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J Med Microbiol 56 (2007), 1309-1316; DOI: 10.1099/jmm.0.47160-0
© 2007 Society for General Microbiology
ISSN 1473-5644

Multiple genus-specific markers in PCR assays improve the specificity and sensitivity of diagnosis of brucellosis in field animals

Falguni Mukherjee1, Jainendra Jain1, Vipul Patel1 and Mrinalini Nair2

1 Research and Development, National Dairy Development Board, Anand 388 001, Gujarat, India

2 Biotechnology Program, Department of Microbiology and Biotechnology Centre, MS University, Baroda 390002, Gujarat, India

Correspondence
Mrinalini Nair
mnair{at}msubiotech.ac.in
or
mnair_in{at}yahoo.com

Received 9 January 2007
Accepted 18 April 2007

Brucella-specific nucleotide sequences encoding the BCSP 31 kDa protein, Omp2 and the 16S rRNA were employed in three independent diagnostic PCR assays. Results of the three PCR assays on six reference strains of Brucella were in complete agreement. The results of PCR assays based on bcsp and omp2 on 19 Indian field isolates (human, bovine and murine tissues) also agreed completely. However, when the 16S rRNA gene was employed as the diagnostic target in the PCR, only 14 out of these 19 isolates and 2 out of 7 bovine milk isolates were identified as the genus Brucella. The bovine blood samples were insensitive to 16S rRNA PCR. The antibody-detecting ELISA results of field samples (n=87) from a serologically positive herd in India were compared separately with omp2 and bcsp PCRs of blood (n=62). While the bcsp PCR was the most sensitive, the degree of association of ELISA with omp2 blood PCR ({kappa}=0.37 at P <0.05) was similar to that with the bcsp blood PCR ({kappa} =0.34 at P <0.05). An improvement in the correlation between ELISA and blood PCR was noticed ({kappa} =0.5 at P <0.05) when a consensus result of omp2 and bcsp blood PCR was considered for comparison with ELISA. The use of more than one marker-based PCR gave increased sensitivity and higher specificity and appears to be a more reliable molecular diagnostic approach for screening of field animals.

Abbreviations: OPA, overall proportion of agreement.


   INTRODUCTION
TOP
 INTRODUCTION
METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Brucellosis causes infertility and abortion in bovines (Radostits et al., 1994; Corbel, 1997) and undulant fever in humans (Corbel & Brinley-Morgan, 1984). Bovine brucellosis is usually caused by Brucella abortus, and less frequently by Brucella melitensis and Brucella suis (OIE, 1996). Among the six recognized species of Brucella, B. abortus, B. melitensis, B. suis and Brucella canis can potentially infect humans (Nicoletti, 1980) while Brucella ovis and Brucella neotomae have not been isolated from humans.

Accurate diagnosis of brucellosis requires bacteriological isolation and detection of the pathogen in the laboratory, which is impractical for regular screening of large populations (Alton et al., 1988; Lulu et al., 1988; Radostits et al., 1994; Yagupsky, 1994). Serological tests can be nonspecific owing to cross-reaction or subsensitive or high immunity reactions, depending on subclinical or endemic prevalence of the disease (Ariza et al., 1992; Weynants et al., 1996; Godfroid et al., 2002). Numerous PCR-based assays have been developed for the identification of the genus Brucella from cultures, animal/human tissues and animal products. These employ the gene encoding the 31 kDa Brucella cell surface salt extractable protein (BCSP), omp2, 16S rRNA, IS711 and other gene targets (Baily et al., 1992; Leal-Klevezas et al., 1995; Da Costa et al., 1996; Rijpens et al., 1996; Bricker, 2002; Morata et al., 2003; Bogdanovich et al., 2004; Mukherjee et al., 2005; O'Leary et al., 2006). Real-time PCRs for high sensitivity detection (Redkar et al., 2001; Probert et al., 2004; Navarro et al., 2006; Queipo-Ortuno et al., 2005) and differential/multiplex PCRs for strain typing based on locus-specific variations (Ewalt & Bricker, 2000; Bardenstein et al., 2002; Probert et al., 2004; Mukherjee et al., 2005; Ferrao-Beck et al., 2006; Marianelli et al., 2006) or variable tandem repeats (Bricker & Ewalt, 2006; Le Fleche et al., 2006) of Brucella isolates have been reported.

