EcoHealth DOI: 10.1007/s10393-015-1011-x

Ó 2015 International Association for Ecology and Health

Short Communication

Serological Evidence of Coxiella burnetii Infection in Cattle and Goats in Bangladesh Najmul Haider,1 Md. Shafiqur Rahman,1 Salah Uddin Khan,1 Andrea Mikolon,2 Muzaffor G. Osmani,3 Emily S. Gurley,1 Ireen Sultana Shanta,1 Suman Kumer Paul,1 Laura Macfarlane-Berry,1 Ariful Islam,4 Ausraful Islam,1 James Desmond,4 Jonathan H. Epstein,4 Rachael A. Priestley,5 Gilbert J. Kersh,5 Mohammed Ziaur Rahman,1 Peter Daszak,4 Stephen P. Luby,1,6 Robert F. Massung,5 and Nord Zeidner1,5 1

Zoonotic Diseases Research Group, Centre for Communicable Diseases, International Centre for Diarrheal Diseases Research, Bangladesh (icddr,b), Dhaka, Bangladesh 2 United States Department of Agriculture, La Cienega, Hawthorne, CA 3 Department of Livestock Services, Ministries of Fisheries and Livestock, Dhaka, Bangladesh 4 EcoHealth Alliance, New York, NY 5 Centers for Disease Control and Prevention, Atlanta, GA 6 Woods Institute of the Environment, Stanford University, Stanford, CA

Abstract: We tested 1149 ruminant sera conveniently collected from three districts of Bangladesh to identify the serological evidence of Coxiella burnetii infection in cattle and goats by enzyme-linked immunosorbent assay. We found that 0.7% (8/1149) of ruminants had detectable immunoglobulin G for C. burnetii: 0.65% (4/620) in cattle and 0.76% (4/529) in goats. A sub-set of ruminant samples was retested and confirmed by immunofluorescence assay (18/112). Although we cannot rule out false-positive reactions, our study suggests the presence of C. burnetii in cattle and goats in Bangladesh. Further studies are required to estimate disease burden at the population level and identify risk factors for Q fever in ruminants in Bangladesh. Keywords: seroprevalence, C. burnetii, Q fever, cattle, goat, Bangladesh

Q fever is a zoonotic disease caused by the bacterium Coxiella burnetii and has near global distribution (ArricauBouvery and Rodolakis, 2005; Vaidya et al., 2010; Porter et al., 2011; Georgiev et al., 2013). Although reported in the South Asian region, there have been no previous reports of Q fever in Bangladesh. Cattle and goats are common ani-

Electronic supplementary material: The online version of this article (doi:10.1007/ s10393-015-1011-x) contains supplementary material, which is available to authorized users. Correspondence to: Najmul Haider, e-mail: [email protected]

mal reservoirs of C. burnetii (Arricau-Bouvery and Rodolakis, 2005; McCaughey et al., 2011). Animals often show no signs of clinical disease; however, Q fever can cause abortion storms in ruminants and can lead to infertility (Porter et al., 2011). Thus, C. burnetii infection can have a significant economic impact on animal production (Porter et al., 2011). Infected animals contaminate the environment by shedding the organism in milk, feces, urine, saliva, and most importantly, in vaginal secretions, placenta, and other by-products of birth (Porter et al., 2011). Ruminants can also be infected by inhalation, tick bite, or through contact

Najmul Haider et al.

Figure 1. Study locations of the veterinary hospitals for sampling cattle and goats in Bangladesh

with heavily infected tick feces (Aitken, 1989). Hard ticks Hyalomma anatolicum and Rhipicephalus sanguineus are vectors for this disease and are common in Bangladesh (Ahmed et al., 2007). We designed a study to detect emerging infections among ruminant livestock brought for medical care to government veterinary hospitals in disparate regions of Bangladesh. We screened ruminant blood samples for

diseases which we suspected were circulating in Bangladesh, and these samples were also used to establish serological evidence of Q fever in cattle and goats in Bangladesh. We evaluated animals from May 2009 to August 2010 in three government veterinary hospitals of Bangladesh within the districts of Netrokona, Dinajpur, and Chittagong. We developed a map of combined cattle and goat population density distribution for Bangladesh using

Coxiella burnetii Infection in Cattle and Goats, Bangladesh

publicly available data from FAO, GeoNetwork (Robinson et al., 2007) and plotted the three surveillances sites (Fig. 1). Sampling locations were selected based on government veterinary hospital figures of high ruminant caseloads (>20 ruminants/day)(Haider et al., 2014) and proximity to the Indian border where livestock trading is common. The veterinarian at each hospital enrolled cattle and goats into the study with one or more of the following clinical signs: diarrhea, respiratory distress, and/or fever. They also interviewed the animal’s owner using a standard questionnaire to collect demographic information and nutritional status of the animals. The veterinarians then collected 5–10 ml of blood from the jugular vein of each ruminant. Blood samples were left to clot for 30 min at room temperature, and serum was obtained by centrifugation at 1500 9 g for 15 min. Serum samples were then transported in liquid nitrogen and stored at -80°C and analyzed at the animal BSL-2 laboratory at icddr,b. This protocol was approved by icddrb’s research review committee and the ethical and animal experimentation ethics review committees. Antibody-capture enzyme-linked immunosorbent assay (ELISA) (Chekit, IDEXX, USA) was used to detect C. burnetii immunoglobulin (IgG) as directed by the manufacturer (Agger et al., 2010). A serum dilution of 1:400 was used according to manufacturer’s recommendation, and samples were considered IgG positive if the calculated optical density (OD) was 40% (Agger et al., 2010). Samples were declared negative for ODs at 30% and were noted as suspect positive if ODs were between 30% and 40% (Agger et al., 2010). All suspect positive samples were retested. The samples that had OD 10% in ELISA (n = 97) were selected for an immunofluorescence assay (IFA) test. In addition, 20 samples were randomly selected with OD values 80% sensitivity and >90% specificity (Kersh et al., 2013). In another study conducted on ruminants using IFA as the standard, it was found that the ELISA had 74% sensitivity and 98% specificity (Vaidya et al., 2010). These results suggest that the ELISA test rarely generates a false-positive reaction. Still, some false positives and negatives may have occurred in our study, and the results are interpretable with this caveat. Furthermore, we found four of six samples in agreement (67%) with the same results in both tests which argues against all of the positives being false positives. That said, we found evidence of C. burnetii-specific IgG antibodies in cattle and goats in Bangladesh indicating past exposure to C. burnetii. Animals recently infected may not have seroconverted, and since IgM was not measured, we may not have identified all current infections. The prevalence of C. burnetii antibodies varies by country. Studies from India demonstrated seropositivity in 11% of cattle and 6% of goats, from Thailand in 5% of cattle, and from China in 10% of cattle (Vaidya et al., 2010; Hong-

