Title: Molecular and serological prevalence of Babesia bovis and Babesia bigemina in water buffaloes in the north region of Brazil Author: Jenevaldo Barbosa da Silva Marcos Rogério André Adivaldo Henrique da Fonseca Cinthia Távora de Albuquerque Lopes Danillo Henrique da Silva Lima Stefano Juliano Tavares de Andrade Carlos Magno Chaves Oliveira José Diomedes Barbosa PII: S0304-4017(13)00343-9 DOI: http://dx.doi.org/doi:10.1016/j.vetpar.2013.05.020 Reference: VETPAR 6858 To appear in: Veterinary Parasitology Received date: 25-2-2013 Revised date: 22-5-2013 Accepted date: 24-5-2013 Please cite this article as: Silva, J.B., André, M.R., Fonseca, A.H., Lopes, C.T.A., Lima, D.H.S., Andrade, S.J.T., Oliveira, C.M.C., Barbosa, J.D., Molecular and serological prevalence of Babesia bovis and Babesia bigemina in water buffaloes in the north region of Brazil, Veterinary Parasitology (2013), http://dx.doi.org/10.1016/j.vetpar.2013.05.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2 Molecular and serological prevalence of Babesia bovis and Babesia bigemina in water buffaloes in the north region of Brazil 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Jenevaldo Barbosa da Silva 1 *, Marcos Rogério André 1, Adivaldo Henrique da Fonseca 2, Cinthia Távora de Albuquerque Lopes 3, Danillo Henrique da Silva Lima 3, Stefano Juliano Tavares de Andrade 3, Carlos Magno Chaves Oliveira 3 and José Diomedes Barbosa 3 1 Laboratório de Imunoparasitologia, Departamento de Patologia Veterinária, Faculdade de Ciências Agrárias e Veterinárias FCAV-UNESP, Via de Acesso Prof. Paulo Donato Castellane s/n, 14884-900, Jaboticabal, SP, Brasil. E-mail: jenevaldo@hotmail.com 2 Laboratório de Doenças Parasitárias, Departamento de Epidemiologia e Saúde Pública, Universidade Federal Rural de Rio de Janeiro (UFRRJ), Br 465, Km 7, 23890-000, Seropédica, RJ, Brasil. E-mail: adivaldo@ufrrj.br 3 Instituto de Medicina Veterinária, Universidade Federal do Pará, Rodovia BR 316 Km 61, Bairro Saudade, 68740-970, Castanhal, PA, Brasil. E-mail: diomedes@ufpa.br Abstract Bovine babesiosis is a tick-borne disease caused mainly by B. bovis and B. bigemina, which are associated to considerable economic losses in cattle herds worldwide. Approximately 60% of buffalo herds in South America are located in Northern Brazil. Little is known about the impact of babesiosis on buffalo herds in 22 23 24 25 Brazil. The present work aimed to verify the occurrence of B. bovis and B. bigemina in 542 water buffaloes in the state of Pará, Northern Brazil, using molecular and serological techniques. The percentage of seropositive animals for B. bovis and B. bigemina was 41.2% and 19.0%, respectively, by ELISA. B. bovis and B. bigemina 1 Page 1 of 13
26 27 DNA were detected in 15 and 16% of sampled buffaloes, respectively. A high correlation (Kappa index of 0.9) between serological and molecular tests suggests that 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 the combination of the utilized techniques in the present study is suitable for babesiosis diagnosis in an endemic unstable area. Significantly difference of positivity for serological and molecular assays was verified to localities and reproductive status of sampled animals, but not between buffalo breeds. The immune status of sampled buffaloes associated to the circulation of babesiosis agents in sampled population suggests that the studied area is at risk to clinical babesiosis outbreaks. Furthermore, this study demonstrated that this region can be classified as endemically unstable. Keywords: Babesia, Brazil, ELISA, npcr, Water bufalloes 1. Introduction Brazil has the biggest water buffalo herds in the Latin America, where approximately 65% of animals is located in the Northern region of the country (IBGE, 2012). Since buffalo breeding has become an economically important activity, studies