural procedures. The technology can also be used on oocytes harvested from older ovulating or nonovulating cows, or cows with physical injuries or having an abnormal cervix.

Oocytes from live donor cows are an important source of known quality genetic material for use in in vitro production of embryos repeatedly (Akshey et al., 2004; Velez et al., 2012). The in vitro embryo production is effi cient in order to exploit the female gamete as steps to shorten the generation interval in a modern animal husbandry. Many studies have been done to understand the development of the follicles in order to predict the availability of follicle sizes which is suitable for oocyte harvesting assisted by OPU.

The cattle used for OPU can be either hormone stimulated or non-stimulated (Pieterse et al., 1989; Pieterse et al., 1992; Walton, 1993; Bungartz et al., 1995), clinically infertile animals (Looney et al., 1994) or from young prepubertal heifers

and early postpartum cow. Thus, the technology is an aid of getting oocytes for in vitro embryo production and can also be used for germplasm preservation of endangered exotic species.

Oocytes from live donors are usually used in in vitro embryo production or for in vitro culture system. The oocytes retrieved could differ in their growth phase, cell morphology, age and the stage of atresia corresponding to the follicle population on ovary stroma. The size of follicles plays a role in OPU because it mirrors the activity of the follicles and only oocytes of befi tting size could be impaled, and aspirated through OPU technique. The morphological classifi cation of oocyte is associated with oocyte quality and the meiotic competence of the oocytes. Thus, it is benefi cial to observe the relationship between the distribution of the follicles and the retrieval rate of immature oocytes in a two consecutive days of OPU in order to identify if OPU could be done repeatedly.

Therefore, the aim of this study was to characterize follicular recruitment and distribution in response to the repeated removal of follicles, and thus, to observe the availability and quality of immature oocyte retrieved repeatedly in two consecutive days of OPU in beef cows.


Animal Selection And Management

A total of 13 Kedah-Kelantan crossbred beef cows were selected from a breeding


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herd managed at MARDI Research Centre, Kluang, Johor. The cows ranged from 3 to 4 year in age and body weight from 250kg to 350 kg with an average Body Condition Score of 4.5 kg (scale 1: severely thin, 4: moderate, 8: obese) adapted with slight modification of the method described by Richard et al. (1986). The cows were assigned into two treatments: two consecutive; 24 (24-h) and 48 (48-h) of repeated OPU after removal of controlled internal drug releasing device (CIDR; Pfi zer New Zealand Ltd).

During the study, the cows were managed in a semi-intensive system, whereby cows were released for grazing in the morning and maintained indoor for the rest of the day in individual pens and fed with cattle pellets and had free excess to water. The cattle pellets contained 15.0% crude protein and 17.6 megajoule calculated gross energy (GE) were fed to cows based on the maintenance requirement of beef cows (ARC, 1980) at a rate of 1 kg per 100 kg body weight per day in addition to the estimated intake of feed from grasses.

Oestrous Synchronisation And Hormonal Treatment

The cows were on feed adaptation period for 14 days before oestrous synchronization. All cows at random stages of oestrous cycle were inserted intravaginally with CIDR containing 1.38 g progesterone for 7 d, followed with an intramuscular injection of 500 μg cloprostenol of prostaglandin synthetic analogue (PGF2α; Estrumate

Schering-Plough Animal Health, Australia) at day 5. Superovulation was done using a total of 17 ml follicle stimulating hormone (FSH; Ovagen®, InterAg, New Zealand) and was administered twice a day at decreasing doses for four consecutive days (3.5 ml x 2, 2.5 ml x2, 1.5 ml x 2, and 1 ml x 2), beginning on day 3 post CIDR insertion.

Ultrasonography Of Follicle Development Study

Both ovaries were scanned using a 5.0 MHz mechanical annular array transrectal (AAS) transducer (Pie Medical, Maastricht, Netherlands) attached to an ultrasonographic scanner (Pie Medical Scanner 250+, Maastricht, Netherlands). The transducer lubricated with an aseptic gel was inserted into the vagina. The cows were given 2 ml of an epidural anaesthesia injection of 2% lignocaine (Troy Laboratories, Limited, Australia) before the transducer insertion into the vagina.

The transducer was adhered along the wall of the vagina in order to visualize the follicles situated on the surface of ovary medulla, through an ultrasound monitor. The image of the ovary was frozen, and the diameter of follicles was measured using an electronic caliper of the ultrasound device. The diameter of a follicle of unequal shape was measured twice and the average was taken as the diameter of the follicle. Then, the diameter of follicles was categorized


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into small (≤ 4 mm), medium (> 4.1 mm – 8.0 mm) and large (≥ 8.1 mm) follicles.

Preparation Of The Cows

The cows were administered a local anaesthesia of 3-5 ml of 2% lignocaine (Troy Laboratories, Limited, Australia) at the upper epidural region in order to prevent abdominal straining and give better manipulation of the ovaries during the palpation. The cow was restrained to the head crushed on the standing positioned. The perineum then was cleaned with hibiscrub before the guidance and probe insertion into the vagina.

Oocyte Retrieval And Selection

The OPU was conducted immediately after measuring the follicle’s diameter. A guidance system was incorporated into the OPU device alongside the transducer of an ultrasonographic scanner (Pie Medical Scanner 250 plus, Maastricht, The Netherlands) in order to visualize the ovaries images. A 19 G 19G x 2” 1.1 × 50 mm luer type disposable, single lumen needle (Terumo Neolus, Belgium) was connected to a stainless steel connector for oocyte aspiration. The silicone tubing was attached to the stainless steel connector and passed through along the stainless steel tube. Then it was placed alongside a mechanical annular array sector (AAS) multi-angle dual frequency transducer (Pie Medical, Maastricht, The Netherlands) in a stainless steel casing with a holder with

a diameter of 45 mm as described by Bols et al. (1995).

The transducer in a stainless steel casing was lubricated and inserted into the vagina through the perineum. During aspiration, the ovaries were pushed against the head of transducer and a needle in stainless steel casing was pushed through the vaginal wall and impaled into the follicle to be punctured. The aspiration was done by means of a footstep connected to a vacuum pump and collecting bottle by a silicone coated tubing that was controlled by the foot of the technician.

One follicle was impaled and aspirated each time. When the aspiration completed, the needle was withdrawn and fl ushed together with the suction set using complete fl ushing medium (Vigro®, AB Technology Inc., Pullman, USA) consisting of phosphate buffer saline (PBS) with 10% heparin, serum and antibiotic. The procedure was discontinued when follicle size ≥ 4 mm was not visible on the ultrasound monitor. The collecting bottle containing oocytes then was brought to the laboratory for oocyte examination and evaluation.

The collected f luid containing immature oocytes was poured into several 60 mm petri dishes. Subsequently all oocytes were examined and separated into three morphological classifi cation under a stereomicroscope. The morphological classifi cation of oocyte quality based on cumulus-oocyte complex (COC) followed the method described by Wurth and Kriup (1992), that is, “A” for presence of a clear


47

and compact cumulus and translucent ooplasm, “B” for dark and compact cumulus and dark ooplasm, and “C” for dark and expanded cumulus and dark ooplasm.

Statistical Analysis

Data were analysed using independent t-test of SPSS version 21. The three categories of follicles of each period of 24-h and 48-h were analysed with one-way analysis of variance (ANOVA), where as data of follicle numbers, follicle sizes categories, and oocyte quality in a 24-h and 48-h of two consecutive days of OPU were analysed using ANOVA for a repeated measure. A confi dence level of 95% or more was considered to be statistically signifi cant.


The ultrasonography for follicle categories showed the mean of both medium and large follicles observed to be higher in

24 hours compared to 48 hours OPU (p<0.05) (Table 1). However, both groups did not show any difference in proportion of follicles of small diameter (p>0.05). In this study, more oocytes can be retrieved from medium and large follicles on 24-h and 48-h repeated OPU.

The retrieval rate of oocyte was 51.22% and 38.17% in 24-h and 48-h of repeated OPU, respectively (Figure 1). It shows the retrieval rate of oocytes that has been impaled in two consecutive days of OPU was 13% higher in 24-h compared to 48-h OPU. Similarly, the mean total number of follicles in 24-h OPU was approximately 34.01% higher (P<0.05) compared to 48-h OPU (Figure 1). Subsequently, the morphological quality classification of oocytes indicated that 24-h oocyte retrieval presented higher number of immature oocytes, which might be suitable candidates for in vitro embryo production (in vitro culture system: in vitro maturation, fertilization and culture) technologies.

Table 1. The number of small, medium and large follicles (mean ± SEM) following ultrasanography of two consecutive days of transvaginal oocyte retrieval (OPU) in beef cows

Period of oocyte retrieval1

Mean no. of follicles observed2

No. of follicles Mean no. of oocytes retrieved3

Small (≤ 4 mm)

Medium(>4 – 8 mm)

Large(≥ 8.1 mm)

24-h 13.76±1.2 x 2.23±0.5 a x 4.84±0.7 b x 5.69±0.7 b, x 7.38±1.69 x

48-h 9.08±1.5 y 1.62±0.6 a x 2.15±0.5 a y 3.31±0.8 a, y 3.54±1.0 y

1 Period of oocyte retrieval: 24-h = 24 h after CIDR removal; 48-h = 48 h after CIDR removal 2 Mean of follicle observed = Number of follicle/Number of cattle3 Mean of oocyte retrieval = Number of oocyte recover/Number of animalsa,b Values within rows with different superscripts are signifi cantly different at p<0.05x,y Values within with columns different superscripts are signifi cantly different (p<0.05)


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The results showed that more immature oocytes could be selected as candidates for in vitro embryo production technologies. The study demonstrated that OPU technology is an appropriate tool that can be used to exploit female reproductive capacity although they are differing in oestrous cycle phases.

Oocytes from live donors are a good source in order to obtain quality and valuable genetic materials for in vitro embryo production technologies. Thus, the present study is a pioneering effort in getting fresh good quality oocytes from live donors that can be offered in large numbers. In the study, it was shown that the follicles developed in an equal proportion

of small, medium and large sizes at 24-h OPU. Similarly, 48-h OPU gave equal proportions of small, medium and large sized follicles.

In cattle, antral follicles of 3-8 mm in diameter are the main suppliers of oocytes used for in vitro embryo production by OPU (Joel et al., 2002). In a follicular development, the follicles develop in a wave of 4 to 12 follicles at a time, and a cow usually has two to four waves of these growing follicles in one oestrous cycle.

A follicle wave involved the synchronous development of a group of follicles from a pool of growing follicles (Driancourt, 1991). The largest follicle which is gonadotrophin-dependent

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²Recovery rate

Figure 1. The rate at which follicles are available and oocytes are retrieved in two consecutive days of transvaginal oocyte retrieval (OPU). 1 Follicle number percentage = Total number of follicles observed (24 or 48-Hr) / Grand total number of follicles x 1002 Retrieval rate = Number of oocyte retrieved / Total number of follicles x 100


49

from this pool is then recruited into a population which grows continuously, and hence inhibits the other surrounding or subordinate follicles from growing and thus undergo atresia.

However, in OPU cattle, the retrieval opposes the development of dominant follicle and this gives a space for the subordinate follicle to grow and increase in size. Thus in this study, enhancing the availability of a relatively homogeneous and equal proportions of small, medium and large follicles allows the suitable harvesting of oocytes at 48-h OPU.

Although the OPU could be done in two consecutive days, the availability of mean number of follicles suitable for retrieval, and number of oocytes recovered on 48-h OPU reduced approximately 30% to 50% of actual total number of follicles available and oocyte retrieval of 24-h OPU. This could probably be due to the duration of OPU that was too short and the time lapse was insuffi cient to recruit the development of new follicles, and unable to increase the diameter size.

Although the two consecutive days of OPU increased the retrieval rate from 51.22% to 89.39% (Figure 1), it produced

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24-Hr48-Hr

Figure 2. The morphological of immature oocyte classifi cation (mean ± SEM) following ultrasonography in two consecutives days of transvaginal oocyte retrieval (OPU) in beef cattle. The morphological classifi cation of oocyte was followed as described by Wurth and Kriup (1992); (A) presence of a clear and compact cumulus and translucent ooplasm, (B) dark and compact cumulus and dark ooplasm, and (C) dark and expanded cumulus and dark ooplasm.


50

an erroneous effect on animal behaviour. Therefore, the repeated OPU of two consecutive days of the present study was determined to be not suitable. It could be suggested to extend the time of oocyte retrieval from 48-h to 72-h or 96-h interval in order to conduct OPU.

The other factors that contributed to the lower number of oocyte retrieved during the 48-h OPU was the degree of calmness and stage of passiveness of the animals. This could probably be related to their experience during the 24-h OPU which led to the animals’ discomfort and uneasiness, and difficulty in handling, even though they were treated with local anaesthesia. Thus, the condition led to the reduced retrieval rate of the immature oocytes. The two consecutive days of OPU would only showed a benefi cial effect for 24-h OPU and increased the degree of discomfort and the animals tended to struggle in 48-h OPU.

The number of follicles developing within each ovary did not differ. The repeated OPU on two consecutive days did not increase further the total number of follicles. It only resulted in a decrease in number of medium and large follicles (p<0.05). The results of our investigation also demonstrated that 62% of the oocytes recovered from individual animals via OPU were suitable for use in in vitro embryo production (Figure 2).

The availability of follicles and immature oocytes is higher at 24-h than 48-h of repeated OPU. Due to the reduction in number of follicles and the recovery rate

in 48-h OPU, it is suggested that OPU be carried out later than 48 hours so that the follicle has more time to increase in size. Further studies should be carried out to examine the ultra structure quality in order to determine the stages of meiosis of those immature oocytes aspirated from these animals for in vitro embryo production.

CONCLUSION

The retrieval rate obtained in the present study described an application of the technology under fi eld conditions. Repeated retrieval at short intervals is possible but it may produce uncertainty in animal behaviour. However, the aspirated immature oocytes obtained are viable and could potentially be used for in vitro embryo production technology. In addition, the immature oocytes can be obtained from cows irrespective of the differences in oestrous phases.

Repeated removal for two consecutive days of OPU has altered the follicular recruitment and distribution by avoiding the development of dominant follicles. Thus, this condition gave an atmosphere to the subordinate follicles to continue to grow in an equal proportion of small, medium and large sized follicles. However, the lower percentage of oocytes that can be used in the in vitro culture system upon OPU suggests further research.


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REFERENCES

1. Akshey Y.S., Palta P., Manik R.S., Gupta V. and Chauhan M.S. (2004). Effect of removal of follciles through repeated transvaginal follicle aspiration on subsequent follicular populations in Murrah Buffalo (Bubalus bubalis. Asian-Aust. J. Anim. Sci. 18(5): 632-636.

2. Agricultural Research Council (ARC). (1980). The Nutr ient Requirements of Ruminant Livestock. Commonwealth Agricultural Bureaux, Slough, pp 351.

3. Bols P.E.J., Vandenheede J.M.M., Van Soom A. and de Kruip A. (1995). Transvaginal ovum-pick up in the cow: A new disposable needle guidance system. Veterinary Vision, Vol 3: 6.

4. Bungartz L., Lucas-Hahn A., Rath D., and Niemann H. (1995). Collection of oocytes from cattle via folllcular aspiration aided by ultrasound with or without gonadotropin pretreatment and in different reproductive stages. Theriogenology 43: 667-675.

5. Callesen H., Greve T. and Christensen F. (1987). Ultrasonically guided aspiration of bovine follicular oocytes. Theriogenology 27: 217 abstr.

6. Driancourt M.A. (1991). Follicular dynamics in sheep and cattle. Theriogenology 35: 55-79

7. Joel A., Carter Shane Bellow, Marius Meintjes, Oscar Perez, Edward Ferguson and Robert A. Godke. (2002). Transvaginal ultrasound-guided oocyte aspiration for production of embryos in vitro. Arch. Tierz., Dummerstorf 45 (1): 99-108

8. Kruip, Th. A.M., Pieterse M.C., van Beneden Th.H., Vos P.L.A.M., Wurth Y.A. and Taverne M.A.M. (1991). A new method for bovine embryo production: A potential alternative to superovulation. Vet. Rec. 128: 208-210.

9. Looney C.R., Lindsey B.R., Gonseth C.L. and Johnson D.L. (1994). Commercial aspects of oocyte retrieval and in vitro fertilization (IVF) for embryo production in problem cows. Theriogenology 41: 67-72.

10. Pieterse M.C., Kappen K.A., Kruip Th. A.M. and Taveme M.A.M. (1988). Aspiration of bovine oocytes during transvaginal ultrasound scanning of the ovaries. Theriogenology 30: 751-762.

