Comparative Efficacy of Serological Tests and Molecular Analysis of Bovine Brucellosis in Western Uttar Pradesh

Brucellosis is one of the most important contagious and communicable bacterial diseases, with a worldwide distribution. It is reported from almost 86 countries and is classified as re-emerging though neglected zoonosis with high rates of morbidity and lifetime sterility stated by Trujillo et al., (1994). Though it has been eradicated in many developed countries of Europe, Australia, Canada, Israel, Japan and New Zealand  Geering et al., (1995) yet remains highly endemic in Africa, Mediterranean, Middle East, parts of Asia and Latin America (Refai, 2002) due to poor management, limited resources (Khan and Zahoor, 2018), increased trade and frequent movement of livestock  Renukaradhya et al., (2002). Schwabe, a WHO consultant estimated an annual loss in India to be Rs 240 million in livestock, Rs 120 millions in human beings and brucellosis costs Rs 350 million in the form of food animals. There is a several routes for Brucellosis transmission which includes mother to offspring before or at the birth, through milk, by sexual contact, direct physical contact, from polluted environments and from eating  spoiled raw meat mainly placenta and birth products. Brucellosis is caused by members of genus Brucella which are small, non-motile, facultative aerobic, intracellular, Gram-negative coccobacilli. The ability of Brucella to replicate and persist in host cells is directly associated with its capacity to cause persistent disease and to circumvent innate and adaptive immunity (Fichi, 2003). The classical species of Brucella and their major hosts are Br. abortus (cattle), Br. Melitensis (goats), Br. suis (swine) and Br. ovis (sheep). In bovines, the disease is clinically characterized by metritis, mastitis, repeat breeding, abortion in the last trimester of pregnancy, retention of placenta and reduced milk production in the females and epididymitis, orchitis and sterility in males Radostits et al., (2000). In humans, brucellosis is considered to be an occupational disease that mainly affects slaughter house workers, butchers and veterinarians. The presence of brucellosis in India was first established early in the previous century and since then it has been endemic in almost all the states (Sehgal and Bhatia, 1990; Renukaradhya et al., 2002). Livestock production is a vital source of providing dietary protein for the rapidly growing human population and it is therefore, important to define clear and effective strategies for controlling infectious diseases like brucellosis that are undermining the livestock industry and are a cause of major economic loss to our country.

Source and place of samples collection
 
The present study was conducted in four districts viz., Aligarh, Firozabad, Hathras and Mathura located in western part of Uttar Pradesh, India, from October 2017 to July 2018. These districts were chosen because of the presence of large number of smallholder dairy producers, gaushalas and good animal husbandry techniques. During the present study, a total of 470 samples (serum, whole blood, milk) were aseptically collected from the selected animals (cattle and buffaloes) from the above mentioned areas and processing of sample performed at the department of Epidemiology and Preventive Medicine, DUVASU, Mathura, U.P. Total 262 serum samples were collected for serological diagnosis and 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. Care was taken to avoid shaking the samples during transportation to prevent the destruction of the RBCs.  Serum was collected from the clotted blood after centrifugation at 4000 rpm for 10 min. A total of 108 whole blood samples were collected for PCR assay in EDTA vaccutainers (BD, USA). 100 milk samples were collected in sterile polypropelene tubes 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, blood sample and milk sample were stored at -20°C till tested.
 
Prevalence based on different serological tests
 
RBPT and STAT antigens were procured from BP Division, Indian Veterinary Research Institute, (IVRI), Izatnagar, India. RBPT antigen was 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 described by Alton et al., (1988). To perform STAT in a serum samples, heat killed phenolised suspension of Brucella abortus strain 99 plain antigen was taken. The ELISA test kit was purchased from National Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI) (Formerly PD-ADMAS) Hebbal Bengaluru, Karnataka, India and the contents were stored at 4-8°C until use. The test was performed as per the manufacturer’s protocol.
 
Molecular characterization of Brucella by PCR
 
Bacterial DNA from whole blood and serum 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 sec 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.

