Background and Aim: In the course of our Indian Council of Medical Research project on coxiellosis in Puducherry and Tamil Nadu, 5.64% goat, 1.85% sheep, 1.06% buffaloes, and 0.97% cattle were positive for Coxiella burnetii antibodies by enzyme linked immunosorbent assay kit (IDEXX, Liebefeld, Switzerland). In this preliminary study, we have proceeded to look for C. burnetii DNA in those antibody positive specimens employing an imported commercial C. burnetii polymerase chain reaction (PCR) kit.

Materials and Methods: Blood samples were collected during slaughtering. All 15 blood samples of antibody positive ruminants and three antibody negative samples were subjected to conventional Trans-PCR assay with a commercial PCR kit (Genekam Biotechnology AG, Duisburg, Germany). An in-house Trans-PCR was included in the study for comparison.

Results: A total of 15 antibody positive and three antibody-negative serum samples belonging to 11 goat, 4 sheep, 1 cattle, and 2 buffaloes were tested in duplicate for the presence of C. burnetii DNA by the commercial agar gel PCR kit and an in-house Trans-PCR. Only one buffalo serum sample was positive for C. burnetii with a band at 243 bp in in-house Trans-PCR.

Discussion: Seropositivity for C. burnetii need not necessarily translate into infectivity status of the animal. Conversely, seronegative ruminants can shed C. burnetii. Rapid disintegration of C. burnetii DNA during the storage period is an important impediment in QF-PCR research. This is the first time the performance of this commercial PCR kit is being validated in India.

Conclusion: Commercial PCR kit, Genekam did not identify any positive sample, probably because it targeted a larger amplicon of 687 bp.

Keywords: Coxiella burnetii DNA, coxiellosis, Trans-polymerase chain reaction.

1. Kovacova, E. and Kazar, J. (2002) Rickettsial diseases and their serological diagnosis. Acta Virol., 46(5-6): 193-210. [PubMed]

2. World Organization for Animal Health. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals; 2013. Available from: http://www.oie.int/international-standard setting/terrestrial-manual/access-online. accessed on 13-06-2016.

3. Hilbink, F., Penrose, M., Kovacova, E. and Kazar, J. (1993) Q fever is absent from New Zealand. Int. J. Epidemiol., 22(5): 945-949. [Crossref]

4. Vaidya, V.M., Malik, S.V.S., Bhilegaonkar, K.N., Rathore, R.S., Kaur, S. and Barbuddhe, S.B. (2010) Prevalence of Q fever in domestic animals with reproductive disorders. Comp. Immunol. Microbiol. Infect. Dis., 33(4): 307-321. [Crossref] [PubMed]

5. Vaidya, V.M., Malik, S.V.S., Kaur, S., Kumar, S. and Barbuddhe, S.B. (2008) Comparison of PCR, IF, pathology and isolation for Q fever in humans with spontaneous abortion. J. Clin. Microbiol., 46(6): 2038-2044. [Crossref] [PubMed] [PMC]

6. Das, D.P., Malik, S.V.S., Rawool, D.B., Das, S., Shoukat, S., Gandham, R.K., Saxena, S., Singh, R., Doijad, S.P. and Barbuddhe, S.B. (2014) Isolation of Coxiella burnetii from bovines with history of reproductive disorders in India and phylogenetic inference based on the partial sequencing of IS1111 element. Infect. Genet. Evol., 22: 67-71. [Crossref]

7. Das, D.P., Malik, S.V.S., Mohan, V., Rawool, D.B. and Barbudhe, S.B. (2013) Screening of fecal droppings of wild birds for coxiellosis by a duplex PCR targeting Com1 and IS1111 genes of Coxiella burnetii. J. Food Borne Zoonotic Dis., 1(1): 14-20.

8. Prasad, B.N., Chandiramani, N.K. and Wagle, A. (1986) Isolation of Coxiella burnetii from human sources. Int. J. Zoonoses, 13(2): 112-117. [PubMed]

9. Kargar, M., Rashidi, A., Doosti, A., Najafi, A. and Ghorbani-Dalini, S. (2015) The sensitivity of the PCR method for detection of Coxiella burnetii in the milk samples. Zahedan J. Res. Med. Sci., 17: 29-32. [Crossref]

10. Stephen, S., Chandrashekara, I. and Rao, K.N. (1980) Natural occurrence of Coxiella burnetii in the brown dog tick Rhipicephalus sanguineus. Indian J. Med. Res., 71: 865-869. [PubMed]

11. Stephen, S. and Rao, K.N.A. (1979) Q fever in South Kanara district: Natural occurrence of Coxiella burnetii in the tick (Aponomma gervaisi)--preliminary report. Indian J. Med. Res., 69: 244-246. [PubMed]