For large-scale field screening, identification of Brucella by genus-specific PCR tends to be simple and adequate. The diagnostic PCRs so far employed in field animals for direct screening (Fekete et al., 1992; Leal-Klevezas et al., 1995; Amin et al., 2001; Leyla et al., 2003; O'Leary et al., 2006) and comparative evaluation against serology (Romero et al., 1995b; Sreevatsan et al., 2000) or isolation have relied on single gene targets. The sensitivity and specificity of diagnostic assays can influence effective prevention and control of zoonoses as well as aid in selection of animals for breeding, etc. There are few comparative studies on the specificity of the different genus-specific PCRs and their correlation with serological diagnosis. This study analyses the sensitivity and specificity of the three established genus-specific PCRs of bcsp, omp2 and 16S rRNA gene sequences, and further evaluates their comparative efficiencies for the simple detection of the genus Brucella directly from blood samples, in a large-scale screening of individual animals from serologically positive Indian field buffaloes/cattle herds. Further, a correlation between the diagnostic specificities of PCRs and an antibody-detecting blood ELISA is assessed employing kappa statistics.


   METHODS
TOP
 INTRODUCTION
 METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Brucella isolates and blood samples. Brucella strains were isolated from experimentally infected murines (n=2), field isolates of human blood (n=4) and bovine milk (n=7) and uterine discharge (n=6). Bovine blood samples (n=87) used for serology and PCR studies were from two serologically positive herds of western India.

Source and maintenance of strains. The details of the origin of the standard strains are given in Table 1Go. Brucella reference and field strains were identified and maintained as per the standard protocols (Alton et al., 1988). Escherichia coli ATCC 3616 was propagated on nutrient agar at 37 °C; Yersinia enterocolitica O : 3 and O : 9 were maintained on brain heart infusion agar (HI Media) at 28 °C; and Vibrio cholerae O : 1 Inaba and Ogawa strains were grown on Terrestrial Yeast Extract medium at 25 °C (Baumann et al., 1984). Mycobacterium tuberculosis H37Rv (ATCC 25618) was maintained on glycerol-supplemented Löwenstein–Jensen medium according to the ATCC Catalogue of Bacteria and Bacteriophages.


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  		Table 1. Bacterial strains studied by three Brucella-specific PCRs

 
ELISA. An indirect Brucella antibody detection avidin–biotin ELISA (Animal Disease Monitoring and Surveillance, Indian Council for Agricultural Research, Bangalore, India) was used to assess the serological status of the two herds.

Extraction of genomic DNA from bacteria. Brucella grown for 72 h was washed twice in PBS (pH 6.4), pelleted by centrifugation at 3000 g for 20 min and suspended in 500 µl Tris/EDTA (pH 8.0). For the other bacteria except M. tuberculosis H37Rv (ATCC 25618), 24–48 h cultures were washed in PBS. The suspension was subjected to three cycles of snap freezing at –196 °C in liquid nitrogen and boiling at 95 °C for 10 min to obtain crude cell lysates. The lysates were sequentially treated with lysozyme (1 mg ml–1) at 37 °C for 1 h, proteinase K (1 mg ml–1) and sodium dodecyl sulfate (1 %) at 50 °C. The lysates were subsequently extracted with standard phenol/chloroform, and the genomic DNA was precipitated, dried, suspended in 50 µl TE and stored at –20 °C.

DNA was extracted from 3–4-week-old cultures of M. tuberculosis H37Rv according to a previously described protocol (Cousins et al., 1993).

Extraction of DNA from blood samples. DNA was extracted from bovine blood samples using a slight modification of a protocol published by Leal-Klevezas et al. (1995). Heparinized blood (500 µl) was centrifuged at 1500 g for 3 min. Cell pellets were suspended in erythrocyte lysis buffer (155 mM ammonium chloride, 10 mM sodium bicarbonate, 100 mM disodium EDTA, pH 7.4) and centrifuged at 1500 g for 3 min; the cycle was repeated two to three times until the red colour due to the erythrocytes was minimal. The pellet was then treated with leukocyte lysis buffer (2 % Triton X-100, 1 % sodium dodecyl sulfate, 100 mM NaCl, 10 mM Tris/HCl, pH 8.0), centrifuged at 3000 g for 5 min and the pellet was digested with 10 µl proteinase K (10 mg ml–1) for 40 min at 50 °C. Following enzymic digestion, the samples were extracted with phenol/chloroform and processed as above

