Comparative Efficacy of Serological Tests and Molecular Analysis of Brucellosis in Dairy Cattle of Mathura District

Brucellosis is a zoonotic problem of great animal welfare and economic implications worldwide. Brucellosis poses a major public health threat by the consumption of non- pasteurized milk and milk products produced by unhygienic dairy farms in endemic areas. It is of high endemicity in the regions of Africa, Mediterranean, Middle East, parts of Asia and Latin America (Refai, 2002). Although developed countries like Europe, Australia, Canada, Israel, Japan and New Zealand have already eradicated the disease (Geering et al., 1995). In India, brucellosis causes an average loss of US $18.2 per buffalo followed by 6.8 per cattle, 0.7 per sheep, 0.6 per pig and 0.5 per goat (Singh et al., 2015). Brucellosis is caused by members of genus Brucella. The Brucella organisms are small, non-motile, non-spore forming, aerobic, facultative intracellular, Gram-negative coccobacilli which are arranged singly or in pairs or short chains. Also, are catalase, oxidase and urea positive. The capacity of causing persistent disease and circumventing innate and adaptive immunity are key secrets of replication and persistence in the host by Brucella. Depending upon the specific natural host affinity, there are six classical Brucella species, viz., B. abortus (cattle and buffaloes), B. melitensis (goats), B. suis (pigs), B. ovis (sheep), B. neotomae (desert wood rats) and B. canis (dogs) (Osterman and Moriyon, 2006). It is stated that more than 80% of dairy cows will abort if they are exposed during the time of gestation. In males orchitis and epididymitis with sterility are prominent (Radostitis et al., 2000). Humans become infected with Brucella species by ingesting organisms, getting contaminated with mucous membranes or through abraded skin. Animal abortion products, unpasteurized dairy products, uncooked or undercooked meat or meat products, unpasteurized milk and cheese prepared from infected unpasteurized milk (Pappas et al., 2005) play a crucial role in its transmission. Symptoms in human brucellosis can be highly variable, ranging from non–specific, flu-like symptoms (acute form) to undulant fever, arthritis, intermittent or irregular fever, orchitis and epididymitis (Smith and Kadri, 2005).

A total of 405 serum samples were collected from cattle in and around various blocks of Mathura district. These consist of  female (372) and  male (33) serum samples were collected from various blocks of dairy farms and gaushalas of Mathura district, within the time period of February, 2023 to middle of November, 2023 and processing of samples performed at the department of veterinary public health and epidemiology. Total 405 serum samples were collected for serological diagnosis and 5 stomach content samples of aborted calf for PCR assay. About 5-10 ml of blood was collected by puncturing the jugular vein in sterile disposable syringes (Dispovan) or plain vaccutainers (BD, USA) for serum collection. Basic precautions was taken to avoid shaking the samples during transportation to prevent the destruction of the RBCs. Serum were collected from the clotted blood after centrifugation at 4000 rpm for 10 min. A total of 5 stomach content samples were collected for PCR assay. The samples were transported immediately to the laboratory on ice pack. The individual animals in the present study were identified by their respective identification numbers or names. None of the animals was vaccinated against brucellosis. All the serum samples were stored at -20°C till tested.
       
Rose Bengal Plate Test (RBPT) and Standard Tube Agglutination Test (STAT) antigen were procured from Indian Veterinary Research Institute, (IVRI), Izatnagar, Bareilly, India. For serum I-ELISA, kit for cattle procured from ICAR National Institute of Veterinary Epidemiology and Disease Informatics (ICAR-NIVEDI), Bangalore, Karnataka. RBPT antigen is an 8% suspension of pure smooth killed cells of Brucella abortus strain 99 phenolised and stained with rose Bengal dye. Rose Bengal Plate Test is a single dilution serum agglutination test. It was performed on glass slides according to the method presented by (Alton et al.,1988). To detection of STAT in a serum samples, Brucella abortus plain antigen were taken which was heat killed phenolised suspension of Brucella abortus strain 99 and it show 50% agglutination at 1/500 final dilution of serum with Indian standard. In cattle serum samples antibody was detected by indirect ELISA (I-ELISA) kit procured from Svanova (Biotech-AB), Uppasala, Sweden. Briefly, each of the kit contained 96 flat bottom polystyrene with Brucella abortus precoated antigen wells. Competitive ELISA (C-ELISA) was performed using separate kits based on IgM for large ruminants procured from SVANOVIR (Sweden).
       
