Ticks and Tick-borne Diseases 5 (2014) 100–104
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Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Original article
Molecular and serological prevalence of Anaplasma marginale in water buffaloes in northern Brazil Jenevaldo Barbosa da Silva a,∗ , Wagner Marcelo Sousa Vinhote b , Carlos Magno Chaves Oliveira b , Marcos Rogério André a , Rosangela Zacarias Machado a , Adivaldo Henrique da Fonseca c , José Diomedes Barbosa b a Laboratório de Imunoparasitologia, Departamento de Patologia Veterinária, Facudade de Ciências Agrárias e Veterinárias FCAV-UNESP, Via de Acesso Prof. Paulo Donato Castellane s/n, 14884-900 Jaboticabal, SP, Brazil b Instituto de Medicina Veterinária, Universidade Federal do Pará, Rodovia BR 316 Km 61, Bairro Saudade, 68740-970 Castanhal, PA, Brazil c Laboratório de Doenc¸as Parasitárias, Departamento de Epidemiologia e Saúde Pública, Universidade Federal Rural de Rio de Janeiro (UFRRJ), Br 465, Km 7, 23890-000, Seropédica, RJ, Brazil
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Article history: Received 25 May 2013 Received in revised form 5 September 2013 Accepted 11 September 2013 Available online 15 November 2013 Keywords: Anaplasmosis Brazil ELISA PCR Water buffaloes
a b s t r a c t Bovine anaplasmosis, caused by Anaplasma marginale, occurs in tropical and subtropical regions throughout the world and is a major constraint on cattle production in many countries. Approximately 60% of the buffalo herds in South America are located in northern Brazil. However, compared with the research on cattle, research on buffaloes has been neglected. Therefore, the present study was conducted to investigate the distribution of A. marginale in water buffaloes in northern Brazil. A total of 500 buffalo blood samples was randomly collected from 16 provinces and was analyzed using both nPCR assay and ELISA techniques. The percentage of animals that were seropositive for A. marginale according to ELISA was 49% (245/500). The main risk factors associated with seroprevalence were the region (p = 0.021; OR = 1.2) and the reproductive status (p = 0.0001; OR = 1.6). Anaplasma marginale DNA was detected in 5.4% (27/500) of the sampled buffaloes. Our data provide information about the incidence of A. marginale infection in water buffaloes and may guide future programs aimed at controlling the disease in the northern region of Brazil. Although these water buffaloes are exposed to A. marginale, a low rate of A. marginale PCR-positive animals was found, which could be explained by the habitat in which the sampled animals live because they exhibited a low rate of attached ticks on their skin. © 2013 Elsevier GmbH. All rights reserved.
Introduction Anaplasma marginale, a tick-borne obligate intracellular ␣ proteobacterium belonging to the order Rickettsiales, family Anaplasmataceae, is the agent of bovine anaplasmosis (Dumler et al., 2001). Anaplasma marginale is the most prevalent tick-borne pathogen of cattle worldwide, with an endemic occurrence in several regions of the Americas, Africa, Asia, and Australia (Kocan et al., 2010). Brazil is a developing agricultural country located in South America, where the development of the livestock industry has been hampered by the high occurrence of tick-borne diseases, particularly bovine anaplasmosis (Vidotto et al., 1998). Water buffaloes are located predominantly in the northern part of the country, where
they meet many important human needs by providing meat, milk, and leather, plowing the land and transporting people and crops (IBGE, 2012). Although water buffaloes are raised with cattle and are potential carriers of Anaplasma, only Corrêa (2011) and Silva et al. (2013) have reported the occurrence of A. marginale among the water buffaloes in Brazil. Therefore, epidemiological surveys of A. marginale infections in water buffaloes are expected to be beneficial in reducing the economic losses of the Brazilian livestock industry. For this reason, we investigated the molecular and serological prevalence of A. marginale infection in water buffaloes in the northern region of Brazil.
