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Seroprevalence and risk factors associated with exposure of water buffalo (Bubalus bubalis) to Neospora caninum in northeast Thailand
Veterinary Parasitology 207 (2015) 156–160
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short Communication
Seroprevalence and risk factors associated with exposure of water buffalo (Bubalus bubalis) to Neospora caninum in northeast Thailand Chanya Kengradomkij a , Tawin Inpankaew a , Ketsarin Kamyingkird a , Kannika Wongpanit b , Sirichai Wongnakphet c , Thomas J. Mitchell d , Xuenan Xuan e , Ikuo Igarashi e , Sathaporn Jittapalapong a,∗ , Roger W. Stich f a
Department of Parasitology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand Faculty of Natural Resource and Agro-Industry, Chalermphrakiat Sakon Nakhon Province Campus, Kasetsart University, Thailand c Department of Veterinary Diagnostic Services, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand d Animal Health Laboratories, Department of Agriculture and Food Western Australia, 3 Baron-Hay Court, South Perth, Western Australia 6151, Australia e National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-13, Inada-cho, Obihiro, Hokkaido 080-8555, Japan f University of Missouri, Department of Veterinary Pathobiology, 201 Connaway Hall, Columbia, MO 65211, USA b
a r t i c l e
i n f o
Article history: Received 14 February 2014 Received in revised form 31 October 2014 Accepted 31 October 2014 Keywords: Neospora caninum Water buffalo IFAT Seroprevalence Neosporosis Thailand
a b s t r a c t Water buffalo are important draft animals for agriculture in resource-restricted areas worldwide. Water buffalo were shown to be experimentally susceptible to infection with Neospora caninum, potentially affected by neosporosis, and naturally exposed to the parasite in Asia. Although enzootic to Thailand, the distribution of N. caninum among Thai water buffalo is unclear. The objectives of this study were to determine the seroprevalence of N. caninum among water buffalo of northeast Thailand and to identify risk factors associated with their exposure to N. caninum. Sera from 628 water buffalo from 288 farms were tested with an indirect fluorescent antibody test (IFAT). A total of 57 samples from 48 herds contained antibodies to N. caninum, indicating overall seroprevalence of 9.1% and 16.7% among individual animals and herds, respectively. The overall seroprevalence was highest in provinces located in the Khorat Basin in the southern part of the region tested. Host age was also associated with seroprevalence, with the greatest seroprevalence (16.1%) among buffalo over 10 years of age, followed by 5–10 years of age (13.4%), 3–5 years (9.2%), and less than 3 years (1.2%). These results collectively suggested that horizontal transmission from canine definitive hosts was an important route of water buffalo exposure to N. caninum. These results also verified the importance of risk factor analysis for effective bovine neosporosis control strategies at the local level. © 2014 Elsevier B.V. All rights reserved.
1. Introduction ∗ Corresponding author at: Department of Parasitology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10903, Thailand. Tel.: +66 2 942 8438; fax: +66 2 942 8438. E-mail address: [email protected] (S. Jittapalapong). http://dx.doi.org/10.1016/j.vetpar.2014.10.034 0304-4017/© 2014 Elsevier B.V. All rights reserved.
Approximately 1.2 million water buffalo (Bubalus bubalis) are indigenous to Thailand, where they serve important roles as draft animals for agriculture. These smallholder farms are often in close proximity, without
C. Kengradomkij et al. / Veterinary Parasitology 207 (2015) 156–160
barriers between water buffalo, wildlife and companion animals (Kyaw et al., 2004). Additionally, Thai buffalo are often reared on public pastures, increasing their exposure to other animals, including dogs. Neospora caninum is a heteroxenous apicomplexan parasite that undergoes sexual reproduction in the alimentary canal of canine definitive hosts and that infects a diverse range of intermediate hosts, including bovids, by horizontal transmission of environmentally resistant oocysts shed in canine feces and by vertical transmission to host offspring (Dubey et al., 2007). This parasite is the etiologic agent of bovine neosporosis, an important cause of bovine fetal abortion, stillbirth, birth of weak calves and reduced milk yields, often leading to culling of infected animals due to the lack of effective alternatives for the prevention and control of this disease (Kyaw et al., 2004). Although water buffalo have different behavior and physiology than dairy and beef cattle, they can serve as reservoirs for infectious disease agents of cattle, and buffalo are considered natural intermediate hosts of N. caninum (Chryssafidis et al., 2014b). Histopathology and PCR assays of placental and fetal tissues from naturally and experimentally infected dams indicated that N. caninum can also contribute to reproductive failure among water buffalo (Auriemma et al., 2014; Chryssafidis et al., 2014a,b; Konrad et al., 2012). Therefore understanding the prevalence and risk factors associated with exposure to N. caninum are critical to effective prevention and control of neosporosis among these important draft animals. Seroprevalence of N. caninum among water buffalo has been reported in Asia and surrounding areas, which was estimated at 1.5% in Vietnam, 3.8% in the Philippines, 54.7% in Pakistan and 68% in Egypt (Chryssafidis et al., 2014b). Anti-N. caninum antibodies were also reportedly detected from 4.5% and 73.3% of Thai water buffalo sampled in two separate studies, and from 5.5 to 70% of Thai cattle in other studies (Chanlun et al., 2007; Chryssafidis et al., 2014b; Jittapalapong et al., 2008; Kyaw et al., 2005; Nam et al., 2012; Wiengcharoen et al., 2010). Although there is evidence that N. caninum is enzootic to Thailand, in addition to reports suggesting that this parasite can be detrimental to water buffalo (Auriemma et al., 2014; Chryssafidis et al., 2014a,b; Konrad et al., 2013), the distribution of this parasite among Thai water buffalo and the importance of horizontal and vertical transmission of this parasite to water buffalo in Thailand are unclear. Identification of risk factors for host exposure to N. caninum is important for control of neosporosis, especially in the absence of effective immunoprophylactic or therapeutic regimens, because putative risk factors can vary among different regions. The objectives of this study were to measure the individual and herd-level seroprevalence of N. caninum among water buffalo in northeast Thailand and to identify putative risk factors associated with water buffalo exposure to this parasite. Sera were collected from 628 water buffalo at 288 farms over a 14-month period, and were assayed for antibodies to N. caninum with the indirect fluorescent antibody test (IFAT). The potential risk factors tested for seropositivity included physiography, geography, herd size, sex, and age.
