Prevalence Studies on Brucellosis in Buffalo Population in Nearby Region of Hyderabad, Telangana

Brucellosis is highly contagious zoonotic infection with economic significance, accounting approximately $3.4 billion annual losses in India (Singh, 2015). The Brucella organisms are gram negative, facultative, and intracellular bacteria significantly affecting domestic, wild animals and human population causing the infectious disease called brucellosis (Mathur, 1971). In animals, brucellosis widely affects reproductive system and mostly accompanied with fertility disorders, reduces the survivability of newborn calves and diminishes milk yield while in human the symptoms of disease are joint and muscle pain, headache, undulant fever, hepatomegaly, splenomegaly, night sweats and chills, marked asthenia and anorexia (Hugh-Jones, 2000). The illness has a significant negative socioeconomic impact, particularly in rural areas where the main sources of income are cattle rearing, milk production and preparation of dairy products. Despite wide usages of various serodiagnosis tests like Rose Bengal precipitation Test (RBPT), Lateral Flow Technology (LFT), Enzyme Linked Immunosorbent Assay (ELISA). These tests were used to detect Brucella antibodies in serum/blood in diagnosing the brucellosis, disease is still widespread and becoming more common in many poor nations as these tests  are widely preferred since, its on par less costly and user friendly. Animals sometimes may be seronegative but can act as consistent source infection affecting healthy flocks. Specificity and sensitivity of tests play s important role to diagnose higher percentage of positive reaction. Numerous diagnostic methods, including molecular assays (PCR), serological (RBPT, Agar Gel Immunodiffusion Test (AGID), ELISA, LFT etc.) and cultural methods are available to determine the prevalence of brucellosis. PCR technique is highly precise, sensitive and targeting two major genetic targets BCSP31 and 16S – 23S r RNA it needs sophisticated laboratory facility and equipment. Sandwich ELISA will be more specific in their action by employing primary and secondary antibodies. Lateral flow assay is a rapid diagnostic method usually based upon detection of IgM or IgG antibodies, which was modified in the present study to detect brucella antigen in the clinical samples as pen side diagnostic kit, highly applicable to field conditions. In the present study, sensitivity, reliability and specificity of various diagnostic tests has been analysed, based upon PCR as gold standard technique.

Collection of samples
 
The experimental study was undertaken for 12 months and collected the vaginal swabs from suspected buffalo population from the end of May 2019 to June 2020 from the nearby private farms in Hyderabad region. The collected samples were processed in the Department of Veterinary Public Health and Epidemiology, College of Veterinary Science, PVNRTVU, Rajendranagar, Hyderabad, Telangana, India.

All samples were processed according to the guidelines of WOAH (2023). The samples were collected carefully and cross-contamination was avoided during collection and transportation. The vaginal secretions from suspected buffaloes were collected using sterile swabs. The swabs were inserted into the vagina and once secretions adhered, the swab was removed and placed in a sterile tube. Each swab tube was aseptically packed, properly labeled, transported to laboratory and processed as early as and can be preserved for 2 hours at 4°C. Vaginal swabs were collected from 204 buffaloes from Kachiguda slaughterhouse and private dairy farms located in and around the Greater Hyderabad Municipal Corporation.
 
Detection of Brucella organisms in vaginal swab samples
 
Sandwich  ELISA
 
Sandwich ELISA test procedure was followed with slight modification recommended by Hans et al., (2020). Poultry antibodies (IgY) and rabbit IgG antibodies used as primary and secondary antibodies. The vaginal swab (field) samples were added to wells and kept at 37°C for 45 minutes in incubator. Finally, 100 µl of stop solution was added and OD values were recorded at 605 nm wavelength in UV Spectrophotometry.
 
Lateral flow Assay
 
The lateral flow assay kit has been prepared and subjected for usage after standardization of conjugation process of polyclonal antibodies raised in rabbit with gold nanoparticles to enhance the specificity of test. The conjugate of Gold nano particle -Poly clonal antibody (rabbit) was sprayed on conjugation pad, poultry antibodies (IgY) as primary antibody were adhered at test line and antirabbit antibodies were adhered at control line on nitrocellulose membrane. The sample pad and absorbant pad were flanked at the ends of strip followed by conjugation pad, test line and control line which ensured single flow of direction of sample was supported by Poly Vinyl Chloride (PVC) material placed in plastic cassettes which serves as kit for diagnosis of brucellosis. The collected Vaginal swab samples were immersed in 2ml of sterile distilled water and   properly mixed and 0.5ml of this mixture is placed on the sample pad of kit, after 2-5 minutes the development of red colour at control and test line were indicative for a positive result.
 
PCR assay
 
The PCR assay considered as referral test for brucellosis where the vaginal samples were processed and centrifuged at 2,348× g for 5 minutes. Supernatant was discarded, and the pellet was resuspended in 100 μl of nuclease free water and placed in a boiling water bath for 10 minutes. After heat treatment, the cell lysate was immediately kept on ice for 10 minutes followed by centrifugation at 2,348×g for 5 minutes. The supernatant containing the DNA was aliquoted into a sterile tube and stored at -20°C until further use (Vijaya Kumar, 2015).

A set of (B4/B5) primers derived from Bcsp31 genes  (Bailey et al., 1992) synthesized by Saha Gene, Hyderabad was used for PCR amplification of Brucella organism up to genus level shown in Table 1 and Primers were dissolved in nuclease-free water to obtain a concentration of 100pmol /µl (stock solution) and it was further diluted to obtain 10 picomoles/µl as working Solution.