Materials and methods ∗ Corresponding author. Tel.: +55 21 2682 29 40. E-mail addresses: [email protected] (J. Barbosa da Silva), marcos [email protected] (M.R. André), [email protected] (R.Z. Machado), [email protected] (A.H. da Fonseca), [email protected] (J.D. Barbosa). 1877-959X/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ttbdis.2013.09.007
Design and population studies A cross-sectional serological and molecular study was conducted from January to December 2011 in buffalo herds in
J. Barbosa da Silva et al. / Ticks and Tick-borne Diseases 5 (2014) 100–104
16 provinces located in the state of Pará in the northeastern region of Brazil. We randomly selected a total of 7 provinces on Marajó Island (Soure, Salvaterra, Muaná, Chaves, Ponta de Pedras, Cachoeira do Arari, and Santa Cruz do Arari) and 9 provinces on the continent (Abaetetuba, Ipixuna do Pará, Marapanim, Moju, Nova Timboteua, Tailândia, Paragominas, Santa Izabel do Pará, and São Caetano de Odivelas). On the continent, the vegetation is predominantly provided by the Amazon rainforest (tropical rainforest). The buffaloes are raised on land and are managed predominantly with beef cattle (Nelore). The buffaloes are vaccinated against brucellosis and foot-and-mouth disease, and they periodically receive endectocidal drugs (ivermectin). Furthermore, these animals are handled in pens using a production system that aims to profit by marketing them. In contrast, large areas of bog and grassland along the floodplains of rivers are found on Marajó Island. In this area, buffaloes are reared in the wetlands. Although they are vaccinated against the same agents as those reared on the continent, endo- and ectoparasite control is rarely used. These animals are reared using an extensive subsistence system. Sampling The number of samples required to assess the prevalence of A. marginale in the water buffaloes in the northern region of Brazil was determined using the formula recommended by the Pan American Zoonosis Center (CEPANZO, 1979) for the study of chronic diseases, as follows: N=
p(100 − p)Z 2 (dp/100)
2
where N is the number of samples; p is the expected prevalence; Z is the confidence level, and d is the error margin. The estimated prevalence of A. marginale-infected buffaloes was 40%. A confidence level of 95.0% and an error margin of 5.0% were established. We estimated that 500 samples should be analyzed. Thus, we randomly selected all of the female water buffaloes on at least 3 farms to provinces, which included 260 buffaloes from Marajó Island and 240 buffaloes from the continent, with 255 belonging to the Murrah breed and 245 to the Mediterranean breed. Regarding their reproductive status, 252 females were pregnant and 248 were not. Whole blood samples were collected from the caudal or jugular veins of individual water buffaloes. To prepare the serum samples, blood samples collected without the anticoagulant EDTA were incubated at room temperature for 1 h and then centrifuged at 1000 × g; then, the sera were collected and stored at −20 ◦ C until use. EDTA-containing blood samples were also stored at −20 ◦ C until they were used for DNA extraction for the PCR assays. Enzyme-linked immunosorbent assay (ELISA) An Anaplasma marginale isolate from a calf in Jaboticabal in the state of São Paulo, Brazil, generously supplied by Professor Rosangela Zacarias Machado (Unesp-Jaboticabal), was used to infect a calf for crude ELISA and IFAT antigen production. For this purpose, a 3-month-old splenectomized calf was inoculated with 200 mL of A. marginale-infected blood (107 infected erythrocytes/mL). The rickettsemia peak (107 A. marginale-infected erythrocytes/mL) was observed 7 days after the experimental infection. After the blood was collected and processed for crude ELISA antigen production (Machado et al., 1997), the experimentally infected animal was treated with oxytetracycline (200 mg/kg). Briefly, 100 l of antigen diluted in 0.05 M carbonate/bicarbonate buffer, pH 9.6, was added to each well of a
101
