566 Indian Journal of Public Health Research & Development, March 2020, Vol. 11, No. 03 Development of Rapid, Sensitive and in-expensive Point of Care Diagnostic Method for Brucellosis in Dairy at Resource-Limited Areas

Revathi Poonati1, Prudhvi Chand Mallepaddi2, Rudrama Devi Punati1, Soumendra Nath Maity3, Krishna Satya Alapati4, Kavi Kishor B. Polavarapu5, Rathnagiri Polavarapu6

1Research Scholar, 2Research Associate, Department of Biotechnology, Acharya Nagarjuna University, , , 3Research Associate, 4Assist. Professor, 5Professor, 6Senior Scientist, Department of Clinical Microbiology, Genomix Molecular Diagnostics Pvt. Ltd, Hyderabad, Department of Veterinary Microbiology, GenomixCARL Pvt. Ltd, Pulivendula, Andhra Pradesh, , Genomix Biotech Inc, 2620 Braithwood Road, Atlanta, GA 30345, USA

Abstract It is an emerging zoonotic disease spread all over the world, but its diagnosis is rather complex. Though a number of serological tests are currently available for diagnosis, none of them are useful at POC diagnosis. Further, they are not user-friendly and reproducible. Precisely for this reason, present study was aimed to develop a simple, inexpensive, field applicable POC diagnostics using protein G-based milk dipstick and indirect enzyme-linked immunosorbent (iELISA) assays. These are useful for rapid detection of brucellosis using a handheld ELISA reader at resource-limited POC areas near pen side. Overall, 4998 raw milk and whole blood samples were collected from organized dairy farms, where animals are reared (Goshalas), and villages in different districts of Andhra Pradesh and Telangana states, India. Collected samples used to evaluate diagnostic performances of these assays in comparison with milk ring test (MRT) and Rose Bengal Plate Test (RBPT) serological assays. The developed milk dipstick and iELISA showed 87.37%, 99.67% and 98.23%, 100% sensitivity and specificity respectively. Results proved that the developed assays can be used as potential diagnostics for rapid field diagnosis of Brucellosis from infected animals using raw milk and whole blood samples at resource-limited settings.

Keywords: MRT (Milk Ring Test), RBPT (Rose Bengal Plate Agglutination test), Point of Care (POC), Polymerase Chain Reaction (PCR), Milk dipstick, iELISA (indirect Enzyme-Linked Immunosorbent Assay).