Master mix was prepared by adding DNTP buffer@ 1.25 units (containing 50-200 µmole DNPT’s,1-2 mM Magnesium chloride, 2.5 µl 10X assay buffer for Taq polymerase), forward and reverse primers to Milli Q water and in one vial positive control (heat killed vaccine) and in another vial no vaccine was added (negative control). DNTP’s are added at 1.25 units (volume 10 µl) per vial. Primers are added in an amount of 2 picomoles (Volume 2 µl) per vial. Template was added (concentration 10⁹CFU/2ml) @ 2µl to positive control vials. in negative control no template was added and final volume was made up to 20µl for both positive and negative control.  The optimization and standardization of PCR steps as per conditions mentioned in Table 2.


 
Visualization of PCR products by agarose gel electrophoresis
 
To confirm the targeted PCR amplification, five µl of PCR products per tube were mixed with one µl of loading buffer and electrophoresed along with DNA molecular weight marker (Gene Ruler, MBI Fermantas). On a 1% agarose gel containing ethidium bromide (0.5 µg/ml) at constant 50mV for 45 minutes in 0.5X TBE buffer. The amplified product was visualized as a single compact band of expected size under UV light and documented by gel documentation system (Bio-Rad Gel Documentation and Imaging System, USA).

The study was carried out on 204 suspected buffalo vaginal swab samples and various diagnostic methods were adopted and percentage of positive and negative were displayed in Table 3. Out of 204 samples, the results on sensitivity of ELISA were on higher side 45.1% with 92 samples shown positive reaction. Almost similar findings were reported with a prevalence of 43%, 44% and 46.6% as reported by Raheela Akhtar et al., (2010), Chauhan et al., (2000) and Jain et al., (2013) respectively. Lower prevalence of 19.4% (Abdel Hamid et al., 2008), 18.20% (Sarkar et al., 1987), 10.42% (Zahid et al., 2002), 9.58% (Hussain et al., 2008), 8.15% (Patel, 2007) and 5.67% (Kanani, 2007).  3.5 % (Samaha et al., 2008), 2.4% (Lodhi et al., 1995) 1.9% (Rahman et al., 2019) and 1.1% (Nawal and Ahmed 2008) were reported than the prevalence in the present study. Very high prevalence of   88.9% was reported by Kang’ethe et al., (2000). Nasir et al., (2014) observed higher prevalence (18.53%) in private dairy farms compared to government farms (14.70%) using ELISA. The high prevalence of brucellosis in buffaloes from private farms in the present study might be due to frequent introduction of new high yielding animals without proper serological tests and high prevalence of abortions (Nasir et al., 2004).



LFA shown lesser 42.64% of positive reaction accounting 87 samples. Abdoel et al., (2008) reported very high prevalence of 90% in bovines using the kit developed by him. Shome et al., (2018) developed lateral flow assay for diagnosis of brucellosis and reported 6.6% prevalence which was less than the prevalence observed in present study.

PCR considered as preferred tool for definitive diagnosis for brucella DNA (Ulu-Kilic et al., 2013) similarly, PCR shown higher side with 113 samples positive reaction accounting 55.4% of positive reaction in the present study. Lower prevalence of 28.3%, 35.8% and 34.3% in buffaloes using vaginal swabs, blood, milk samples respectively were reported by AL-Shemmari (2018) by PCR assay. Very high prevalence of 87.5% using vaginal samples was reported by Romero et al. (1995) compared to prevalence in the present study. A very low prevalence of 2.73% in buffaloes was reported by Patel et al., (2022).
 
Sensitivity and specificity of ELISA and LFA compared with PCR assay
 
The sensitivity and specificity of the tests on comparison with PCR as referral test was shown on Table 4. Out of 204 vaginal samples PCR assay has given 113 Positives whereas LFA and ELISA has given 92 and 87 positives. All the samples positive by the LFA and ELISA were positive by PCR. The sensitivity of the LFA and ELISA compared to PCR assay was 77 and 81.42% respectively. By LFA and ELISA  117 and 112 samples were negative whereas 91 samples were negative by PCR assay. All the PCR assay negatives were negative by ELISA and LFA assay. Hence the specificity of both LFA and ELISA was 100%.

Brucellosis is one of the important zoonotic infection that has to be diagnosed as early as possible since it has huge impact on productivity of the animal and also affecting the human population severely. For detection of Brucellosis there are so many reliable techniques available like PCR, ELISA and AGID etc, but reliability and efficiency has to be graded on basis of most reliable technique i.e PCR. In the present study, the prevalence of brucellosis in the Hyderabad region were analysed by employing various serological test like ELISA and LFA. Based upon the sensitivity and specificity the techniques were evaluated with gold standard technique on PCR. Out of 204 vaginal swabs tested for PCR assay, ELISA and LFA based 113, 92 and 87 samples were positive for Brucella accounting 55.4%, 45.1% and 42.64% prevalence respectively. The sensitivity of LFA and ELISA were 76.99% and 81.4% respectively whereas specificity was 100% for both tests.

The authors are grateful to PVNRTVU, Rajendranagar, Hyderabad, Telangana and IgY private ltd, India for providing laboratory facilities to perform ELISA, PCR assay and providing animal house facility to raise poly clonal antibodies in rabbits and poultry and to carry forward the research in successful manner. 

Authors have no competing interests that are directly or indirectly related to the work submitted for publication.