micro-ELISA plate (Immulon; Dynatech Laboratories Inc., Alexandria, VA), and the protein concentration was adjusted to 10 g/mL. The plates were sealed and incubated overnight at 4 ◦ C. The plates were blocked with 3% ovalbumin in carbonate/bicarbonate buffer for l h at 37 ◦ C in a humid chamber. After 5 washes with PBS-Tween (phosphate-buffered saline, pH 7.2, containing 0.05% Tween 20), 100 l of diluted bovine sera (1:400) in PBS-Tween plus 5% normal rabbit serum was added in duplicate to the ELISA plate. The plates were incubated at 37 ◦ C in a humid chamber for 90 min and then washed 5 times with PBS-Tween. A 100-l aliquot of a 1:10,000 dilution of alkaline phosphatase-conjugated anti-bovine IgG (Sigma Chemical Co.) was added to each well, and the plates were incubated at 37 ◦ C under the same conditions for 90 min. The plates were washed 5 times with PBS-Tween. The appropriate substrate (p-nitrophenyl phosphate) was added, and the plates were sealed and incubated for 40 min at room temperature. Finally, the absorbance at a wavelength of 405 nm was read using a micro-ELISA reader (B.T.-100; Embrabio, São Paulo, Brazil). The cut-off values were calculated using receiver operating characteristic (ROC) analysis of the values for 30 non-Anaplasma-infected water buffaloes’ sera, using MedCalc statistical software (Version 11.4; http://www.medcalc.be) (Terkawi et al., 2011). Thirty sera samples obtained from foals before they suckled colostrum were used as negative controls. A positive reference group consisting of serum samples from 30 buffaloes that tested positive for A. marginale (titers of 1280 by IFAT) were used as positive controls for the serological assays. Semi-nested PCR DNA was extracted from 200 L of each of the 500 EDTA wholeblood samples using the QIAamp DNA Blood Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Initially, the presence of A. marginale DNA was detected using 1.0 l each (20 pmol) of primers targeting part of the MSP-5 gene, namely Amar msp-5 eF (5 GCA TAG CCT CCG CGT CTT TC 3 ) and Amar msp5 eR (5 TCC TCG CCT TGG CCC TCA GA 3 ), previously described by de Echaide et al. (1998) and optimized by Singh et al. (2012). The PCR protocol was at 94 ◦ C for 5 min, followed by 34 repetitive cycles of 94 ◦ C for 1 min, 58 ◦ C for 1 min, and 72 ◦ C for 1 min, followed by a final extension at 72 ◦ C for 10 min (Singh et al., 2012). Each sample of extracted DNA was used as a template in 25L reaction mixtures containing 10× PCR buffer, 1.5 mM MgCl2 , 0.5 mM deoxynucleotide triphosphate (dNTPs) mixture, and 1.0 U of Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA). The amplified products of the first reaction were used for another PCR using the primers Amar msp5 eR (5 TCC TCG CCT TGG CCC TCA GA 3 ) and Amar msp5 iF (5 TAC ACG TGC CCT ACC GAG TTA 3 ) (Singh et al., 2012). The cycling conditions were the same as for the PCR using the Amar msp-5 eF and Amar msp5 eR primers. Ultra-pure sterile water was used as the negative control. To prevent PCR contamination, the DNA extraction, reaction setup, PCR amplification, and electrophoresis were performed in separate rooms. Statistical analysis An individual animal served as the unit of analysis. The serological and molecular results (positive or negative) were the response variables for A. marginale in this study. Crude odds ratios (OR) were calculated for each risk factor. The exposure variables were the locations (island and continent), provinces (the 16 provinces studied), breed (Murrah or Mediterranean), and reproductive status (pregnant or nonpregnant). The confidence intervals of the overall and stratified individual buffalo’s seroprevalence for each variable evaluated were calculated as the proportion of ELISA-positive animals divided by the
J. Barbosa da Silva et al. / Ticks and Tick-borne Diseases 5 (2014) 100–104
total number of buffaloes tested and adjusted for clustering within the herds and provinces. An ANOVA with a Bonferroni adjustment for multiple pairwise comparisons was used to compare the mean prevalence among the 4 variables evaluated in individual buffaloes. The kappa coefficient was calculated to evaluate the agreement among the results of the nPCR assay and the ELISA. The chi-squared test was used to evaluate the significance of the differences (p < 0.05) in the infection rates of animals from different breeds, reproductive status, and locations. The analytical procedures were performed using the R version 2.2.1 statistical package (R Development Core Team, 2005).