PCR assays. Three genus-specific PCR assays were performed for the identification of Brucella. (i) The PCR for the genus-specific Brucella cell surface salt extractable (BCSP) 31 kDa protein gene (Bricker et al., 1988; Mayfield et al., 1988) was performed on bacterial lysates and DNA extracts of isolates from experimentally infected mouse spleen, bovine blood and milk isolate samples, employing forward primer B4 (5' TGG CTC GGT TGC CAA TAT CAA 3') and reverse primer B5 (5' CGC GCT TGC CTT TCA GGT CTG 3') as described by Baily et al. (1992). (ii) The omp2 gene (GenBank accession no. M26034) (Fitch et al., 1989) was amplified from reference and field strains of Brucella in a 25 µl reaction mixture using primers JPF (5' GCG CTC AGG CTG CCG ACG CAA 3') and JPR (5' ACC AGC CAT TGC GGT CGG TA 3') as per Leal-Klevezas et al. (1995). However, for amplification of DNA from field blood samples, the above protocol was modified by using 100 pmol of each primer and 4 mM Mg2+ in the PCR. (iii) The 16S rRNA gene (EMBL accession no. X13695) (Dorsch et al., 1989) was amplified from reference and field strains of Brucella by modifying the PCR protocol of Romero et al. (1995b). The PCR employed 0.5 µM each of a forward F4 (5' TCG AGC GCC CGC AAG GG 3') and a reverse R2 (5' AAC CAT AGT GTC TCC ACT AA 3') primer (Romero et al., 1995a). The 25 µl reaction mixture consisted of IX PCR reaction buffer (Promega), 200 µM dNTPs and 0.5 units Taq polymerase. The PCR cycling parameters were: 30 cycles of denaturation at 95 °C for 30 s, annealing at 54 °C for 90 s and extension at 72 °C for 90 s, preceded by heating at 95 °C for 5 min and followed by a final extension at 72 °C for 6 min. For amplification of the 16S rRNA from blood samples from the field, elevated magnesium ion concentrations at an increment of 2.5, 3.0 and 3.5 mM and a primer concentration of 1.0 µM were used.

The PCR products of the bcsp and omp2 gene targets were electrophoresed on 1.5 % agarose while the amplicons from the 16S rRNA gene targets were electrophoresed on a 2 % agarose gel, stained with ethidium bromide and visualized under UV light.

Comparison of results and statistical analysis. Comparisons were made within the results of: (1) the three different PCRs on reference and 19 field isolates of Brucella; (2) the blood/serum ELISA and the PCR based on the bcsp/omp2 gene on blood from 87 field samples; (3) the consensus of blood PCR based on the bcsp and omp2 gene with the antibody detection ELISA on 62 blood samples from field. The overall proportion of agreement (OPA) and the proportion of agreement beyond chance ({kappa} value) between different blood PCR protocols and antibody detection ELISA were analysed employing the software WinEpiscope 1.0 (EPIDECON) at 95 % confidence intervals.


   RESULTS AND DISCUSSION
TOP
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Although serological tests are the major diagnostic tools for screening of animal brucellosis in the field, they are neither fully sensitive nor specific due to insufficient immunity or serological cross-reactivity. Bacteriological isolation of Brucella on the other hand is regarded, because of its specificity, as the gold standard for diagnosis. Since this procedure is laborious for large-scale diagnosis and since detection of specific DNA is a true indication of the presence of a pathogen, we wanted to compare the applicability of different established PCRs against serology for rapid, sensitive and specific detection of Brucella in animals from a large population under endemic situations. Few reports have described the application of diagnostic PCR on field samples (Amin et al., 2001; Fekete et al., 1992; Romero et al., 1995b; Sreevatsan et al., 2000) and there are no studies correlating results of PCR from multiple gene targets with those of serological diagnosis.

The study was designed in two components: (1) evaluation of the specificity of the genus-specific bcsp PCR and its comparison with other established diagnostic PCRs on the omp2 (Leal-Klevezas et al., 1995) and 16S rRNA (Romero et al., 1995b) genes using standard Brucella strains, serologically related non-Brucella organisms as well as Brucella isolates from human and bovine infections; (2) PCR with confirmed specificity was then used as a positive indicator of infection to screen bovine blood samples from sero-positive herds and compare with ELISA.