11. Pieterse M.C., Kappe K.A., Kruip T.A.M. and Taverne M.A.M. (1989). Aspiration of bovine oocytes during transvagrnal ultrasound scanning of ovaries. Theriogenology 30: 751-762

12. Pieterse M.C., Vos P.L.A.M., Kruip T.A.M., Wurth Y.A., Van Beneden T.H., Willemse, A.H. and Taverne M.A.M. (1991). Transvaginal ultrasound-guided follicular aspiration of bovine oocytes. Theriogenology 35: 19-24

13. Pieterse M.C., Vos P.L.A.M., Kruip T.A.M., Wurth Y.A., Van Beneden T.H., Willemse A.H. and Taverne M.A.M. (1992). Repeated transvaginal ultrasound-guided ovum pick-up in ECG treated cows. Theriogenology 37: 273 abstr.

14. Richard, M.W., Spitzer, J.C. and Warner, M.B. (1986). Effects of varying levels of postpartum nutrition and body condition at calving on subsequent reproductive performance in beef cattle. J. Anim. Sci. 62: 300-306.

15. Velez J.C., Arnold C., Jacobson C.C., Norris J.D., Choi Y.H., Edwards J.F., Hayden S.S. and Hinrichs K. (2012). Effects of repeated transvaginal aspiration of immature follicles on mare health and ovarian status. Equine Veterinary Journal 44(43): 78-83.

16. Walton S.J., Christie K.A. and Stubbings R.B. (1993). Evaluation of frequency of ultrasound guided follicle aspiration on bovine ovarian dynamics. Theriogenology 39: 336 abstr.

17. Wurth Y. A . and Kruip T.A.M. (1992). Bovine embryo production in vitro after selection of the follicles and oocytes. In: Proceedings of the 12th International Congress of Animal Reproduction (ICAR). The Hague, The Netherlands. 1: 387-389.


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ABSTRACT. Brucellosis is a zoonotic and widely contagious infection in animals. Cattle, water buffalo, and bison are animals that are highly susceptible to Brucella abortus infection. A group of forty-two Murrah and Nili-Ravi buffaloes, which were positively diagnosed for brucellosis, were found dead a week after an antibiotic therapy regime using long acting tetracycline and streptomycin. The antibiotic therapy was applied to control the shedding of Brucella organisms from host animals. Death started to occur after 8 days of treatment and continued until day 23 post-treatment. A holistic investigation was done to determine the cause of death. A total of 182 samples were taken from 31 carcasses and sent for bacteriology, haematology, histopathology, parasitology, virology, serology, clinical chemistry, feed and mineral analysis, and water quality investigation and analysis. It was concluded that the antibiotic therapy regime carried out on the immunocompromised buffaloes

that were infected with B. abortus may have attributed to the death of the buffaloes.

Keywords: Brucella abortus, antibiotic therapy, immunocompromised.

INTRODUCTION

Brucella is one of world’s widespread zoonotic diseases causing infectious abortion (Boschiroli et al., 2001), except cats which are resistant to Brucella infection (Gul & Khan, 2007) B. abortus has been eradicated from Japan, Canada, Northern Europe, Australia and New Zealand. Brucellosis has been known to survive and replicate in different host species, which can lead to infection from animal reservoir to human host (Godfroid et al., 2011). Human brucellosis is commonly caused by accidental contamination from infected animal or animal product (Boschiroli et al., 2001). According to Smits & Kadri ﴾2005﴿, the brucellosis transmission to human population has been signifi cantly decreased due to effective vaccination –

MULTIPLE FACTORS CAUSING DEATH IN BRUCELLA-POSITIVE MURRAH AND NILI-RAVI BUFFALOES: A CASE REPORT MAIZATUL AZLINA A.M.1, MASWATI M.A2, NORAZURA A.H.3, MOHD. FAIZ M.K.1, SHAHAZA O.1, BOHARI M.J. 3, NORHAFIZA H.3, JAMNAH O. 3, SYAMSUL A. 2, KAMARUL RIZAL M.I.4, RUZANAH ASMAH A.S4. AND SABARIAH B.1

1 Malaysia Veterinary Institute, Kluang, Johor

2 Regional Veterinary Laboratory Bukit Tengah, Pulau Pinang.

3 Veterinary Research Institute, Ipoh, Perak.

4 Department of Veterinary Services, Wisma Tani, Presint 4, 62630 Putrajaya

* Correspondence author: [email protected]

RE# MJVR -0035-2014


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based control and prevention programmes, but remain as uncontrolled problems in regions of high endemicity such as Asia, Mediterranean, Middle East, Africa, and Latin America. The pathological lesion in domestic animals is different and infl uenced by species and strain of Brucella, immune status of animal host, exposure route, sexual maturity, infection rate and the virulence of the organism ﴾Ahmed et. al., 2012﴿

Buffalo is the largest milk producer in Asia especially Pakistan and India. These breeds, Nili-Ravi and Murrah have been established as top milk producing buffalo breeds. In January 2010, a farm received 31 Murrah buffaloes (30 females and 1 male) from India, and subsequently, 70 female and 8 male Nili-Ravi buffaloes from Pakistan in December 2010. Serological screening in Kuala Lumpur International Airport Quarantine Station showed that 4 out of 78 Nili-Ravi buffaloes were positive for Brucella abortus and 5 more positive for tuberculosis (TB). All of these positively infected animals were isolated from non-infected animals. However, they were not culled but quarantined in the farm compound. According to Ahmed et al. (2012﴿, a serological survey conducted indicated that the incidence of Brucella infections among buffaloes can range from as low as 0.13%-1.49% to as high as 5.29%-25.49%. On May 2011, the farm received another 34 pregnant Murrah females from a neighbouring farm for milk production.

Results from the disease-screening programme in the farm showed that there was an increase in the numbers of Brucella abortus positive buffaloes from 5.13% (4/78) in 2011 to 71.8% (84/117) in 2012. Another source of infection for herdmates is when an infected water buffalo expelled the bacterium during abortion and the subsequent ingestion of virulent B.abortus caused its infection. Therefore, all the positively infected buffaloes were segregated from the negatively infected buffaloes in separate paddocks. Fagiolo et al. stated that, generally Bubalus bubalis is a healthy animal, but when removed from their natural habitat of hot and humid regions, they become susceptible to most diseases and parasites which infect cattle and become favourable for micro-organisms and parasite proliferation.

This study was conducted to study the effect of antibiotic treatment for animals infected with brucellosis.

MATERIAL AND METHODS

Animals and Study Area

In this study, two breeds of buffaloes namely Murrah and Nili-Ravi were selected for study. This was based on the occurrence of mortality in the same farm and enclosure. The study was conducted in Kluang, a district located near the middle of the state of Johor in the southern part of Peninsular Malaysia.


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Experimental Antibiotic Therapy

Due to the high genetic value of the animals, the management of the farm decided not to cull the buffaloes as proposed in culling procedures. Instead, the buffaloes were quarantined inside the compound of the farm. An antibiotic treatment was scheduled to control the shedding of Brucella abortus in all the seropositive buffaloes (Appendix 1). Treatment was streptomycin and long-acting oxytetracycline (streptomycin 25 mg/ kg, SID, intramuscularly for 8 days and oxytetracycline 25 mg/kg, q3d, intramuscularly for 42 days). The purpose of this treatment was to reduce shedding of the Brucella abortus in water buffalos kept in the Kluang Farm, using a treatment regimen described in cows (Radwan et al., 1993) with some modifi cations. Next, all the treated animals were subjected to serological screening by Rose Bengal Plate Tests (RBPT) and the positive sera were sent to the Veterinary Research Institute (VRI) for Complement Fixation Tests (CFT) every 2 months starting from day 1 post-treatment. Last sampling was scheduled 18 months after the post-treatment. The success of the treatment should show the animal to be negative for brucellosis by using CFT consecutively for 18 months.

There were two groups of animals being treated which were divided into Group 1 (n=45) and Group 2 (n=51). The treatment was scheduled simultaneously for the 2 groups. The division into two

groups was the solution for a shortage of manpower and limitation of space in the holding yard for performing the treatment regime. The pregnancy status of these buffaloes in both groups was unknown. On day 5 of treatment, two buffaloes were found weak and were on sternal recumbency. Deaths were seen on day 8 treatment. Treatment was stopped totally on day 15. However, death still occurred until day 23 post-treatment. Total mortality was 42 buffaloes and 2 buffaloes, which were then slaughtered for disease investigation.

Clinical Signs

Clinical signs manifested by the buffaloes during and after treatment were emaciation and dehydration, with body score 1 out of 5 (Figure 1), weak (Figure 2), inappetence, bloody urine (Figure 3), hyper salivation, ruminal stasis, sternal recumbency and melena (Figure 4), rectal prolapsed and abortion.

Samples and Laboratory Analysis

A total of 182 samples (grass, water, blood, vaginal swab, and organ samples) were taken and sent to the laboratories (Appendix 2). These samples were sent for bacteriology (routine bacterial culture, salmonella, brucella, brucella and Q fever (IHC),histopathology, virology, serology, haematology, chemical, feed and mineral analysis, and water quality tests (Appendix 2).


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A total of 51 blood and serum samples from buffaloes in Group 2 (untreated group) were also taken for serology, virology, parasitology and haematology tests. The buffalo fatality data was recorded for epidemiological analysis.

Post-mortem

Post-mortem was conducted on the 42 buffaloes. Samples were taken from 31 buffaloes. Post-mortem samples were sent for disease investigation at Veterinary Research Institute (VRI) and Regional Veterinary Laboratoty, Bukit Tengah (RVL BT).

Statistic Analysis

In this study, antibiotic was the risk factor. This study used a relative risk method to determine the possibility of death in buffaloes given antibiotic compared to buffaloes not receiving treatment. Odd ratio method was used to determine that death might occur in treated buffaloes given antibiotic treatment and buffaloes not given antibiotic treatment. The statistic was calculated using a standard formula for relative risks and odds ratio.

RESULTS

Pathological Results

Macroscopic Findings

From the post-mortem conducted, there were frothy f luid found in the trachea lumen and haemorrhagic tracheal cartilage; serosanguineous fl uid in abdominal cavity with presence of helminth, hepatization and severe haemorrhage of lungs; enlarged mesenteric lymph nodes; fatty liver ; patchy and pulpy liver; friable spleen; congestion in small intestine especially in jejunum and ileum; bleeding in small intestinal mucosa; necrotic enteritis, necrotic infl ammation in cotyledons and endometrium of uterus; and presence of Paramphistomum sp. in rumen were observed in 2 slaughtered animal and 7 dead animal on the fi rst day investigation being conducted. Almost similar postmorten changes were seen day 2 investigation being done in another 5 dead buffaloes.

Histopathological Findings

Histopathology examination showed that most organ tissues of liver, spleen, heart, kidney, lungs, intestines and uterus had severe congestion with thrombosis in blood vessels. There were necrosis of hepatocytes near the central hepatic vein in liver tissue and bacterial colonies were seen in the blood vessel (Figure 11). Splenic cells had undergone degeneration with depleted spaces and kidney tubular cells also


57

had undergone diffuse tubular necrosis. Histopathologically, lungs showed congestion and proteineous oedema in alveolar spaces. Intestinal tissues showed severe lymphocytic enteritis with necrosis in villus causing the mucosal layer to slough off (Figure 12). Uterus showed neutrophilic infl ammation with bacterial colonies in the intestinal mucosa.

Laboratory Tests

Bacteriology

E. coli was the most abundant bacteria isolated from 21 out of 31 cases (67.74%) followed by Staphylococcus epidemidis and Klebsiella pneumonia with the percentage of 19.36% (6 cases) each. Salmonella sp. was also isolated from intestinal samples with S. albany isolated from 4 cases (12.90%) and S. typhimurium from 1 case (3.22%). The isolated E. Coli was serotyped and polymerase chain reaction (PCR) with detection of virulent gene showed that the bacteria were not from a pathogenic strain. The antibiotic sensitivity test showed that the E. coli strain were resistant to streptomycin.

Virology

Isolation and detection for bovine viral diarrhoea (BVD) or infectious bovine rhinotracheitis (IBR) were conducted using tissue culture tests and PCR. Results from Group 2 showed 48/51 (94%) positive for

IBR and 10/51 ( 20%) positive for BVD via serum neutralisation tests (SNT).

Parasitology

In Gro up 1, a total of 7/15 cases (46.67%) were posit ive for blood protozoa Eperythrozoon and Babesia, while Anaplasma were detected from 6/15 cases (40% Setaria sp. were isolated from 5/15 cases (33.33%) which were tested for helminthiasis. Psoroptes mites dan Hematopinus sp. also isolated in 3 out of 15 (20%). In Group 2, buffaloes were negative for blood protozoa.

Serology

CFT serology tests on 10 serum samples from Group 1 showed that 7 (70%) of the animals were positive for Brucella abortus and 2 (20%) were positive for leptospirosis. Meanwhile, Group 2 showed 34/51 (65%) were positive against Brucella abortus and 17/51 (33%) were positive for leptospirosis.

Immunohistochemistry test

Seven cases showed positive results for Brucella abortus and Q Fever whereas 4 cases were positive for B. abortus only in Group 1.

Feed and mineral analysis

Proximate analysis done on Guinea grass samples showed the crude protein value was very low for values suggested for


58

cattle (suggested value >16, sample value 8.2). Percentage of calcium, phosphorus, and crude fat also showed a value below the standard value suggested for feed pellet (Table 1).

Water quality

Two drinking water samples were tested. Water from House A was from weaner house while water from House B was taken from Group 1 house . Analysis results showed that the water from both houses were safe for drinking.

Clinical chemistry

Seven serum samples were tested for clinical chemistry. Results showed that the buffaloes had damaged skeletal and cardiac muscles observed from the increased levels of AST, CK and LDH enzymes. All serum samples tested also

showed an increase in parameters of AST, CK, LDH, phosphorus and magnesium due to the increase in the stress level of buffaloes. Untreated buffaloes from Group 2 showed that the animals were receiving low protein diet, low urea-N and albumin. Buffaloes from Group 1 showed signifi cant increased of urea-N and decrease of albumin parameter which indicates kidney failure. Liver function tests especially liver enzymes results showed that there was liver failure characterised by increase of liver enzymes (AST and LDH). Decrease in the cholesterol parameter were observed in Group 1 buffaloes.

Haematology

There were no haematology results from Group 1 since no blood samples were taken. Haematology analysis (PCV) from Group 2 showed that 33/51 (67%) buffaloes had anaemia.

Table 1. Feed and mineral test results

Sample type

Guinea grass PKE Pellet Soil

Nutritive Suggested

Value

As received

basis Dry matter

As received

basis Dry matter

As received

basis

As received

basis Dry matter

Crude protein, % 3.50 8.23 15.97 - 1.26 2.33 > 16.00

Calcium, % 0.08 0.18 0.62 - 1.35 2.51 2.40

Phosphorus, % 0.08 0.18 0.62 - 0.00 0.00 1.00

Crude fat, % 0.77 1.80 1.37 - 0.01 0.01 > 6.00

Total Ash, % 3.47 8.17 4.89 - 42.00 77.89 < 7.00

Crude fi bre, % 13.31 31.30 20.30 - 0.92 1.70 >12.00

Moisture, % 57.49 - 7.71 - 46.08 - <12.00


59

Epidemiology analysis

This analysis was done to relate the risk factors between Group 1 (treated group) and Group 2 (untreated group). Analysis done was to determine the relative risk and odds ratio which measures the relation between risk factors and death.

The analysis result was made based on: Risk factor – antibiotic.

Attack rate a/a = b × 100 : 88% Relative risk (how many possibilities of death in buffaloes given antibiotic compared to buffaloes not receiving treatment )= a/a + b / c/c + d= 40/45 / 24/111= 0.8888 / 0.2162= 4.11 The possibility of death in treated buffaloes was 4 times higher than untreated buffaloes.

Odd ratio a/b/c/d (determine odd ration death might be occur in treated buffaloes compared to untreated buffaloes)

= a × d / c × b= 40 × 87 / 24 × 5= 3,480 / 120= 29 Ratio of death caused by antibiotic treatment was 29 times greater than death in untreated buffaloes.

DISCUSSION

Results of laboratory investigations indicate that there were many factors that caused deaths in Group 1 (treated) buffaloes.