Introduction of livestock causing severe morbidity, playing an enormous impact on economic losses17,31. Prevalence Brucella is a facultative, intracellular, Gram negative of brucellosis is not clear and is changing continuously pathogenic bacteriacauses brucellosis. It is a neglected, because of improper usage of sanitary method, highly infectiousunder reported zoonotic disease20. intensification of farming, socio-economic status of Brucella infects dairy animals, small ruminants, canine, farmers and international movement of animals11,36. swine, humans24, andis a highly contagious disease Recent studies pointed out an increased prevalence of brucellosis in dairy livestock of India, becoming geographical hotspotcausing an economic loss of US $3.4 billion per annum9,26,32. Lactating animals excrete Corresponding Author: bacterium into milk throughout their lives means that Dr. Kavi Kishor B. Polavarapu bacteria are localized in lymph nodes and mammary Genomix Molecular Diagnostics Pvt. Ltd, 5-36/207, glands in infected animals13,14, 19. Though, India is one Prashanthnagar, Kukatpally, Hyderabad-500072, India among largest milk producers in world, there is still a Indian Journal of Public Health Research & Development, March 2020, Vol. 11, No. 03 567 huge demand for milk and dairy products. Since growth of brucellosis free herds.21 of dairy industry depends on productive and reproductive health of dairy animals, it is crucial not only to upkeep Culture: Collected fresh milk samples were but also to prevent bovine diseases. centrifuged at 3000xg for 10mins. Pellet was placed onto tryptose soy agar (Ref # M290, Himedia) media MRT is inexpensive technique for screening and supplemented with antibiotics15,25. monitoring of brucellosis2,24,30. It may give false positive reactions with milk samples such as colostrums or milk Antigen Preparation: Brucella abortus strain 99 at the end of lactation period, milk from cows suffering was obtained from Indian Veterinary Research Institute, from a hormonal disorder19. Serological diagnostics for Izatnagar, Indiawas grown onsoybean casein digest brucellosis infection are RBPT, Slow Agglutination Test agar medium and harvested as per OIE recommended 23 (SAT), Complement Fixation Test (CFT) forprimary protocol . screening of brucellosis. However, agglutination Development of Dipstick Assay: Dipstick strips are 2 techniques may have limitations in sensitivity . easy to perform for quick sero-diagnosis of brucellosis Lateral flow immunochromatography showed better due to its robustness, simplicity and are highly suitable 29 sensitivity and specificity compared to RBPT . Existing for application under field conditions10. Capillary action diagnostics are not well established for field diagnosis of dipstick resulted in formation of colored bands as and are time consuming; require expensive laboratory shown in Figure 2. with expertise. Thus, there is a need to develop highly specific, sensitive, inexpensive, POC diagnostics for Development of indirect ELISA: Protein G-based rapid detection of brucellosis. So, present study is aimed iELISA using sLPS was carried out with a modified to develop and evaluate milk dipstick and iELISA with protocol to increase the accuracy6. a portable hand held ELISA reader for the detection of brucellosis at resource-limited areas. Hand-held ELISA Reader: Battery-operated, portable, hand-held ELISA reader is easy to carry from Materials and Method one place to another with external facilities. Study site and animals: A total of 4,998 individual Cut Off Value: iELISA assay cut off value between milk and whole blood samples were collected from positive and negative samples was derived by calculating third and fourth lactating cows from organized dairy mean OD values obtained from 450 negative samples farms, Goshalas and villages in Kadapa, Kurnool, plus three times of standard deviation28. Ananthapuram, Chittoor, Prakasam, Krishna, Guntur, Cut off = X + 3 SD (X- mean, SD- standard West Godavari, and Nellore districts in Andhra Pradesh; deviation). Khammam, Kareemnagar, Nalgonda, Sangareddy and Rangareddy districts in Telangana, India. The Statistical Analysis: Collected samples used for study samples include Holstein Friesians cows (1057), validation of milk dipstick and iELISA in comparison Jersey (909), Shahiwal (701), Ongole (813), Gir (512), with MRT, RBPT, and culture. Calculation of specificity, Punganur (311) and Murrah buffaloes (695) as a source sensitivity, PPV, NPV and efficiency were carried for sample collection. out using statistical analysis software NCSS 12 and McNemars Chi square test used to calculate significance Sample collection and processing: A total of 20 ml level and p-value < 0.05 was used as significant. volumes of midstream milk wascollected directly from all teats into a sterile 50ml falcon tube without preservative Results: Results were presented in figure no 1-4 and from selected individual cows. Approximately, 5ml table no 1-2. While dipstick showed 87.37%, 99.67% volume of whole blood was collected from jugular vein sensitivity and specificity, iELISA displayed 98.23% of animal in Lithium-Heparin coated BD Vacutainer sensitivity and 100% specificity. Statistical data in terms tubes (REF 367820) for serological studies with consent of sensitivity, specificity, PPV, NPV and efficiency were of farm owners and farmers under supervision of a significantly differing with standard values (Table 2). veterinarian. Chi square statistical value is 54.3028 and the p-value is <0.00001, so, obtained results are significant at p<0.05 Milk Ring Test: MRT is standard, most common level. method for identifying infected animalsfor surveillance 568 Indian Journal of Public Health Research & Development, March 2020, Vol. 11, No. 03

Fig. 1: Silver staining gel image of sLPS obtained from Brucella abortus S99 strain. Lane M is Bio- Rad precision plus protein standard marker, Lane 1 to 4 are four different lots of sLPS and lane 5 is concentrated sLPS using lyophilization by pooling all the four lots.

Fig. 2: Dipstick lateral flow test strip tested with brucellosis positive and negative samples. Indian Journal of Public Health Research & Development, March 2020, Vol. 11, No. 03 569

Fig. 3: Diagrammatic representation of OD values for 450 unvaccinated animal samples for defining the negative cut off value. While X-axis shows the number of animals, Y- axis deals with the OD of 450 negative samples at 450 nm.

