Brucellosis (DS..22.8.14)

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INTRODUCTION Brucellosis is an important zoonotic disease with a worldwide distribution. The prevalence of brucellosis infection varies greatly from one country to another and between regions within a country. The highest prevalence is seen in dairy cattle. In India, brucellosis was first recognized in 1942 and is now endemic throughout the country. The disease has been reported in cattle, buffaloes, sheep, goats, pigs, dogs and humans (OIE, 2004). Economic losses because of brucellosis in animals are due to abortions, premature births, and decreased milk production as well as due to repeat breeding and may lead to temporary or permanent infertility in infected livestock. This disease poses a significant health hazard to in contact human beings (Cottorello et al., 2002 ). The genus Brucella comprises of six recognized species: B._melitensis, B. abortus, B. suis, B. ovis, B. canis and B. neotomae (Corbel and Brinley-Morgan, 1984). This nomenclature was established on the basis of differences in pathogenicity, host preference, growth and biochemical characteristics (Corbel and Brinley-Morgan, 1984). Brucella produces generalized infection with a bacteremic phase followed by localization in the reproductive organs and reticulo endothelial system. Brucellosis is essentially a disease of sexually matured animals and have predilection for ungulates placentae,

Transcript of Brucellosis (DS..22.8.14)

Page 1: Brucellosis (DS..22.8.14)

INTRODUCTION

Brucellosis is an important zoonotic disease with a worldwide

distribution. The prevalence of brucellosis infection varies greatly from one

country to another and between regions within a country. The highest

prevalence is seen in dairy cattle. In India, brucellosis was first recognized in

1942 and is now endemic throughout the country. The disease has been

reported in cattle, buffaloes, sheep, goats, pigs, dogs and humans (OIE,

2004). Economic losses because of brucellosis in animals are due to

abortions, premature births, and decreased milk production as well as due to

repeat breeding and may lead to temporary or permanent infertility in infected

livestock. This disease poses a significant health hazard to in contact human

beings (Cottorello et al., 2002 ).

The genus Brucella comprises of six recognized species:

B._melitensis, B. abortus, B. suis, B. ovis, B. canis and B. neotomae (Corbel

and Brinley-Morgan, 1984). This nomenclature was established on the basis

of differences in pathogenicity, host preference, growth and biochemical

characteristics (Corbel and Brinley-Morgan, 1984).

Brucella produces generalized infection with a bacteremic phase

followed by localization in the reproductive organs and reticulo endothelial

system. Brucellosis is essentially a disease of sexually matured animals and

have predilection for ungulates placentae, foetal fluids, mammary gland, joints

and testes of bulls, rams, boars and male dogs. The disease is manifested by

reproductive failure, which includes abortion during mid to late pregnancy,

birth of unthrifty calves and retained placentae in female animals. Localization

may also occur in mammary tissues with excretion in the milk (Corbel, 1988).

Lesions in Brucella infected male are largely confined to the genital organs

including testicles, seminal vesicles and epididymes (Morgan and MacKinnon,

1979).

Bovine brucellosis is found worldwide, however, it has been eradicated

from many countries (Romero et al., 1995a) as there are stringent regulations

like test and slaughter policy, but it is one of the most serious diseases in

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developing countries. The rates of infection vary greatly from one country to

another and between regions within a country. The highest prevalence is seen

in dairy cattle. Despite the advances made in the diagnosis and therapy,

brucellosis is still wide spread and its prevalence in many developing

countries is increasing (OIE, 2004). Economic losses by brucellosis in animals

are due to abortions, premature births, decreased milk production and due to

repeat breeding and may lead to temporary or permanent infertility in infected

livestock. Economic losses due to brucellosis in livestock are considerable in

an agrarian country like India (Mehra et al., 2000; Renukaradhya et al., 2002

and Sarumathi et al., 2003) accounting to U.S.$58.8 million (Kollannur et al.,

2007) per year. Probably the main route of portal of entry is the ingestion.

Transmission via the teat canal has also been suggested as a route of

infection but laboratory results and extensive field experience have not

confirmed this as an important route. The practice of sharing equipment

between various farms is also a potential danger. It has also been observed

that calves fed on infected milk harbour infection and excrete Brucella

organisms in their faeces for up to 4 weeks after the cessation of feeding.

Artificial insemination,.infected AI guns, infected bulls semens, secretions and

excretions (Neilsen and Duncan., 1990)

The organism produces humoral antibody response (Khatun et al.,

2009). Depending upon this property various serological techniques for

diagnosis of brucellosis are being evolved. The test recommended by WHO

are isolation or demonstration of the organism in tissues or fluids, and

serological tests and agglutination tests on milk or seminal plasma, and gold

standard being isolation and identification of the organisms (Eaglesome and

Garcia., 1997). Conventionally, above mentioned serological tests are used to

screen, or to confirm the disease. These screening tests are inexpensive, fast

and highly sensitive but not necessarily highly specific. The most widely used

serological tests for diagnosis of brucellosis in animals are Rose Bengal Plate

Test (RBPT), Standard Tube Agglutination Test (STAT) and Enzyme Linked

Immunosorbent Assay (ELISA). The diagnostic value may be questionable on

individual basis because of cross reacting antibodies but for screening of herd

these tests remain ideal. Other than serum, Brucella antibodies are also

excreted in milk. The Milk Ring Test (MRT) is often used as a herd test to

know the prevalence of Brucella_infection & and screening of herd. The MRT

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can also be used to test individual milk samples to identify the infected animal

in the herd but, it may give false-positive results shortly after parturition, near

the end of lactation and when mastitis is present (Alton et al.,1988). To

overcome this problem Milk-ELISA is employed on individual milk samples to

detect Brucella antibodies.