Histopathological fi ndings showed that there were circulatory disturbance caused by disseminated intravascular coagulopathy (DIC). It was indicated by severe congestion formation with thrombosis in blood vessels in vital organs. This condition results from constriction or blockage of blood supply to related tissues which leads to cell necrosis and distruption of organ functions, subsequently leading to death. The condition matched with the post-mortem fi ndings which showed hepatization and severe congestion in

Table 2. Number of dead and surviving animals in two distinct groups, (i) buffaloes given antibiotic treatment and (ii) buffaloes not given antibiotic treatment

Dead Not dead Total

Given antibiotic treatment (treated)

40 (a) 5 (b) 45 (a+b)

Not given antibiotic treatment (untreated)

24 (c) 87 (d) 111 (c+d)

Total 64 (a+c) 92 (b+d) 156 (N)


60

lungs, pulpy and haemorrhagic kidney, and congestion in intestines, necrotising enteritis and friable spleen.

At the same time, feed supplies were low in protein and minerals. The buffaloes having hypoproteinaemia and it was observed as edematous lungs and trachea, and also fatty liver. In malnutrition state the liver will become fatty liver due to fat degeneration.

DIC was suspected to be caused by substances or septicaemic condition which can result in thrombosis. Toxemia or septicaemia occurred due to long-term antibiotic treatment which disturbed the function of normal intestinal microfl ora. It also gave chances to resistant pathogens to multiply in the intestines. These opportunistic pathogens infiltrated via blood vessels to other vital organs. The findings of necrotic intestines showed lymphocytes infi ltration which indicates the buffaloes were burdened with viral infection before the treatment started. Even though there were no virus isolated from tissue samples in Group 1, SNT results for Group 2 showed 94% were positive for IBR and 20% positive for BVD. SNT results of Group 1 taken before treatment started, showed overall that buffaloes in Group 1 were 92% positive for IBR and 13% positive for BVD (farm data).

One of the possibilities of the occurrence of DIC was infection from non-pathogenic bacteria E. coli. The E. coli in this case was not from a pathogenic strain and was assumed to be the contaminant in normal condition.

The immunocompromised buffaloes had low immune system response which was not suffi cient to fi ght multiple infections. Furthermore, antibiotic sensitivity tests showed that the E. coli isolates were resistant to streptomycin. Toxin from E. coli could cause damage to the endothelium cells of the blood vessels and could promote thrombosis formation.

Concurrent infection of blood protozoa (Eperythrozoon, Babesia and Anaplasma) also worsened the condition in Group 1 buffaloes because the blood protozoa caused damage in red blood cells, stimulated blood coagulation leading to thrombosis and DIC.

The antibiotic treatment itself played a major role in circulatory disturbances and deaths of the animals. According to epidemiology analysis using antibiotic treatment as a risk factor, the relative risks of buffaloes treated were 4 times greater in Group 1 (treated buffaloes) than Group 2 (untreated buffaloes). Meanwhile, the odd ratio analysis showed that the treated buffaloes have 29 times greater death possibility. This analysis indicates that the antibiotic treatment have a role as an enhancer to cause of death.

There are not many literature published on the adverse effect or toxicity of oxytetracyclines and streptomycin. There is a possiblity that the infection could occur from bacteria which are resistant to the antibiotics (Lee, 2009). This can result in disturbances of the digestive system by interrupting the normal microfl ora activity leading to an


61

increase incidence of concurrent infection. Infection of opportunistic microorganism such as fungi, yeasts, and resistant bacteria is deemed as a possibility when broad-spectrum antibiotics are used. Severe and even fatal diarrhoea can occur in horses receiving tetracyclines, especially if the animals are severely stressed or critically ill (Merck Veterinary Manual Online, 9th edition). An increase in BUN can be expected because tetracyclines interfere with protein synthesis even in the host cells and therefore tend to be catabolic. The combined use of glucocorticoids and tetracyclines often leads to a signifi cant weight loss, particularly in anorectic animals (Merck Veterinary Manual Online, 9th edition).

Regarding treatment of brucellosis infection, histopathology fi ndings and IHC specifi c tests for detection of brucella antigen showed positive detection of brucella antigen in death buffaloes’ uterus (Group 1), indicating that the treatment was not effective in controlling brucella infection. Meanwhile, prevalence of brucellosis and leptospirosis were high in Group 2 (65% and 33% respectively). The treatment was not effective in Group 1, due to the condition of the animals which is critically ill, immunocompromised, stressed and malnutrition. The treatment might be successful if done in brucella seropositive animals without other concurrent multiple infections. The farm management should improve the food nutrition, stable condition and parasite control before starting the treatment. Buffaloes in Group 1 were

brucella seropositive to start with, and some buffaloes were having concurrent multiple infections of BVD, IBR, Q Fever, helminthiasis, blood protozoa infections and leptospirosis.

There is no available vaccine for the prevention of brucellosis in human. Thus, knowledge of the disease prevalence and reporting of brucellosis to health authorities is extremely important.

CONCLUSION

Streptomycin and oxytet racycline antibiotic treatment which were given to the buffaloes in order to treat brucellosis did not give positive result. The antibiotic treatment had resulted in a disturbance of the digestive system and ruminal microfl ora activity, causing severe damage to the animals. There was also secondary bacterial infection by antibiotic resistant bacteria. With the multiple infection, digestive system disturbances and feed which did not meet the physiological needs of the animals, the animals failed to survive due to an immunocompromised state and multiple organs failure.

REFERENCES

1. Ahmed, Y.F., Sokkar, S. M., Desouky, H. M. and Madbouly, A. A. (2012). Pathological Studies on Buffalo-Cows Naturally Infected with Brucella melitensis. Global Veterinaria 9 2012, (6): pp 663-668.

2. Boschiroli, M.L., Foulongne, V. and O’Callaghan, D. (2001). Brucellosis: A world wide zoonosis. Current Opinion in Microbiology 2001, 4: pp 58–64


62

3. Godfroid, J., Scholzc, H.C., Barbierd, T., Nicolasd, C., Wattiaue, P., Fretine D., Whatmoref A.M., Cloeckaertg A., Blascoh J.M., Moriyoni I., Saegermanj C., Mumak J.B., Al Dahoukl S., Neubauern H., Letesson J.-J. (2011). Brucellosis at the animal/ecosystem/human interface at the beginning of the 21st century. Preventive Veterinary Medicine 102. pp 118 – 131.

4. Gul, S.T. and Khan, A. (2007). Epidemioogy and Epizootology Of Brucellosis: A review. Pakistan Vet. J, 145-151.

5. The Merck Veterinary Manual Online, 9th Edition. www.merchmanuals.com/vet/pharmacology/antibacterial_agents/tetracycline.html (Accessed on Dec 23th, 2014)

6. Lee, J.H. (2009). Antimicrobial resistance of Escherichia coli O26 and O111 isolates from cattle and their characteristics. Veterinary Microbiology 135 (2009). pp 401–405

7. Radwan, A.I., Bekairi, S.I., Al-Bokmy, A.M., Prasad, P.V.S., Mohamed, O.M. and Hussain S.T. (1993). Successful therapeutic regimens for treating Brucella melitensis and Brucella abortus.Rev. sci. tech. Off. int. Epiz., 1993,12 (3), 909-922

8. Smits, H. L.and Kadri, S. M. (2005). Brucellosis in India: A deceptive infectious disease. Indian J Med Res 122, November 2005, pp 375-384.

Appendix 1. Activity Schedule For Brucellosis Treatment In 96 Heads Of Murrah & Nilli Ravi Buffaloes

NO. ACTIVITY2012 2013

Nov Dec Jan Feb Mac Apr May June July Aug Sept Oct Nov Dis

1.

Purchase materials

and consumables

2. Serological testing

Ist sam-pling

2nd sam-pling

3rd sam-pling

4th sam-pling

5th sam-pling

3. Report writing

NO. ACTIVITY2014

Jan Feb Mac Apr May June July Aug Sept Oct Nov Dis

1.

Purchase materials

and consumables

2. Serological testing

6th sam-pling

7th sam-pling

8th sam-pling

9th sam-pling

3. Report writing


63

ACKNOWLEDGEMENT. The authors would like to thank Director General of Department of Veterinary Services, Director of Institut Veterinar Malaysia, Director of Regional Veterinary Laboratory Bukit Tengah, Director of Veterinary Research Institute, and all staff of Institut Veterinar Malaysia who involved directly or indirectly in completing this study. The authors would like to thank the students of Faculty of Veterinary Medicine, Universiti Malaysia of Kelantan for their help in preparing this article.

Appendix 2

No. Samples Laboratory Tests

1. Internal organs (5 organs–liver, spleen , kidney , lungs, heart)

VRI (12 buffaloes/ 60 samples)MVKBT (14 buffaloes/ 51 samples)

1. Routine culture2. Salmonella3. Impression Smear4. Histology5. Brucella & Q-Fever (IHC)

2. Reproductive organs(uterus, cotyledon, testicle)


1. Routine culture2. Brucella3. Brucella & Q-Fever (IHC)

3. Digestive system(intestines)


1. Routine culture2. Salmonella3. Impression Smear4. Histology5. Brucella & Q-Fever (IHC)

4. Blood (EDTA) VRI (7 buffaloes/ 14 samples)

1. Blood protozoa2. Complete Blood Count3. PCV4. Virus isolation

5. Blood (plain tube) VRI (10 buffaloes/ 60 samples)

1. Brucella (CFT& RBPT)2. Melioidosis (CFT)3. Leptospirosis (LMAT)4. BVD (SNT)5. IBR (SNT)6. Clinical Chemistry

6. Skin scraping, insects collection VRI (2 skin scraping, 4 bags of insects, 1 worm) 1. Direct smear2. Species Identifi cation

7. Lymph nodes, brain, swabs (vaginal, nasal)

VRI(Lymph nodes – 1 buffaloes/3 samples)Brain – 2 buffaloes/ 6 samplesNasal swab – 2 buffaloes/3 samplesVaginal swabs – 8 buffaloes/8 samples

1. Routine culture2. Salmonella3. Virus isolation

8. Culture plate VRI (7 buffaloes/ 8 samples) 1. Brucella isolation

9. Feed (Total Mix Ration, Guinea grass), soil VRI (3 samples) 1. Proximate analysis

2. Mineral tests

10. Water VRI (2 samples) 1. Water Quality Test


65


ABSTRACT. The aim of this study was to investigate the bacteria found in different parts of the genital system in cows and the susceptibility to different types of antibiotics. The genital systems of sixteen cows were collected from Al-Hilla, Iraq slaughterhouse. Isolation and identifi cation of bacteria were made for each part of the genital system and antibiotic susceptibility tests was conducted to the isolated bacteria. The results of this study indicated that there were several types of bacteria present in the genital system sof cows. Different species of bacteria were isolated from the samples including, Escherichia coli (28.97)%, Klebsiella spp. (16.82)%, Salmonella spp. (14.95)%, Proteus spp. (13.08)%, Staphylococcus aureus (11.21)%, Staphylococcus epidermidis (8.41)% and Streptococcus spp. (6.54)%. In vitro susceptibility towards different types of antibiotic indicated high susceptibility of Escherichia coli to antibiotic group impenem and ciprof loxacin, while Klebsiella spp. was found to be most susceptible to ciprofl oxacin and amikacin. Both Escherichia coli and Klebsiella spp. showed resistance to piperacillin and tetracycline. It was concluded that

Escherichia coli was the most predominant bacteria in genital system of cows and were most susceptible to antibiotic impenem and ciprofl oxacin.

Keywords: cow, genital system, E. coli, antibiotic susceptibility, ciprofl oxacin

INTRODUCTION

Infection of uterus by different types of bacteria in cows affects on reproductive effi ciency with decrease in milk yield and mainly affects on cost of treatment (Wang et al., 2013; Sheldon et al., 2006). Previous study by Al-jebori (2013) in Iraq reported that the percentage of pathological cases of uterus in cow consist about 29.4% distributed as either acute or subacute and also causes chronic infl ammation, which represent as adenomyoma and fi broma. Endometritis, pyosalpinx, hemosalpinx, salpingit is, mucometra, pyometra, permetritis, parametritis, uterine abscess, these are the most pathological cases observed in genital systems in cows and buffalo cows (Azawi et al., 2008; Mansor and Majeed, 2005). Hameed et al. (2010) revealed that Escherichia coli, Streptococcus spp. and Klebsiella spp. were

ANTIBIOTIC SUSCEPTIBILITY OF BACTERIA ISOLATED FROM THE GENITAL SYSTEM OF COWS IN AL-HILLA, IRAQSULAKE FADHIL AL-ZUBAIDISurgery and Obstetric Department, Veterinary Medicine College, Al-Qasim Green University, Iraq

Corresponding author: [email protected]

RE#MJVR-0033-2014


66

the most isolated bacteria from aborted fetuses of cows under stress due to other infection. Bacteria that cause infl ammation of uterus have been considered as uterine pathogens, potential uterine pathogens or opportunistic contaminant bacteria which could affect ovarian activity (Sheldon et al., 2002a; Williams et al., 2005; Williams et al., 2007). These bacteria include Arcanobacterium pyogenes, Escherichia coli, Clostridium perfringens, Corynebacterium spp., Staphylococcus aureus, Streptococcus uberis, Proteus mirabilis, Pseudomonas aeroginosa, Klebsiella pneumonia and Bacillus spp. (Dolezel et al., 2010; Udhayavel et al., 2013; Dini et al., 2012; Mshelia et al., 2012; Mshelia et al., 2014a; Šiugždaitė et al., 2013 and Burfeind et al., 2014). Studies have also been carried out to investigate the susceptibility to antibiotics for bacteria responsible for infection of the genital system in domestic animals (Udhayavel et al., 2013; Mshelia et al., 2014b; Tel, 2011 and Silva et al., 2011). The aim of this study was to investigate the bacteria found in different parts of the genital system in cows and the susceptibility to different types of antibiotics.


Collection of samples

The genital system of sixteen cows were collected from an Al-Hilla slaughterhouse in the period from October 2013 to April 2014. The collection consisted of 112

samples of different parts of the system including vagina, cervix, uterine body, uterine horns and oviducts. Each part was dissected using a surgical blade and the mucosae gently swabbed for bacteriological studies. Each swab was cultured immediately or stored in a transport medium until cultured.

Isolation and identifi cation of bacteria

The culture media used for isolation and purifi cation of bacteria included nutrient agar, blood agar, MacConkey agar, Mannitol salt agar, SS agar medium (for Salmonella) and Eosin methylene blue (EMBA) agar. Inoculated media were incubated aerobically at 37ºC for 24 hours.

All the isolates were stored in brain heart infusion broth with 15% glycerol at -20ºC until further use.

The bacteria isolates were identifi ed by culture morphology and biochemical character is t ics. For the cu lt u re characteristics, discrete colonies on the agar surface were observed. The shape, size, consistency and colour were studied. Gram-stained slides of the isolates were examined microscopically as a study of its cellular morphology. Biochemical tests were catalase, oxidase, IMVIC test (indol production, methyl red, vogas-proskauer and citrate utilization), TSI (triple sugar iron). Individually isolated colonies of the same morphology were inoculated on appropriate culture media prepared according to standard protocols as described by (Forbes et al., 2007).


67

Antibiotic susceptibility testing

Antimicrobial susceptibility tests of the isolates on antibiotics was determined by the disc diffusion technique on Muller Hinton agar using commercially available discs following CLSI guidelines (2010). Sterile swabs were used to inoculate the suspension by streaking on the prepared and dried Mueller Hinton agar plate evenly. It was then allowed to stay for 3-5 minutes. Sterile forceps were used to place the antimicrobial discs on the inoculated plates. The plate was incubated at 37°C for 18-24 hours within 30 minutes after applying the disc. The diameter of each zone of inhibition was measured in millimeters using a meter rule on the underside of the plate. The zone diameter of each isolate was compared with CLSI Published Limits and its chart was then used to interpret the zone sizes of inhibition. Results were recorded as susceptible, intermediate susceptible or resistant, based on the zone size of each antimicrobial disc used. The results were then interpreted according to CLSI documentation (CLSI, 2010).


From the 112 samples collected, only 20 samples did not show any bacterial growth, while the other 92 samples showed growth of more than one bacteria. A total of 107 isolates were identifi ed. 79 isolates (73.83%) were Gram-negative bacteria distributed as Escherichia coli (28.97)%, Klebsiella spp. (16.82)%, Salmonella

spp. (14.95)% and Proteus spp. (13.08)%, while the remaining 28 isolates (26.16%) were Gram-positive bacteria identified as Staphylococcus aureus (11.21)%, Staphylococcus epidermidis (8.41)% and Streptococcus spp. (6.54)%. Table 1 shows the distribution of bacteria species in different parts of the genital system.