Amplification of bcsp, omp2 and the 16S rRNA from reference strains and Indian field strains

The three independent PCR assays resulted in the amplification of 223, 193 and 905 bp amplicons, respectively, from the bcsp, omp2 and 16S rRNA gene PCRs from all six Brucella reference strains but not from other serologically related strains (Table 1Go). All PCRs were repeated twice. All the three categories of PCR products from the field isolates were confirmed for specificity by Southern hybridizations using non-radioactive probes prepared by labelling of corresponding amplicons from standard strains (data not shown). The 905 bp amplicon from the 16S rRNA gene in the Brucella reference strain was obtained after modification of the protocol of Romero et al. (1995b) by changing the Mg2+ concentration from the recommended 1.0 mM to 1.5 mM and the annealing temperature from 54 to 50 °C.

All 19 Indian field strains were identified as belonging to the genus Brucella by the PCR assays based on both bcsp and the omp2 gene (Fig. 1Go). These were therefore concluded as specific for Brucella and taken further for analysis of blood samples. However, 16S rRNA gene specific amplification was obtained only in 14 of the 19 isolates. Of the 19, 7 were bovine milk isolates of which only 2 were identified as Brucella by the 16S rRNA PCR (Fig. 2Go).


Figure 1
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  		Fig. 1. Brucella-specific BCSP 31 kDa gene (Baily et al., 1992) (a), omp2 (Leal-Klevezas et al., 1995) (b) and 16S rRNA gene (Romero et al., 1995b) (c) PCRs on isolates from field cattle from the Indian Veterinary Research Institute (IVRI), Izzatnagar. Lanes: 1, DNA molecular size markers [marker V (Boehringer Mannheim) in (a); phiX174 HaeIII digest (Promega) in (b) and (c)]; 2 and 3, Brucella abortus 544 (ATCC) and Brucella melitensis (ATCC), respectively; 4–13, human and bovine isolates from IVRI (10/98, 24/97, 3/97, 5/97, 20/97,6/86, 5/98, 25/97 and 7/97, respectively); 14 and 15, B. abortus isolates from mice liver and genital tissue (a) and M. tuberculosis H37Rv and water (b, c). Ten microlitres of amplicon was separated by electrophoresis, treated with ethidium bromide, and visualized under UV light.

 

Figure 2
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  		Fig. 2. Brucella-specific PCR on isolates from bovine milk. (a) BCSP 31 kDa PCR; (b) omp2 PCR; (c) 16S rRNA PCR. Lanes: 1, DNA molecular size markers [marker V (Boehringer Mannheim) in (a); phiX174 HaeIII digest (Promega) in (b) and (c)]; 2, Brucella abortus 544 (ATCC); 3–9, isolates from bovine milk; 10, water control; 11 and 12 in (c), B. abortus isolates from mice liver and genital tissue. Ten microlitres of amplicon was separated by electrophoresis, treated with ethidium bromide, and visualized under UV light.

 
Isolation of B. abortus biovars 1, 3, 4, 6 and 9, B. melitensis biovars 1, 2 and 3, B. suis biovar 1 and B. canis has been reported from India (Sen & Sharma, 1975) and there is enough evidence from previous work by others (Leal-Klevezas et al., 1995; Romero et al., 1995b; Da Costa et al., 1996; Sifuentes-Rincon et al., 1997; Lopez-Goni, 2001; Bricker, 2002; Probert et al., 2004) that PCR assays based on bcsp, omp2 and the 16S rRNA are able to detect all species and biovars of the genus Brucella. However, even with modifications only two out of seven bovine milk isolates yielded 16S rRNA PCR products. All Brucella 16S rRNA gene sequences have been reported to be identical (Gee et al., 2004). However, our subsequent multiple alignment analysis of 16S rRNA sequences of various Brucella spp. to the primers used (Romero et al., 1995a) showed alignment of the primer F4 with 100 % identity to the sequence of a single B. abortus (Dorsch et al., 1989; accession no. X13695), while with other species and other B. abortus-like sequence (Gee et al., 2004), alignment was seen only for the first 10 of the 17 nucleotides in the primer owing to the presence of an additional base ‘C’ in these strains that was absent in the said B. abortus (Fig. 3Go). This lack of alignment beyond 10 bp would have resulted in defective annealing at the temperature used yielding no PCR products. The reverse primer too showed an identity of 18/20 for strains other than B. abortus. These do not, however, explain why the standard strains yielded expected PCR products albeit at a lower annealing temperature than recommended. A new primer that is fully and specifically conserved in all Brucella species is required for genus-specific 16S rRNA PCR. In a study using 16S rRNA target (Lopez-Goni, 2001), milk culture and PCR results have been negatively correlated with each other where 26 out of 31 B. melitensis biovar 3 isolates (from bovine milk) could be identified as Brucella.