The present scenario of seroprevalence of bovine brucellosis indicate

8.8 percent % seroprevalance in India and 3.7 percent % in Maharashtra

Isloor et al.,(1998).Similarly 22.56% and 10.40% seroprevalance was

recorded in goat and sheep in Maharashtra region (Raju et al., 2004) which

clearly indicate the need of knowing status of brucella infection in bovine and

caprine species of Maharashtra.

The control of brucellosis depends upon reliable methods for detection

of the infection in livestock, wildlife and humans. Several diagnostic strategies

have been developed which when used in concert, have been instrumental in

decreasing the incidence of the disease. Considering the above facts the

present study is planned with following objectives:

11. OBJECTIVE:-

1. To determine the area wise sero-prevalence of brucellosis in Livestock

of Western-Maharashtra.

REVIEW OF LITERATURE:

Seroprevalence of Brucellosis in different species of animals

Kim et al. (1988) compared serological tests in 84 brucellosis reactors

and 44 healthy controls. The PAT resulted in 3.1% of false positive and 1.6%

of false negative reactions in comparison with that of STAT. The agreement

between both the tests was found to be 61.7%. Among the Riv, 2 MET,

ELISA, RBPT and CFT, the ELISA and CFT resulted in to very sensitive

reactions, while the Riv test revealed the most specific reactions.Patiet al.

(2000) tested 23 sera from buffaloes (male 2, female 21) of the village

Danpur, Distt. Moradabad (U.P) by applying RBPT, STAT and ELISA. They

concluded that ELISA was more sensitive than the RBPT and STAT.

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Bright and Smith (1897) described perhaps for the first time

agglutination test for the diagnosis of Malta fever in British soldiers stationed

at Malta, which helped to distinguish it from typhoid fever, since then there

have been reports in the literature regarding the use of various serological

tests for the diagnosis of brucellosis in animals and man.

Badnjevic and Barjrovic (1981) have screened 2212 blood samples

from man, cattle, pigs, horses, sheep and goats by agglutination, CF and

RBPT tests for brucellosis. Bachhetal., (1987) Screened sera samples of

unvaccinated 294 sheep and 46 goats in Kashmir by Plate Agglutination Test.

(PAT), Acid Plate Agglutination Test (APAT), Standard Tube Agglutination

Test (STAT), Heat Inactivation Test (HIT) 9 and Complement Fixation Test

(CFT). The overall prevalence of brucellosis in sheep and goats was 4.6 and

7.6 percent respectively.

Radwan et al., (1992) conducted serological and bacteriological study

of brucellosis in camels in Saudi Arebia. Sera from 2,630 apparently normal

adult camels raised in central Saudi Arabia wereexamined serologically by the

Rose Bengal and standard United States ofAmerica Brucellaplate

agglutination tests.Of the 2,630 serum samples tested, 212 were found to be

brucellosis seropositive,giving an overall prevalence of 8%.

Hadad and Jamalludeen (1992) screened out 2006 cattle sera for

Brucella antibodies by RBPT, TAT, MET and CFT. 117 (5.8 percent) sera

gave positive reaction to RBPT and CFT, and 91 (7.78 percent) sera gave

positive results with TAT, whereas, 73 (6.24 percent) gave positive result with

MET.

Desai et al., (1995) screened 653 sheep, 630 goats and 102 human

sera samples in Bidar area of Karnataka by rapid plate test and RBPT. The

incidence of brucellosis was 4.9, 7.6 and 5.9 percent respectively in sheep,

goats and humans.

Sharma and Saini (1995) tested a total of 573 sera samples from

different herds of cattle and buffaloes and flock of sheep and goats by RBPT.

The prevalence of brucellosis among cattle, buffaloes, sheep and goats was

8.69, 14.61, 15.45 and 1.75 percent respectively.

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Sharma and Saini (1995) tested 573 sera samples of cattle and

buffaloes by RBPT and STAT and found 8.69 and 14.91 percent positive by

STAT in cattle and buffaloes respectively.

Isloor et al., (1998) reported the data of serological survey of

brucellosis in cattle and buffaloes in 23 states of India. The prevalence rate of

Brucella antibody was 1.9 percent in cattle and 1.8 percent in buffaloes using

RBPT and STAT.

Isloor et al., (1998) conducted serological survey on bovine brucellosis

in India. Survey of brucellosis in cattle and buffalo was performed in 23 States

of India. A total of 30,437 bovine samples, comprising 23,284 cattle and 7,153

buffalo (Bubalusbubalis), were screened. The screening initially used the rose

bengal plate test. The RBPT results were interpreted as either positive or

negative on the basis of presence or absence of agglutination reaction,

respectively. Doubtful and positive samples were then titrated in the serum

tube agglutination test.

Kim et al. (1988) evaluated comparative efficacy of different serological

tests viz., RBPT, STAT, ELISA, CFT, ME and Riv in diagnosis of brucellosis.

The study conducted on 84 brucellosis reactors and 44 healthy controls

revealed that the Plate Agglutination (PA) test produced 3.1% false positive

and 1.6% false negative results in comparison to STAT. The agreement

between these tests was found to be 61.7%. Among the other tests evaluated

during study, ELISA and CFT were found to be highly sensitive while the Riv

test proved to be most specific.