Out of the sixteen genital systems tested, only two cows were in pregnancy condition. The distribution of bacteria isolated from these samples were illustrated in Table 2. Pathological cases were found in the dissection of uterine horns and uterine body where inf lammation, pus formation or degeneration of endometrium were observed.

Escherichia coli and Klebsiella spp. isolates subjected to antibiotic sensitivity profi le are shown in Figure 1. It shows that Escherichia coli were highly sensitive to antibiotic group impenem and Ciprof loxacin. Whereas Klebsiella spp. showed a high level of sensitivity to amikacin, ciprofl oxacin with different degrees of sensitivity to another type of antibiotics.

The most predominant bacteria isolated were Escherichia coli and Klebsiella spp. for which sensitivity tests were conducted. Escherichia coli have been reported by other authors to be the most predominant bacteria in cow, ewe and doe genital systems neither microfl ora nor pathogenic cases (Wang et al., 2013; Mshelia et al., 2014b; Otero et al., 2000 and Al-Hilali and Al-Delemi, 2001) in cow


68

Table 1. Bacteria distribution in different parts of the genital system of cows

PartStaphy-lococcus aureus

Strepto-coccus

spp.

S.epidermid

E. ColiKlebsiella

sppProteus

sppSalmonel-

la spp.Total(%)

Right Salpinx

4 - - 3 2 2 314

(13.08)

Left Salpinx

2 1 - 7 - 3 114

(14.08)

R. Uterine horn

1 1 3 3 4 - 416

(14.95)

L. Uterine horn

2 2 1 4 2 3 317

(15.88)

Uterine body

1 1 2 - 3 1 210

(9.34)

Cervix 1 2 - 6 4 3 117

(15.88)

Vagina 1 - 3 8 3 2 219

(17.75)

Total(%)

12(11.21)

7(6.54)

9(8.41)

31(28.97)

18(16.82)

14(13.08)

16(14.95)

107(100)

Table 2. Distribution of bacteria in genital systems of pregnant cows

PartStaphy-lococcus aureus

Strepto-coccus

spp.

S. epidermid

E. ColiKlebsiella

sppProteus

sppSalmonel-

la spp.Total (%)

Right Salpinx

4 - - 3 2 2 314

(13.08)

Left Salpinx

2 1 - 7 - 3 114

(14.08)

R. Uterine horn

1 1 3 3 4 - 416

(14.95)

L. Uterine horn

2 2 1 4 2 3 317

(15.88)

Uterine body

1 1 2 - 3 1 210

(9.34)

Cervix 1 2 - 6 4 3 117

(15.88)

Vagina 1 - 3 8 3 2 219

(17.75)

Total(%)

12(11.21)

7(6.54)

9(8.41)

31(28.97)

18(16.82)

14(13.08)

16(14.95)

107(100)

R.S. = Right Salpinx, L.S. = Left Salpinx, R.U.H. = Right Uterine horn, L.U.H. = Left Uterine horn, U.B. = Uterine body, C=Cervix, V=Vagina.


69

and also in ewe (El-Arabi et al., 2013 and Al-Zubaidi et al., 2013).

The most predominant bacteria were E-coli and Klebsiella spp for which sensitivity test was made. Regarding E-coli, many authors reported it to be the predominant bacteria in cow, ewe and doe genital systems neither microf lora nor pathogenic cases (Wang et al., 2013; Williams et al., 2005; Williams et al., 2007; Dolezel et al., 2010; Udhayavel et al., 2013; Dini et al., 2012; Mshelia et al., 2012; Mshelia et al., 2014a; Šiugždaitė et al., 2013; Otero et al., 2000; Sheldon et al., 2002b; Sheldon et al., 2009; Sheldon et al., 2010; Martins et al., 2009; Oliveira et al.,

2013; Penna et al., 2013 and Mshelia et al., 2014b). Regarding E-coli, ciprofl oxacin and imipeneme have been found to be the most sensitive among the battery of antibiotics used in the in vitro study. The sensitivity of E-coli to ciprofl oxacin is in accordance to the fi nding by Mshelia et al., 2014a; Zinnah et al., 2008; Gani et al., 2008; Goncuoglu et al., 2010; Romanus et al., 2012 and Parul et al., 2014. However, Udhayavel et al. (2013) reported low sensitivity of E-coli to ciprofl oxacin. None of the isolates were found to be sensitive to tetracycline. Moges et al. (2013) observed resistance of E-coli to tetracycline in reverse to Klebsiella spp. which is highly sensitive to tetracycline,

0

20

40

60

80

100

IMP AZM CIP PY CAR AK S CN TE

E.coli

Klebsiella

Antibiotic

Sen

siti

vity

%

Figure 1. Antibiotics susceptibility patterns of most predominant bacteria (E. coli and Klebsiella spp.) isolated from the genital system of cows. IMP: imipenem, AZT : aztreonam, CIP: ciprofl oxacin, PY: piperacillin, CAR: carbenicillin, AK: amikacin, S: streptomycin , CN: gentamicin, TE: tetracycline.


70

whereas the sensitivity to gentamycin for E-coli and Klebsiella spp. related to this study. In the present study, Klebsiella spp. was found to be highly sensitive to amikacin and ciprofl oxacin. Udhayavel et al. (2013) demonestrated low sensitivity of Klebsiella spp. to ciprofl oxacin. Rajeev et al. (2010) reported moderate sensitivity of Klebsiella spp. to amikacin.

REFERENCES

1. Al-Hilali, H.A & Al-Delemi, D.H. (2001). The uterine bacterial fl ora of normal reproductive tract, non-pregnant Iraqi cows. Vet., 11: 112-120.

2. Al-jebori, Kareem kadhim Radhi. (2013). Comparison study of pathophysiological conditions of genitalia in cows by rectal and pathological examination. Thesis Ms.C, College of Veterinary Med., Baghdad University, Iraq.

3. Al-Zubaidi, S. F., Hasson, S. O., Ajeel. (2013). Isolation and identification of microf lora species at different levels of the ewe genital tract. Journal of Agriculture and Veterinary Science 6(3):54-57.

4. Azawi. O. I., A. J. Ali and E. H. Lazim. (2008). Pathological and anatomical abnormalities affecting buffalo cows reproductive tracts in Mosul. Iraqi Journal of Veterinary Sciences, Vol. 22, No. 2 (59-67).

5. Burfeind, O., Bruins, M., Bos, A., Sannmann, I., Voigtsberger, R. and Heuwieser, W. (2014). Diagnosis of acute puerperal metritis by electronic nose device analysis of vaginal discharge in dairy cows. Theriogenology Volume 82, Issue 1, Pages 64-70.

6. Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS). (2010). Performance standards for antimicrobial susceptibility testing : Seventeenth informational supplement.

7. Dini, P., Amarloie, O. A., Moghadam, M. F., Moosakhani, F., Mottaghian, P., and Barin, A. (2012). The comparison of antagonistic effects of normal vaginal lactobacilli and some commonly used antibiotics on isolated bacteria of uterine infections in dairy cows. International Journal of Animal and Veterinary Advances 4(6): 358-362.

8. Dolezel, R., Palenik, T., Cech, S., Kohoutova, L., Vyskocil, M. (2010). Bacterial contamination of the uterus in cows with various clinical types of metritis and endometritis and use of hydrogen peroxide for intrauterine treatment. Veterinarni Medicina, 55 (10): 504–511.

9. El-Arabi, A. A., Taylor, D. J., Logue, D. N. and Benothman, M. (2013). Isolation and Identifi cation of Bacterial Flora from Reproductive Tracts of Normal Ewes in Glasgow. J Vet Adv, 3(10): 275-280.

10. Forbes, B. A., Sahm, D. F., and Weissfeld, A. S. (2007). Bailey and Scotts’ Diagnostic microbiology, 12th ed. Elsevier.

11. Gani, M.O., Amin, M.M., Alam, M. G. S., Kayesh, M. E. H., Karim, M.R., Samad, M. A. , Islam, M. R. (2008). Bacterial fl ora associated with repeat breeding and uterine infections in dairy cows. Bangladesh J Vet Med 6:79–86

12. Goncuoglu, M., Seda, F., Ormanci, B., Ayaz, N.D. and Erol, I. (2010). Antibiotic resistance of Escherichia coli O157:H7 isolated from cattle and sheep. Ann Microbiol 60:489–494.

13. Hameed H. A. Abdul, H. R. Abass, M. Y. Obeed. (2010). Study the incidence of abortion and retained placenta in pregnant cows with ephemeral fever and its relation with treatment and bacterial causes. AL-Qadisiya Journal of Veterinary Medicine Science, Vol. 9 No.2 (33-37).

14. Mansor A. R. and A. F. Majeed. (2005). Anatomical and pathological abnormalities of female genitalia of cows. (2005). Al-Anbar Journal of Agriculture Sciences, Vol. 3, No. 2 (141-151).

15. Martins, G., Figueira, L., Penna, B., Brandão, F., Varges, R., Vasconcelos, C. and Lilenbaum, W. (2009). Prevalence and antimicrobial susceptibility of vaginal bacteria from ewes treated with progestin-impregnated intravaginal sponges. Small Ruminant Research. Volume 81, Issue 2 , Pages 182-184.

16. Moges, N., Regassa, F., Yilma, T. and Unakal, C. G. (2013). Isolation and Antimicrobial Susceptibility of Bacteria from Dairy Cows with Clinical Endometritis. Journal of Reproduction and Infertility 4 (1): 04-08.

17. Mshelia, G. D., Abba, Y., Voltaire, Y. A. C., Akpojie, G., Mohammed, H., & Aondona, D. U. (2012). Comparative uterine bacteriology and pathology of camels (Camelus dromedarius) and cows in north-eastern Nigeria. Comp Clin Pathol DOI 10.1007/s00580-012-1549-8.

18. Mshelia, G. D., Bilal, V. T., Maina, V. A., Okon, K., Mamza, S. A., Peter, I. D. & Egwu, G. O. (2014a). Microbiological studies on genital infections in slaughtered ewes from tropical arid zone of Nigeria. Sokoto Journal of Veterinary Sciences, Volume 12 (Number 1).

19. Mshelia, G. D., Okpaje, G., Voltaire, Y. A. S. and Egwu, G. O. (2014b). Comparative studies on genital infections and antimicrobial susceptibility patterns of isolates from camels (Camelus dromedarius) and cows (Bos indicus) in Maiduguri, north-eastern Nigeria. SpringerPlus, 3:91.

20. Otero, C., Saavedra, L., Silva de Ruiz, C., Wilde, O., Holgado A. R. and Nader-MacõÂasb, M. E. (2000). Vaginal bacterial microfl ora modifi cations during the growth of healthy cows. Applied Microbiology, 31, 251-254.


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21. Oliveira, J. K., Martins, G., Esteves, L. V., Penna, B., Hamond, C., Fonseca, J. F., Rodrigues, A. L., Brandão, F. Z. and Lilenbaum, W. (2013). Changes in the vaginal fl ora of goats following a short-term protocol of oestrus induction and synchronisation with intravaginal sponges as well as their antimicrobial sensitivity. Small Ruminant Research, volume 113, Issue 1 , Pages 162-166.

22. Parul, B. B., Sharma, B. and Jain, U. (2014). Virulence associated factors and antibiotic sensitivity pattern of Escherichia coli isolated fromcattle and soil, VeterinaryWorld 7(5): 369-372.

23. Penna, B., Libonati, H., Director, A., Sarzedas, A. C., Martins, G., Brandão, F. Z., Fonseca, J. and Lilenbaum, W. (2013). Progestin-impregnated intravaginal sponges for estrus induction and synchronization infl uences on goats vaginal fl ora and antimicrobial susceptibility. Animal Reproduction Science. volume 142, Issue 1 , Pages 71-74.

24. Rajeev, R., Gupta, M. K., Singh, S. and Kumar, S. (2010). Current trend of drug sensitivity in bovine mastitis. Veterinary World Vol.3, No.1, January.

25. Romanus, I. I., Chinyere, O.E., Amobi, N.E., Anthonia, O.E., Ngozi, A.F., Chidiebube, N.A. and Eze, A.T. (2012). Antimicrobial resistance of Escherichia coli isolated from animal and human clinical sample. Glo. Res. J. Microbiol. 2 (1):85-89.

26. Sheldon I.M., Noakes, D.E., Rycroft, A.N., Pfeiffer, D.U., Dobson, H. (2002a). Infl uence of uterine bacterial contamination after parturition on ovarian dominant follicle selection and follicle growth and function in cattle. Reproduction, 123:837–845.

27. Sheldon, I. M., Noakes, D. E., Rycroft, A. N., Dobson, H. (2002b). Effect of postpartum manual examination of the vagina on uterine bacterial contamination in cows. Veterinary Record 151, 531-534.

28. Sheldon IM, Lewis GS, LeBlanc S, Gilbert RO. (2006). Defi ning postpartum uterine disease in cattle. Theriogenology, 65:1516–1530.

29. Sheldon, I.M., Cronin, J., Goetze, L., Donofrio, G., & Schuberth, H-J. (2009). Defi ning Postpartum Uterine Disease and the Mechanisms of Infection and Immunity in the Female Reproductive Tract in Cattle. Biology of reproduction, 81, 1025–1032

30. Sheldon, I.M., Rycroft, A.N., Dogan, B., Craven, M., Bromfi eld, J.J., et al. (2010). Specifi c Strains of Escherichia coli Are Pathogenic for the Endometrium of Cattle and Cause Pelvic Infl ammatory Disease in Cattle and Mice. PLoS ONE 5(2): e9192. doi:10.1371/journal.pone.0009192.

31. Silva, V.F., Damasceno, T.E.F., Souza, N.J.D., Franco, I. & Costa, M.M. (2011). Cervical-vaginal microbiota of crossbred sheep in Petrolina/PE, Brazil, and its susceptibility to antibiotics. Pesq. Vet. Bras. 31(7):586-590.

32. Šiugždaitė, J., Juodžentis, V., Petkevičius, S. (2013). Bacterial contamination of the uterus in different lactation cows on endometr it is. Veter inarija ir zootechnika. T. 61 (83).

33. Tel, O. Y. (2011). Aerobic bacteria and their antibiotic susceptibility of sheep with vaginitis due to intravaginal sponges application. Etlik Vet Mikrobiyol Derg, 22, 7-10.

34. Udhayavel, S., Malmarugan, S., Palanisamy, K., and Rajeswar, J. (2013). Antibiogram pattern of bacteria causing endometritis in cows, Vet World 6(2): 100-102.

35. Wang, Y., Ametaj, B. N., Ambrose, D. J., & Gänzle, M. G. (2013). Characterisation of the bacterial microbiota of the vagina of dairy cows and isolation of pediocinproducing Pediococcus acidilactici. BMC Microbiology, 13:19.

36. Williams EJ, Fischer DP, Pfeiffer DU, England GCW, Noakes DE, Dobson H, Sheldon IM. (2005). Clinical evaluation of postpartum vaginal mucus refl ects uterine bacterial infection and the immune response in cattle. Theriogenology, 63:102–117.

37. Williams, E. J. , Fischer, D. P. , Noakes, D. E. , England, G.C.W., Rycroft, A., Dobson, H., Sheldon, I. M. (2007). The relationship between uterine pathogen growth density and ovarian function in the postpartum dairy cow. Theriogenology 68(4): 549–559.

38. Zinnah, M.A., Haque, M.H., Islam, M.T., Hossain, M.T., Bari, M.R., Babu, S.A.M., Rahman, M. T. and Islam, M.A. (2008). Drug sensitivity pattern of Escherichia coli isolated from samples of different biological and environmental sources. Bangl. J. Vet. Med. 6 (1): 13–18.

ACKNOWLEDGMENTS. I wish to thank Veterinarians in Al-Hilla slaughterhouse for their efforts, specialized pharmacist Ruaa F. Abbas and Dr.Hameedh H. Ajeel for their help.


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ABSTRACT. The antimicrobial drug resistance pattern of Salmonella enterica serovar Typhimurium of various meats received in Veterinary Research Institute (VRI) was evaluated for the period of 2009 to 2012. A total of 64 strains of Salmonella typhimurium were isolated from beef, pork and chicken meat. All isolates were tested for resistance against 12 different antimicrobial agents. Antimicrobial susceptibility of the isolates was conducted using the disk diffusion technique according to the standards of Clinical and Laboratory Standards Institute (CLSI, 2007). 49 isolates (76.5%) showed multidrug resistance (MDR) and they originated from pork (100%), chicken (75.0%) and beef (58.8%). Salmonella typhimurium isolates from beef and pork showed multiple antimicrobial resistance to tetracycline, streptomycin, nalidixic acid and ampicillin, while isolates from chicken meat showed common profile of multiple antimicrobial resistance to sulphamethoxazole, sulphonamides and tetracycline. Ceftriaxone and cefotaxime

recorded as most sensitive drug for Salmonella typhimurium isolates.