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2. Materials and methods 2.1. Study area Six provinces with the highest water buffalo densities in northeast Thailand were chosen for this study. The areas sampled primarily consisted of hilly terrain with small mountainous or highland areas, and are physiographically divided into the Sakon Nakhon and Khorat basins, each of which are further divided into political provinces. The northern Sakon Nakhon basin includes the Sakon Nakhon and Roi Et provinces, and the southern Khorat basin contains Ubon Ratchathani, Surin, Buri Ram and Si Sa Ket provinces (Supplemental Fig. 1). Geographic locations were expected to be representative of interfaces between animals subjected to different cultures, resources, and animal care practices.
2.2. Sample size and distribution The survey was conducted on 288 farms. Prior surveys for anti-N. caninum antibodies among cattle and water buffalo in Thailand reported seroprevalence values ranging from 2.4 to 73.3% with a median of 24.65% (Chanlun et al., 2007; Dubey et al., 2007; Jittapalapong et al., 2008; Nam et al., 2012; Wiengcharoen et al., 2012, 2010). Using this average to estimate the prevalence (p) of N. caninum among water buffalo in Thailand, a 95% confidence level (t) and 5% margin of error (m), the minimal sample size (n) of 67 was calculated based on the equation, n = t2 × p(1 − p)/m2 . Farms selected for sample collection were distributed throughout the study area to avoid geographic clustering. Assuming a design effect of 4, primarily due to possible seasonal variations, we decided to test ≥268 farms. Access to these buffalo was acquired through local veterinarians and provincial staff that arranged for owners to bring their buffalo to a meeting place before releasing them for the day to graze in pastures, harvested rice fields or harvested cornfields. Thus, information was limited to that available for the water buffalo sampled, and dogs from these farms and grazing areas were not available for samples. Because they are draft animals for rice cultivation, buffalo per farm was expected to reflect plantation size, and buffalo on larger plantations were expected to work greater land areas, increasing the likelihood of interface with more dogs. Buffalo herd sizes sampled in this study were divided into two groups of >5 and ≤5 buffalo per farm.
2.3. Blood samples A total of 628 samples were collected from buffalo representing 288 farms. Blood was collected from jugular veins, and sera stored at −20 ◦ C until analysis. The animals sampled were divided into four age groups: ≤2.9 years (161 animals), 3–4.9 years (217 animals), 5–9.9 years (194 animals) and >10 years (56 animals). Equal sample numbers were collected from each age group present on a farm whenever possible.
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2.4. Detection of antibodies to N. caninum
2.5. Statistical analysis
IFAT was used to examine seroreactivity to intact N. caninum (NC-1 strain) tachyzoites grown in African green monkey kidney (Vero) cell culture. Parasites and host cells were maintained in minimum essential medium (MEM, Sigma, USA) supplemented with 10% heat-inactivated fetal bovine serum, l-glutamine and penicillin-streptomycin at 37 ◦ C in 5% CO2 in air. Infected host cells were scraped from flasks and disrupted by three passages through 25 and 27-gauge needles, followed by 5.0 m ultrafiltration (Millipore, USA), filtrate centrifugation at 1448 × g for 5 min, washing pellets with 10 mL of PBS, and re-centrifugation before pelleted parasites were counted and diluted to 1 × 104 tachyzoites/mL. The tachyzoite suspension was dispensed into 4-mm wells (10 L/well) on teflon-coated slides (Cel-Line Associates, Newfield, NJ), which were then air dried at room temperature, fixed with acetone for 30 min and stored at −20 ◦ C. Samples and control sera were diluted to 1:100 in PBS with 4% bovine serum albumin (BSA), placed onto antigencoated slides, and incubated at 37 ◦ C for 30 min before three washes with PBS and incubation with 1:200 caprine antibovine IgG1, 2 (H&L) FITC conjugate (VMRD, Pullman, WA, USA) in PBS-BSA for 1 h at 37 ◦ C. These slides were washed thrice with PBS before examination with coverslips and a fluorescence microscope. Neospora-positive and negative control sera were obtained from cattle experimentally inoculated with NC-1 or uninfected Vero cells, respectively (VMRD).