Bacterial DNA from stomach content was extracted by Phenol Chloroform Method (Sambrook and Russel, 2001) and from milk sample as per Pokorska et al., (2016) method. PCR analysis for Brucella genus specific bcsp31 gene was carried out according to the method described by Baily et al., (1992) (Table 1). The final reaction volume of 25 μl was obtained by adding 12.5 μl of Master mix (Genie, Banglore), 3 μl of DNA template, 1μl of each of the primers (forward and reverse) made up by adding nuclease free water. For bcsp31 gene amplification, the initial denaturation step was carried out at 95°C for 3 min followed by denaturation at 95°C for 30 sec., annealing at 63°C for 45 sec., extension 72°C for 45 second and a final extension step at 72°C for 10 min. Brucella species specific IS711 gene amplification, the PCR conditions were an initial denaturation step at 95°C for 10 min, denaturation at 94°C for 1 min., annealing 58°C for 1 minute, extension 72°C for 1 min. and followed by a final extension step at 72°C for 7 minute. For each gene 35 amplification cycles were performed. After the amplification, amplicons were separated in 1.5% gel in tris-acetate EDTA (TAE) buffer at 60 volt for 80 min, stained with 0.5% ethidium bromide solution and visualized under ultraviolet light. For species differentiation of positive samples, the method (AMOS-PCR) described by Bricker and Halling (1994) was adopted. The five primers cocktail poised to differentiate Brucella species DNA into B. abortus, B. melitensis, B. ovis and B. suis (Table 2).

Screening of cattle (n=405) in which female (372) and male (33) serum samples were detected by RBPT, STAT and I-ELISA, different outcomes were recorded for the diagnosis of antibodies against Brucella species. On analysis total of 405 serum samples, 49 (13.17%) were found positive by RBPT (Fig 1), whereas 43 (11.55%) by STAT (Fig 2) in dairy cattle. Similarly, I-ELISA revealed 55 (14.78%). The overall seroprevalence of bovine brucellosis was found to be 9.92%, 16.03% and 6.48% by RBPT, STAT and I-ELISA respectively, Bohrey et al., (2022). Out of 405  samples, on analysis of dairy cattle (n=45)  in which 40 sera samples were from female and 5 sera samples were from male cattle from various blocks of Mathura district, having history of vaccination for Brucellosis. C-ELISA test was performed and the result showed that 42 (93.34%) sample came positive in C-ELISA and 3 sera samples came negative. Out of 42 positive sample 6 sera samples were weak positive and 36 sera sample were strong positive. There have been reports of higher prevalence rates in cattle (10.74%). Ahmed et al., (2009) (25.7%) in Jordan, Mishra et al., (2022) in India and Junaidu et al., (2011) in Nigeria in agreement with the results of this study. Studies by Krishnamoorthy et al., (2015) and Trangadia et al., (2015) estimated the prevalence of I-ELISA to be 11.63 per cent in Southern India, 6.8 per cent in Andhra Pradesh, 8.2 per cent in Gujarat and 2.3 per cent in Odisha. Kassahun (2004) observed somewhat lower prevalence rates for intensive (2.5%) and extensive farms (1.7%) in Southern Ethiopia, whereas Berhe et al., (2007) reported slightly higher prevalence rates (3.19%) for extensive agricultural systems in Algeria region there is more gaushalas where culled cattle are kept with healthy cattle without proper testing of brucellosis and these gaushalas are running very unhygienic conditions so that it may be the higher finding of brucellosis.