regarding the occurrence of pathogens circulating among buffaloes raised in Brazil are much needed. Bovine babesiosis is a tick-borne disease caused by protozoa belonging to Phylum Apicomplexa, Order Piroplasmida, and genus Babesia, causing high morbidity and mortality worldwide (McCosker, 1981). Babesia bovis and B. bigemina show a high 47 48 49 prevalence in tropical and subtropical areas, causing economic losses in cattle herds (Bock et al., 2004). On the other hand, the economic impact of babesiosis on buffalo herd has not been evaluated yet (Terkawi et al., 2011). 2 Page 2 of 13
50 51 Although the observation of parasites in blood smears is routinely used in the babesiosis diagnosis, this technique shows a low sensitivity in subclinical and chronic 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 phase of the disease (Terkawi et al., 2011). Molecular tools have shown a higher sensitivity and specificity. On the other hand, serological assays, such as Enzyme- Linked Immunosorbent Assay (ELISA) and Indirect Fluorescent Antibody Test (IFAT), are used aiming to detect specific antibodies in carrier animals in epidemiological surveys. The combination of ELISA and Polymerase Chain Reaction (PCR) is considered a powerful tool in epidemiological surveys, showing high precision and sensitivity in babesiosis diagnosis (Terkawi et al., 2011). Our work aimed to verify the occurrence of B. bovis and B. bigemina in water buffaloes in state of Pará, Northern Brazil, using molecular and serological techniques. 2. Materials and Methods 2.1. Sampling Field samples of blood from water buffalo were collected from different farms in seven provinces in Marajó island and five provinces in Continent, in the northern region of Brazil in December 2011. The number of samples to assess the prevalence of B. bovis and B. bigemina in the north region of Brazil was determined using the formula recommended by the Pan American Zoonosis Center (CEPANZO, 1979) for the study of chronic diseases: 71 72 73 74 N=p.(100-p)Z 2 /(d.p/100) 2, where n, number of samples; p, expected prevalence; Z, confidence level, and d, error margin. An estimated prevalence of buffaloes positive for B. bovis and B. bigemina was 40%, confidence level of 95.0% and an error margin of 5.0%, were established. We estimated that 542 samples should be analyzed by ELISA. 3 Page 3 of 13
75 76 Thus, we randomly selected a total of 382 buffaloes from Marajó Island and 160 buffaloes from Continent, being 299 and 243 of breed Murrah and Mediterranean, 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 respectively. Regarding the reproductive period, 111 females were pregnant and 431 were non-pregnant. However, we only tested 272 buffaloes in PCR assays, because of high costs of performing molecular techniques in all sampled buffaloes. Animal sampling was performed based on proportional stratification. Taking account that the Marajó Island and the continent have approximately herds of 1000000 and 400000 buffaloes, respectively, we select 2.5 times more animals in the island than that in the continent. While 38 buffaloes were sampled in 18 farms from Marajó island, a number of 160 buffaloes were sampled in 8 farms from continents. All sampled animals grazed on Brachiaria decumbens pastures, and were not treated with tick acaricides and anthelmintics. Despite of that, the majority of sampled animals showed a low infestation by ticks, predominantly represented by larvae and nymphs. Whole blood samples were collected from caudal or jugular vein of individual water buffaloes. For serum samples, blood samples without EDTA were incubated at room temperature and then centrifuged at 3000 rpm for 15 min; the sera were collected. DNA was extracted from 200 µl of each 271 EDTA-whole blood sample using the QIAamp DNA Blood Mini kit (QIAGEN, Valencia, California, USA) according to the manufacturer s instructions. 2.2. Enzyme-like immunosorbent assay (ELISA) 96 97 Microtiter plates (Immulon ; Dynatech Laboratories Inc.) were coated overnight at 4 C with each recombinant antigen at a concentration of 5 µg/ml 4 Page 4 of 13