Keywords: Salmonella typhimurium, multidrug resistance (MDR), beef, pork, chicken

INTRODUCTION

Salmonellosis caused by Salmonella enterica serovar Typhimurium is a worldwide food-borne zoonoses with public health concern (Wasyl et al., 2006; Yukino et al., 2011). A variety of foods have been known to harbour potential of transmitting salmonellosis to humans, including poultry, meat products, raw vegetables and fruits (Diana et al., 2012; Se-Yeoun Cha et al., 2013). The incidence of Salmonella has been studied recently in animal farms, meats, the environment including slaughter houses in many countries (Hanson et al., 2002; Frederick and Nurul Huda, 2012; Wang et al., 2013; Se-Yeoun Cha et al., 2013). Fresh produce has been identifi ed as a potential transmission of pathogens to cause human illness where common practice in agricultural irrigation of using

ANTIMICROBIAL RESISTANCE OF Salmonella enterica SEROVAR TYPHIMURIUM FROM VARIOUS MEATS RECEIVED IN VRIKHOO E.*, ROSELIZA R, KHOO L.L, NAFIZAH M., SAIFU NAZRI R., HASNAH Y., NORAZARIYAH M.N., ROSNAH Y., ROSNA D., SITI NOR HANANI R. AND. RAMLAN M.Veterinary Research Institute, 59 Jalan Sultan Azlan Shah, 31400 Ipoh, Perak

* Correspondence author: [email protected]

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animal waste fertilizer and wastewater contributed to the main source of pathogen contamination (Madden, 1992; Lee et al., 2009).

Antimicrobial resistance is a common issue regarding Salmonella from meat products as reported by Roseliza et al., 2011. The main concern is the transfer of antibiotic resistance to human pathogens and this has been emphasized by the World Health Organization on the increasing number of non-typhoid Salmonella strains. The livestock and poultry industry in Malaysia has been closely monitored and developed by the Malaysian government in order to achieve self-suffi cient domestic supply of food. Thus, well being and food security has come into the spotlight where constant monitoring of meat products is necessary. The aim of this study is to determine the antimicrobial resistance of Salmonella typhimurium isolated from various meat products in the country.


The Salmonella typhimurium strains used in this study were received from Veterinary Public Health Laboratory and Regional Veterinary Laboratory in VRI for diagnosis over the period of 2009 to 2012. Antimicrobial susceptibility of the isolates was tested using the disc diffusion technique with commercially available antibiotic discs on Mueller-Hinton agar (Oxoid) according to the standards of Clinical and Laboratory Standards Institute (CLSI, 2007). The following

antibiotic discs were used (Oxoid): ampicillin (10 μg), cefotaxime (30 μg), ceftriaxone (30 μg), cephalothin (30 μg), chloramphenicol (30 μg), gentamicin (10 μg), nalidixic acid (30 μg), streptomycin (10 μg), sulphamethoxazole (25 μg), sulphonamides (300 μg), tetracycline (30 μg), and trimethoprim (5 μg). The inhibition zones were measured and scored as sensitive, intermediate susceptibility and resistant, according to CLSI guidelines (CLSI, 2007).

RESULTS

A total of 64 strains of Salmonella typhimurium were tested for resistance against 12 different antimicrobial agents. 17 strains (26.6%) were isolated from beef, 15 strains (23.4%) were from pork while 32 isolates (50%) were from chicken meat. 49 isolates (76.5%) showed multidrug resistance (MDR) where they exhibited resistance to three or more antimicrobial classes used in this study. For the MDR strains, 15 isolates (100%) were isolated from pork meat, 24 isolates (75%) from chicken meat and 10 isolates (58.8%) from beef (Table 1). Salmonella typhimurium isolates from beef and pork showed multiple antimicrobial resistance to ampicillin, streptomycin, tetracycline and nalidixic acid, while isolates from chicken meat showed a common profi le of multiple antimicrobial resistance to sulphamethoxazole, sulphonamides and tetracycline. Cefotaxime and Ceftriaxone


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recorded as the most sensitive drug for Salmonella typhimurium isolates.

DISCUSSION

The presence of Salmonella typhimurium in beef, pork and chicken were found in this study. This indicates that the meat or our food source might serve as a potential route to disseminate the multidrug resistant (MDR) strain of Salmonella typhimurium to the human population, and this is in agreement with other researchers (Lee et al., 2009; Adetunji and Isola, 2011; Diana et al., 2012; Kusumaningrum et al., 2012). The dissemination of MDR strains

of Salmonella typhimurium to human population may be via contaminated food products or direct contact with infected animals. The multiple resistances observed were to those antimicrobials frequently employed and readily available in veterinary practices either as growth promoters in farm animals or to control infectious disease (Ali et al., 2009; Adetunji and Isola, 2011). Antibiotic treatment in farm animals may promote the emergence and spread of antibiotic resistant microorganisms to human population.

Antimicrobial resistance is the biggest challenge in virtually all areas of infectious disease, including viral, bacterial, fungal

Table 1. Percentage of Salmonella isolates resistant to antibiotics from various meat sources

Antibiotic% of resistant isolates from:

Beef (n=17) Pork (n=15) Chicken (n=32) All meats (n=64)

Ampicillin 29.4 73.3 40.6 59.4

Cefotaxime 5.9 13.3 15.6 12.5

Ceftriaxone 0.0 6.7 6.3 4.7

Cephalothin 11.8 40.0 56.3 48.4

Chloramphenicol 0.0 13.3 21.9 18.8

Gentamicin 5.9 20.0 25.0 23.4

Nalidixic acid 29.4 80.0 40.6 60.9

Streptomycin 35.3 86.7 46.9 65.6

Sulphamethoxazole 23.5 40.0 81.3 70.3

Sulphonamides 17.6 46.7 62.5 60.9

Tetracycline 35.3 100.0 62.5 73.4

Trimethoprim 0.0 20.0 21.9 18.8

Multiresistant (Resistant to more than 3 classes of antibiotic)

58.8 100.0 75.0 76.5


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and parasitic diseases (Weber and Courvalin, 2005). The signifi cant health implication in this scenario is the loss of therapeutic usefulness of antibiotics especially in clinical cases. Currently, public health impact of multidrug resistance is often highlighted because resistance has shadowed the success of treatment in outbreaks of infectious diseases. Public awareness and health education on the negative effects from overuse of antibiotics other than for therapeutic purposes in farms should be assessed and carried out. Many articles and publications in several countries have discussions on the issue of antimicrobial resistance where infection control and reducing the discretionary use of antimicrobial drugs wherever possible is helpful although resistance will continue to evolve and spread as an evolutionary process (Harbarth and Samore, 2005). The drug resistant patterns reported for Salmonella typhimurium is important to constantly monitor the resistant pattern to provide suitable guidelines for treatment or better infection control. Government should make efforts to draw a guideline to strictly control the use of antibiotics in livestock, agriculture, community and healthcare settings. Moreover, the use of antibiotics in animal feed needs to be regulated to minimize the exposure for bacteria to develop resistance (Thi Thu Hao Van et al., 2007). A database for microbial resistance patterns can be designed to monitor the resistance pattern of antibiotics over time to aid the selection of antibiotics by clinical practitioners.

Public awareness and health education to the layman through mass media on the side-effect of the abuse of antibiotics in farms should be carried out in efforts to reduce the dissemination of multidrug resistant bacteria. Consumers can play their role to ensure good food hygiene practices and thorough cooking to reduce or eliminate the risk of antibiotic resistant pathogens originating from animal products.

CONCLUSION

Salmonella typhimurium isolates from beef and pork showed multiple antimicrobial resistance to ampicillin, streptomycin, tetracycline and nalidixic acid, while isolates from chicken meat showed multiple antimicrobial resistance to sulphamethoxazole, sulphonamides and tetracycline. Cefotaxime and ceftriaxone recorded as most sensitive drug for Salmonella typhimurium isolates.

REFERENCES

1. Adetunji, V. O., and Isola, T. O. (2011). Antibiotic resistance of Escherichia coli, Listeria and Salmonella isolates from retail meat tables in Ibadan municipal abattoir, Nigeria. African Journal of Biotechnology, 10(30):5795 – 5799.

2. Ali Akond, M., Saidul, A., Hassan, S.M.R. and Shirin, M. (2009). Antibiotic Resistance of Escherichia coli Isolated from Poultry and Poultry Environment of Bangladesh. Internet Journal of Food Safety, 11:19-23.

3. Clinical and Laboratory Standards Institute. (2007). Performance Standards for Antimicrobial Susceptibility Testing, 17th Informational Supplement. CLSI Document M100-S17.

4. Diana, J. E., Pui, C. F. and Son, R. (2012). Enumeration of Salmonella spp., Salmonella Typhi and Salmonella Typhimurium in fruit juices. International Food Research Journal, 19(1):51 – 56.


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5. Frederick, A.G.R. and N. Huda (2012). Prevalence and Antibiotic Resistance of Salmonella Serovars in Ducks, Duck Rearing and Processing Environments in Penang, Malaysia. Food Research International. 45(2): 947 – 952.

6. Harbarth, S. and Samore, M. H. (2005). Antimicrobial Resistance Determinants and Future Control. Emerging Infectious Diseases, 11(6):794-801.

7. Kusumaningrum, H.D., Suliantari and Dewanti-Hariyadi, R. (2012). Multidrug Resistance Among Different Serotypes of Salmonella Isolates from Fresh Products in Indonesia. International Food Research Journal. 19 (1): 57 – 63.

8. Lee, L.H., Cheah, Y.K., Shiran, M.S., Sabrina, S., Noor Zaleha, A.S., Sim, J.H., Khoo, C.H. and Son, R. (2009). Molecular Characterization and Antimicrobial Resistance Profiling of Salmonella enterica subsp. enterica Isolated from ‘Selom’ (Oenanthe stolonifera). International Food Research Journal, 16:191-202.

9. Madden, J.M. (1992). Microbial Pathogens in Freah Produce – The Regulatory Perspective. Journal of Food Protection, 55:821-823.

10. Roseliza, R., Maswati, M.A., Hasnah, Y. and Ramlan, M. (2011). Identifi cation of Salmonella Serotypes Isolated from Meat Samples in Malaysia. Malaysian Journal of Veterinary Research. 2(1):59 – 64.

11. Se-Yeoun Cha, Min Kang, Ran-Hee Yoon, Choi-Kyu Park, Oun-Kyoung Moon and Hyung-Kwan Jang (2013). Prevalence and Antimicrobial Susceptibility of Salmonella Isolates in Pekin Ducks from South Korea. Comparative Immunology, Microbiology and Infectious Diseases. 36(5): 473 – 479.

12. Thi Thu Hao Van, George, M., Taghrid, I., Linh, T.T., and Peter, J.C. (2007). Detection of Salmonella spp. in Retail Raw Food Samples from Vietnam and Characterization of Their Antibiotic Resistance. Applied and Environmental Microbiology. 73(21): 6885 – 6890.

13. Wang, H., Ye, K., Wei, X., Cao, J., Xu, X. and Zhou, G. (2013). Occurrence, Antimicrobial Resistance and Biofi lm Formation of Salmonella Isolates from a Chicken Slaughter Plant in China. Food Control. 33(2): 378 – 384.

14. Wasyl, D., Sandvang, D., Skov, M.N. and Baggesen, D.L. (2006). Epidemiological characteristics of Salmonella Typhimurium isolated from animals and feed in Poland. Epidemiol. Infect. 134: 179 – 185.

15. Weber, J.T. and Courvalin, P. (2005). An Emptying Quiver: Antimicrobial Drugs and Resistance. Emerging Infectious Diseases, 11(6):791-793.

16. Yukino, T., Ikuo, U., Kiyoshi, T., Hizuru, O., Satoru, T., Hideki, H., Natsumi, K., Sou-ichi, M., Masato, K., Takayuki, K., Toru, K., Shinichi, H., Ryoko, I., Eiji, H., Hironari, Y., Yuuji, N. and Masato, A. (2011). Molecular Epidemiology of Salmonella enterica Serovar Typhimurium Isolates from Cattle in Hokkaido, Japan: Evidence of Clonal Replacement and Characterization of the Disseminated Clone. Applied and Environmental Microbiology, 77(5):1739 – 1750.

ACKNOWLEGDEMENT. This work is done by using the funding from Veterinary Research Institute (VRI) and Department of Veterinary Services (DVS) Malaysia. The author would like to thank all the people that contributed in this study.


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ABSTRACT. Seminomas are one of the common testicular tumours that occur mostly in dogs above fi ve years of age. A case of nine-year-old male German Shepherd dog, presented to University Veterinary Hospital, Kokkalai with a history of swelling on the scrotum for the past two months and its successful surgical management is discussed. The right testicle was found to be highly enlarged and hard while the other atrophied. Histopathological examination following its surgical removal, confirmed it as seminoma.

Keywords: seminoma, dog, histopathology

INTRODUCTION

Seminoma is one of the testicular neoplasia that occurs in dogs. Testicular neoplasia is rare in the bull, ram and boar and, although common in dogs, rarely act as a cause of infertility. Interstitial cell tumours are the most common tumours of the dog. Seminomata, the next most common canine testicular tumour, are also occasionally found in bulls (and stallions), while Sertoli cell tumours are

rarely seen in species other than dogs. Overall, testicular tumours account for over 10% of tumours in male dogs, with a considerably increased incidence in animals with cryptorchid testes (Arthur, 2001). Seminomas are testicular tumours originating from germinal epithelium of seminiferous tubules (Roberts, S. J., 1971). They contribute about 35% of canine testicular tumours and are mostly seen in dogs above fi ve years. Seminomas may become large but are generally innocuous in scrotal testes. They often grow slowly for long periods but may occasionally undergo a sudden increase in the rate of growth. The tumours may become necrotic or haemorrhagic, whereupon affected dogs may exhibit lameness, pain, crouching or hunching. Occasionally, they metastasize to local lymph nodes. Surgical removal of affected testis is indicated in case of seminoma.

History and Clinical observation

A nine-year-old male German Shepherd dog weighing 30 kg, was presented to the University Veterinary Hospital, Kokkalai with a history of swelling on its scrotum

SURGICAL MANAGEMENT OF UNILATERAL SEMINOMA IN A DOG BINI JOY*, JOHN MARTIN K.D. AND NARAYANAN M.K.Department of Veterinary Surgery and Radiology, College of Veterinary and Animal Sciences,

Mannuthy Thrissur, Kerala 680 651


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for the past two months. The animal had been treated with antibiotics for the same and no improvement was noticed. On examination, generalised swelling over the scrotal sac was noticed. Erythematous lesions and pigmentation were noticed all over the scrotal skin. On palpation, the right testicle was found greatly enlarged, about three times than normal, with the surfaces smooth. The other testicle was smaller than usual and was atrophied. Surgical removal of testicles was decided.

Treatment and discussion

The dog was premedicated with intramuscular injections of atropine su lphate (Atrowok, 0 .6 mg/ml , Wockhardt Ltd) at the rate of 0.045 mg/kg body weight, followed by xylazine hydrochloride (Xylaxin, 23.22 mg/ml, Indian Immunologicals Ltd) at the rate of 1.5 mg/kg bodyweight at 15 min interval. Anesthesia was induced with ketamine hydrochloride (Aneket, 50 mg/ml, Neon Laboratories Ltd.) given at a rate of 5 mg/ kg bodyweight intramuscularly. Anesthesia was maintained with Helibron mixture and diazepam (Calmpose, 5 mg/ml, Ranbaxy Diagnostic Ltd) intravenously to effect.

Surgical procedure

The animal was controlled in dorsal recumbency. The enlarged testis was held tensed against the dorsal scrotal skin. A single bold incision parallel to the median raphae, cutting through the skin, dartos

and tunica vaginalis was made and the testis was exposed. The scrotal vessels were found highly enlarged. The least vascular part of the spermatic cord was separated. The vessels were clamped and ligated using polyglactin No. 1 (Vicryl, polyglactin 910 suture, Ethicon Inc) and the spermatic cord was severed and removed from the testicle (Figures 1 and 2). Through the same scrotal incision, the other testis which was atrophied was also removed by incising the median scrotal septum (Figure 3). Since the scrotum sac was highly pendulous and with ischemic changes, scrotal ablation was performed after orchiectomy (Figure 4). Curvelinear incisions were made on both sides of scrotum at its base, dissected deep and transected. Hemorrhage was controlled by ligation. The subcutis was sutured using polyglactin No. 1 in continuous lockstitch pattern. Skin suture was applied using Nylon in horizontal mattress pattern. Tincture benzoin seal was applied over the suture line.