Molnar et al. (1998) investigated 878 serum samples of cattle and

buffaloes employing Buffered Plate Agglutination Test, STAT, CFT, i–ELISA

and c-ELISA. The i-ELISA in general yielded higher numbers of positive

results however for samples derived from the Marajo Island, c-ELISA proved

to be more sensitive. The sensitivity of the classical tests like agglutination

and complement fixation tests was found to be markedly lower than that of the

ELISAs.

Rao et al. (1999) investigated the abortions in buffaloes and cross-

bred cowsemploying Rapid Plate Agglutination Test (RPAT), STAT and dot-

ELISA. A total of 160 serum samples were processed for detection of anti-

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Brucella antibodies. Highest proportion of positive animals were detected by

dot-ELISA (buffaloes - 16.25% and cross-bred cows - 31.25%) followed by

RPAT (buffaloes - 11.5% and cross-bred cows - 16.25%) and STAT (buffaloes

- 8.75% and cross-bred cows - 15.00%).

Kalorey et al., (2000) reported seroprevalence of brucellosis among

cow in Vidarbha region in Maharashtra by using RBPT, STAT and dot-ELISA

and overall prevalence was reported to be 20.0 and 14.16 percent by RBPT

and STAT respectively

Chakraborty et al., (2000) had screened 141 sera samples of cattle for

brucellosis antibodies by ELISA, SAT and RBPT and seroprevalance was

56.02 percent 50.33 percent and 33.33 percent respectively.

Chauhan et al., (2000) tested 59 sera samples (50 from aborted

buffaloes and 9 from pregnant buffaloes) for bovine brucellosis in North

Gujarat by using RBPT and STAT and 28 (17,46 percent) and 23 (38.98

percent) were found positive by RBPT and STAT respectively.

Lodhi et al. (1995) carried out seroprevalence study of brucellosis by

colleting 208 serum samples of adult buffaloes and cows in and around

Faisalabad by RBPT and STAT. They found 12.98% and 2.40% of

seroprevalence by RBPT and SAT, respectively. They also revealed that 5

animals positive to STAT, 3 (60%) gave positive results with RBPT.

Prahlad et al. (1999) carried out seroprevalence study of brucellosis in

buffaloes. Of the 296 serum samples collected at an abattoir in Delhi 7.09%,

2.70%, 11.14% and 8.10% were found to be positive by RBPT, STAT, CFT

and dot-ELISA, respectively. Seroprevalence of brucellosis in Punjab was

found higher (21.39%) than in Uttar Pradesh (11.32%). RBPT showed the

highest relative sensitivity (33.33%) using CFT as an indicator test. All the

tests showed relative specificity of >90%.

Sarumathi et al. (2003)compared efficacy of avidin-biotin ELISA (AB-

ELISA), RBPT and STAT in detectingBrucellaantibodies in 1541 serum

samples of cattle with and without the history of reproductive failures in

Andhra Pradesh. AB-ELISA, RBPT and STAT showed specificities of 100%,

88.22% and 90.59% respectively. The AB-ELISA was suggested to be a

reliable screening test for detecting antibodies to Brucella in cattle.

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Rajesh et al. (2003) assessed seroprevalence of brucellosis in 719

cattle of Kerala (India) using RBPT and STAT. Of these samples, 9 were

found positive by RBPT but 5 gave a doubtful reaction, whereas all 14

samples were positive in STAT. They found that the overall seroprevalence

was 1.95% and greater in adult cattle. They also concluded that seropositivity

was higher in heifers and pregnant animals. The efficacy of AB-ELISA, RBPT

and STAT in detecting antibodies to Brucella of 1541 serum samples from

cattle with a history of reproductive failures and in healthy cattle from farms in

Andhra Pradesh was compared by Sarumathi et al. (2003b). AB-ELISA, RBPT

and STAT gave specificities of 100%, 88.22% and 90.59%, respectively. They

also found AB-ELISA as a reliable screening test for detecting antibodies to

Brucella in cattle.

Varasada (2003) tested 344 cattle and 251 buffaloes for brucellosis.

The results revealed that 68(19.76%), 57(16.57%) and 83(24.12%) of cattle

were positive by RBPT, STAT and IELISA, respectively. Whereas 32(12.75%),

28(11.16%) and 48(19.12%) of buffaloes were positive by RBPT, STAT and I-

ELISA, respectively. In an overall seroprevalence study of brucellosis in cattle

and buffaloes of central Gujarat, 16.80%, 14.03% and 22.01% of animals

were found positive by RBPT, STAT and I-ELISA, respectively.

Barbuddhe et al. (2004) investigated prevalence of brucellosis in

organized farms with abortion storms in Goa region. Out of 107 serum

samples tested for brucellosis, 40 (37.38%), 39 (36.45%) and 43 (40.18%)

were found positive for antibodies against Brucella by RBPT, STAT and AB-

ELISA, respectively.

Nielsen et al. (2004) evaluated the sum of the sensitivity and specificity

values for each test was averaged to give a performance index (PI) and allow

for a comparison between the different methodologies. A score of 200 was

perfect. Based on the PI, the buffered antigen plate agglutination test (BPAT)

rated highest (PI = 193.1) among the conventional tests. This indicates better

accuracy than the other conventional tests including the Rose Bengal test (PI

= 167.6) and the complement fixation test (PI = 172.5). Overall, the primary

binding assays, including the fluorescence polarisation assay (PI = 196.4), the

indirect enzyme linked immunosorbent assay (PI = 189.8) and the competitive

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enzyme-linked immunosorbent assay (PI = 188.2), were more accurate than

the conventional tests, except for the BPAT.