Figure 3
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  		Fig. 3. Multiple alignment of 16S rRNA sequences of various Brucella spp. indicating differences between B. abortus (X13695) and other Brucella spp. in sequence conservation corresponding to the forward primer F4 (a) and reverse primer R2 (b) used for PCR as per Romero et al. (1995a). The source, accession number and strain name for each sequence are indicated on the left.

 
Results of 16S rRNA PCR on blood samples

Although Mg2+ was elevated and the primer concentration increased to 1 µM, the 905 bp specific amplicon could not be detected directly from any of the blood samples tested on repeated attempts under various PCR conditions, probably owing to the reasons described above. Hence the 16S rRNA was not used for comparison with bcsp and omp2 PCRs. The sensitivity of 16S rRNA primers has been previously reported to be affected by the presence of human DNA (Navarro et al., 2002).

Correlation studies between bcsp and omp2 gene based blood PCR on field samples

The observed proportion of agreement (OPA=0.71 at P <0.05) was good and the degree of association ({kappa}=0.45 at P <0.05) was moderate between the two diagnostic blood PCR assays. The two PCR assays disagreed in 28.7 % of cases (Table 2Go). The bcsp was more sensitive as it could detect 24 % more samples (21/87) as positive than omp2. Such a difference was not seen in PCR carried out on bacterial isolates. The present studies seemed to indicate that the presence of host DNA could affect the sensitivity of primers for the detection of Brucella in bovine blood as observed previously (Navarro et al., 2002). The sensitivity of the detection system could also be affected by modification of the original blood PCR protocol (Romero et al., 1995a, b; Leal-Klevezas et al., 1995) in our hands. Finally it cannot be ruled out that the bcsp gene sequence is better conserved than the omp2 sequence in the genus Brucella. Variation in the omp2 sequence has been used as a basis for typing strains (Bardenstein et al., 2002; Ferrao-Beck et al., 2006).


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  		Table 2. Correlation of blood PCRs and their comparison with antibody-detecting ELISA

 
Correlation studies between antibody-detecting ELISA and blood PCRs

ELISA versus bcsp PCR. Correlation studies on 87 bovine/buffalo blood samples from the field showed that although the overall proportion of agreement between the two tests (0.66 at P <0.05) was strong, the degree of association was weak ({kappa}=0.34 at P <0.05) (Table 2Go). The PCR could detect 20.6 % more blood samples as positive compared to ELISA. Brucella genus specific sequence signatures were detected from 47 % (24/51) of serologically negative animals. Also this PCR was unable to detect Brucella in 6.8 % (6/87) of samples that were ELISA-positive.

Increased sensitivity of BCSP PCR over serology has been observed by Queipo-Ortuno et al. (2006), where seroagglutination was inconclusive in 30 % of cases whereas real-time PCR assay was positive in 90 % of samples of human brucellosis.

ELISA versus omp2-based PCR. The omp2 gene could be amplified from blood samples by a modified PCR protocol. The 193 bp amplicon was obtained only after increasing the concentration of Mg2+ from 3.0 to 4.0 mM and that of the primers from 50 to 100 pmol in the reaction. The overall proportion of agreement (OPA=0.70 at P <0.05) and the degree of association ({kappa}=0.38 at P <0.05) between the two tests were similar to those in ELISA versus bcsp PCR (Table 2Go). The ELISA identified 41.4 % (36/87) of samples as positive for brucellosis; in comparison, the omp2 blood PCR detected Brucella in 38 % (32/87) of samples. From serologically negative samples 21.6 % (11/51) were PCR-positive while 17.2 % (15/87) of ELISA-positive samples yielded negative results for omp2 blood PCR.

omp2-based PCR has been used previously (Leal-Klevezas et al., 1995) for identification of Brucella from blood of naturally infected caprines where 86.3 % (19/22) were identified as positive by PCR in comparison to 63.6 % (14/22) by serology. The reduced sensitivity of the omp2 PCR against ELISA on blood samples might be a mere reflection of the high immune response in the bovine system as compared to the caprine system.