98 99 emulsified in a coating buffer. The assay was then performed as earlier described (Terkawi et al., 2011). 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 2.3 IFAT Briefly, a 10µl field serum sample diluted in PBS (1:80) was applied as the first antibody on the fixed smears and then incubated for 1 h at 37 ºC in amoist chamber. The assay was then performed as earlier described (Terkawi et al., 2011). The samples were tested at 1:80, 1:160, 1:320; 1:640 dilutions. 2.4. nested PCR A. marginale DNA was extracted from infected blood and the 18S rrna gene were amplified by PCR as previously reported (Terkawi et al., 2011). PCR products (primary as well as nested) were checked for amplification by electrophoresis on a 2.0% agarose gel and visualized using gel documentation system (Syngene, UK). 2.5. Statistical analysis The kappa coefficient was calculated to evaluate the agreement among the npcr assay and ELISA. The chi-square test was used to evaluate significant differences (P < 0.05) of infection rate in animals of different breed, reproductive status, and locations. The operational procedures were done using the R statistical software (R Foundation for Statistical Computing, version 2.12.2, 2011). 119 120 3. Results 121 5 Page 5 of 13
122 123 The prevalence of positive buffaloes in PCR assays for B. bovis, B. bigemina and both parasites was 15.1% (41/271), 16.2% (44/271) and 8.9% (24/271), 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 respectively. The number of positive animals in PCR assays for B. bovis and B. bigemina was correlated to sampling area, breed and reproductive status. In Marajó island, the number of PCR positive buffaloes for B. bovis (25/191) and B. bigemina (26/191) was significantly lower (p=0.003 and 0.001, respectively) than the number of PCR positive buffaloes for B. bovis (16/80) and B. bigemina (18/80) in Continent. The number of positive buffaloes at B. bovis and B. bigemina-pcr assays did not show difference between Murrah and Mediterranean breeds (p=0.09 e p=0.07, respectively). Pregnant female buffaloes were more likely to show B. bovis (p=0.0001) and B. bigemina (p=0.0001) positive PCR results when compared to non-pregnant females. IgG antibodies to B. bovis and B. bigemina was detected in 41.2% (223/542) and 19.0% (103/542) of sampled buffaloes, respectively. Furthermore, 18% (97/542) of sampled animals showed IgG antibodies to both parasites. Among seropositive buffaloes, 43.3% (97/229) showed seropositivity to both B. bovis and B. bigemina. The percentage of seropositive buffaloes for B. bovis and B. bigemina in continent localities (46% and 50%, respectively) were higher (p=0.0001) than that found among animals from Marajó island (39% and 16%, respectively). Pregnant female buffaloes were more likely (p=0.0001) to show antibodies to Babesia sp. when compared to non-pregnant female buffaloes. The seropositivity for studied parasites did not shown significantly difference between Murrah and Mediterranean breeds. 143 144 145 Eighty-one (30%) and 75 (28%) out of 271 buffalo serum samples submitted to both ELISA and PCR assays showed positive results in at least one test for B. bovis and B. bigemina, respectively (Figure 1). The number of animals positive to ELISA or PCR 6 Page 6 of 13