Singh et al. (2004) performedserological survey in 6 organized dairy

farms in Punjab using RBPT, STAT and AB-ELISA. To compare the sensitivity

and specificity of RBPT and STAT, AB-ELISA was used as the gold standard.

The study revealed that the sensitivity of RBPT (88.46%) was higher when

compared with STAT (46.15%), while specificity of STAT (98.31%) was

slightly higher than RBPT (97.75%). In a comparative study for detection of

Brucella antibodies, AB-ELISA detected antibodies in 43(11.94%), RBPT in

37(10.28%) and STAT in 29(8.05%) samples out of 360 bovine serum

samples tested in Assam (Bhattacharya et al., 2005).

Genc et al. (2005) collected and tested sera from 163 aborted dairy

cattle that had no history of vaccination against brucellosis. They detected B.

abortus antibodies in these serum samples as 68.1, 65.6, 58.9 and 55.2%,

respectively, by the C-ELISA, CFT, RBPT and STAT. A total of 859 cattle and

133 buffaloes of organized sector (Goshala and Tabela) and unorganized

sector of Jodhpur region were screened by Kachhawaha et al. (2005) using

RBPT. The positive samples were subjected to STAT. The prevalence of

brucellosis was found much higher in cattle (41.79%) than in buffaloes

(25.56%) and also more in cattle of organized sector (Goshala) in comparison

to unorganized sector.

Mittal et al. (2005) compared three serological tests namely RBPT,

STAT and ELISA by testing 217 cattle sera and 67 buffalo sera from the

district Udham Singh Nagar, Uttranchal. They found that ELISA was more

sensitive followed by RBPT and STAT when applied to cattle sera, whereas

RBPT was more sensitive followed by STAT and ELISA when applied to

buffalo sera.

Sunder et al. (2005) found 13.83% of the samples positive in RBPT

while 10.4% of the samples positive in AB-ELISA in sero screening analysis of

cattle belonging to Andaman and Nicobar islands.

Mishra et al. (2005) examined 579 cows and 407 buffaloes employing

STAT and I-ELISA. The i-ELISA was found to be more sensitive and could

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detect 3.11% cows and 4.18% of buffaloes as positive. The STAT detected

1.55% cow and 1.97% buffalo sera positive.

Ganesan and Anuradha (2006) compared dot-ELISA and RBPT for

diagnosis of bovine brucellosis. Out of the total 81 samples tested 11.11%

and 13.59% were found positive by RBPT and dot-ELISA, respectively.

Agrawal et al. (2007) compared three serological tests namely RBPT,

STAT and ELISA by applying to the 142 cattle and 61 buffalo sera of

Bageshwari district of the state Uttaranchal. They found that ELISA was more

sensitive followed by RBPT and STAT.

Mitat et al. (2007) examined 626 cattle sera derived from 27 herds with

a history of abortions and recorded a greater proportion of samples positive by

ELISA (39.45%) than RBPT (35.30%) and STAT (32.92%).

Berhe et al., (2007) performed serological test against bovine

brucellosis. Of the 816 sera examined, 27 (3.3%) were seropositive to RBPT

out of which 26 (3.19%) reacted positively to CFT with a titer>1:20. The entire

seropositive animals were female animals. Among the 12 Districts included in

the study, Brucella antibodies were detected in 6 districts.

Upadhyay et al. (2007) screened 1034 serum samples of animals from

17 randomly selected districts of Uttar Pradesh state for brucellosis employing

AB16 ELISA, STAT and RBPT. The AB-ELISA detected higher proportion of

positive animals (7.25%) as compared to STAT which could detect 4.73% and

RBPT that detected 2.90% positive animals.

Chachra et al. (2009) evaluated comparative efficacy of 3 sero

diagnostics tests RBPT, STAT, and Dot ELISA in detecting anti-Brucella

antibodies on a total of 28 serum samples including 18 from brucellosis

suspected and 10 from healthy cattle. Out of 18 sera from suspected cattle,

only 1 (5.55%) was found positive by STAT whereas 9 (50%) samples proved

positive by RBPT. The Dot ELISA however could detect antibodies in all the

18 (100%) samples.

Azar Khan et al. (2009) studied the sero-prevalence of brucellosis in

buffaloes and humans in Swat Valley in Pakistan using RBPT and i-ELISA. A

total of450 samples including 400 buffalo and 50 human sera were examined.

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The overall prevalence in buffalo and human recorded by RBPT was 4.75%

and 0% respectively whereas; it was 5.5 and 0% respectively with ELISA. The

RBPT and ELISA were suggested to be equally efficient in screening of

animals for brucellosis.

Bertu et al. (2010) conducted a sero epidemiological study of

brucellosis in small ruminants in Nigeria. A total of 1347 serum samples

including sheep (496) and goats (851) collected from nine randomly selected

local government areas were examined for presence of Brucellaantibodies by

RBPT and STAT the result revealed brucellosis prevalence of 14.5% in sheep

and 16.1% in goats.

Poester et al.,(2010) evaluated Sensitivity, Specificity and

Performance Index of the Serological Tests for Brucellosis where he studied

several serological tests and their sensitivity. In all tests including SAT, RBT,

BPAT, RIV, 2ME, CFT, i-ELISA, c-ELISA and FPA sensitivity percentage is

higher in c-ELISA showed 97.5-100% sensitivity.