Individual and farm-level characteristics were analyzed in relation to seroreactivity to identify putative risk factors associated with water buffalo exposure to N. caninum. These characteristics included basin, province, herd size, sex, and age, which were analyzed with the chi-square (2 ) test in Number Cruncher Statistical System (NCSS) version 2000 (Kaysville, UT). These factors were assessed for association with exposure to N. caninum at the 95% confidence interval with WinEpiscope software version 2.0 (Thrusfield et al., 2001). 3. Results Individual host and herd-level seroprevalence values for anti-N. caninum-positive samples are shown in Table 1. Seroprevalence among individual water buffalo ranged from 3.9 to 16.7% in the six provinces sampled, with an overall seroprevalence of 9.1% among individual hosts. The highest host seroprevalence was among samples from Ubon Ratchathani province, followed by Si Sa Ket and Surin. Herd-level seroprevalence ranged from 6.9 to 36.4%, with an overall value of 16.7% (48/288); the highest herd seroprevalence was in Si Sa Ket province followed by Ubon Ratchathani and Buri Ram. Risk factors for seroprevalence among individuals included host age, herd size and basin region in northeast Thailand (Table 1). A statistically significant association was not observed between seroprevalence and sex. Farms
Table 1 Detection of antibodies to N. caninum from water buffaloes in northeast Thailand. Parameter Individual prevalence Province Ubon Ratchathani Si Sa Ket Surin Buri Ram Roi Et Sakon Nakhon Total Basin Sakon Nakhon Khorat Age group <3 years 3–5 years 5–10 years >10 years Sex Male Female Herd prevalence Province Si Sa Ket Ubon Ratchathani Buri Ram Surin Sakon Nakhon Roi Et Total Herd size ≤5 animals >5 animals
IFAT+/No. tested (%)
Statistical parameters
95% CI
2 = 20.4, df = 5, p = 0.001 23/138 (16.7) 9/61 (14.8) 7/73 (9.6) 6/70 (8.6) 4/81 (4.9) 8/205 (3.9) 57/628 (9.1) 2 = 15.2, df = 1, p = 0.0001 12/286 (4.2) 45/342 (13.2)
0.2–0.5 1.9–6.5 2 = 19.7, df = 3, p = 0.0002
2/161 (1.2) 20/217 (9.2) 26/194 (13.4) 9/56 (16.1) 2 = 2.83, df = 1, p = 0.09 4/91 (4.4) 53/537 (9.9)
0.2–1.2 0.9–6.6 2 = 24.9, df = 5, p = 0.001
8/23 (36.4) 18/59 (30.5) 5/22 (22.7) 6/28 (21.4) 7/98 (7.1) 4/58 (6.9) 48/288 (16.7) 2 = 1.3, df = 1, p = 0.2 45/278 (16.2) 3/10 (30.0)
0.1–1.8 0.6–8.6
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with >5 animals had higher seroprevalence (30%) than those with ≤5 (16.2%), but these values were not statistically significant (p = 0.2). The highest seroprevalence was measured among water buffalo >10 years old, followed by 5–10 years, 3–5 years and <3 years of age. A significantly higher seroprevalence was measured from the southern Khorat basin than from the northern Sakon Nakhon basin. 4. Discussion The results of this investigation indicated that water buffalo are exposed to N. caninum in northeastern Thailand, with an overall seroprevalence of 9.1% among individual hosts and an overall herd prevalence of 16.7%. To the best of our knowledge, this is the first report of risk factors associated with water buffalo exposure to N. caninum or of herd-level seroprevalence of the parasite among water buffalo in Asia. Putative risk factors for exposure to the parasite included physiographic area (basin), province, and host age. All of the provinces with the highest individual and herd-level seroprevalence were located in the southern Khorat Basin of northeastern Thailand, which had more than a three-fold higher seroprevalence than the Sakon Nakhon Basin to the north. The 9.1% overall individual seroprevalence from the present study was less than half the median value based on previous reports, which was used to estimate sample size for the current study. Previous reports of N. caninum seroprevalence among water buffalo included 73.3% from a single farm in Chachoengsao province with cELISA (Wiengcharoen et al., 2010) and 4.5% in northeast Thailand with iscom ELISA (Nam et al., 2012). All of these investigations used different methods for detection of anti-N. caninum antibodies, which could partially explain the discrepant results. Additionally, the study reporting 73.3% seroprevalence was focused on a single farm, while the present report focused on smallholder farms and required us to sample regions rather than individual farms with different management practices. Notably, a seroprevalence of 4.2% among provinces in the northern Sakon Nakhon Basin during the current study was similar to the overall individual host seroprevalence of 4.5% measured for the same basin in one of the previous reports (Nam et al., 2012). In Sakon Nakhon Province, the only province sampled in both studies, the previous and current investigations detected 5.1% and 3.9% seroprevalence among individual water buffalo, respectively, further indicating agreement between these reports where the study areas overlap. Thus, these studies collectively suggest that the higher seroprevalence in the Khorat Basin is an important observation that underscores the importance of risk factor analysis for identification of potential management issues. Additional work is warranted to further investigate differences between water buffalo in these basins and surrounding areas. There are at least two possible explanations for the higher seroprevalence in the Khorat Basin. First, exposure to N. caninum could be associated with the presence of more rivers and their branches. These rivers are commonly used for agriculture, livestock husbandry, human and animal consumption, and for transportation. Therefore it is possible that N. caninum oocysts in these areas are less