146 147 was higher (p=0.05 and p=0.04, respectively) than the number of positive animals in only one of the techniques utilized (Figure 1). 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 The number of samples positive to B. bovis and B. bigemina to both tests was 9.2% and 10.0%, number positive to PCR/negative to ELISA was 5.9% and 6.3%, number negative to PCR/positive to ELISA was 32.1% and 8.8% and number negative to both tests was 53.1% and 75.3% (Table 1). These results showed a high correlation between molecular and serological tests used in the present study (kappa index of 0.9). 4. Discussion The combination between ELISA and PCR has been considered useful tool in epidemiological surveys regarding the occurrence of Babesia infections in buffaloes (Terkawi et al., 2011). The present work showed a molecular occurrence of B. bovis and B. bigemina higher than that found in Thailand (1.7 and 4.1 times higher, respectively) (Terkawi et al., 2011) and China (24 and 28 times higher, respectively) (He et al., 2012). Herein, the percentage of co-positivity for B. bovis and B. bigemina (18.75%) was higher than that found among buffaloes from China (3.9%) (He et al., 2012). In Argentina, an occurrence of B. bovis-pcr positive buffaloes ranging from 34% a 61% was observed (Ferreri et al., 2008). In India, buffaloes rarely show clinical signs of babesiosis when submitted to natural conditions because are refractory to infection due to their natural immunological resistance (Roychoudhury and Gautam, 167 168 169 170 1979). However, B. bigemina has been showing up as an agent of major concern in Indian cattle herds, parasitizing both cattle and buffaloes (Muraleedharan et al., 1984). In additional buffaloes might serve as reservoirs for the infections of cattle, since they are together in the pastures and continue to infect the tick vector (Bock et al., 2004). 7 Page 7 of 13
171 172 The molecular occurrence of B. bovis and B. bigemina found in the present study was higher (1.3 and 4.4 times, respectively) than that found among buffaloes in 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 Thailand (Terkawi et al., 2011). In Brazil, despite a high seroprevalence for Babesia sp. have been found among buffaloes from Rio of Janeiro, only 1% of sampled animals were positive at PCR targeting the studied agents (Côrrea, 2011). On the other hand, molecular surveys carried out among cattle in Brazil have found a molecular prevalence of 90% for B. bovis and B. bigemina (Oliveira-Sequeira et al., 2005). A high correlation was observed between molecular and serological tests used in the present study (kappa index of 0.9). While these findings are similar to that found by Terkawi et al. (20011), they differed significantly from those found by Corrêa (2011). In a nutshell, our results showed that a combination of both molecular and serological techniques is useful tool in for epidemiological investigations with high accuracy in the diagnosis of Babesia infection among buffaloes in Northern Brazil. The slightly higher rate of infection detected in pregnant female is most likely because the physiology of pregnancy and lactation period, which are associated with hormonal and immunological changes, and in a non-specific immunosuppression. The magnitude and timing of immunosupression depend on many factors such as inadequate hygienic and sanitary management, inappropriate feed and housing, and genetic differences (Bonizzi et al., 2003). 5. Conclusion 192 193 194 195 This is the first epidemiological study investigating the occurrence of B. bovis and B. bigemina in water buffalo from Brazil using both molecular and serologic tests. These data provide information about the incidence of B. bovis and B. bigemina infections in water buffaloes and may guide future control programs of the disease. 8 Page 8 of 13