Mohammed Yesuf et al. ( 2010) studied the seroprevalance of ovine

brucellosis in Ethiopia. A total 800 sheep above six months age were

screened for presence of Brucella antibodies by RBPT and CFT. Over all

seroprevalence of 1.5% (12 of 800) was observed. Seroprevalence was

higher in female sheep compared to male sheep.

Kaoud et al. (2010) carried out epidemiological studies on brucellosis

in ruminants in Egypt. Serum samples (1670) were collected from 126 Herds /

Flocks of sheep, goats and cattle and analyzed using RBPT and iELISA test.

The results pointed out that, prevalence of brucellosis among herds/flocks of

sheep, goats and cattle were; 26.66%, 18.88% and 17.22% respectively.

Aher (2010) screened serum samples of 74 animals including 68 cattle

and 6 buffaloes from different locations in Maharashtra for presence of

Brucellaantibodies by RBPT, STAT and i-ELISA. Highest proportion of positive

samples were detected by i-ELISA (56.75 %) followed by RBPT (54.05 %) and

STAT (47.29 %).

Montasser et al., (2011) studied efficacy of serological test for

detection of Brucellosis in ruminants at south Provices of Egypt. A total of

2138 serum samples(715 from cattle, 1323 from sheep and 100 from goats)

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from differentdistricts in Assuit governorate, was tested for the detection of

antibodies against Brucellaspp. Results obtained by Buffer acidified plate

antigen test (BAPAT) and Rose bengal test (RBT) as screening tests indicated

a positive reactors percentage of 4.6-5.3, 4.4-7.6 and 10-15% followed by

overall brucellosis incidence of 4.5, 5.2 and 5.0 % in case of cattle, sheep and

goats, respectively. It was reported that no single test can identify all infected

animals at all stages of the diseases and therefore a combination of

serological test should be included to reduce the number of both false

negative and false positive serological reactions. It was also reported that

BAPAT and RBPT serological tests revealed the highest rate of sensitivity that

guide to use these test as screening test on animal brucellosis.

Munir et al., (2011) studied seroprevalence of bovine brucellosis at

farms under different management conditions. A total of 3029 serum samples

from adult cattle and buffaloes were collected between the years 2007 to

2009, from three types of farm categorie.Sera were tested by an indirect

ELISA and Rose Bengal Precipitation Test (RBPT). I-ELISA detected sero-

conversion in 15.2% buffaloes and 9% cattle, whereas, by using RBPT it was

8 and 6.5% in buffaloes and cattle, respectively. In cattle, more abortions were

recorded at private farms (17.86%) followed by gawala colonies (11.61%) and

government livestock farms (8.92%). Also, I-ELISA detected sero-conversion

against brucellosis in 13.2% female and 1.3% male animals, while by using

RBPT it was 7.9% in female and 1.6% in male animals, respectively.

Garadi et al., (2011) detected Brucella melitensis in blood samples

collected from goats. For the study 288 blood and sera samples were

collected from goat farm in Kedah state of Malaysia which was suspected for

brucellosis. The RBPT and CFT were detected 23.3% and 25.3% of samples

positive to B. melitensis respectively.

Mai et al., (2012) collected serum samples of 4,745 cattle from 271

herds and were tested using the Rose-Bengal plate-agglutination test (RBPT).

the positives samples were confirmed using a competitive enzyme-linked

immunosorbent assay (c-ELISA).Prevalence estimates were calculated by

adjusting for sampling weights and where possible for test sensitivity and

specificity. Thirty-seven % of all animals were RBPT positive, and after

confirmation with c-ELISA the overall animal-level prevalence, adjusted for

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sampling weights, was 26.3%. Of the herds sampled, 210 had at least one

animal positive to both tests. Overall animal-level seroprevalences of 29.2% (n

= 1,827), 23.3% (n = 1,870) and 26.7%(n = 1,048) were observed in

Adamawa, Kaduna and Kano states, respectively (P = 0.496). A significantly

higher seroprevalence was found in males (38.2%) than in females (24.7%)

and in non-pregnant females (27.8%) than in pregnant females (17.2%).

Mohamed et al., (2012) conducted studies on molecular and

serological detection of Brucellaspecies in cattle and buffaloes. Out of 32 cow

serum sample examined by RBPT 30 samples (93.8%) were positive and out

of 18 buffalo serum samples examined 16 (88.9%) were positive by RBPT.

Mustafa et al., (2012) collected a total of 40 sheep blood serum

sample aseptically from animal under investigation. Buffered Acidified Plate

Antigen Test (BAPA), Rose Bengal Plate Test (RBPT), Rivanol Test and

Competitive Enzyme Linked Immunosorbent Assay (c-ELISA) were

performed. Results shown that c-ELISA test gives accurate result at 90 days

or more post vaccination.

Amin et al., (2012) studied serological and molecular diagnosis of

bovine brucellosis. The animals included in this study were180 naturally

infected non vaccinated cows in governmental farm (group 1), 125

brucellafree cows in which strain 19 vaccination had never been practiced

(group 2) and 530 strain 19 vaccinated cows (group3). Sera from these

animals were examined for brucellosis using RBPT, BAPAT, Riv.T, TAT, CFT.

For cows suspected to be infected with brucellosis, the results revealed that

the percentage of positive reactors for RBPT, BAPAT, Riv.T, TAT and CFT

were 139(77.2%), 143(79.4%), 130(72.2%), 146(81.1%) and 131(72.8%)

respectively. While for brucella free cows, the percentage of positive reactors

were 2(1.6%), 4(3.2%), 1(0.8%), 5(4%) and 1(0.8%) respectively.

