Journal of Veterinary Medicine and Health
Open Access

Brucellosis has long been a major threat to live stock and one of the world’s major zoonotic problems. It is an infectious disease caused by gram negative bacterial organisms of the genus Brucella [1]. There are six common species of Brucella in nature (each comprising several biovars) and are classified according to biochemical, antigenic properties, habitat and pathogenicity for humans. These are B. abortus, B. melitensis, B. suis, B. ovis, B. canis, B. netomae [2]. Each species has a characteristic, but not an absolute, predilection to infect certain animals. The three most common species in terms of worldwide prevalence and causing disease in human are B. abortus, B. melitensis and B. suis. B. abortus is least virulent but is able to produce tissue damage with abscess formation. Species and biovars are differentiated by their CO₂ requirements, ability to use glutamic acid, lysine, ribose; hydrogen sulfide production. Recently, analysis of fragment lengths of DNA (cut by various restriction enzymes) has also been used to differentiate Brucella groupings [3,4].

B. abortus is a bacterium that can cause abortions in cattle and undulant fever; that is disease characterized by intermittent fever, fatigue, in humans. These bacteria are non-sporulating, lacking capsule and are non-motile (aflagellate) coccobacilli. In vivo, found within the cytoplasm of cells in closed packed clusters [5]. Brucella abortus was discovered by Bang in 1897. The disease (brucellosis) since then has been recorded from almost all the countries of world.

Brucellosis is included in the OIE (Office International dis Epizootics) list of notifiable disease as a multiple specie disease. It is list B disease. Brucella organisms are classified as class III pathogens. Animals may transmit Brucella organisms during abortion, at the time of slaughter and in their milk [6]. Cell wall surface Lypopolysaccharide (LPS) is the main antigenic and immunogenic structure. Different strains of Brucella abortus are classified to rough and smooth based on the LPS they express. B. abortus smooth lipopolysaccharide (as same as the most gram negative bacteria) consist of three domains: Lipid A, core oligosaccharide and O-specific polysaccharide (O-antigen). The lipid A of B. abortus S-LPS consists of special fatty acids (different from other gram negative bacteria) and a unique structure (16 carbons long). Further the O-antigen from smooth organisms contains an unusual sugar perosamine (4, 6-dideoxy-4-formamide-d-mannopyranosyl). Since LPS is the main expressed antigen on the surface of Brucella the serologic responses following Brucella infection are directed against LPS. Thus in animals, the serological diagnosis of brucellosis are mainly based on the detection of specific anti-LPS antibodies [7,8].

Diagnostic tools of B. abortus include isolation and identification from clinical samples, detection of antigen, genome and antibodies i.e. culturing on selective media (serum dextrose agar), antigen detection by ELISA, genome detection by PCR, antibodies detection by agglutination test (RBPT, SAT). AGID test has also been reported to be a simple and rapid assay to demonstrate antibodies eliciated against Brucella antigen specially LPS [9].

Control of brucellosis in animals depends on elimination of seroreactors combined with immunization of non-reactors (susceptible). In many parts of the world vaccination of cattle is done by inoculating cows with Brucella abortus S-19, an attenuated strain [10].

Objectives

• Surveillance of brucellosis in different districts of Punjab.
• To check the results of the test with local and imported antigen.
• To check the efficacy of the 3 serological tests RBPT, SAT and ELISA.

Salmani, et al., carried out a study to evaluate the potency of modified extracted Lipopolysaccharide (LPS) of B. abortus to elicit specific anti-Brucella antibodies in animal model (Rabbit). In their study the specificity of synthesized antibodies in the sera of immunized animals for B. abortus S99 LPS was assayed by agarose gel-immunodiffusion method in comparison to E. coli LPS. Pooled serum of immunize animals react rapidly with B. abortus S99 LPS (sharp precipitation line). A thin line of precipitation between pooled serum and E. coli LPS was observed after 3 h. Since the animals had been injected with B. abortus S99 LPS (not E. coli LPS) they concluded that the relative reaction might be a sign of some structural similarities between B. abortus S99 LPS and E. coli LPS. In their study, they showed that this method would be efficiently used to detect positive serum samples for smooth strains of Brucella such as B. abortus S99 [11].