       
Molecular detection
 
The DNA was extracted directly from the 4 tissue samples (T= stomach content of aborted fetus) collected from the 4 different dairy cattle which were detected anti-Brucella antibodies by I-ELISA test. These were then subjected to PCR using Brucella genus specific primer pair B4/B5 targeting bcsp31 gene (Baily et al., 1992). Amplicon size of 223 bp was obtained only in 1 tissue samples (T=1), out of 4 tissue samples were found carrying Brucella organisms. (Fig 3). None of the other samples yielded DNA which implied that Brucella organism was not present in the tissues of those animals even though the antibody titre was quite high leading to positive results in serology Singh et al., (2015). Various PCR procedures have been developed for the detection of Brucella (Probert et al., 2004; Tanmay, 2007; Zamanian et al., 2015). B. abortus is an intracellular bacteria and this poses a problem for selection of a suitable sample (Wattam et al., 2009).


               
For species differentiation of Brucella organisms, AMOS-PCR using five primers which was developed by Bricker and Halling (1994) was employed. This assay was able to detect B.abortus, B.melitensis, B.ovis and B.suis. However, it was unable to detect B.canis, B.neotomae, B.pinnipedialis, B.ceti and some biovars of B.abortus and B.suis. Again, 4 extracted DNA samples were subjected to this assay using four specific primers of four different species along with one IS711 primer. Amplicon of 976 bp and 731 bp was seen in 1of the featal stomach content sample which indicates presence of B.ovis and B. melentensis in the sample. An amplicon of 976 bp and 731 bp was seen in 2 of the tissue (featal stomach content) sample which indicates presence of B. ovis and B. melentensis in the sample. An amplicon of 498 bp and 285 bp was seen in 3 of the tissue (featal stomach content) sample which indicates presence of B.abortus and B.suis in the sample (Fig 4). No other amplicons at 498 bp, 731 bp, 976 bp and 285 bp were observed in 4 tissue (featal stomach content) sample indicating absence of B. abortus, B. melitensis, B. ovis and B. suis respectively. Among the other tissue samples, none of such amplicons were detected. Similar study was done by Kurmanov et al., (2022) Cases are reported annually across the range of known infectious species of the genus Brucella. Globally, Brucella melitensis, primarily hosted by domestic sheep and goats, affects large proportions of livestock herdsand frequently spills over into humans. Same study was done by Gumma et al., (2020), duplex recombinase polymerase amplification (Duplex RPA) assay for the specific detection of Brucella melitensis and Brucella abortus was developed in this study. Primers were designed targeting hypothetical protein genes and membrane transporter genes of B. melitensis and B. abortus, respectively. Brucella melitensis biovar 3 was isolated from 86 (84.31%) cows. Additionally, universal PCR showed the presence of Brucella DNA in all tissue extracts from which Brucella melitensis has been isolated as well as DNA extracts of their cultures.

Among the different samples viz. serum were tested by serological tests and stomach content of aborted foetus of cattle were used for confirmation of active infection of brucellosis by PCR assay. As the percent positivity by serological tests was found to be 14.78% (55/372) in female cattle and 30.30% (10/33). So combination of RBPT, STAT and I-ELISA and confirmation by PCR assay using tissue samples was found to be the most suitable combination for the confirmatory diagnosis of brucellosis in cattle in absence of isolation of organisms. The incidence of brucellosis cases is increasing over the recent years especially in developing countries due to poor management, limited resources and increased trade and frequent movement of livestock. The disease’s higher prevalence in this location raises the likelihood of zoonotic transmission, posing a substantial threat to the human population as well as a significant economic impact due to lost production and animal population.

The authors are highly thankful to Indian Council of Agricultural Research, New Delhi and Dean, College of Veterinary Science and Animal Husbandry, Uttar Pradesh Pandit Deen Dayal Upadhayay Pashu Chikitsa Vigyan Vishvidhyalaya Ewam Go-Anusandhan Sansthan (DUVASU), Mathura, U.P., India, for providing necessary funds and facilities to carry out the investigations.

The authors declare that there is no conflict of interest.