Post-operatively the dog was treated with ceftriaxone (Intacef, 500 mg injection, Intas Pharmaceuticals Ltd) at a rate of 25 mg/kg bodyweight and meloxicam (Melonex BP, 5 mg, Intas Pharmaceuticals Ltd) at the rate of 0.2 mg/kg bodyweight, both intravenously for fi ve days. Skin sutures were removed on the seventh postoperative day. The dog had an uneventful recovery.

The histopathology of the cut section of the enlarged testis revealed the testicular


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Figure 1

Figure 3

Figure 2

Figure 4

tumour – seminoma (Figure 4 and Figure 5).

DISCUSSION

Testicular tumors have been reported to have a higher incidence in older dogs

(Mitchell, R., 2002), and it is the second most commonly seen tumours in male dogs. Among the three testicular tumours, viz., interstitial cell tumour, seminoma and sertoli cell tumour, seminomas are second common in incidence. Seminomas arise from the germinal epithelium of


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seminiferous tubules and are typically benign tumours, with a metastatic rate of 5-10%. When functional, seminomas produce more amounts of androgen, aprostatic enlargement and perianal adenomas are likely to occur. Clinical signs include increased fi rmness, generalised enlargement of testis and scrotum, swelling of one or both testes and pain. Signs of feminization like hyperpigmentation are also reported (Grieco et al., 2008). The definitive diagnosis of testicular cell tumours can be reached by an excisional wedge biopsy of the testis away from the epididymis. In the present case, histopathology of the excised testicle revealed the presence of proliferating cells arising from tubular epithelium (Figure 5) and hyperchromatic vesicular nuclei with scarce cytoplasm.

Individual necrotic cells were found in the proliferating sheets of cells, which had a typical star sky pattern (Figure 6), suggestive of benign seminomas. Neoplastic cells were round to polyhedral in shape with distinct cell borders and mild to marked basophilic cytoplasm. Areas of neutrophilic infi ltration were also evident. Marked anisocytosis and anisokaryosis with prevalent karyomegalic cells were noticed (Masandet al.,2013). For the vast majority of testicular tumours, orchiectomy is suggested as the treatment (Dhaliwal et al., 1999).

SUMMARY

Seminomas are the second most common testicular tumours in dogs above five years. In this report, the case of a nine-

Figure 5 Figure 6


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year-old male German shepherd dog with the clinical signs of generalised swelling of the scrotum for the past two months, unilateral enlargement of right testis and atrophy of left testis is presented. The dog was subjected to bilateral orchiectomy and scrotal ablation under general anesthesia. Histopathology of the enlarged testis confi rmed it as a case of seminoma.

REFERENCES

1. Mitchell, R. 2000. Reproductive oncology In: Slatter, D. H. ed. Text Book of Small Animal Surgery. 3rd Edn. Saunders Company, Philadelphia. pp. 2443

2. Arthur, G. H. 2001. Arthur̀ s Veterinary Reproduction and Obstetrics, 8th ed. Saunders Company. Elsevier Ltd. pp.735

3. Dhaliwal, R.S., Kithcell, B.E. Knight, B.L. and SchmidtB. R. 1999. Treatment of aggressive testicular tumors in four dogs.J. Am. Anim. Hosp. Assoc.35: 311-318

4. Grieco, V., Riccardi, E. and Greppi, G.F. 2008. Canine testicular tumours - A study on 232 dogs. J. Comp. Path. 138: 86-89

5. Roberts, S.J., 2004. Veterinary Obstetrics and Genital Diseases. 2nd Ed.CBS Publisher. pp.776.

6. Masand,A., Kumar,N., Patial,V. 2013. Unilateral diffuse spermatocytic seminoma in a dog.Vet. Res. International.October-December.1: 46-48

7. Takiguchi, M., Iida, T.,Kudo, T., and Hashimoto,A.. 2001.Malignant seminoma with systemic metastases in a dog.J. of Sm. Anim. Pract. 42:7: 360–362.


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ABSTRACT. Histopathological changes were studied in Swiss albino mice (N:36) which were challenged with the South Indian local strain of Trypanosoma evansi. Each animal was infected with 5×105 trypanosomes intraperitoneally. The animals were examined daily for development of clinical signs and infection status by wet blood-fi lms made from the tail veins. The infected mice were dull and depressed from two days post-infection (DPI) onwards. Systematic post-mortem examination of the infected mice was performed and pathological changes were recorded. The different tissue samples were collected in 10% formalin and were used to study the histopathological changes. Post-mortem examination from 3-4 DPI (the maximum period of observation) revealed splenomegaly, hepatomegaly, marked congestion of lungs, presence of fl uid in peritoneal cavity. Histopathologically, heart muscles showed hyaline degenerative changes and haemorrhages. Liver parenchyma revealed congestion of central vein and sinusoids, binucleated hepatocytes and fatty change of hepatic cells. Thickening of interstitial space with mononuclear infi ltration, areas of collapse, areas of emphysema, edema and dilated

and congested blood vessels were the histopathological changes noticed in the lungs of the infected mice. In the spleen, giant cells aggregation, hyperplasia, thickening of capsule and trabecule were the changes indicating irreversible degeneration. The affected kidney showed inter-tubular hemorrhages in the cortex, medullary hemorrhages, congested glomerulus, at rophied glomerulus, desquamated tubular epithelium and disruption of renal tubules at some places.

Keywords: histopathology, mice, Trypanosoma evansi

INTRODUCTION

Out of all the pathogenic trypanosomes, Trypanosoma evansi has the widest host range and worldwide geographical distribution. T. evansi is also highly pathogenic to laboratory animals (rat, mice and rabbit) (Sivajothi et al. 2013a). The parasite utilizes glucose and oxygen for its growth and multiplication resulting in depletion of these metabolites leading to degenerative changes in the host. Further changes develop in the organs either due to cellular damage caused by toxicants released by the parasite, or

EXPERIMENTAL Trypanosoma Evansi INFECTION IN ALBINO MICE – A HISTOPATHOLOGICAL STUDYS. SIVAJOTHI*, V.C. RAYULU1, K. SUJATHA2 AND B.SUDHAKARA REDDY3

College of Veterinary Science, Proddatur, Andhra Pradesh, India.


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due to immunological reactions. Though T. evansi is a haemoprotozoa, visceral forms have been reported in heart, optic lobes, cerebrum, liver, kidney and lungs (Bal et al., 2012). Haematological abnormalities and different prevalence status with different diagnostic tests was recorded previously in the Andhra Pradesh (Sivajothi et al., 2012 and Sivajothi et al., 2014a). Pathology of the disease by different species of trypanosomes in laboratory animals has been documented (Virmani et al., 2004 and Sivajothi et al., 2013b), yet the information on pathology of the visceral organs in mice induced by south Indian isolate of T. evansi is very little. Hence, the present investigation was intended to study pathology of the visceral organs in mice induced by south Indian (local) isolate of T. evansi.


The virulent strain of T. evansi was isolated from a cattle suffering from clinical surra. The strain was maintained in vivo in albino mice through serial passages. Thir ty-six albino mice were maintained in a fl y-proof house with clean and hygienic environment. Mice were kept on ad libitum feed and fi ltered water was provided to these animals throughout the experimental study. Thirty-six mice were infected with 5×105 trypanosomes intraperitonially. Twelve mice were kept as a control group. The blood of infected mice was collected by tail clipping and examined daily from the second day of

post-inoculation. After four (maximum) days of infection, mice were sacrifi ced and detailed post-mortem examination was carried out and representative tissue samples from liver, lungs, heart, kidneys and spleen were collected and preserved in 10% formalin for histopathological examination. These tissues were processed by routine conventional methods and sections were cut at 4 μ thickness and stained with haematoxylin and eosin stain (Sivajothi et al., 2014b).

RESULTS

The infected mice were dull and depressed from two DPI onwards. Examination of wet blood films (WBF) revealed the presence of motile trypanosomes after two DPI. Post-mortem examination from 3-4 DPI (the maximum period of observation) revealed splenomegaly, hepatomegaly (Figure 1), marked congestion of lungs, presence of f luid in peritoneal cavity. Histopathologically, hear t muscles showed hyaline degenerative changes and haemorrhages (Figure 2). Liver parenchyma revealed congestion of central vein and sinusoids, binucleated hepatocytes and fatty change of hepatic cells (Figure 3). Thickening of interstitial space with mononuclear infi ltration, areas of collapse, areas of emphysema, edema and dilated and congested blood vessels were the histopathological changes noticed in the lungs of the infected mice (Figure 4). The affected kidney showed inter-tubular hemorrhages in the cortex, medullary


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Figure 1. Post-mortem examination from 3-4 DPI (the maximum period of observation) revealed splenomegaly and hepatomegaly.

Figure 2. Heart muscles showing hyaline degenerative changes and haemorrhages.


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Figure 3. Liver parenchyma showing congestion of central vein and sinusoids, binucleated hepatocytes and fatty change of hepatic cells.

Figure 4. Lungs showing thickening of interstitial space with mononuclear infi ltration, areas of collapse, areas of emphysema, edema and dilated and congested blood vessels.


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Figure 5. Kidney showed inter-tubular hemorrhages in the cortex, medullary hemorrhages, congested glomerulus, atrophied glomerulus, desquamated tubular epithelium and disruption of renal tubules at some places

Figure 6. The spleen showing giant cells aggregation, hyperplasia, thickening of capsule and trabecule.


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hemorrhages, congested glomerulus, atrophied glomerulus, desquamated tubular epithelium and disruption of renal tubules at some places (Figure 5). In the spleen, giant cells aggregation, hyperplasia, thickening of capsule and trabecule were the changes indicate irreversible degeneration (Figure 6).

DISCUSSION

The clinical signs and gross lesions in the present study were gained the support from Virmani et al., (2004). They observed clinical signs were dull and depressed mice from two DPI till four DPI. Splenomegaly, marked congestion of lungs, petechiae on the serous surfaces and liver were the gross leisions observed. According to Tizard (1998), diseases caused by trypanosomes induce the formation of high levels of systemic antigen antibody immune complexes, and their consequent deposition in the heart, liver, brain and kidneys may possibly play a role in tissue damage. However, some reports indicated that trypanosomes can cause tissue infl ammation directly as a result of the infection (Damayanti, 1993). Enlargement of spleen might be due to increased activity of mononuclear phagocytic system resulting in destruction of trypanosomal antigen coated RBCs. The RBCs destruction was corroborated by hemosiderosis of the spleen. Splenomegaly followed by hyperplasia and hypersplenigism are very much pronounced as the disease progressed (Singla et al., 2001).

Histopathologically, heart muscles showed hyaline degenerative changes and haemorrhages. The parasite utilizes glucose and oxygen for its growth and multiplication resulting in depletion of these metabolites leading to changes in the host. Further developmental changes are either due to toxins released by the parasite or to immunological reactions (Bal et al., 2012). The degenerative changes in heart may be due to anemia and hypoglycemia.

Microscopically, the liver revealed severe fatty change, focal areas of dilated and congested sinusoids, congested blood vessels and periportal infiltration of mononuclear cells. Fatty changes in liver, manifested by lipid accumulation inside hepatocytes due to tissue hypoxia resulted from the present anemia and vascular damage (Derakhshanfar et al., 2010). Fatty degeneration followed by necrosis of hepatocytes was considered the common cytological findings associated with T. evansi infection as a result of nutritional impairment and the asphyxia (Uche and Jones, 1992). Histopathological changes such as necrosis and haemorrhages within the sinusoids of the liver with fatty degeneration in hepatic cells of the bandicoat rat infected with T. evansi were also noticed by Biswas et al. (2001). Suryanarayana et al., 1986 observed congestion, haemorrhages and fatty degeneration of hepatocytes and concluded that it may be due to hypoglycemia leading to starvation of the cells and anoxia due to anaemia in T. evansi infected animals. Damayanti et al., 1994 noticed congestion


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in the liver following necropsy in goat and buffalo infected with T. evansi. Congestion observed in the present study was also in agreement with fi ndings of Virmani et al. (2004).

Thickening of interstitial space with mononuclear infi ltration, areas of collapse, areas of emphysema, oedema and dilated and congested blood vessels were the histopathological changes noticed in the lungs of the infected mice. Congestion and edema of lungs were mainly due to infl ammatory response of the lungs to the parasite resulting in vasodilatation and exudation. Histopathological changes in lungs supported the results of Virmani et al., 2004 who observed alveolar oedema, congestion of alveolar blood vessels and massive areas of haemorrhages along with focal areas of infi ltration with polymorphs. Ngeranwa et al. (1993) observed marked cellular infiltration in lungs of small African goats.

The affected kidney showed inter-tubular hemorrhages in the cortex, medullary hemorrhages, congested glomerulus, at rophied glomerulus, desquamated tubular epithelium and disruption of renal tubules at some places. Pulmonary congestion and chronic interstitial nephritis developed by infected mice may be due to immune complex deposition and complement cascade reaction (Tizard, 1998). In the spleen, giant cells aggregation, hyperplasia, thickening of capsule and trabecule were the changes indicative of an immunological response by the infected mice. Virmani et al., 2004

observed that the spleen was haemorrhagic with pronounced haemosiderosis and RE cell proliferation. It has been reported that changes in kidneys are mainly due to toxins produced the parasite and accumulation of immune complexes which impair the structure and function of the kidney (Ngeranwa et al., 1993).

Initial changes in the spleen may be due to immediate hypersensitivity to T. evansi. Biswas et al. (2001) noticed the haemorrhages, congestion, absence of germinal centres, hemosiderosis, increase in follicular cells, focal necrosis and formation of giant cells due to aggregation of histiocytes during the progression of the disease. Suryanarayana et al. (1986) observed that the stimulation given by the presence of T. evansi or their toxic metabolites result in varying degrees of anaemic anoxia, which may induce splenic damage. Reduced proliferative activity followed by increase in the number of macrophages and multinucleated giant cells with severe disruption in the splenic architecture.

It was evident in this study that T. evansi was able to invade all the visceral organs examined. Previous work had also shown that T. evansi was capable of invading these organs. Similar observation was also been made by Lawal et al. (2007) in experimentally T. brucei infected rats. The invasive ability of T. evansi therefore, seems to be in line with that of the brucei group, to which T. evansi belongs (Losos, 1980). However, despite the fact that the organs enhance multiplication


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of the parasites, they certainly provide the mechanism for destroying them as evidenced by the presence of numerous degenerated forms. The mononuclear phagocytic system might be responsible for the attack on the parasites. T. evansi is highly pathogenic to laboratory animals as observed in the previous studies also (Sivajothi et al., 2014b). Consumptions of oxygen by trypanosomes for their multiplication lead to hypoxemic state as a result of which animal tissues are deprived of oxygen and it results in degenerative changes in all the vital organs.

REFERENCE

1. Bal, M. S, L. D. Singla, H. Kumar, Ashuma Vasudev, K. Gupta and P. D. Juyal, 2012. Pathological studies on experimental Trypanosoma evansi infection in Swiss albino mice. JPD, 36 (2): 260-264.

2. Biswas, D., Choudhury, A., Misra, K.K. 2001 .Histopathology of Trypanosoma evansi infection in bandicoot rat Visceral organs. Experimental Parasitology. 99(3),148-59.

3. Damayanti, R. 1993. Identification of Trypanosoma evansi in infected rat tissues by immunohistochemical methods. Penyakit Hewan, v.25, p.11-13.

4. Damayanti, R., Graydon, R.J., Ladds, P.W. 1994. The pathology of experimental TRypanosoma evansi infection in the Indonesian buffalo (Bubalus bubalis).Journal of Comparative Pathology 110(3), 237-52.

5. Derakhshanfar, A., A. Mozaffari and A. Zadeh. 2010. An Outbreak of trypanosomiasis (Surra) in camels in the southern fars Province of Iran: Clinical, hematological and pathological fi ndings. Res J Parasitol, 5: 23-26.

6. Lawal, A.I., Ameya, F., Ajanusi, O.J., Jatau, I.D., & Musa B. (2007). Distribution and morphological forms of Trypanosoma brucei in tissues/organs of experimentally infected albino rats. Proceedings of 44th Annual Congress of the Nigerian Veterinary Medical Association (NVMA). Pp 257-260.

7. Losos, G.J. (1980). Disease caused by Trypanosoma evansi, A review Veterinary Research Communication, 4: 165 – 181.

8. Ngeranwa, J. J. N., Gathumbi, P. K., Mutiga, E. R., Augumbah, G. J. O. 1993. Pathogenesis of Trypanosoma evansi in small African goats. Research in Veterinary Science 54,283-289.