Chachra, et al., evaluated the comparative efficacy of 3 serological tests (RBPT, STAT and Dot ELISA) on a total of 28 serum samples from cattle including 18 from cattle suspected of brucellosis and 10 samples from brucellosis free cattle from different parts of the Punjab state of India [12]. All the serum samples were subjected to the common serological tests RBPT, STAT and Dot ELISA. In their study on 18 serum samples from cattle suspected of brucellosis, 9 (50%) samples were found to be positive while 9 (50%) samples were found to be negative by RBPT. Only 1 (5.55%) out of 18 samples was positive by STAT and showed a titer of 1:40. All the 9 RBPT positive samples (100%) showed negative results by STAT. All the 18 samples (100%) showed positive results with Dot ELISA. It was, therefore, suggested that for an accurate and fool proof diagnosis of brucellosis, a combination of RBPT and ELISA should be followed. Interestingly, RBPT commonly believed to be cruder and less sensitive test could detect 58.82% samples negative by STAT.

Abbas and Adleewan tested the raw milk samples in Iraq. 120 cows and 120 buffalo’s milk samples were collected randomly through August 2006 to July 2007 from different sites of province in Iraq (Basra) [13]. Upon subjection on MRT 31 samples of cow’s milk and 42 of buffalo were positive. Then they did bacterial culturing. For classification of Brucella species and biotypes CO₂ requirement for growth, hydrogen sulphide production was performed. The results of bacterial isolation revealed that total isolates in cows were 12 and 19 in buffaloes. Among that B. abortus was 9 and 14 in cows and buffalos samples respectively. Brucella abortus was the most dominant among recovered Brucella species. Among the B. abortus isolates the biotypes were 2, 3, 4. All Brucella isolates showed an ability to grow within the temp range 18°C-44°C and at pH 4-9 upon subjection on different pH values and temperature.

Brahmabhatt, et al., compared the sensitivity and specificity of serological tests RBPT, SAT with i-ELISA. The study was carried out to find out the seroprevalence of Brucella spp [14]. in central Gujrat, Anand, Kaira, Ahmedabad and Vadodara districts of Gujrat (India). These districts were one of the biggest pockets of milk production in India. A total of 251 (230 female and 21 male) sera samples from buffaloes were collected. The seroprevalence was found to be 12.75 percent, 11.16 percent and 19.12 percent by RBPT, STAT and i- ELISA, respectively. The highest (23.26%) prevalence was found in Ahemdabad district. In their study, RBPT showed 64.58% sensitivity and 99.50% specificity when compared with i-ELISA. STAT showed 56.25% sensitivity and 99.50% specificity when compared with i- ELISA. Thus, conclusion was made by them that i-ELISA test in conjunction with other serological tests can give more reliable diagnosis.

Ibrahim, et al., investigated and identified the status of bovine brucellosis in Ethiopia and potential risk factors respectively by conducting seroprevalence study on 1,595 cattle in Jimma zone, Ethiopia [15]. Sera samples were analyzed using Rose Bengal Plate Test (RBPT) and Complement Fixation Test (CFT). The overall individual and herd level seroprevalences were 3.1% (n=1595) and 15.0% (n=227), respectively. Moreover, significantly higher seroprevalence was observed in herds of larger sizes. Individual animal seroprevalence was also positively associated with the occurrence of abortion (26.98 and 1.54% in those with and without previous history of abortion, respectively). Generally, the seroprevalence of bovine brucellosis found in Jimma area was not high and the seroprevalence was closely associated with some of the risk factors considered at individual animal and herd level.

Rose Bengal Plate Test (RBPT)

Rose Bengal Plate Test (RBPT) is simple and rapid test by which early infection can be detected. This test can be used for the screening of herd. The assay is performed by testing the unknown sera with Brucella abortus strain, colored with Rose Bengal dye. The presence or absence of visible agglutination indicates the presence or absence of antibodies in samples tested. The test depends on the ability of antibody in the serum to agglutinate the stained bacterial antigen. When this occurs the aggregates become clearly visible to the naked eye. Blood samples were collected from 25 districts of Punjab (Table 1). Sera from these blood samples were obtained through centrifugation, putted in separate tubes and stored at -20°C till used. These were then examined for the presence of B. abortus antibodies. First of all antigen and sera were kept at room temp for 30 mins-60 mins. Then one drop of antigen (25 ul or 0.03 ml) was placed on the glass plate. One drop of serum (25 ul or 0.03 ml) was placed along the side of the antigen (Table 2). Antigen and serum were then mixed thoroughly with wooden applicator stick. The plate was then placed on the rocking platform or mixed manually for 4 minutes. Test was examined for agglutination in a good light (Table 3).

Table 1: No. of samples tested.

Table 2: Total number of positive samples through RBPT.

Table 3: Results of the antigen used for RBPT.