Table 1. A comparative diagnostic evaluation data of five different diagnostic assays for detection of brucellosis

Total No. of Samples Assays Sample Type Positive Negative TP TN FP FN MRT Milk 958 4040 285 3929 673 111 RBPT Serum/plasma 894 4104 299 4007 595 97 Culture Milk 396 4602 396 4602 - - Dipstick Milk/Whole blood 361 4637 346 4587 15 50 Indirect ELISA Milk/Whole blood 389 4609 389 4602 - 7

*Note: TP- True positive (Reactive), TN- True negative (Non-reactive), FP- False positive, FN- False negative. Table 2. Statistical analysis of brucellosis diagnostic assays and comparison with different parameters

Assay Sensitivity % Specificity % PPV % NPV % Accuracy % MRT 71.97 (0.00) 85.38 (0.75) 29.75 (15.27) 97.25 (3.09) 84.31 (0.60) RBPT 75.51 (0.03) 87.07 (0.63) 33.45 (12.82) 97.64 (2.07) 86.15 (0.49) Culture 100.00 (0.07) 100.00 (0.31) 100.00 (4.66) 100.00 (1.07) 100.00 (0.28) Dipstick 87.37 (0.02) 99.67 (0.22) 95.84 (5.01) 98.92 (1.10) 98.70 (0.30) Indirect ELISA 98.23 (0.30) 100.00 (0.18) 100.00 (4.51) 99.85 (0.48) 99.86 (0.04)

Values are represented in terms of percentage. Values in parenthesis represent 95% confidential intervals. Note: PPV- Positive predictive value, NPV- negative predictive value. 570 Indian Journal of Public Health Research & Development, March 2020, Vol. 11, No. 03

Fig. 4: The correlation and regression between two ELISA readers

Discussion than that of dipstick and ELISA22. Because of this, sLPS- based diagnostics were developed and preferentially These are widely used diagnostic assays for used for detecting Brucella specific antibodies. RBPT is detection of Brucella specific antibodies in raw milk routinely used for multiple livestock species in endemic as well as in whole blood samples6, 34. Present study countries including India. It is a simple, cost effective, describes difference in accuracy of diagnostic method but requires refrigeration of antigen and has limitations for detection of Brucella specific antibodies in collected of false positive results due to cross reacting antibodies samples. Relative performance of different diagnostics against many gram negative bacteria7,27. Therefore, and obtained data by these method were shown in table there is a great demand for development of simple tests 1. In-house developed method showed high sensitivity that can be used in field with higher sensitivity and and specificity values that are also statistically significant specificity. over existing method. The study proved that developed milk dipstick and Our results reveal that sensitivity and specificity iELISA are preferred as alternative methodin field since of MRT was 71.97% and 85.38%, respectively. existing method have limitations for individual screening However, it is limited by milk quality and results may of livestock affected with brucellosis12. Use of sLPS as be false negative or false positive because of low ab an antigen candidate possible to detect brucellosis very concentrations, lack of fat-clustering factors25 and rapidly and accurately and these clinical findings and contains colostrums or milk obtained from vaccinated results obtained were corroborate previous reports3,6,10. or mastitis cows. Our results are in agreement with 35 study carried out by Vanzini etal., (2001) showed that Conclusion MRT gives inaccurate results leading to misdiagnosis of brucellosis and its sensitivity and specificity is lower In present study, developed kits are very simple, Indian Journal of Public Health Research & Development, March 2020, Vol. 11, No. 03 571 effective, inexpensive, user friendly, with high potential 7. Franco M P et al. Human Brucellosis. Lancet for POC diagnosis. Dipstick and iELISA showed Infect Dis. 7, 775-786. DOI: 10.1016/S1473- 87.37%, 99.67% and 98.23%, 100% sensitivity and 3099(07)70286-4, 2007 specificity respectively in comparison with culture as 8. Goknur terzi et al. Detection of Brucella antibody standard assay. 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