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subject to desiccation due to moisture in the soil, that ungulates are more likely to be exposed to N. caninum oocysts when drinking and grazing in areas near a water source that is shared by canine definitive hosts, and that transportation on these rivers can facilitate exposure to canine feces from a broader geographic area. Second, N. caninum seroprevalence might be associated with animal migration from other geographic areas that border the Thai Khorat Basin to the south and to the east. Thus, both definitive and intermediate hosts could enter from neighboring areas that may contain different host populations or where different resources or policies may be in place for livestock disease control. Notably, only Thai provinces surround the provinces surveyed in the Sakon Nakhon Basin, while all of the provinces sampled in the Khorat Basin have at least one international border. Ubon Ratchathani, the province with the highest individual (16.7%) and second highest herd (30.5%) seroprevalence, has two different international borders. However, to the best of our knowledge, the seroprevalence of N. caninum among water buffalo in these neighboring areas has not been reported. The risk of exposure to N. caninum appeared to increase with age. This observation could be due to differences in immune systems among water buffalo age groups. However, some have reported seroprevalence rates that appeared less dependent of age (Dubey et al., 2007; Nasir et al., 2011), while others also measured the lowest seroprevalence among the youngest age group tested (Dubey et al., 2007; Konrad et al., 2013) and others reported trends that were similar to the current report (Campero et al., 2007; Dubey et al., 2007). Interestingly, one of the latter studies also reported higher seroprevalence at herd-level (82%) than among individual hosts (34.6%) (Dubey et al., 2007). These reports collectively suggest that seroprevalence differences among water buffalo age groups are not due to immune system differences alone, but that these differences may reflect the frequencies of horizontal and vertical transmission of N. caninum to water buffalo. For example, although vertical transmission can occur in buffalo and frequently occurs in cattle (Dubey et al., 2007), the increased risk of exposure with water buffalo age can be attributed to horizontal transmission of N. caninum from definitive canine to intermediate buffalo hosts in enzootic regions. This conclusion is also supported by a previous report that the presence of dogs on farms is an important risk factor bovine exposure to N. caninum in Thailand (Arunvipas et al., 2012). In conclusion, the seroprevalence of N. caninum was confirmed among water buffalo in northeast Thailand. Location in the Khorat Basin and older host age were identified as putative risk factors, which, along with a greater herd than individual host prevalence, collectively suggest that horizontal transmission from canine definitive hosts is currently the most important means of exposure of water buffalo to N. caninum in northeast Thailand. These results underscore the importance of N. caninum prevention and control in regions of Southeast Asia where buffalo are important draft animals and the importance of risk factor analysis for effective control of neosporosis in different regions. Further investigations are warranted to evaluate the effects of river systems, animal management and the
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presence of infected dogs on water buffalo exposure to N. caninum in Thailand and neighboring countries. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgments This research was financially supported by The Graduate School, Kasetsart University, Kasetsart University Research and Development Institute (KURDI), and a scholarship from Faculty of Veterinary Medicine, Kasetsart University. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.vetpar.2014.10.034. References Arunvipas, P., Inpankaew, T., Jittapalapong, S., 2012. Risk factors of Neospora caninum infection in dogs and cats in dairy farms in Western Thailand. Trop. Anim. Health Prod. 44, 1117–1121. Auriemma, C., Lucibelli, M.G., Borriello, G., De Carlo, E., Martucciello, A., Schiavo, L., Gallo, A., Bove, F., Corrado, F., Girardi, S., Amoroso, M.G., Degli Uberti, B., Galiero, G., 2014. PCR detection of Neospora caninum in water buffalo foetal tissues. Acta Parasitol. 59, 1–4. Campero, C.M., Perez, A., Moore, D.P., Crudeli, G., Benitez, D., Draghi, M.G., Cano, D., Konrad, J.L., Odeon, A.C., 2007. Occurrence of antibodies against Neospora caninum in water buffaloes (Bubalus bubalis) on four ranches in Corrientes province. Argentina. Vet. Parasitol. 150, 155–158. Chanlun, A., Emanuelson, U., Frossling, J., Aiumlamai, S., Bjorkman, C., 2007. A longitudinal study of seroprevalence and seroconversion of Neospora caninum infection in dairy cattle in northeast Thailand. Vet. Parasitol. 146, 242–248. Chryssafidis, A.L., Canton, G., Chianini, F., Innes, E.A., Madureira, E.H., Gennari, S.M., 2014a. Pathogenicity of Nc-Bahia and Nc-1 strains of Neospora caninum in experimentally infected cows and buffaloes in early pregnancy. Parasitol. Res. 113, 1521–1528.