Reagent

Brucella abortus antigen stained with Rose Bengal dye. The reagent was stained, killed Brucella abortus bacteria. RBPT antigen for Brucella was obtained from Veterinary Research Institute Lahore and Peshawar. The reagents were stored at 2°C to 8°C.

SAT

Positive serum samples from RBPT were then subjected to SAT. Six conical agglutination tubes were placed in the rack for each test serum sample (Table 4). Then 0.8 ml of normal saline solution containing 0.5% phenol was added to the first tube and 0.5 ml in the remaining four. 0.2 ml of serum was added in 1st test tube and mixed thoroughly, to make 1/5 dilution. 0.5 ml from the 1st tube was taken and transferred into the 2^nd tube. After mixing well, 0.5 ml was carried to the 3^rd and so on up to the 5^th tube where after mixing, 0.5 ml was discarded. Now the dilution in each tube was 1/5, 1/10, 1/20, 1/40, 1/80 respectively. 0.5 ml of the standardized Brucella abortus concentrate antigen (1:10) was added to each tube containing, serum dilution, giving a series of final dilutions from 1/10, 1/20, 1/80 and 1/60 respectively. Serum and antigen suspension were mixed thoroughly from highest dilution to the lowest i.e. 1/60 to downward. Known positive and negative sera were kept as controls. Rack was incubated at 37°C for 18 hours-20 hours. Results were based on clearing of the suspensions along with clumping of the organisms and permanency of sediments upon gentle shaking.

Table 4: SAT results.

Reagents

Normal saline containing 0.5% phenol

100 ml distilled water+0.9 g NaCl+0.5 g Phenol. In 100 ml deionized water 0.9 g NaCl was properly dissolved then 0.5 g of phenol was added to solution and autoclaved.

c-ELISA

After SAT the positive and suspected serum samples from rose Bengal plate test, samples were then processed with c-Elisa. Samples and controls were diluted in the plate. 45 ul of sample dilution buffer was put in each well used for serum samples and serum controls. 5 ul of positive, weak positive and negative serum controls were added in 1^st column of the plate and run in duplicate. 4 ul of sample dilution buffer as conjugate control was added in 2 wells of the 1^st column. 5 ul of test sample added to appropriate wells (run in duplicate). 50 ul of mAb solution added to all wells used for controls and samples. Plate was then sealed and sides of the plate were tapped. Incubation was then done at room temp for 30 mins. Washing was done with PBSTween buffer 4 times. 100 ul of conjugate solution was added to each well. Then incubation at 18°C to 25°C for 30 mins. Again plate was rinsed with PBS-Tween 20 for four times. Plate was then sealed. 100 ul substrate solutions were added to each well. Incubation was done for 10 mins at room temp. 50 ul of stop solution to each well was added and mixed thoroughly.

Optical density of controls and samples was then measured within 15 minutes at 450 nm in a micro plate photometer and the results were noted (Figure 1).

Figure 1: c-ELISA test results for B. abortus

Reagents

• PBS-Tween solution 20 x concentrate was diluted 1/20 in distilled water. 500 ml per plate was prepared by adding 25 ml PBST solution to 475 ml distilled water and mixed thoroughly. Crystal precipitation in the bottle was avoided. Crystals when present, bottle was then incubated for 1 min-2 mins at 37°C.
• Freeze dried mAbs were reconstituted with 6 ml sample dilution buffer. It was prepared immediately before use and mixed gently. All the reagents and kit were stored at 2°C-8°C (35°F to 45°F).

Serodiagnostic tests

Three types of serodiagnostic tests were applied for anti-Brucella antibodies detection; RBPT as screening test, SAT and Brucella specific ELISA as confirmatory tests because cross reactivity is present in RBPT.

RBPT

800 serum samples (400 of buffaloes and 400 of cattle) were screened for seropositivity first through qualitative test RBPT with three types of stained Brucella abortus antigen (Table 5).

• Rose Bengal Ag (Imported)
• Rose Bengal Ag (Lahore VRI)
• Rose Bengal Ag (Peshawar VRI)

In cattle, RBPT indicated 3.25% (n=13) positive reactors while in buffaloes 3.75% (n=15). The high number of cases were recorded in Faisalabad district 12.5% (n=4) (Table 6). On RBPT, the overall positive percentage in cattle and buffaloes was 3.5% as RBPT recorded 28 positive samples (Tables 7 and 8).

The incidence recorded is in line with observation of Nasir who found seroprevalence of brucellosis in 1377 cattle through RBPT as 3.84% (53) in Punjab but not in agreement with the study of Nasir, et al., who reported 424 cattle and buffaloes sereopositive through RBPT out of 2463 samples in Punjab [16]. Lower incidence of Brucellosis through RBPT during current study might be due to segregation or culling of the positive reactors which resulted in lower prevalence of this disease.