Jagapur et al., (2013) conducted seroprevalence studies on bovine

brucellosis from three states of India (Karnataka, Utter Pradesh and

Uttarakhand). For study total of 1005 sera samples were collected and tested

for bovine brucellosis using ELISA Kit; IDEXX, CHEKIT, Brucellose serum,

BrucellaabortusAntibody Test Kit. Sera from 5 organized farms in Karnataka

were collected for seroprevalence studies. Out of 417 animals, 191 (45.80%)

animals were found positive by i-ELISA. A total of 361 serum samples were

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collected from 5 unorganized farms or villages, of which 82 (22.71%) were

positive. From Uttar Pradesh, bovine serum samples were collected from 3

organized farms. Out of 192 animals, 43 (22.39%) animals were found

positive for brucellosis. Similarly, sera collected from a single organized farm

from Uttarakhand, showed 3 (8.57%) positivity among 35 animals. On the

whole, 319 (31.74%) animals were found positive for brucellosis among the 3

states taken for study, which includes 138 (27.21%) cattle and 181 (36.34%)

buffaloes.

Senthil et al., (2013) conducted seroprevalence study of bovine

brucellosis in slaughter house. For the study total of two hundred and ten sera

samples collected from unvaccinated bulls slaughtered at slaughter house in

Chennai over a period of one year in 2010 and stored at -20oC till further use.

Sera samples were subjected to Rose Bengal Plate Test (RBPT) and

Standard Tube Agglutination Test (STAT) using Rose Bengal Plate antigen

(IVRI, Izat Nagar) and plain Brucellaabortusantigen respectively. The indirect-

ELISA test was carried out using kits supplied by Defence Research

Development Establishment (DRDE), Gwalior. Of these 11 (5.23%), 7 (3.3%),

24 (11.4%) were positive by RBPT, STAT and i-ELISA respectively. It is found

that i-ELISA test was more sensitive and specific when compared to other two

tests viz RBPT and STAT.

12.2) Serological tests.

Rose Bengal Plate Test (RBPT)

Allan et al., (1976) in their study found that the RBPT antigen used at a

pH of 3.65. Prevents some agglutination by IgM and encourages agglutination

by IgG1 thereby reducing non-specific interactions. This test is considered as

a screening test however, some cross-reacting antibodies have been detected

by this test and false negative reaction may occur mostly due to prozoning

(OIE, 2004).

Islam et al., (2013) conducted study on serological test for diagnosis of

brucellosis in buffaloes. Out of 178 samples tested 81 were found positive for

brucellosis by RBPT.

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Ghodasara N,S. et al., (2010) performed comparative study of RBPT,

STAT and ELISA for detection of antibody against bovine brucellosis. From

180 samples 11.21% were positive for brucellosis in cow and 9.59 % were

positive in buffalo.

Rajesh et al., (2003) assessed seroprevalence of brucellosis in 719

cattle of Kerala (India) using RBPT and STAT. Of these samples, 9 were

found positive by RBPT but 5 gave a doubtful reaction, whereas all 14

samples were positive in STAT. They found that the overall seroprevalence

was 1.95% and greater in adult cattle. They also concluded that sero positivity

was higher in heifers and pregnant animals.

Bertu et al. (2010) conducted a seroepidemiological study of

brucellosis in small ruminants in Nigeria. A total of 1347 serum samples

including sheep (496) and goats (851) collected from nine randomly selected

local government areas were examined for presence of Brucella antibodies by

RBPT and STAT the result revealed brucellosis prevalence of 14.5% in sheep

and 16.1% in goats.

Enzyme Linked Immunosorbent Assay (ELISA)

Enzyme immunoassays have replaced the traditional serological tests in

diagnosis of brucellosis over the past few years. The indirect ELISA (I-ELISA)

that is very sensitive in detection of brucellosis.

Molnar et al., (1998) carried out a comparative study on total of 878

selected serum samples from cattle and buffaloes in the Amazonian region by

5 serological tests (BPAT, STAT, CFT, I-ELISA, competitive ELISA) by The I-

ELISA yielded the highest number of positive results, except in samples

derived from the Marajo Island, for which the competitive ELISA was found to

be the most sensitive. They found sensitivity of the classical tests

(agglutination and complement fixation) markedly lower than that of the

ELISAs.

Sarumathi et al., (2003) compared. AB-ELISA, RBPT and STAT and

gave specificities of 100%, 88.22% and 90.59%, respectively. They also found

AB-ELISA as a reliable screening test for detecting antibodies to Brucellain

cattle.

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Ghodasara N,S .et al., (2010) performed comparative study of RBPT,

STAT and ELISA for detection of antibody against bovine brucellosis. From

180 samples 24.30 % were positive for cows and 26.03% were positive for

buffalo by ELISA

Islam et al., (2013) conducted study on serological test for diagnosis of

brucellosis in buffaloes. Out of 178 samples 102 were found positive by

ELISA.

The indirect ELISA (I-ELISA) that is highly sensitive in detection of

brucellosis is prone for false positive serological reactions (FPSR). Further,

the test may not be able differentiate between antibodies induced by S19 and

wild Brucella strains. It has therefore been suggested to be suitable screening

test rather than confirmatory test (OIE, 2004).

Mishra et al. (2005) examined 579 cows and 407 buffaloes employing

STAT and I-ELISA. The i-ELISA was found to be more sensitive and could

detect 3.11% cows and 4.18% of buffaloes as positive. The STAT detected

1.55% cow and 1.97% buffalo sera positive.

