Brucellosis (DS..22.8.14)
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Transcript of 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
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
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.
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.
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-
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.
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
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
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.
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)
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
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
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.
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.
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
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
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.
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
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
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
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
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.
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
PP = Average OD value of test serum X 100
Median OD value of positive control sera