9. Singla LD, Juyal PD, Sandhu BS (2001) Clinico-pathological response in Trypanosoma evansi infected and immuno-suppressed buffalo-calves. In: 18th International conference of WAAVP, WAAVP, Stresa, 26–30.

10. Sivajothi S, Rayulu VC., Reddy BS (2012). Development of Slide Enzyme Linked Immunosorbent Assay (SELISA) for Detection of Trypanosoma evansi Infection in Bovines. Journal of Advanced Veterinary Research. Volume 2 (2012) 15-17.

11. Sivajothi S, Rayulu VC, Malakondaiah P, Sreenivasulu D (2013a) Colloidal Dye Immunobinding Assay for Detection of Trypanosoma evansi Antibodies in Animals, International Journal of Livestock Research. Vol 3(3), 48-56.

12. Sivajothi S, Rayulu VC, Sudhakara Reddy B (2013b) Haematological and biochemical changes in experimental Trypanosoma evansi infection in rabbits. Journal of parasitic diseases. DOI 10.1007/s12639-013-0321-6.

13. Sivajothi S, Rayulu VC, Malakondaiah P, Sreenivasulu D (2014a). Diagnosis of Trypanosoma evansi in bovines by indirect ELISA. J. Parasit. Dis. DOI 10.1007/s12639-014-0465-z.

14. Sivajothi S, Rayulu V.C., Sujatha, K., Sudhakara Reddy B (2014b). Study of Histopathological Changes in Experimental Trypanosoma evansi Infected Rats. Proc. Zool. Soc. DOI 10.1007/s12595-014-0104-9.

15. Suryanarayana, C., Gupta, S.L., Singh R.P., Sadana, J.R. (1986). Pathological changes in donkeys (Equus asinus) experimentally infected with T. evansi. Indian Vet. Med. J. 6:57–59.

16. Tizard, I.R. Imunologia veterinária: uma introdução. 5ed São Paulo: Roca, 1998. p.331-332.

17. Uche, U., Jones, T. 1992. Pathology of experimental Trypanosoma evansi infection in rabbits. J. Comp. Pathol., 106: 299-30.

18. Virmani, N., Sengupta, P. P., and Panisup, A. S. (2004). Pathomorphological changes in rats following infection with Trypanosoma evansi isolatedfrom a heterologous host. Indian J. Anim. Res., 38 : 29 – 32.

ACKNOWLEDGEMENTS. The authors are thankful to Sri Venkateswara Veterinary University for providing various facilities to carry out the research work in the Department of Parasitology, College of Veterinary Science, Tirupati.


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ABSTRACT. Many parasitology laboratories practiced the McMaster technique as a method in obtaining the quantitative diagnosis of Strongyle eggs burden in farm animals especially ruminants. The McMaster technique also play a crucial role in faecal egg count reduction test (FECRT) for anthelmintic resistance identifi cation. Some laboratories recommend two-chamber counting method while some recommend single chamber counting method. This study focuses on the comparison between single and double counting in McMaster technique for detection of Strongyle egg count. In this study, it is shown that there is no signifi cant diffe rence between both methods based on the p-value obtained which is p>0.05 from 127 fresh goat faecal samples. The techniques practised during the study follow the standard established technique. Single chamber counting is suitable for a large number of faecal samples from big herds because it is faster, less laborious and produces sensitive and reliable results in Strongyle egg count. As more commercial

farms are set up, there is a need to conduct a fast and effi cient test to help farmers evaluate their livestock worm burden.

Keywords: McMaster technique, EPG, single chamber, double chamber

INTRODUCTION

The McMaster technique is a quantitative method to demonstrate and count Strongyle eggs in faeces of herbivores. The eggs are fl oated in a known volume of faecal suspension and counted microscopically on a McMaster slide. McMaster technique utilizes faecal sample to determine faecal egg count enabling the detection of parasitic element (e.g. helminths egg and larvae, protozoa oocysts and cysts) which is a widely used method to count parasite eggs (Cringoli et al., 2010). Faecal egg count is done for monitoring helminthiasis burdens in herd and fl ocks, determining the degree of pasture contamination and in epidemiological studies or anthelmintic resistance identifi cation (Coles et al., 1992).

MCMASTER METHOD OF WORM EGG COUNT FROM FAECAL SAMPLES OF GOATS: A COMPARISON OF SINGLE AND DOUBLE CHAMBER ENUMERATION OF WORM EGGSCHANDRAWATHANI P.1*, PREMAALATHA B.1, JAMNAH O.1, PRISCILLA F.X.2, ERWANAS A.I.1, LILY ROZITA M.H.1, JACKIE P.3 AND JOSEPHIN S.J.A.L.3

1 Veterinary Research Institute, 59 Jalan Sultan Azlan Shah , 31400 Ipoh, Perak

2 Faculty of Agro Based Industry, University Malaysia Kelantan, Kelantan

3 Faculty of Veterinary Medicine, University Malaysia Kelantan, Kelantan


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The McMaster technique is developed and improved at the McMaster laboratory of the University of Sydney (Gordon and Whitlock, 1939) which is the most universally used technique for estimating the number of helminth eggs in faeces. In literature, many variations of the McMaster technique can be found and a lot of modifi cations is done to this method (Cringoli et al., 2004). Different McMaster method modifi cations use various weight of faeces examined, volumes and types of fl oatation solutions, sample dilutions, fl oatation times, applications of additional centrifugation, duration and speeds of centrifugation, numbers of sections of McMaster slide counted and different coeffi cients for interpretation (Cringoli et al., 2004). These variation factors can limit the accuracy and signifi cance of diagnostic faecal egg count in the McMaster technique. The choice of single and double chamber counting in McMaster can also affect the reliability of the McMaster technique in estimating real egg per gram (EPG) from faeces.

Thus, the aim of this study is to compare the sensitivity and relationship between single chamber and double chamber counting in the McMaster technique by assessing the number of strongyle eggs per gram (EPG) in fresh faecal sample of goats.

METHODOLOGY

A total of 127 faecal samples were collected from goats of various ages (three months

to three years) from nine smallholder goat farms around the vicinity of Ipoh. About fi ve grams of faeces was collected using plastic gloves, per recta of each goat, selected randomly from the f lock. The faecal sample was processed immediately within two hours of collection. An amount of three grams of faeces from each goat was weighed, placed in a jar, and saturated salt (sodium chloride) solution was added. The saturated salt solution was prepared by diluting common salt in distilled water in an electric mixer until it cannot be dissolved anymore. The ratio of faeces to the sodium chloride solution is 1 g:15 ml. The faeces and sodium chloride mixture was then poured into a mortar through a tea-sieve and ground to a paste using a pestle. The waste produced in the tea-sieve is discarded while the liquid in the mortar is poured back into a clean jar. The jar is gently spun to stir the sediment in it. Using a clean pipette, the sediment is pipetted by placing the pipette in the centre of the jar to the centre of the overall depth of the sediments. Next, this sediment is pipetted into the chambers of the McMaster slides in which one chamber is for single reading and the other two-chamber is for double chamber reading where the eggs from both chamber is counted and an average faecal egg count (FEC) is estimated. For example, number of eggs found in chamber A of a McMaster slide is 300 and for chamber B is 200, so the total number of eggs for that faecal samples is 500 EPG. From this value, only the average FEC is calculated and used in the data, which is 250 EPG.


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The McMaster slide is then immediately observed under the microscope to prevent crystallization of the salt solution. This study focused only on strongyle eggs (MAFF, 1986).

The counted strongyle eggs will be calculated using the following formula to obtain the EPG value:

EPG = Total number of eggs counted × 100

For example, if 2 eggs were counted, the EPG is 200.

RESULTS

Statistical analysis was conducted using a 20.0 version SPSS system where direct calculation of mean, median, variance, and standard variation for 127 sample sizes including a histogram were presented in Table 1, Table 2, Table 3, Table 4, and Figure 1, Figure 2 and Figure 3. Kolmogorov-Smirnov and Shapiro-Wilk tests were used for normality test of data. A nonparametric Mann-Whitney U test was chosen by the system as suitable method for this experiment base on data provided and sample size. It evaluates

Table 1. Descriptive statistic of single chamber data.

Worm Egg Count (WEC) Statistic Std. Error

Mean 1086.61 218.834

95% Confi dence Interval for MeanLower Bound 653.55

Upper Bound 1519.68

5% Trimmed Mean 669.73

Median 100.00

Variance 6081803.525

Std. Deviation 2466.131

Minimum 0

Maximum 16500

Range 16500

Interquartile Range 1100

Skewness 4.123 .215

Kurtosis 19.941 .427

Table 2. Test of Normality.

Kolmogorov-Smirnova Shapiro-Wilk

Statistic df Sig. Statistic df Sig.

WEC .330 127 .000 .483 127 .000a. Lilliefors Signifi cance Correction


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Table 3. Descriptive statistic of double chamber data.

WEC Statistic Std. Error

Mean 932.68 195.628

95% Confi dence Interval for MeanLower Bound 545.54

Upper Bound 1319.82

5% Trimmed Mean 554.13

Median 100.00

Variance 4860332.458

Std. Deviation 2204.616

Minimum 0

Maximum 15400

Range 15400

Interquartile Range 800

Skewness 4.473 0.215

Kurtosis 23.970 0.427WEC: Worm Egg Count

Figure 1. Data for single chamber method presented in histogram and the bell shape graph showing the normality of the data skewed to the right.


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Table 4. Tests of Normality

Kolmogorov-Smirnova Shapiro-Wilk

Statistic df Sig. Statistic df Sig.

WEC .336 127 .000 .460 127 .000a. Lilliefors Signifi cance Correction

Figure 2. Data for double chamber method presented in histogram and the bell shape graph showing the normality of the data skewed to the right.

Figure 3. Nonparametric Tests. Asymptotic signifi cances are displayed. The signifi cance level is 0.05


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the statistical differences among the two conducted McMaster methods. Based on Mann-Whitney U test, p-value = 0.950 > x=0.05, hence we accept H0. Meaning that there is no signifi cant difference in egg per gram (EPG) value when single or double chamber McMaster counting is used. In this case, there is no signifi cant reduction in EPG value when either one of the McMaster method is being used.

DISCUSSION

There are many factors in the methodology of the McMaster faecal egg count technique, that can affect the result of both counting techniques. One of the most important factors that affects the result is the weight of faeces examined. Variation in amount of faecal sample will affect the amount of strongyle eggs seen and counted. Precise measurement of faecal egg count can be determined using electronic balance to weigh the faeces. The next factor in methodology that can affect the accuracy of both counting methods in McMaster technique is sampling error arising from the fact that the eggs are not evenly distributed through the faeces, especially if the consistency of faeces is variable; where faeces may be soft, diarrhoeic, hard, normal or constipated.

Other factors that can contribute to variation of results during experiments are bias in pipetting the mixture; which can be avoided by pipetting the sample from the centre of the jar after stirring its content to allow even distribution of helminth

eggs in the jar. Moreover, pipetting at the centre of the jar instead of at the base of the jar will prevent pipetting up of heavier eggs sedimented at the base of the jar and almost no strongyle eggs as it is lighter and might have fl oated.

Apart from that, other factors that can infl uence strongyle egg count in both counting methods include parasite biology (fecundity of the species, parasite number, prepatent period, arrested development), host physiological status (history of prior exposure to parasite, nutrition), host management factors (treatments, anthelmintic resistance, herd or f lock density) or technician’s skill level (Vadlejch et al., 2011).

Both single and double chamber counting methods in the McMaster technique can be used in determination of helminth eggs. The mean egg count of double chamber counting gives the same reading of strongyle eggs when compared with single chamber counting. Single chamber counting method is faster, less laborious and produces sensitive and reliable results of strongyle egg count with repeated measurement over time.

CONCLUSION

In conclusion, the fi ndings of the present study proved that there is no difference on results obtained when single chamber counting or double chamber counting is being used. Hence, this experiment has aided the laboratory practitioner in choosing a suitable method for their EPG


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counting which is time-saving especially when it involves many samples.

REFERENCES

1. Coles, G. C., Bauer, C., Borgsteede, F. H. M., Geerts, S., Klei, T. R., Taylor, M. A. and Waller, P. J. (1992). World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P) Methods for the detection of anthelmintic resistance in nematodes of veterinary importance. Vet Parasitol 44:35-44

2. Cringoli, G., Rinaldi, L., Veneziano, V., Capelli, G. and Scala, A. (2004). The infl uence of fl oatation solution, sample dilution and the choice of McMaster slide area (volume) on the reliability of the McMaster technique in estimating the faecal egg counts of gastrointestinal strangles and Dicrocoelium dentritium in sheep. Vet Parasitol 123:121-131

3. Cringoli, G., Rinaldi, L., Maurelli, M. P., Utzinger, J. (2010). FLOTAC: New multivalent techniques for quantitative copromicroscopic diagnosis of parasites in animals and humans. Nat Protoc 5:503-515

4. Gordon, H. and Whitlock, H. (1939). A new technique for counting nematode eggs in sheep faeces. J. Counc. Sci. Ind.Res 12, 50-52

5. MAFF. 1986. Fisheries and Food, Reference Book, Manual of Veterinary Parasitological Laboratory Techniques, Vol.418, Ministry Of Agriculture, HMSO, London, 5pp.

6. Vadlejch, J., Petrtyl, M., Zaichenko, I., Cadkova, Z., Jankovska, I., Langrova, I. and Moravec, M. (2011). Which McMaster egg counting technique is the most reliable? Parasitol Res 109:1387-1394


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ABSTRACT. Biting fl ies are important vectors in disease transmission in ruminants such as cattle and buffaloes where pathogenic organisms such as Trypanosoma evansi are transmitted. The Nzi and Vavoua traps were applied to survey the population of biting fl ies such as stable fl ies (Muscidae: Stomoxyinae) and horse fl ies (Tabanidae) which are the vectors for this disease. Results indicated the presence of biting f lies as well as Musca sp. and Haematobia sp. in several livestock farms in Perak that can transmit Trypanosomiasis. Vector control has been suggested to limit the occurrence of the disease especially during rainy season when there is high morbidity and mortality in herds. These traps can be used as an eco friendly method to control fl ies in farms as well as a tool to collect vectors of veterinary importancefor research purposes.

Keywords: biting fl ies, vector control, Tabanus sp., Stomoxys sp., Haematobia sp.

INTRODUCTION

Flies are signifi cant as disease vectors for human and animal health (Truc et al., 2013).

Malaysia in endemic with Trypanosomiasis caused by Trypanosoma evansi (Cheah et al, 1999). Cheah et al. (1996 and 1997) reported that Trypanosomiasis in cattle can cause severe losses in milk production and bodyweight. Stable fl ies (Stomoxys calcitrans) and horse fl ies (Tabanus sp.) can transmit Trypanosomiasis (Trypanosoma evansi) or commonly known as Surra, in cattle, buffalo and deer in Malaysia.

In 2013, there was a disease outbreak with high mortality and mobidity reported in Perak state at Infoternak, Sungai Siput and several private pig farms in surrounding areas (Nurulaini et al., 2013) and Sani et al. (1990 and 1995) has also reported Trypanosomiasis in cattle. The outbreak of trypanosomiasis was diagnosed by the Parasitology Section of the Veterinary Research Institute (VRI) in early 2012. A total of 86 whole blood samples from deer, cattle and buffaloes from a government farm near Sungai Siput, Perak and 16 whole blood samples from a commercial pig farm in Jalong, Perak were submitted for analysis. Sixty-three samples were found to be positive for the Trypanosoma sp. A detailed epidemiological investigation

VECTORS OF VETERINARY IMPORTANCE IN MALAYSIA: A SURVEY OF BITING FLIES IN RELATION TO TRYPANOSOMIASIS IN PERAKERWANAS A.I.*, MASRIN A., CHANDRAWATHANI P., JAMNAH O., PREMAALATHA B. AND RAMLAN M.Veterinary Research Institute, 59 Jalan Sultan Azlan Shah, 31400 Ipoh, Perak


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was conducted to prevent future outbreaks (Nurulaini et al., 2007). In the South-east Asian countries, vectors for surra have been long studied. In a study based on horse fl ies and deerfl ies collected by the Phillipine Zoological Society Phillipines, a total of 50 species of Tabanus have been recorded (Philip, 1959). In Thailand, Papp et al. (2006) reported 99 Diptera families. Tumrasvin in 1978 reported the Tabanus sp. and its distribution, as it was deemed important in the transmission of trypanosomiasis in livestock. In Malaysia, Philip also reported in 1960 on Tabanidae and new species of Haematopota. As such there is keen interest in local species of biting f lies as trypanosomiasis in livestock is a continuing threat causing low productivity.