Aher (2010) screened serum samples of 74 animals including 68 cattle

and 6 buffaloes from different locations in Maharashtra for presence of

Brucella antibodies by RBPT, STAT and i-ELISA. Highest proportion of

positive samples were detected by i-ELISA (56.75 %) followed by RBPT

(54.05 %) and STAT (47.29 %).

Kaoud et al. (2010) carried out epidemiological studies on brucellosis

in ruminants in Egypt. Serum samples (1670) were collected from 126 Herds /

Flocks of sheep, goats and cattle and analyzed using RBPT and iELISA test.

The results pointed out that, prevalence of brucellosis among herds/flocks of

sheep, goats and cattle were; 26.66%, 18.88% and 17.22% respectively

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CHAPTER III

3. MATERIALS AND METHODS

3.1 Materials

3.1.1 Glassware and plastic ware

During course of study, properly cleaned, neutral standard glassware and

plastic ware compatible for research work were used.

3.1.2 Chemicals, Buffers and Reagents

The details of chemicals, buffers used during course of study are given in

Appendix.

3.1.3 Equipments

Micropipettes, ELISA reader, ELISA plate washer etc. all these instruments

are available in dept of Veterinary Microbiology, KNP College of Veterinary

Sci. and CIF, KNP College of Veterinary Science.

3.2 Collection of samples

A total of 1000 serum samples were collected from different district of western

Maharashtra region, the sample comprised of 250 samples each of cattle,

buffalo, sheep and goat. About 9 ml of blood was collected aseptically from

the jugular vein of individual animal in a vacuette with serum clot activator

(Greiner bio-one, Austria). The vacuettes were kept in upright position at room

temperature for about 2 h. The separated serum was collected in a screw

capped plastic vials and transported to the laboratory. The serum samples

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were heat inactivated at 56ºC for 30 min and merthiolate (1:10,000) was

added in all vials as a preservative. The sera were stored at -20ºC till further

use. Collected serum samples were subjected to Rose Bengal Plate Test

(RBPT), and Enzyme Linked Immunosorbent Assay (ELISA

Details of serum samples collected for research purpose.

Table please.

3.3 Rose Bengal Plate Agglutination Test

3.3.1 RBPT Antigen

The antigen obtained from the Indian Veterinary Research Institute (I.V.R.I.),

Izatnagar, Uttar Pradesh was used for the test.

3.3.2 Procedure

The test was performed according to the manufacturer's literature. Serum

samples and RBPT antigen were brought to the room temperature and then

one drop (0.03 ml) of serum was taken on a clean, dry and non greasy glass

slide by micropipette. The antigen bottle was shaken well to ensure

homogenous suspension and then one drop (0.03 ml) of the antigen was

added. The antigen and serum were mixed thoroughly with the spreader and

then the slide was rotated for four min. The result was noted immediately after

four min.

3.3.3 Observation of Result

Definite clumping/agglutination was considered as positive reaction, where as

no clumping/agglutination was considered as negative.

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3.4 Protein G based indirect ELISA for Bovine brucellosis

Detection of antibodies against Brucellsis using protein G based indirect

ELISA kit obtained from PD-ADMAS Banglore and test was performed as per

protocol outlined in user’s manual supplied with kit.

3.4.1 Contents ofProtein G based indirect ELISA for

Bovine/Caprine/Ovine brucellosis

a. ELISA polysorp uncoated microtitre plates

b. sLPS antigen

c. protein-G HRP conjugate

d. Positive control serum

e. Negative control serum

f. Sodium carbonate

g. Sodium bicarbonate

h. Bovine gelatin

i. Tween 20

j. Psosphate Buffered saline

k. Chromogen (5 mg)

l. Hydrogen Peroxide (30%)

m. Stopping solution (1 Msulphuric acid)

3.4.2 Preparation of reagents

3.4.2.1 Preparation of Coating Buffer

Solution A: sodium carbonate 1.06 gm

Distilled water 50 ml

Solution B sodium bicarbonate 0.84 gm

Distilled water 50 ml

To prepare 25 ml coating buffer (sufficient for coating 2 plates)

following solutions were mixed together.

Solution A 1.75 ml

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Solution B 4.25 ml

Distilled water 19 ml

The pH of solution was checked and it is adjusted to 9.6. this

solution is prepared freshly every time.

3.4.2.2 Phosphate buffered saline (1X)

NaCl 7.0 gm

KCl 0.2 gm

NaH2PO4 0.353 gm

Na2HPO4 1.09 gm

Distilled water 1000 ml

The PBS was prepared and stored at 40C

3.4.2.3 Preparation of washing buffer

PBS (1 X) 500 ml

Tween 20 0.25 ml

The washing buffer was prepared freshly every time.

3.4.2.4 Preparation of blocking buffer

Bovine gelatine 2.0 gm

PBS (1X) 100 ml

This quantity was sufficient for performing 2 plates. It is kept in

water bath at 370C until bovine gelatine is dissolved completely for

15-20 min. after taking out from water bath 50 µl of tween 20 was

added.

3.4.2.5 Preparation of stopping solution

Conc. H2SO4 5.5 ml

Distilled water 94.5 ml

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Total 100 ml

This quantity of solution was sufficient for performing 2 plates.

3.4.2.6 Hydrogen peroxide

30 % Hydrogen Peroxide 10 µl

Distilled water 90 µl

Total 100 µl

This quantity was sufficient for performing 2 plates.