12. Mediannikov, O., Fenollar, F., Socolovschi, C., Diatta, G., Bassene, H. and Molez, J.F., Sokhna, C., Trape, J.F. and Raoult, D. (2010) C. burnetii in humans and ticks in rural sengal. PLoS. Negl. Trop. Dis., 4(4): e654. [Crossref] [PubMed] [PMC]

13. Muskens, J., Engelen, E.V., Maanen, C.V., Bartels, C. and Lam, T.J.G. (2011) Prevalence of Coxiella burnetii infection in Dutch dairy herds based on testing bulk tank milk and individual samples by PCR and ELISA. Vet. Rec., 168(3): 79. [PubMed]

14. Kim, S.G., Kim, E.H., Lafferty, C.J. and Dubovi, E. (2005) Coxiella burnetii in bulk tank milk samples, United States. Emerg. Infect. Dis., 11(4): 619-621. [Crossref] [PubMed] [PMC]

15. Anderson, P.K. and Kalra, S.L. (1954) Q fever studies in India: A case of human Q fever. Indian J. Med. Res., 42: 307-313.

16. Ghosh, B. and Rao, K.N.A. (1956) 'Q' fever - A case report. AF. Med. J. India, 12: 27-33.

17. Kalra, S.L. and Taneja, B.L. (1954) Q fever in India: A serological survey. Indian J. Med. Res., 42(3): 315-318. [PubMed]

18. Padbidri, V.S. and Gupta, N.P. (1978) Riskettsiosis in India: A review. J. Indian Med. Assoc., 71(4): 104-107. [PubMed]

19. Stephen, S. and Rao, K.N.A. (1980) Q fever in India: A review. J. Indian Med. Assoc., 74(10): 200-202. [PubMed]

20. Randhawa, A.S., Dhillon, S.S. and Jolley, W.B. (1973) Serologic prevalence of Q fever in the state of Punjab, India. Am. J. Epidemiol., 97(2): 131-134. [Crossref]

21. Sodhi, S.S., Joshi, D.V., Sharma, D.R. and Baxi, K.K. (1980) Seroprevalence of brucellosis and Q fever in dairy animals. Zentralbl. Veterinarmed. B., 27(8): 683-685. [Crossref]

22. Joshi, M.V., Padbidri, V.S., Rodrigues, F.M. and Gupta, N.P. (1979) Prevalence of Coxiella burnetii infection among humans and domestic animals of Rajasthan State, India. J. Hyg. Epidemiol. Microbiol. Immunol., 23(1): 67-73. [PubMed]

23. Shanmugam, J., Raveendranath, M. and Sukumaran, M. (1978) Seroprevalence of Q fever infection in human beings from southern region of Kerala State. Indian J. Med. Res., 67: 217-220. [PubMed]

24. Joshi, M.V., Menon, R.D., Padbidri, V.S. and Manjrekar, S.L. (1975) A note on the serological evidence of 'Q' fever in sheep from Karnataka State. Indian J. Anim. Sci., 45(1): 54-55.

25. Stephen, S., Indrani, R. and Rao, K.N.A. (1978) Q fever antibodies in domestic animals in South Kanara - A preliminary report. Indian J. Med. Res., 68: 39-43. [PubMed]

26. Stephen S and Rao KN. (1979) Coxiellosis in reptiles of South Kanara district, Karnataka. Indian J Med Res. 70:937-41. [PubMed]

27. Stephen, S., Chandrashekara, I., Rao, H.L., Rao, K.G. and Rao, K.N. (1980) Prevalence of human Q fever in South Kanara district, Karnataka. Indian J. Med. Res., 71: 510-515. [PubMed]

28. Yadav, M.P. and Sethi, M.S. (1979) Sero-epidemiological studies on coxiellosis in animals and man in the state of Uttar Pradesh and Delhi (India). Int. J. Zoonoses, 6(2): 67-74. [PubMed]

29. Padbidri, V.S., Rodrigues, F.M., Vidyasagar, J. and Joshi, M.V. (1981) Prevalence of antibodies to Coxiella burnetii among the domestic animal population in Orissa. Indian J. Med. Vet., 5(3): 99-103.

30. Rao, R. (2003) Seroprevalence of human coxiellosis Q-fever in Bikaner district of Rajasthan. J. Vet. Public Health, 1: 135-139.