ELISA versus consensus of PCR based on bcsp and omp2. Only 62 out of 87 samples were screened by ELISA and bcsp and omp2 PCR. The consensus data of the bcsp and omp2 blood PCR showed the best overall proportion of agreement (OPA=0.74 at P <0.05) and a fair improvement in the degree of association ({kappa}=0.5 at P <0.05) with ELISA as compared to ELISA versus bcsp or ELISA versus omp2-based PCR (Table 2Go). The consensus of two PCRs identified 6 % more samples (30/62) than ELISA (26/62) as positive and detected Brucella infection in 27.7 % (10/36) of animals that were serologically negative, but eliminated 9.7 % (6/62) animals that were ELISA-positive.

In the absence of the availability of an antigen-detecting ELISA we had used the antibody detection ELISA, widely used for serological monitoring programmes in India, despite the fact that antibody status did not always indicate disease under the chronic endemic situation that exists in the country. Evidence from human brucellosis indicates that the expression of anti-brucella antibodies does not correlate with the status of the disease (Elfaki et al., 2005). Therefore, we wanted to study how the degree of association ({kappa} value) between two tests systems was affected when the diagnostic gene targets in the PCR assays were altered. As observed in this study, the change in the gene target did not affect the nature or degree of association between ELISA and blood PCR. However, the closest and the best degree of statistical association ({kappa}=0.5 at P <0.05) was achieved when consensus results of bcsp and omp2 PCR were compared with those from ELISA. Therefore, we believe that consensus PCR is a more reliable diagnostic approach.

In an overall analysis of differential detection rates by ELISA and the two blood PCRs, the bcsp PCR was the most sensitive (92.72 %) followed by omp2 PCR (61.81 %) and ELISA (55.55 %). These values were calculated taking into consideration that, in the absence of pathogen isolation, a positive PCR for bcsp or omp2 from the blood samples was regarded as a true indication of infection, as their specificities were confirmed by the PCRs on field isolates. Thus bcsp and omp2 PCRs together indicated 55 true infections (Table 2Go), wherein bcsp showed maximum sensitivity (51/55). This assumption did not permit any consideration of false positive by PCR. Moreover, neither PCR gave false positives with non-brucella cultures. Thus both PCRs exhibited a specificity and positive predictive value of 100 % while ELISA showed 81.8 % specificity and 83.3 % positive predictive value. bcsp PCR also gave a higher negative predictive value (88.88 %) than the omp2 PCR (61.81 %) and ELISA (55.55 %). Since anti-brucella antibodies do not always indicate disease, evaluation of ELISA was done using bcsp PCR results as a reference (Table 2Go). ELISA was positive for 30 out of 54 bcsp-positive samples, making it the least sensitive test. The consensus of PCRs detected 6 % more samples as positive than ELISA and perhaps it should be considered most specific, as it seemed to be neither under- nor over-detecting Brucella infection. The consensus of PCRs also seemed to show a low degree of failure since it was unable to detect infection on 9.7 % of occasions from ELISA-positive animals. Compared to the PCR consensus the omp2 system failed on 17.2 % occasions. The most favourable factor regarding the omp2 PCR was that it had the best agreement of 78 % (40/51) with the ELISA-negative samples in comparison to other PCR systems. We finally conclude that the use of more than one marker-based PCR gave increased sensitivity and higher specificity and appears to be a more reliable molecular diagnostic approach for screening of field animals.


   ACKNOWLEDGEMENTS
 
This work was supported by funds kindly provided by the National Dairy Development Board, Anand, under a collaborative research project between the National Dairy Development Board and the Department of Microbiology and Biotechnology Centre, M. S. University, Baroda. We want to thank Dr G. K. Sharma from the National Dairy Development Board for providing Brucella isolates from bovine milk samples from the field and Mrs S. A. Patel for maintenance and propagation of these strains and for performance of Brucella antibody detection ELISA on serum samples from the field.


   REFERENCES
TOP
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
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