Chryssafidis, A.L., Canton, G., Chianini, F., Innes, E.A., Madureira, E.H., Soares, R.M., Gennari, S.M., 2014b. Abortion and foetal lesions induced by Neospora caninum in experimentally infected water buffalos (Bubalus bubalis). Parasitol. Res., http://dx.doi.org/ 10.1007/s00436-014-4178-0 (in press). Dubey, J.P., Schares, G., Ortega-Mora, L.M., 2007. Epidemiology and control of neosporosis and Neospora caninum. Clin. Microbiol. Rev. 20, 323–367. Jittapalapong, S., Sangwaranond, A., Inpankaew, T., Phasuk, C., Pinyopanuwat, N., Chimnoi, W., Kengradomkij, C., Saengow, S., Pumhom, P., Arunwipat, P., Anakewit, T., Robertson, I.D., 2008. Seroprevalence of Neospora caninum infections of dairy cows in the northeast of Thailand. Kasetsart J. Nat. Sci. 42, 61–66. Konrad, J.L., Campero, L.M., Caspe, G.S., Brihuega, B., Draghi, G., Moore, D.P., Crudeli, G.A., Venturini, M.C., Campero, C.M., 2013. Detection of antibodies against Brucella abortus, Leptospira spp., and Apicomplexa protozoa in water buffaloes in the northeast of Argentina. Trop. Anim. Health Prod. 45, 1751–1756. Konrad, J.L., Moore, D.P., Crudeli, G., Caspe, S.G., Cano, D.B., Leunda, M.R., Lischinsky, L., Regidor-Cerrillo, J., Odeon, A.C., Ortega-Mora, L.M., Echaide, I., Campero, C.M., 2012. Experimental inoculation of Neospora caninum in pregnant water buffalo. Vet. Parasitol. 187, 72–78. Kyaw, T., Suwimonteerabutr, J., Virakul, P., Lohachit, C., Kalpravidh, W., 2005. Seronegative conversion in four Neospora caninum-infected cows, with a low rate of transplacental transmission. Vet. Parasitol. 131, 145–150. Kyaw, T., Virakul, P., Muangyai, M., Suwimonteerabutr, J., 2004. Neospora caninum seroprevalence in dairy cattle in central Thailand. Vet. Parasitol. 121, 255–263. Nam, N.H., Aran, C., Kwankate, K., Suneerat, A., 2012. Seroprevalence of Neospora caninum in swamp buffaloes and beef cattle in the northeast of Thailand. Thai J. Vet. Med. 42, 213–218. Nasir, A., Ashraf, M., Khan, M.S., Yaqub, T., Javeed, A., Avais, M., Akhtar, F., 2011. Seroprevalence of Neospora caninum in dairy buffaloes in Lahore District, Pakistan. J. Parasitol. 97, 541–543. Thrusfield, M., Ortega, C., de Blas, I., Noordhuizen, J.P., Frankena, K., 2001. WIN EPISCOPE 2.0: improved epidemiological software for veterinary medicine. Vet. Rec. 148, 567–572. Wiengcharoen, J., Nakthong, C., Mitchaothai, J., Udonsom, R., Sukthana, Y., 2012. Toxoplasmosis and neosporosis among beef cattle slaughtered for food in Western Thailand. Southeast Asian J. Trop. Med. Public Health 43, 1087–1093. Wiengcharoen, J., Thuchadaporn, C., Suvarin, P., Nattaya, J., 2010. High prevalence of antibodies to Neospora caninum in water buffaloes (Bubalus bubalis) from a local farm in Chachoengsao province. In: Fourth Mahanakorn University of Technology Veterinary Annual Conference, Bangkok, Thailand, November 26, 2010, pp. 117–118.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short Communication
Seroprevalence and risk factors associated with exposure of water buffalo (Bubalus bubalis) to Neospora caninum in northeast Thailand Chanya Kengradomkij a , Tawin Inpankaew a , Ketsarin Kamyingkird a , Kannika Wongpanit b , Sirichai Wongnakphet c , Thomas J. Mitchell d , Xuenan Xuan e , Ikuo Igarashi e , Sathaporn Jittapalapong a,∗ , Roger W. Stich f a
Department of Parasitology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand Faculty of Natural Resource and Agro-Industry, Chalermphrakiat Sakon Nakhon Province Campus, Kasetsart University, Thailand c Department of Veterinary Diagnostic Services, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand d Animal Health Laboratories, Department of Agriculture and Food Western Australia, 3 Baron-Hay Court, South Perth, Western Australia 6151, Australia e National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-13, Inada-cho, Obihiro, Hokkaido 080-8555, Japan f University of Missouri, Department of Veterinary Pathobiology, 201 Connaway Hall, Columbia, MO 65211, USA b
a r t i c l e
i n f o
Article history: Received 14 February 2014 Received in revised form 31 October 2014 Accepted 31 October 2014 Keywords: Neospora caninum Water buffalo IFAT Seroprevalence Neosporosis Thailand
a b s t r a c t Water buffalo are important draft animals for agriculture in resource-restricted areas worldwide. Water buffalo were shown to be experimentally susceptible to infection with Neospora caninum, potentially affected by neosporosis, and naturally exposed to the parasite in Asia. Although enzootic to Thailand, the distribution of N. caninum among Thai water buffalo is unclear. The objectives of this study were to determine the seroprevalence of N. caninum among water buffalo of northeast Thailand and to identify risk factors associated with their exposure to N. caninum. Sera from 628 water buffalo from 288 farms were tested with an indirect fluorescent antibody test (IFAT). A total of 57 samples from 48 herds contained antibodies to N. caninum, indicating overall seroprevalence of 9.1% and 16.7% among individual animals and herds, respectively. The overall seroprevalence was highest in provinces located in the Khorat Basin in the southern part of the region tested. Host age was also associated with seroprevalence, with the greatest seroprevalence (16.1%) among buffalo over 10 years of age, followed by 5–10 years of age (13.4%), 3–5 years (9.2%), and less than 3 years (1.2%). These results collectively suggested that horizontal transmission from canine definitive hosts was an important route of water buffalo exposure to N. caninum. These results also verified the importance of risk factor analysis for effective bovine neosporosis control strategies at the local level. © 2014 Elsevier B.V. All rights reserved.
1. Introduction ∗ Corresponding author at: Department of Parasitology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10903, Thailand. Tel.: +66 2 942 8438; fax: +66 2 942 8438. E-mail address: [email protected] (S. Jittapalapong). http://dx.doi.org/10.1016/j.vetpar.2014.10.034 0304-4017/© 2014 Elsevier B.V. All rights reserved.