196 197 The present work showed that water buffaloes from state of Pará, Northern Brazil, are exposed to bovine babesiosis agents, although do not show clinical signs of the disease. 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 The lower rate of exposure to Babesia sp. verified among water buffaloes could be explained by the habitat where sampled animals live, where ticks show a lower rate of attachment to animal skin. Acknowledgements - We are grateful to Dr. Rosangela Zacarias Machado by kindly supplied the antigen used. We also thank the Coordination for the Improvement of Higher Level of Education Personnel (CAPES) for financial support. Reference Bock, R., Jackson, L., de Vos, A., Jorgensen, W., 2004. Babesiosis of cattle. Parasitology 129, 247 269. Bonizzi, L., Menandro, M.L., Pasotto, D., Lauzi, S. 2003. Transition Cow: Non-specific Immune Response. Veterinary Research Communications, 27, 137-142. Cepanzo, 1979. Procedures for sampling studies prevalence. Pan American Zoonoses Center, Technical Note 18, Rev. 1, Ramos Mejia, Buenos Aires, p. 35. Corrêa, F.N. Estudo Epidemiológico de Borrelia burgdorferi, Babesia bovis, Babesia bigemina e Anaplasma marginale em Búfalos (Bubalus bubalis) do Estado do Rio de Janeiro. Tese de Doutorado, Universidade Federal Rural do Rio de Janeiro, Seropédica, Rio de Janeiro. 99p., 2011. 217 218 219 220 Ferreri, L. Benitez, D. Dominguez, M. Rodriguez, A. Asenzo, G. Mesplet, M. Florin- Christensen, M. & Schnittger, L. 2008. Water buffalos as carriers of Babesia bovis in Argentina. Animal Biodiversity and Emerging Diseases: Ann. N.Y. Acad. Sci. 1149:149 151. 9 Page 9 of 13
221 222 He, L., Feng, H.H., Zhang, W.J., Zhang, Q.L., Fang, R., Wang, L.X., Tu, P., Zhou, Y.Q., Zhao, J.L., Oosthuizen, M.C. 2012. Occurrence of Theileria and Babesia 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 species in water buffalo (Bubalus babalis, Linnaeus, 1758) in the Hubei province, South China. Vet Parasitol, 186, 490-496. Instituto Brasileiro de Geografia e Estatística, IBGE. 2012. Disponível em: <www.ibge.gov.br > Acesso em: 05 december 2012. McCosker, P.J., 1981. The global importance of babesiosis. In: Ristic, M., Krier, J.P. (Eds.), Babesiosis. Academic Press, New York, pp. 1 24. Muraleedharan, K., Syed Ziauddin, K., Gopalaswamy, K., Muraleedhar, T., Seshadri, S.J. 1984. Some observations on clinical cases of Babesia bovis (BABES, 1888) Starcovici, 1893, in buffaloes (Bubalus bubalis). Ind. Vet. J, 61, 76-79. Oliveira-Sequeira, T.C.G., Oliveira, M.C.de.S., Araújo Jr., J.P., Amarante, A.F.T., Oliveira, H.N., 2005. PCR-based detection of Babesia bovis and Babesia bigemina in their natural host Boophilus microplus and cattle. Int. J. Parasitol. 35, 105 111. Roychoudhury, G. K. and Gautam, O. P. 1979. Experimental studies on the pathogenicity of Babesia bigemina in buffalo calves. Trop Anim Health Prod, 11, 91-93. Terkawi, M.A., Huyen, N.X., Shinuo, C., Inpankaew, T., Maklon, K., Aboulaila, M., Ueno, A., Goo, Y.K., Yokoyama, N., Jittapalapong, S., Xuan, X., Igarashi, I. 2011. Molecular and serological prevalence of Babesia bovis and Babesia bigemina in water buffaloes in the northeast region of Thailand. Vet. Parasitol, 178, 201 207. 242 243 244 245 Legend of tables and figures Figure 1. Serological and molecular detection of Babesia bovis and Babesia bigemina in water buffaloes from north region of Brazil. 10 Page 10 of 13
246 247 Table 1. Summary of the molecular and serological detection of B. bovis and B. bigemina using npcr assay and ELISA. 248 249 250 251 11 Page 11 of 13
251 252 Table 1 253 254 255 256 257 258 259 260 261 Agent npcr a ELISA b (+) (-) Babesia bovis (+) 41 25 16 ( ) 231 87 144 272 112 160 Babesia bigemina (+) 44 27 17 ( ) 228 24 204 272 51 221 a Number of positive and negative buffaloes in npcr assays. b Number of positive and negative buffaloes in both npcr and ELISA assays. 12 Page 12 of 13
261 262 263 264 265 Fig. 1 Percentage of positive water buffaloes (%) 50 40 30 20 10 0 * (p=0.017) ns (p=0.053) * (p=0.033) Babesia bovis * (p=0.045) * (p=0.022) Babesia bigemina ELISA + npcr ELISA npcr * (p=0.047) 13 Page 13 of 13