Table 5: An overall seroprevalence of brucellosis through RBPT.

Table 6: Seroprevalence of brucellosis through SAT.

Table 7: Seroprevalence of brucellosis through ELISA.

Table 8: District wise prevalence.

Comparison between local and imported antigen

Local antigen was much better as it gave clear cut positive results when the samples were checked with that as compared to UK antigen. The reason might be due to some change in the staining procedure. Preparation of antigen (UK) for staining might have their own standards of preparation and local have their own standards. There might be chances of contamination that’s why differences were observed during screening of samples with both the antigen.

SAT

RBPT positive samples were then subjected to confirmatory test SAT. 20 samples were processed through SAT and their antibody titer was observed. The results of agglutination in SAT were recorded by reading the degree of clearing and sedimentation.

• Tube agglutination test cut off value 1:10 was considered to be negative.
• More than 1:40 was considered negative.
• More than 1:80 was considered to be positive.
• 1:160 also positive.
• High titer was 1:320.

Among these 20 samples 8 were proved to be positive giving titer from 1:80 to 1:320 Nasir, et al., carried out serum agglutination test on 424 serum samples of cattle and buffaloes which were RBPT positive and recorded 8% seroprevalence through SAT while in current study there was 40% seropositivity. It means that RBPT did not detect high number of false positive samples in the current study (Figures 2-4).

Figure 2: Agglutination results of foreign and local antigen (RBPT).

Figure 3: Agglutination of antibodies in serum (RBPT).

Figure 4: Serum tube agglutination test results.

c-ELISA

A ratio of positive and suspected samples from RBPT was then processed through c-ELISA. 44 samples (positive and suspected through RBPT) were analyzed for Brucella abortus positive test confirmation. 26 RBPT positive samples were subjected to c-ELISA. It was performed in 96 well flat bottom microtiter plate coated with B. abortus Lipopolysaccharide (LPS) antigen.

Results of the c-ELISA indicated 13 samples as seropositive. Inferences were made on the basis of colour development. Brucella specific antibodies present in sample compete for epitopic sites and inhibited the subsequent colour development. The observations were recorded on ELISA reader at 450 nm wavelength (filter). The observations in the form of Optical Density (OD) were recorded after 15 minutes interval to get the optimum results. The improper incubation time may result in high number of false positive and inappropriate estimation of positive samples.

Mean OD value for each of the controls and samples were calculated. Percent Inhibition values (PI) for controls as well as samples were calculated using the following formula.

Most of the seropositive samples were obtained from Faisalabad (4). One of the Faisalabad sample resulted positive by RBPT proved negative by c-ELISA and the one that was suspected by RBPT proved positive by c-ELISA (Figure 5).

Similar study was conducted by Brahambhatt, et al., who reported 19.12% buffalo samples out of 251 in central Gujrat as ELISA positive for Brucellosis while current study indicated 27.5% buffalo samples out of 29 in different districts of Punjab (Pakistan) as ELISA positive. Brahambhatt studied only 4 villages that are why lower percentage was detected by him.

Figure 5: Frequency distribution of positive and suspected Brucella samples using suspected ELISA.

Prevalence

Overall prevalence of brucellosis through current study was 3.5 %. Highest prevalence was recorded in Faisalabad i.e. 12.5%. It might be due to poor hygienic conditions prevailing at the place of sampling, improper testing of animals and avoidance of slaughtering of infected animals. Average was in Mandibahauddin i.e. 6.25% and minimum was recorded in Kasoor, Okara, Attock, Pakpatan (3.125%) [17].

RBPT and ELISA results differences

According to Chen, et al., ELISA can be used as a diagnostic test for the screening of antibodies as it is reported to have a sensitivity of 95%–100% [18]. Current results of the serodiagnostic tests indicated that RBPT detected higher percentage of seropositive animals as compared to SAT and ELISA. Some serum samples that showed positivity through RBPT were proved negative by ELISA. Higher percentage through RBPT may be attributed to non-specific agglutination. According to Flad RBPT is rapid, simple and sensitive test but has low specificity [19-20].

Overall study suggested that the rate of infection was higher in buffaloes than cattle. The reason might be due to frequent introduction of new high yielding animals without proper serological testing and avoiding the slaughtering of infected animals. Regular testing in proper hygiene can prevent the spread of disease. Serological evaluation of sera proved that RBPT should not be considered as absolutely reliable test and should be performed with other confirmatory tests for the confirmation of positive results.

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