Approximately 1.2 million water buffalo (Bubalus bubalis) are indigenous to Thailand, where they serve important roles as draft animals for agriculture. These smallholder farms are often in close proximity, without
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barriers between water buffalo, wildlife and companion animals (Kyaw et al., 2004). Additionally, Thai buffalo are often reared on public pastures, increasing their exposure to other animals, including dogs. Neospora caninum is a heteroxenous apicomplexan parasite that undergoes sexual reproduction in the alimentary canal of canine definitive hosts and that infects a diverse range of intermediate hosts, including bovids, by horizontal transmission of environmentally resistant oocysts shed in canine feces and by vertical transmission to host offspring (Dubey et al., 2007). This parasite is the etiologic agent of bovine neosporosis, an important cause of bovine fetal abortion, stillbirth, birth of weak calves and reduced milk yields, often leading to culling of infected animals due to the lack of effective alternatives for the prevention and control of this disease (Kyaw et al., 2004). Although water buffalo have different behavior and physiology than dairy and beef cattle, they can serve as reservoirs for infectious disease agents of cattle, and buffalo are considered natural intermediate hosts of N. caninum (Chryssafidis et al., 2014b). Histopathology and PCR assays of placental and fetal tissues from naturally and experimentally infected dams indicated that N. caninum can also contribute to reproductive failure among water buffalo (Auriemma et al., 2014; Chryssafidis et al., 2014a,b; Konrad et al., 2012). Therefore understanding the prevalence and risk factors associated with exposure to N. caninum are critical to effective prevention and control of neosporosis among these important draft animals. Seroprevalence of N. caninum among water buffalo has been reported in Asia and surrounding areas, which was estimated at 1.5% in Vietnam, 3.8% in the Philippines, 54.7% in Pakistan and 68% in Egypt (Chryssafidis et al., 2014b). Anti-N. caninum antibodies were also reportedly detected from 4.5% and 73.3% of Thai water buffalo sampled in two separate studies, and from 5.5 to 70% of Thai cattle in other studies (Chanlun et al., 2007; Chryssafidis et al., 2014b; Jittapalapong et al., 2008; Kyaw et al., 2005; Nam et al., 2012; Wiengcharoen et al., 2010). Although there is evidence that N. caninum is enzootic to Thailand, in addition to reports suggesting that this parasite can be detrimental to water buffalo (Auriemma et al., 2014; Chryssafidis et al., 2014a,b; Konrad et al., 2013), the distribution of this parasite among Thai water buffalo and the importance of horizontal and vertical transmission of this parasite to water buffalo in Thailand are unclear. Identification of risk factors for host exposure to N. caninum is important for control of neosporosis, especially in the absence of effective immunoprophylactic or therapeutic regimens, because putative risk factors can vary among different regions. The objectives of this study were to measure the individual and herd-level seroprevalence of N. caninum among water buffalo in northeast Thailand and to identify putative risk factors associated with water buffalo exposure to this parasite. Sera were collected from 628 water buffalo at 288 farms over a 14-month period, and were assayed for antibodies to N. caninum with the indirect fluorescent antibody test (IFAT). The potential risk factors tested for seropositivity included physiography, geography, herd size, sex, and age.
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2. Materials and methods 2.1. Study area Six provinces with the highest water buffalo densities in northeast Thailand were chosen for this study. The areas sampled primarily consisted of hilly terrain with small mountainous or highland areas, and are physiographically divided into the Sakon Nakhon and Khorat basins, each of which are further divided into political provinces. The northern Sakon Nakhon basin includes the Sakon Nakhon and Roi Et provinces, and the southern Khorat basin contains Ubon Ratchathani, Surin, Buri Ram and Si Sa Ket provinces (Supplemental Fig. 1). Geographic locations were expected to be representative of interfaces between animals subjected to different cultures, resources, and animal care practices.
2.2. Sample size and distribution The survey was conducted on 288 farms. Prior surveys for anti-N. caninum antibodies among cattle and water buffalo in Thailand reported seroprevalence values ranging from 2.4 to 73.3% with a median of 24.65% (Chanlun et al., 2007; Dubey et al., 2007; Jittapalapong et al., 2008; Nam et al., 2012; Wiengcharoen et al., 2012, 2010). Using this average to estimate the prevalence (p) of N. caninum among water buffalo in Thailand, a 95% confidence level (t) and 5% margin of error (m), the minimal sample size (n) of 67 was calculated based on the equation, n = t2 × p(1 − p)/m2 . Farms selected for sample collection were distributed throughout the study area to avoid geographic clustering. Assuming a design effect of 4, primarily due to possible seasonal variations, we decided to test ≥268 farms. Access to these buffalo was acquired through local veterinarians and provincial staff that arranged for owners to bring their buffalo to a meeting place before releasing them for the day to graze in pastures, harvested rice fields or harvested cornfields. Thus, information was limited to that available for the water buffalo sampled, and dogs from these farms and grazing areas were not available for samples. Because they are draft animals for rice cultivation, buffalo per farm was expected to reflect plantation size, and buffalo on larger plantations were expected to work greater land areas, increasing the likelihood of interface with more dogs. Buffalo herd sizes sampled in this study were divided into two groups of >5 and ≤5 buffalo per farm.
2.3. Blood samples A total of 628 samples were collected from buffalo representing 288 farms. Blood was collected from jugular veins, and sera stored at −20 ◦ C until analysis. The animals sampled were divided into four age groups: ≤2.9 years (161 animals), 3–4.9 years (217 animals), 5–9.9 years (194 animals) and >10 years (56 animals). Equal sample numbers were collected from each age group present on a farm whenever possible.