A
Percentage of Anaplasma marginale PCR-positive buffaloes
102
10.0
5.0
2.5
0.0
Island
Correlation between the serological and molecular assay results The number of samples that were positive for A. marginale using nPCR or ELISA was 50.4% (252/500; Table 2). A low correlation between serological tests and molecular assay was found in the present study (kappa index of 22.3%). Discussion Few studies have examined the occurrence of A. marginale among buffaloes, which are economically highly important in the northern region of Brazil, particularly on Marajó Island. Although previous studies reported the occurrence of Babesia sp. among buffaloes in China and Thailand (Liu et al., 1997; Terkawi et al., 2011), studies regarding the prevalence of A. marginale are scarce. In Pakistan, an examination of buffaloes’ Giemsa-stained blood smears revealed 22% prevalence for A. marginale (Rajput et al., 2005). In another survey using the complement-fixation test, only one out of 10 buffaloes (10%) sampled in Kenya were seropositive for A. marginale (Kimber et al., 2002). In Brazil, although a high seroprevalence (90%) for A. marginale was found among the buffaloes in Rio de Janeiro, only 1% of 295 sampled animals were positive according to PCR assays (Corrêa, 2011).
Percentage of Anaplasma marginale PCR-positive buffaloes
10.0
Mediterranean Murrah 7.5
ns (p=0.843)
5.0
2.5
0.0
Mediterranean
Murrah
C Percentage of Anaplasma marginale PCR-positive buffaloes
ELISA results indicated that IgG antibodies against A. marginale were found in 49% (245/500) of the buffaloes (Table 1). The percentage of buffaloes seropositive for A. marginale in the island localities (53.5%) was higher (p = 0.021; OR = 1.2) than that of the animals from the continent (44.2%). The seropositivity rate of the Murrah and Mediterranean breeds were not significantly different (p = 0.712; OR = 0.9; Table 1). Pregnant female buffaloes were more likely (p = 0.0001; OR = 1.6) to exhibit antibodies to A. marginale compared with nonpregnant female buffaloes.
Continent
B
Detection of Anaplasma marginale genomic DNA in water buffalo blood samples
IgG antibodies to Anaplasma marginale
Continent
7.5
Results
The prevalence of A. marginale-positive buffaloes according to the PCR assays was 5.4% (27/500). The number of animals found to be positive for A. marginale using the PCR assays was correlated with the sampling area, breed, and reproductive status (Fig. 1). The number of A. marginale PCR-positive buffaloes (6.4%; 16/250) on Marajó Island was significantly higher (p = 0.031) than that found on the continent (4.4%; 11/250; Fig. 1a). There was no difference between the number of A. marginale PCR-positive buffaloes of the Murrah and Mediterranean breeds (p = 0.843) (Fig. 1b). Pregnant female buffaloes were more likely (p = 0.0421) to have A. marginalepositive PCR results compared with nonpregnant females (3.0%; 9/302; Fig. 1c).
Island
*
(p=0.031)
10.0
Pregnant No pregnant 7.5
* (p=0.0421)
5.0
2.5
0.0
Pregnant
No pregnant
Fig. 1. Comparisons of the overall nPCR-based prevalence of Anaplasma marginale infection according to the area (A), breed (B), and reproductive status (C) of 500 female water buffaloes in the northern region of Brazil, 2012.