Life cycle of Tabanidae

After a blood meal the female lays batches of several hundred creamy-white or greyish cigar-shaped eggs, 1.0-2.5 mm long, on the underside of vegetation or on stones, generally in muddy or marshy areas. The eggs hatch in 1-2 weeks and the cylindrical, poorly differentiated larvae drop into the mud or water. The larvae, 1.0-6.0 cm long, are recognized as tabanids by their small black retractable heads, the prominent raised rings around the segments. Optimally, larval development takes three months, but if hibernation occurs, may extend for up to three years. Mature larvae pupate partially buried in mud or soil and the adult fl y emerges after

1-3 weeks. The whole life cycle takes a minimum of 4-5 months or longer if larval development is prolonged (Taylor et al., 2007).

Life cycle of Muscidae

Female f lies lay batches of up to 100 creamy-white, 1.0 mm long, banana-shaped eggs in faeces or rotting organic material. Eggs hatch, under optimal temperatures, in 12–24 hours to produce whitish, segmented, cylindrical larvae (maggots), which, anteriorly, are pointed and have a pair of small hooks. The three larval instars feed on decomposing organic material and mature to 1.0 – 1.5 cm long maggots in 3-7 days under suitable conditions. These then move to drier areas around the larval habitat and pupate in the fi nal larval skin which contracts and becomes rigid and dark brown, to form the 6.0 mm long barrel-shaped puparium or pupal case. The adult fl y emerges after 3-26 days depending on temperature (Taylor et al., 2007).

Annual repor t of VRI shows Trypanosomiasis to be commonly isolated in diagnostic cases. In 2012, there were 58 samples of blood positive for Trypanosoma evansi.

The aim of this study is to show the common vectors found in Malaysia that can be a vector for the transmission of Surra in ruminants.


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MATERIALS & METHODS

Survey location

The survey was conducted in 5 districts in Perak.

The fl y traps were set up in several farms in Perak State and during the trapping activities, the following f lies were identified according to keys by Masmeatathip et al. (2006); Foil, 1989; and Phasuk et al., 2013. A total of 29 Tabanus sp. fl ies, 646 Stomoxys sp. fl ies, 50 Haematobia sp. fl ies and 1176 Musca sp. fl ies were collected using the Nzi and Vavoua trap (see Table 1.)

The weather during the trapping activities, carried out in several districts in Perak was bright and sunny which is suitable for trapping activities.

Trapping methods

Two traps were used to evaluate the effectiveness in catching the fl ies in the farm environment. The traps were set up close to animal facilities in VRI and in several commercial livestock farms in Perak State.

Nzi Trap

The Nzi trap (Figure 1) is a simple, safe and economical cloth trap for the capture of biting fl ies. It is a passive killing device that works through the attraction of fl ies to large blue and black objects. Flies enter at the bottom front, fl y into the transparent

Table 1. Location and Animals

Farm Location Species

VRI, Ipoh Goat, Sheep, Cattle

VRI, Ipoh Buffalo, Cattle, Goat

Infoternak Sg. Siput Buffalo, Deer

Gopeng, Perak Goat, Cattle, Chicken



Infoternak Sg. Siput Cattle

Ampang, Kinta Cattle

Kampar, Perak Pig, Chicken

Tambun Horse

Lawan Kuda, Gopeng Horse

Jalong, Sg.Siput Pig

Chemor Cattle, Chicken

Tg. Rambutan Cattle

Taiping Horse

Jeram Cattle

Lenggong Deer

Parit Cattle

netting, and are then trapped when they fl y up through the gap between the netting shelf and the back of the trap. Two blue rectangular “wings” extend out at an angle from the front framing the lower entrance. The top is closed by a netting “cone”, made by cutting a wedge out of a square piece of netting and sewing up the sides. These results in a tetrahedron: a 3-D shape with three triangles joining at the apex (Mihok et al., 2006).


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VAVOUA Trap

The Vavoua trap (Figure 2) was designed as an economical alternative to the pyramidal trap in widespread use for the control of stable fl ies. There are many similar designs that employ hanging blue-black screens. These open designs are excellent for stable fl ies. Traps like the Vavoua are straight forward to sew and assemble. They can be hung from simple wooden supports or trees. Unfortunately, trap styles with this open design are poor for tabanids according to Laveissière & Grébaut (1990).

Identifi cation of fl ies

For identification, keys according to Masmeatathip et al. (2006); Foil, 1989; Phasuk et al., (2013) were used.

Method for Trypanosomiasis diagnosis is by MAFF, 1978

Blood samples were subjected to buffycoat and thin blood smear examination for detection of any blood protozoa (Wahab et al., 2002). Chandrawathani et al., (1998) has screened Trypanosomiasis using ELISA and CATT (Wahab, 2012) to measure the antibody against Trypanosomes. Whereas Cheah et al., (1999) has seen success in conducting PCR for the detection of Trypanosomiasis in a cattle blood. A capillary tube (1/4 of blood) was fi lled with blood from EDTA tube and one side of the capillary tube was sealed with plasticine. The capillary tube was placed in a micro-centrifuge and the sealed side must face out. Each capillary tube was balanced before centrifuging at

Figure 1. NZI fl ytrap Figure 2. Vavoua fl ytrap


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12,000 rpm for 5 minutes. The buffy-coat was examined under 10X of magnifi cation objective by using compound microscope and trypanosomes were observed moving. Trypanosoma evansi was also identifi ed in Giemsa stained thin blood smears as a monomorphic organism with a mean length of 24 um, with subterminal kinetoplast, undulating membrane and free fl agellum (Soulsby, 1982).

RESULTS

Analysis of fl ies captured from local farms.

Results obtained are shown in Table 2 and Figure 3.

From January to December 2013, a total number of 18 animal farms were screened for biting and sucking f lies whereby four species of them were collected using both Vavoua and Nzi traps. The fl ies that were found are Tabanus sp., Stomoxys sp., Haematobia sp. and Musca sp. The results indicated that the farm which has cattle, buffalo and deer harboured four species of fl ies which can be a vector for Trypanosomiasis. The fl y traps were set up around VRI animal facilities, Ipoh, two (2) animal farms in Kuala Kangsar districts including Infoternak Government farm in Sungai Siput (U), four (4) animal farms in Kinta districts, four (4) animal farms in Kampar, one (1) farm in Perak Tengah districts, one (1) farm in Hulu Perak districts and (1) one farm in Larut, Matang & Selama districts. During the trapping

activities, a total of 1176 Musca sp. fl ies, 646 Stomoxys sp. fl ies, 50 Haematobia sp. fl ies and 29 Tabanus sp. fl ies were collected by using the NZI and Vavoua traps. During the time of collection, the weather was sunny and the traps were set up in an open area about 50 metres from farm animals such as buffalo, pig, horse or cattle.

Positive cases of Trypanosomiasis diagnosed in VRI from 2000-2013

Table 3, Figure 4 and Figure 5 show that there were less than 4 positive cases diagnosed in 2000 to 2013. However, in 2013 there were 11 positive cases,

There is a general increase in trend of positive cases diagnosed.

DISCUSSION

Results indicated the presence of biting f lies as well as Tabanus sp., Stomoxys sp., Musca sp. and Haematobia sp. in the farms that can transmit Trypanosomiasis. Infoternak Sg. Siput farm has annual outbreaks of Surra in the deer and cattle herd, and control of these f lies is an important aspect of the epidemiology of Surra. The fl ies that were found are Musca sp., Stomoxys sp. and Haematobia sp. Rainfall f luctuations may be the main climatic factor correlated with population changes of the most abundant species (Phasuk et al., 2011).


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Figure 3.

Table 2

No. Farm Location Species Musca Stomoxys Tabanid Haematobia

1 VRI, Ipoh Goat, Sheep, Cattle 69 42 0 0

2 VRI, Ipoh Buffalo, Cattle, Goat 2 4 0 0

3 Infoternak Sg. Siput Buffalo, Deer 18 160 0 6

4 Gopeng, Perak Goat, Cattle, Chicken 41 8 0 5



7 Infoternak Sg. Siput Cattle 253 3 0 0

8 Ampang, Kinta Cattle 4 50 0 26

9 Kampar, Perak Pig, Chicken 153 0 0 11

10 Tambun Horse 12 7 0 0

11 Lawan Kuda, Gopeng Horse 81 0 2 1

12 Jalong, Sg.Siput Pig 6 1 0 0

13 Chemor Cattle, Chicken 82 164 11 0

14 Tg. Rambutan Cattle 20 3 0 0

15 Taiping Horse 6 0 0 0

16 Jeram Cattle 240 89 1 0

17 Lengggong Deer 47 4 5 1

18 Parit Cattle 125 93 10 0

Total 1176 646 29 50


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0102030405060708090

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013

NO

OF

CASE

YEARFigure 4. Cases of Trypanosomiasis diagnosed in VRI from 2000-2013

Figure 5.


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Table 3. Positive cases of Trypanosomiasis diagnosed in VRI from 2000-2013

Year SpeciesNo. of

SampleNo. of

Positive

2000 Buffalo 4 2

2001 Cattle 19 6

2003 Deer 160 35

2006 Deer 21 12

2007 Deer 22 3

2007 Cattle 13 5

2008 Cattle 3 1

2008 Deer 80 7

2009 Deer 155 23

2010 Cattle 100 9

2010 Deer 94 9

2011 Deer 28 5

2012 Deer 105 41

2012 Cattle 105 19

2012 Buffalo 20 5

2012 Pig 16 13

2013 Deer 35 3

2013 Cattle 8 3

2013 Buffalo 17 7

CONCLUSION

In conclusion, f ly trapping can be an alternative f ly control technique as a sizeable number of fl ies were caught. Other management methods such as reducing water puddles and clearing damp fodder to prevent fl y breeding habitats will further help to alleviate the problem. Many farmers still tend to rely on pesticides as pour on or spray to prevent the fl y nuisance but a more sustainable, green method would be the use of a mechanical fl y trap such as Nzi or Vavuoa in farms.

REFERENCES

1. Chandrawathani, P., Luckins, A. G., Jamnah, O., Adnan, M., Zaini, C. M. & Cheah, T. S. (1998). Serological Prevalence of bovine Trypanosomiasis in peninsular Malaysia. In Proc.: 10th Vet. Assoc. Malaysia Scientifi c Congress 4-6 Sept 1998, Shah Alam, Malaysia. p.99-100 (OP)

2. Cheah, T. S., Sani, R. A., Chandrawathani, P., Bahri, S., & Dahlan, I. (1996). Prevalence of T.evansi in a dairy cattle farm. In Proc: 8th Veterianary Association

3. Cheah, T. S., Sani R.A., Chandrawathani P., Sansul B. & Dahlan, I. (1997). Natural Infection of T. evansi in crossbred calves in a farm in Peninsular Malaysia. In Proc: 9th Veterinary Association Malaysia Scientifi c Congress, 3-5 October 1997, Penang p. 38-40 (OP)

4. Cheah, T. S. & Chandrawathani, P. (1999). Detection of T. evansi by DNA Amplifi cation using PCR. In proc: National Congress on Animal Health and Production (VAM), A’ Formosa Melaka 3-5 Sept. 1999. pp 99 (PP)

5. Cheah, T.S., Sani, R. A., Chandrawathani, P., Bahari, S., & Dahlan, I. (1999). Epidemiology of T. evansi infection in crossbred dairy cattle in Malaysia.Trop.Ani. Hlth. & Prod. 31:25-31

6. Foil, L. D. (1989). Tabanids as vectors of disease agents. Parasitology Today 5, 88-96.

7. Laveissière, C., & Grébaut, P. (1990).The trapping of tsetse Flies (Diptera: Glossinidae). Improvement of a model: the Vavoua trap. Tropical Medical Parasitology.Jun; 41(2):185-92.

8. Mihok S, Carlson DA, Krafsur ES, Foil LD (2006) Performance of the Nzi and other traps for biting fl ies in North America. Bulletin of Entomological Research 96, 387-397.

9. Ministry of Agriculture, Fisheries and Food. (1978). Reference Book 365 & 368: Manual of Veterinary Investigation Laboratory Technique

10. Nurulaini, R., Jamnah, O., Adnan, M., Zaini, C. M., Khadijah, S., Rafi ah, A. & Chandrawathani P. (2007). Mortality of domesticated java deer attributed to Surra. Tropical Biomedicine 24(2): 67-70(2007).


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11. Nurulaini R., Premaalatha B., Zaini C.M., Adnan M., Chandrawathani P., Fazly Ann Z.A., Ernie Aryanti A. & Ramlan M. (2013). Trypanosomiasis Outbreak in Deer, Cattle, Buffaloes and Pigs in Perak, 2013. Vol. 4 No.1 January 2013 pg. 55-58. Malaysian Journal of Veterinary Research (MJVR)

12. Papp L., Merz B. & Foldvari M. (2006) : Diptera of Thailand ; A summary of the families and genera with references to the species representations. Acta Zoologica Academiae Scientiarum Hungaricae 52(2), pp. 97-269, 2006

13. Phasuk J., Tharawoot T., Beaver R.A., Jittapalapong S. (2011). Seasonal abundance of Tabanidae (Diptera) on Dairy Farms in Saraburi Province, Thailand. Thai Journal of Agricultural Science, 2011 44(3): 175-181

14. Phasuk J., Prabaripai A. & Chareonviriyaphap T. (2013): Seasonality and daily f light activity of stable f lies (Diptera: Muscidae) on dairy farms in Saraburi Province, Thailand. Parasite, 2013, 20, 17.

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17. Ro ungthip Masmeatathip, Chitapa Ketavan and Gérard Duvallet (2006) Morphological Studies of Stomoxys spp. (Diptera: Muscidae) Kasetsart J. (Nat. Sci.) 40 : 872 - 881 (2006)

18. Sani, R. A., Chandrawathani, P., & Nuzolazuan, I. (1990). Trypanosoma evansi in W.Malaysia. In Proc. of 26th Annual Scientifi c seminar, Malaysian Society of Parasitology and Tropical Medicine, 22-24 Feb. 1990, USM Pulau Pinang - (PP).

19. Sani, R.A., Chandrawathani, P., Salim, N.B. (1995). Diagnosis of T. evansi infection in cattle and buffaloes in W. Malaysia. In Proc. 7th National Biotech.Seminar.20-22 Nov. Langkawi, Malaysia - (OP).

20. Soulsby, E. J. (1982). Helminths, Arthropods and Protozoa of Domesticated Animals, 7th Edition. Published by Bailliere Tindall, London

21. Taylor, M. A, Coop, R. L., & Wall, R. L. (2007). Veterinary Parasitology, 3rd Edition. Blackwell Publishing Ltd.

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23. Truc, P., Buscher, P., Cuny, G., Gonzatti, M. I., Jannin, J., Joshi, P.,Juyal, P., Lun, J. L., Mattioli, R., Pays, E., Simarro, P. P., Maria, M., Teixeira, G., Touratier, L., Vincendeau, P., and Desquesnes, M. (2013). A Typical Human Infection by Animal Trypanosomes. Plos Neglected Trop. Diss. 7 (9) 2256

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The Malaysian Journal of Veterinary Research (MJVR) is the offi cial journal of the Department of Veterinary Services, Malaysia. The MJVR aims to advance veterinary science by publishing and promoting high quality refereed scientifi c and clinical articles. The MJVR publishes original articles, case reports, short communications, clinical updates, diagnostic challenges, reviews and veterinary history articles. All articles are peer reviewed. English is the offi cial language of the journal. Only manuscripts with substantial scientifi c merit will be reviewed for originality, signifi cance, relevance and quality.

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1. Chaudary F.R., Khan M.F.U. and Qayyum M. (2007). Prevalence of Haemonchus contortus in naturally infected small ruminants grazing in the Potohar Area of Pakistan. Pakistan Vet. J. 27(2): 73-79.

2. Kao R.R., Leathwick D.M., Roberts M.G. and Sutherland I.A. (2000). Nematode parasites of sheep: a survey of epidemiological parameters and their application in a simple model. Parasitology 121(Pt 1): 85-103.

3. Ministry of Agriculture, Fisheries and Food (1986). Manual of Veterinary Parasitological Laboratory Techniques. Her Majesty’s Stationery Offi ce, London, pp 160.

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USE OF PRIMARY QUAIL EMBRYO FIBROBLAST CELLS FOR PROPAGATION AND ASSAY OF AVIAN VIRUSESMaj may 2015

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