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2.4. Detection of antibodies to N. caninum
2.5. Statistical analysis
IFAT was used to examine seroreactivity to intact N. caninum (NC-1 strain) tachyzoites grown in African green monkey kidney (Vero) cell culture. Parasites and host cells were maintained in minimum essential medium (MEM, Sigma, USA) supplemented with 10% heat-inactivated fetal bovine serum, l-glutamine and penicillin-streptomycin at 37 ◦ C in 5% CO2 in air. Infected host cells were scraped from flasks and disrupted by three passages through 25 and 27-gauge needles, followed by 5.0 m ultrafiltration (Millipore, USA), filtrate centrifugation at 1448 × g for 5 min, washing pellets with 10 mL of PBS, and re-centrifugation before pelleted parasites were counted and diluted to 1 × 104 tachyzoites/mL. The tachyzoite suspension was dispensed into 4-mm wells (10 L/well) on teflon-coated slides (Cel-Line Associates, Newfield, NJ), which were then air dried at room temperature, fixed with acetone for 30 min and stored at −20 ◦ C. Samples and control sera were diluted to 1:100 in PBS with 4% bovine serum albumin (BSA), placed onto antigencoated slides, and incubated at 37 ◦ C for 30 min before three washes with PBS and incubation with 1:200 caprine antibovine IgG1, 2 (H&L) FITC conjugate (VMRD, Pullman, WA, USA) in PBS-BSA for 1 h at 37 ◦ C. These slides were washed thrice with PBS before examination with coverslips and a fluorescence microscope. Neospora-positive and negative control sera were obtained from cattle experimentally inoculated with NC-1 or uninfected Vero cells, respectively (VMRD).
Individual and farm-level characteristics were analyzed in relation to seroreactivity to identify putative risk factors associated with water buffalo exposure to N. caninum. These characteristics included basin, province, herd size, sex, and age, which were analyzed with the chi-square (2 ) test in Number Cruncher Statistical System (NCSS) version 2000 (Kaysville, UT). These factors were assessed for association with exposure to N. caninum at the 95% confidence interval with WinEpiscope software version 2.0 (Thrusfield et al., 2001). 3. Results Individual host and herd-level seroprevalence values for anti-N. caninum-positive samples are shown in Table 1. Seroprevalence among individual water buffalo ranged from 3.9 to 16.7% in the six provinces sampled, with an overall seroprevalence of 9.1% among individual hosts. The highest host seroprevalence was among samples from Ubon Ratchathani province, followed by Si Sa Ket and Surin. Herd-level seroprevalence ranged from 6.9 to 36.4%, with an overall value of 16.7% (48/288); the highest herd seroprevalence was in Si Sa Ket province followed by Ubon Ratchathani and Buri Ram. Risk factors for seroprevalence among individuals included host age, herd size and basin region in northeast Thailand (Table 1). A statistically significant association was not observed between seroprevalence and sex. Farms
Table 1 Detection of antibodies to N. caninum from water buffaloes in northeast Thailand. Parameter Individual prevalence Province Ubon Ratchathani Si Sa Ket Surin Buri Ram Roi Et Sakon Nakhon Total Basin Sakon Nakhon Khorat Age group <3 years 3–5 years 5–10 years >10 years Sex Male Female Herd prevalence Province Si Sa Ket Ubon Ratchathani Buri Ram Surin Sakon Nakhon Roi Et Total Herd size ≤5 animals >5 animals
IFAT+/No. tested (%)
Statistical parameters
95% CI
2 = 20.4, df = 5, p = 0.001 23/138 (16.7) 9/61 (14.8) 7/73 (9.6) 6/70 (8.6) 4/81 (4.9) 8/205 (3.9) 57/628 (9.1) 2 = 15.2, df = 1, p = 0.0001 12/286 (4.2) 45/342 (13.2)
0.2–0.5 1.9–6.5 2 = 19.7, df = 3, p = 0.0002
2/161 (1.2) 20/217 (9.2) 26/194 (13.4) 9/56 (16.1) 2 = 2.83, df = 1, p = 0.09 4/91 (4.4) 53/537 (9.9)
0.2–1.2 0.9–6.6 2 = 24.9, df = 5, p = 0.001
8/23 (36.4) 18/59 (30.5) 5/22 (22.7) 6/28 (21.4) 7/98 (7.1) 4/58 (6.9) 48/288 (16.7) 2 = 1.3, df = 1, p = 0.2 45/278 (16.2) 3/10 (30.0)
0.1–1.8 0.6–8.6
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with >5 animals had higher seroprevalence (30%) than those with ≤5 (16.2%), but these values were not statistically significant (p = 0.2). The highest seroprevalence was measured among water buffalo >10 years old, followed by 5–10 years, 3–5 years and <3 years of age. A significantly higher seroprevalence was measured from the southern Khorat basin than from the northern Sakon Nakhon basin. 4. Discussion The results of this investigation indicated that water buffalo are exposed to N. caninum in northeastern Thailand, with an overall seroprevalence of 9.1% among individual hosts and an overall herd prevalence of 16.7%. To the best of our knowledge, this is the first report of risk factors associated with water buffalo exposure to N. caninum or of herd-level seroprevalence of the parasite among water buffalo in Asia. Putative risk factors for exposure to the parasite included physiographic area (basin), province, and host age. All of the provinces with the highest individual and herd-level seroprevalence were located in the southern Khorat Basin of northeastern Thailand, which had more than a three-fold higher seroprevalence than the Sakon Nakhon Basin to the north. The 9.1% overall individual seroprevalence from the present study was less than half the median value based on previous reports, which was used to estimate sample size for the current study. Previous reports of N. caninum seroprevalence among water buffalo included 73.3% from a single farm in Chachoengsao province with cELISA (Wiengcharoen et al., 2010) and 4.5% in northeast Thailand with iscom ELISA (Nam et al., 2012). All of these investigations used different methods for detection of anti-N. caninum antibodies, which could partially explain the discrepant results. Additionally, the study reporting 73.3% seroprevalence was focused on a single farm, while the present report focused on smallholder farms and required us to sample regions rather than individual farms with different management practices. Notably, a seroprevalence of 4.2% among provinces in the northern Sakon Nakhon Basin during the current study was similar to the overall individual host seroprevalence of 4.5% measured for the same basin in one of the previous reports (Nam et al., 2012). In Sakon Nakhon Province, the only province