In this study, a total of 470 samples from 203 cattle and 59 buffaloes were collected including 262 serum samples from 203 cattle and 59 buffaloes which were screened for seroprevalence of bovine brucellosis using RBPT, STAT and I-ELISA. The overall seroprevalence of bovine brucellosis was found to be 9.92%, 16.03%, 6.48% by RBPT, STAT and I-ELISA respectively. The distribution of antibodies against Brucella antigen varied significantly between cattle and buffalo, according to a species-wise seroprevalence investigation (Table 2). By RBPT, STAT and I-ELISA, the seroprevalence in cattle was 11.82 per cent, 16.25 per cent and 8.37 per cent, whereas it was 3.38 per cent, 15.25 percent and 0 per cent in buffalo serum samples. As a result, cattle had a higher prevalence of the disease than buffalo, but there was no difference that was statistically significant (p=0.0565). According to I-ELISA, Kebede et al., (2008) (11%), Eshetu et al., (2005) (10%) and Aggad and Boukra (2006) (9.7%) observed almost identical seroprevalences of bovine brucellosis. There have been reports of higher prevalence rates in cattle (10.74%). Ahmad et al., (2009) (25.7%) in Jordan, Mishra et al. (2022) in India, Junaidu et al., (2011) in Nigeria and Ahmad et al (32.2 per cent ). In agreement with the results of this study, studies by Krishnamoorthy et al., (2015) and Tragandi 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 Gujrat 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.


 
Molecular detection
 
Serum (100 samples), whole blood (108 samples) and milk (100 samples) were subjected to PCR assay and amplicons of 223 bp of Brucella bcsp 31 gene (genus specific Fig 1) Baily et al., (1992) and 498 bp of Brucella species specific IS711 (Fig 2) gene (Bricker and Halling, 1994) were obtained. Out of 100 serum samples, none was by PCR while out of 108 whole blood samples, 3 and from 100 milk samples, 2 were found positive by PCR (Fig 1). None of the serum samples yielded DNA which implied that Brucella organism was not present in the serum of those animals even though the antibody titre was quite high leading to positive results in serology Singh et al., (2010). Various PCR procedures have been developed for the detection of Brucella (Probert et al., 2004; Tanmay, 2007; Zamain et al., 2015). B. abortus is an intracellular bacteria and this poses a problem for selection of a suitable sample (Wattam et al., 2009). Only during acute phase of infection, it circulates in blood, mostly inside the white blood cells and hides itself in mammary organs, genital organs and lymph nodes (Morgan and Mackinnon, 1979) hence sample should be selected according to the phase of Brucella’s life cycle which is not practically possible to find out. The wide variation in the number of samples detected as positive by RBPT (26), STAT (42), ELISA (17) and PCR (5) might be due to many factors. Whole blood was found to be the better sample out of serum, whole blood and milk for DNA extraction to perform PCR assay as DNA might have been in very low or negligible quantity in serum and in milk, presence of fat globules and other proteins might have inhibited the DNA yield. Karthik et al., (2014) also performed bcsp 31 gene based PCR and species specific IS711 gene based PCR using whole blood samples to detect 15.13% positivity. Their results indicated that whole blood can be used for studying the molecular epidemiology of B. abortus in bovine and particularly detecting the active phase of infection. Further, at the time of equilibrium of host parasite interaction, the Brucella may persist in circulation for some time before getting localized in their preferred sites. Similar results that DNA can be extracted from whole blood and used as a sample for screening for brucellosis has been reported by Guarino et al., (2000) in buffaloes, Keid et al., (2010) in dogs and Khamesipour et al., (2013) in cattle and sheep. Nakkas et al., (2002) and Leal-Klevezas et al., (2000) used buffy coat instead of whole blood for DNA extraction as macrophages take up brucellae but it needs additional steps (Mitka et al., 2007) and hence the use of whole blood as such was tried in this study with better results. Use of commercially available kits have been said to have improved the quality as well as the quantity of extracted DNA (Queipo-Ortuno et al., 2008; Keid et al., 2010) as compared to conventional DNA extraction method. Alamian et al., (2017) described a novel PCR assay for detecting Brucella abortus. Daugaliyeva et al., (2016) developed a differential PCR assay for detection of Brucella abortus. Orizil et al., (2016) conducted a multiplex PCR technique for detection of Brucella spp. Hemande and Gandge (2016) showed that PCR-SSCP is more sensitive than PCR-RFLP for detection of polymorphism in bcsp31 gene. Arasoglu et al., (2013) detected Brucella genus specific bcsp31 PCR from tested milk samples. In a study by Rekha et al., (2013), none of the samples of blood, milk and serum were positive for brucellosis. Various factors like time of sample collection, infection status of the animal, condition of farm, number of samples collected can influence the results. The better molecular diagnostic approach for screening of field animals and for increased sensitivity and higher specificity or more than one marker-based PCR could be used.



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Among the suitability of different samples viz. whole blood, serum and milk for PCR assay, whole blood was found to be better than the other two. As the percent positivity by whole blood was found to be 2.8% in comparison to that of milk (2%) and none by serum. So combination of RBPT, STAT and I-ELISA and confirmation by PCR assay using whole blood was found to be the most suitable combination for the confirmatory diagnosis of bovine brucellosis in absence of isolation of the organism. 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.