3.4.2.7 Working solution of conjugate (Protein G-HRP)

Working solution of conjugate (Protein G-HRP)was prepared by

adding 1.5 ul of conjugate to 12 ml of blocking buffer (1:8000 dilution)

3.4.2.8 Chromogen solution

Chromogen solution was prepared by adding 1 OPD tablet (5mg)to

12 ml of distilled water followed by addition of 50 ul of hydrogen

peroxide (3%).

3.4.3 Manufacturers protocol for performing Protein G based indirect

ELISA for Bovine brucellosis

3.4.3.1 Coating of microtitre plates

a. Antigen from stock solution was added at 40 ul/12 ml of coating buffer.

It was mixed properly and then dispensed at 100 ul into each well.

b. Sides of plate were tapped to ensure that antigen was evenly

distributed over the bottom of each well.

c. The plate was then covered with aluminium foil and kept for incubation

overnight at 40C in refrigerator.

d. The plate was washed 3 times with 100 ul of wash buffer after

overnight incubation and tapped on tissue paper to remove residual

wash buffer.

e. 100 ul of blocking buffer was added and plate was incubated for 1 hr.

3.4.3.2 Addition of test and control sera

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a. 5 ul of test and control sera was diluted in 500 ul of blocking buffer in

separate Perspex plate and mixed thoroughly 10 times to ensure

homogeneity before loading to microtitre plate.

b. The diluted 100 ul test sera samples in duplicate wells and two control

sera (positive and negative)along with conjugate samples in

quadruplicate well are transferred from perplex plate to the micro titre

plate.

c. The plate was then incubated at 370C for 1 hr on ELISA plate shaker at

300 rpm.

3.4.3.3 Addition of conjugate

a. The plate was then taken out of shaker and washed three times with

washing buffer as mentioned earlier.

b. 100 ul of working dilution of conjugate (protein G HRP conjugate) was

added to each well and incubated at 370C for 1 hr on shaker at 300

rpm.

3.4.3.4 Addition of substrate/ chromogen

a. The plate was taken out of shaker and washed three times with

washing buffer.

b. 100 ul of substrate was added to each well of microtitre plate.

c. The plate was then incubated at room temperature for 7 min or until a

visible yellow colour develops in strong positive wells by covering with

aluminium foil

3.4.3.5 Addition of stopping solution

a. After colour development 50 ul of stopping solution was added to each

well of plate immediately.

b. The plates are then read immediately in ELISA reader at 492 nm.

3.4.4 Interpretation of results

Per-cent positivity values which are used for diagnostic interpretation are

calculated as follows

PP = Average OD value of test serum X 100

Median OD value of positive control sera

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3.4 Protein G based indirect ELISA for Ovine/Caprine brucellosis

Detection of antibodies against Brucellsis using protein G based indirect

ELISA kit obtained from PD-ADMAS Banglore and test was performed as per

protocol outlined in user’s manual supplied with kit.

3.4.1 Contents of Protein G based indirect ELISA for Bovine brucellosis

a) ELISA polysorp uncoated microtitre plates

b) sLPS antigen

c) protein-G HRP conjugate

d) Positive control serum

e) Negative control serum

f) Sodium carbonate

g) Sodium bicarbonate

h) Bovine gelatin

i) Tween 20

j) Psosphate Buffered saline

k) Chromogen (5 mg)

l) Hydrogen Peroxide (30%)

m) Stopping solution (1 Msulphuric acid)

3.4.3 Manufacturers protocol for performing Protein G based indirect

ELISA for Ovine/Caprine brucellosis

3.4.4.1 Coating of microtitre plates

a. Antigen from stock solution was added at 40 ul/12 ml of coating buffer.

It was mixed properly and then dispensed at 100 ul into each well.

b. Sides of plate were tapped to ensure that antigen was evenly

distributed over the bottom of each well.

c. The plate was then covered with aluminium foil and kept for incubation

overnight at 40C in refrigerator.

d. The plate was washed 3 times with 100 ul of wash buffer after

overnight incubation and tapped on tissue paper to remove residual

wash buffer.

e. 100 ul of blocking buffer was added and plate was incubated for 1 hr.

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3.4.4.2 Addition of test and control sera

a. 5 ul of test and control sera was diluted in 500 ul of blocking buffer in

separate Perspex plate and mixed thoroughly 10 times to ensure

homogeneity before loading to microtitre plate.

b. The diluted 100 ul test sera samples in duplicate wells and two control

sera (positive and negative)along with conjugate samples in

quadruplicate well are transferred from perplex plate to the micro titre

plate.

c. The plate was then incubated at 370C for 1 hr on ELISA plate shaker at

300 rpm.

3.4.4.3 Addition of conjugate

a. The plate was then taken out of shaker and washed three times with

washing buffer as mentioned earlier.

b. 100 ul of working dilution of conjugate (protein G HRP conjugate) was

added to each well and incubated at 370C for 1 hr on shaker at 300

rpm.

3.4.4.4 Addition of substrate/ chromogen

a. The plate was taken out of shaker and washed three times with

washing buffer.

b. 100 ul of substrate was added to each well of microtitre plate.

c. The plate was then incubated at room temperature for 7 min or until a

visible yellow colour develops in strong positive wells by covering with

aluminium foil

3.4.4.5 Addition of stopping solution

a. After colour development 50 ul of stopping solution was added to each

well of plate immediately.

b. The plates are then read immediately in ELISA reader at 492 nm.

3.4.5 Interpretation of results

Per-cent positivity values which are used for diagnostic interpretation are

calculated as follows

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PP = Average OD value of test serum X 100

Median OD value of positive control sera