sampled in both studies, the previous and current investigations detected 5.1% and 3.9% seroprevalence among individual water buffalo, respectively, further indicating agreement between these reports where the study areas overlap. Thus, these studies collectively suggest that the higher seroprevalence in the Khorat Basin is an important observation that underscores the importance of risk factor analysis for identification of potential management issues. Additional work is warranted to further investigate differences between water buffalo in these basins and surrounding areas. There are at least two possible explanations for the higher seroprevalence in the Khorat Basin. First, exposure to N. caninum could be associated with the presence of more rivers and their branches. These rivers are commonly used for agriculture, livestock husbandry, human and animal consumption, and for transportation. Therefore it is possible that N. caninum oocysts in these areas are less
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subject to desiccation due to moisture in the soil, that ungulates are more likely to be exposed to N. caninum oocysts when drinking and grazing in areas near a water source that is shared by canine definitive hosts, and that transportation on these rivers can facilitate exposure to canine feces from a broader geographic area. Second, N. caninum seroprevalence might be associated with animal migration from other geographic areas that border the Thai Khorat Basin to the south and to the east. Thus, both definitive and intermediate hosts could enter from neighboring areas that may contain different host populations or where different resources or policies may be in place for livestock disease control. Notably, only Thai provinces surround the provinces surveyed in the Sakon Nakhon Basin, while all of the provinces sampled in the Khorat Basin have at least one international border. Ubon Ratchathani, the province with the highest individual (16.7%) and second highest herd (30.5%) seroprevalence, has two different international borders. However, to the best of our knowledge, the seroprevalence of N. caninum among water buffalo in these neighboring areas has not been reported. The risk of exposure to N. caninum appeared to increase with age. This observation could be due to differences in immune systems among water buffalo age groups. However, some have reported seroprevalence rates that appeared less dependent of age (Dubey et al., 2007; Nasir et al., 2011), while others also measured the lowest seroprevalence among the youngest age group tested (Dubey et al., 2007; Konrad et al., 2013) and others reported trends that were similar to the current report (Campero et al., 2007; Dubey et al., 2007). Interestingly, one of the latter studies also reported higher seroprevalence at herd-level (82%) than among individual hosts (34.6%) (Dubey et al., 2007). These reports collectively suggest that seroprevalence differences among water buffalo age groups are not due to immune system differences alone, but that these differences may reflect the frequencies of horizontal and vertical transmission of N. caninum to water buffalo. For example, although vertical transmission can occur in buffalo and frequently occurs in cattle (Dubey et al., 2007), the increased risk of exposure with water buffalo age can be attributed to horizontal transmission of N. caninum from definitive canine to intermediate buffalo hosts in enzootic regions. This conclusion is also supported by a previous report that the presence of dogs on farms is an important risk factor bovine exposure to N. caninum in Thailand (Arunvipas et al., 2012). In conclusion, the seroprevalence of N. caninum was confirmed among water buffalo in northeast Thailand. Location in the Khorat Basin and older host age were identified as putative risk factors, which, along with a greater herd than individual host prevalence, collectively suggest that horizontal transmission from canine definitive hosts is currently the most important means of exposure of water buffalo to N. caninum in northeast Thailand. These results underscore the importance of N. caninum prevention and control in regions of Southeast Asia where buffalo are important draft animals and the importance of risk factor analysis for effective control of neosporosis in different regions. Further investigations are warranted to evaluate the effects of river systems, animal management and the
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presence of infected dogs on water buffalo exposure to N. caninum in Thailand and neighboring countries. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgments This research was financially supported by The Graduate School, Kasetsart University, Kasetsart University Research and Development Institute (KURDI), and a scholarship from Faculty of Veterinary Medicine, Kasetsart University. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.vetpar.2014.10.034. References Arunvipas, P., Inpankaew, T., Jittapalapong, S., 2012. Risk factors of Neospora caninum infection in dogs and cats in dairy farms in Western Thailand. Trop. Anim. Health Prod. 44, 1117–1121. Auriemma, C., Lucibelli, M.G., Borriello, G., De Carlo, E., Martucciello, A., Schiavo, L., Gallo, A., Bove, F., Corrado, F., Girardi, S., Amoroso, M.G., Degli Uberti, B., Galiero, G., 2014. PCR detection of Neospora caninum in water buffalo foetal tissues. Acta Parasitol. 59, 1–4. Campero, C.M., Perez, A., Moore, D.P., Crudeli, G., Benitez, D., Draghi, M.G., Cano, D., Konrad, J.L., Odeon, A.C., 2007. Occurrence of antibodies against Neospora caninum in water buffaloes (Bubalus bubalis) on four ranches in Corrientes province. Argentina. Vet. Parasitol. 150, 155–158. Chanlun, A., Emanuelson, U., Frossling, J., Aiumlamai, S., Bjorkman, C., 2007. A longitudinal study of seroprevalence and seroconversion of Neospora caninum infection in dairy cattle in northeast Thailand. Vet. Parasitol. 146, 242–248. Chryssafidis, A.L., Canton, G., Chianini, F., Innes, E.A., Madureira, E.H., Gennari, S.M., 2014a. Pathogenicity of Nc-Bahia and Nc-1 strains of Neospora caninum in experimentally infected cows and buffaloes in early pregnancy. Parasitol. Res. 113, 1521–1528.
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