31. Balakrishnan, N., Menon, T., Fournier, P.E. and Raoult, D. (2008) Bartonella quintana and Coxiella burnetii as causes of endocarditis, India. Emerg. Infect. Dis., 14(7): 1168-1169. [Crossref] [PubMed] [PMC]

32. Stephen, S., Sangeetha, B. and Antony, P.X. (2014) Seroprevalence of coxiellosis (Q fever) in sheep and goat in Puducherry and neighbouring Tamil Nadu. Indian J. Med. Res., 140(6): 785-787. [PubMed] [PMC]

33. Aarthi, P., Bagyalakshmi, R., Mohan, K.R., Krishna, M., Nitin, M., Madhavan, H.N. and Kalyani, S. (2013) First case series of emerging rickettsial neonatal sepsis identified by polymerase chain reaction-based deoxyribonucleic acid sequencing. Indian J. Med. Microbiol., 31(4): 343-348. [Crossref] [PubMed]

34. Gangoliya, S.R., Kumar, S., Alam, S.I., Devi, D.R.G. and Guchhait, P. (2016) First molecular evidence of Coxiella burnetii in patients with atypical pneumonia, India. J. Med. Microbiol., 65(3): 255-256. [Crossref] [PubMed]

35. Chakarabarthy, A., Bhattacharjee, P.K., Sarker, R.R., Rahman, A.K.M., Henning, K., Neubauer, H. and Rahman, M.S. (2016) Prevalence of Coxiella burnetii infection in cattle, black bengal goats and ticks in Bangladesh. Bangladesh J. Vet. Med., 14(1): 65-68. [Crossref]

36. van der Hoek, W., Dijkstra, F., Schimmer, B., Schneeberger, P.M., Vellema, P., Wijkmans, C., Ter Schegget, R., Hackert, V. and van Duynhoven, Y. (2010) Q fever in the Netherlands: An update on the epidemiology and control measures. Eur. Surveill., 15(12): 19520. [PubMed]

37. Fretz, R., Schaeren, W., Tanner, M. and Baumgartner, A. (2007) Screening of various foodstuffs for occurrence of Coxiella burnetii in Switzerland. Int. J. Food Microbiol. 116(3): 414-418. [Crossref] [PubMed]

38. Edalati-Shokat, H., Abbasi-Doulatshahi, E., Hajian-Bidar, H., Gharekhani, J. and Rezaei, A.A. (2015) Q fever in domestic ruminants: A seroepidemiological survey in hamedan, Iran. Int. J. Curr. Microbiol. Appl. Sci., 4(1): 589-596.

39. Mares-Guia, M.A., Rozental, T., Guterres, A., Gomes, R., Almeida, D.N., Moreira, N.S., Barreira, J.D., Favacho, A.R., Santana, A.L. and Lemos, E.R. (2014) Molecular identification of the agent of Q fever-Coxiella burnetii-in domestic animals in State of Rio de Janeiro, Brazil. Rev. Soc. Bras. Med. Trop., 47(2): 231-234. [Crossref]

40. Kilic, A., Kalender, H., Koc, O., Kilinc, U., Irehan, B. and Berri, M. (2016) Molecular investigation of Coxiella burnetii infections in aborted sheep in eastern Turkey. Iran. J. Vet. Res., 17(1): 41-44. [PubMed] [PMC]

41. Bisias, G., Burriel, A.R., Boutsini, S., Kritas, S.K. and Leontides, L.S. (2010) A serological investigation of some abortion causes among small ruminant flocks in Greece. Internet J. Vet. Med., 8(2): 1-7.

42. Panaiotov, S., Ciccozzi, M., Brankova, N., Levterova, V., Mitova-Tiholova, M., Amicosante, M., Rezza, G. and Kantardjiev, T. (2009) An outbreak of Q fever in Bulgaria. Ann. Ist. Super. Sanita., 45(1): 83-86. [PubMed]

43. Raele, D.A., Garofolo, G., Galante, D. and Cafiero, M.A. (2015) Molecular detection of Coxiella burnetii using an alternative Loop-mediated isothermal amplification assay (LAMP). Vet. Ital., 51(1): 73-78. [PubMed]

44. Bellini, C., Magouras, I., Chapuis-Taillard, C., Clerc, O., Masserey, E., Peduto, G., Peter, O., Schaerrer, S., Schuepbach, G. and Greub, G. (2014) Q fever outbreak in the terraced vineyards of Lavaux, Switzerland. N. Microbe. N. Infect., 2(4): 93-99. [Crossref] [PubMed] [PMC]

45. Gunaydin, E., Pekkaya, S., Mustak, H.K. and Dalkilic, B. (2014) Investigation of Q fever in Kilis and Shamil goats by ELISA and touchdown-PCR. Ankara Univ. Vet. Fak. Derg., 61(3): 161-165. [Crossref]