Najmul Haider et al.

Table 1. Distribution of C. burnetii IgG Antibody Among Cattle and Goats Sampled from Government Veterinary Hospitals in Bangladesh During 2009–2010 Variables

Species Goat Cattle Total Sex Female Male Districts Netrokona Chittagong Dinajpur Total Goat breeds Black Bengal Jamunapari Cross (Black Bengal 9 Jamunapari) Total Cattle breeds Local Cross Nutritional status-goat Good Thin Emaciated Total Nutritional status-cattle Good Thin Emaciated Total

Enzyme-linked immunosorbent assay (ELISA)

Immunofluorescence assay (IFA)

# tested

Positive (%)

# tested

Positive (%)

529 620 1149

4 (0.76) 4 (0.65) 8 (0.70)

14 98 112

3 (21) 15 (15) 18 (16)

713 436 1149

5 (0.70) 3 (0.69) 8 (0.70)

72 40 112

12 (17) 6 (15) 18 (16)

418 300 431 1149

4 4 0 8

(0.96) (1.3) (0) (0.70)

65 16 31 112

9 4 5 18

(14) (25) (16) (16)

345 135 49 529

0 2 2 4

(0) (1.5) (4.1) (0.76)

4 10 0 14

0 3 0 3

(0) (30) (0) (21)

528 92 620

3 (0.57) 1 (1.1) 4 (0.65)

86 12 98

226 221 82 529

3 1 0 4

(1.3) (0.5) (0) (0.76)

5 3 6 14

2 1 0 3

281 283 55 619

4 (1.4) 0 (0) 0 (0) 4(0.65)

50 41 7 98

10 4 1 15

Bo et al., 2011; Rodtian et al., 2013). Seroprevalence figures were even higher in Japan (cattle 58.9% and goats 11.8%) (To et al., 1998). However, several of these studies sampled animals that had a history of recent abortion (India, China) (Vaidya et al., 2010; Hong-Bo et al., 2011) or reproductive disorders (Japan) (To et al., 1998), and thus may have been more likely to be infected with Q fever than those selected in our study. We enrolled animals brought to veterinary hos-

12 (14) 3 (25) 15 (15) (40) (33) (0) (21) (20) (9.8) (14.3) (15)

pitals for care of acute illness (fever, respiratory distress, or diarrhea) and did not target animals with reproductive disorders. Bangladesh has one of the highest human and animal population densities in the world, with 1000 persons (World Bank, 2007) and 145 domestic ruminants per square kilometer (Bangladesh Agricultural Research Council, 2010; Haider et al., 2014). The high human–ani-

Coxiella burnetii Infection in Cattle and Goats, Bangladesh

mal density and frequent contact between animals and humans make the country an ideal site for Q fever and other zoonotic diseases to emerge. Rural people in Bangladesh generally have both very close contact to and occupational involvement with cattle and goats providing an opportunity for Q fever transmission. Evidence of Q fever in Bangladesh has important implications for livestock production and public health and warrants further investigation. Veterinarians in Bangladesh should consider Q fever as a differential diagnosis when managing reproductive disorders in cattle and goats. The findings from this study are subject to limitations because the study was hospital-based and sampled only sick animals. Without credible information about sensitivity and specificity of the tests used, we cannot rule out false-positive reactions, but our study suggests the presence of C. burnetii in Bangladesh. Further studies are required to estimate disease burden at the population level and identify risk factors for Q fever in ruminants in Bangladesh. Physicians, who routinely deal with unexplained human cases of febrile illness, hepatitis, meningoencephalitis, and/or endocarditis, should consider Q fever as a differential diagnosis, which may require different first-line antimicrobial treatments. Further studies should be conducted on patients with infectious valvular endocarditis of unknown etiology to identify the burden and specific risk factors of Q fever in humans in Bangladesh.

ACKNOWLEDGEMENTS This research was funded by Google and the Rockefeller foundation through EcoHealth Alliance. icddr,b acknowledges with gratitude the commitment of Google and the Rockefeller foundation to its research efforts. We acknowledge the veterinarians of Department of Livestock Services of Bangladesh as well as Chittagong Veterinary and Animal Sciences University who assisted with various aspects of this project. We are grateful to Ms. Diana DiazGranados, icddr,b, for her constructive support in editing this manuscript.

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Serological Evidence of Coxiella burnetii Infection in Cattle and Goats in Bangladesh.

We tested 1149 ruminant sera conveniently collected from three districts of Bangladesh to identify the serological evidence of Coxiella burnetii infec...
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