In the present study, the percentage of buffaloes positive for A. marginale was 43.4% according to ELISA and 5.4% according to PCR. Silva et al. (2013) found a molecular and serological prevalence of 2% and 25%, respectively, among buffaloes from the northern region of Brazil. In contrast, Guedes et al. (2008) found a seroprevalence greater than 90% among cattle sampled in the same region as the present study. According to de la Fuente et al. (2004), buffaloes are more resistant than cattle to infection upon exposure to A. marginale. Moreover, most of the sampled buffaloes were raised on submerged wetlands, thus avoiding tick attachment (Somparn et al., 2004; Terkawi et al., 2011) and precluding the biological transmission of A. marginale. However, it is worth mentioning the role of blood-sucking flies in the transmission of anaplasmosis (de la Fuente et al., 2004). Although no study has highlighted the importance of such flies in the transmission of A. marginale among buffaloes in the northern region of Brazil, we truly believe that those insects participate in the epidemiology of exposure to A. marginale among the sampled animals. Kessler (2001)
J. Barbosa da Silva et al. / Ticks and Tick-borne Diseases 5 (2014) 100–104 Table 1 ELISA-based serological prevalence of A. marginale infection among 500 female water buffaloes in the northern region of Brazil, 2012. Risk factors
N
P (%)
2
OR
p-Value
– 1.40 3.09 1.35 2.50 0.31 0.66
– 1.2 1.3 1.6 1.0 0.9 1.4
– 0.221 0.037 0.021 0.655 0.757 0.042
40.0% 53.3% 36.7% 46.7% 53.3% 36.0% 44.0% 50.0% 35.0%
– 3.34 0.87 0.23 5.21 1.68 1.21 5.76 1.26
– 0.8 1.1 0.9 0.8 1.1 0.9 0.8 1.1
– 0.046 0.235 0.549 0.032 0.234 0.639 0.029 0.654
260 240
53.5% 44.2%
– 5.38
– 1.2
– 0.041
Breed (1) Mediterraneana (2) Murrah
245 255
47.8% 50.2%
– 0.21
– 0.9
– 0.561
Pregnant (1) Positivea (2) Negative
252 248
60.3% 37.5%
– 30.92
– 1.6
– 0.001
Marajó Island (1) Sourcea (2) Salvaterra (3) Muaná (4) Chaves (5) Ponta de Pedra (6) Cachoeira do Arari (7) Santa Cruz do Arari
40 40 45 30 30 30 40
62.5% 52.5% 46.7% 40.0% 60.0% 73.3% 44.4%
Continent (1) Abaetetubaa (2) Ipixuna (3) Marapanim (4) Mojú (5) Nova Timboteua (6) Tailândia (7) Paragominas (8) Santa Izabel (9) São C. de Odivela
30 30 30 30 30 25 25 20 20
Localization (1) Continenta (2) Island
a Reference value. N, number of animals evaluated; P (%), prevalence of seropositivity to A. marginale; 2 = chi-square; OR, Odds ratio.
Table 2 Summary of the molecular and serological detection of A. marginale infection in female water buffaloes using an nPCR assay and ELISA techniques. The results of nPCR were cross-tabulated with those of ELISA. Anaplasma marginale PCRa (+) (−)
ELISAb (+)
(–)
20 225
7 248
a
The frequencies of positive and negative buffaloes in nPCR. The frequencies of positive and negative buffaloes as results of ELISA crosstabulated with nPCR results. b
experimentally demonstrated the mechanical transmission of A. marginale to cattle by stable flies (Stomoxys calcitrans) in Brazil. The prevalence of A. marginale in buffaloes sampled on Marajó Island was higher than that found among buffaloes from the continent. This finding could be explained by the use of tick and fly control associated with the nutritional management of herds on the continent. In that region, buffaloes are treated with avermectin 4–6 times annually and receive mineral and protein supplements during the dry season because they are raised with cattle. In contrast, on Marajó Island, buffaloes are not subjected to ectoparasite control and do not receive nutritional supplements. The slightly higher rate of infection detected in pregnant females compared with nonpregnant females most likely results from the physiology of pregnancy and the lactation period, which are associated with hormonal and immunological changes (Khansari et al., 1990; Mallard et al., 1998) and result in nonspecific immunosuppression. The magnitude and timing of immunosuppression also depends on many others factors, such as (in)appropriate hygienic and sanitary management, (in)appropriate feed and housing and genetic differences (Bonizzi et al., 2003).
103
Although the use of Bos indicus cattle to control cattle ticks and babesiosis has been advocated since 1912 (Francis, 1966), the evidence of the relative susceptibility of B. taurus and B. indicus cattle to infection by A. marginale is contradictory (Jonsson et al., 2012). We did not observe any difference in the susceptibility of Murrah and Mediterranean buffaloes to parasitism by Rhipicephalus (Boophilus) microplus or to infection with A. marginale. Moreover, the significant differences in the prevalence of anaplasmosis among the different provinces suggest the necessity of further studies of the geographic distribution of tick and blood-sucking fly vectors in the surveyed areas. Conclusion This is one of the first epidemiological studies to investigate the occurrence of A. marginale in Brazilian water buffaloes using both molecular and serologic tests. Our results show that even on Marajó Island, where buffaloes are raised separate from cattle and exhibited a low density of ticks, a high percentage of the animals were seropositive for A. marginale, suggesting that in these areas, bloodsucking flies are the main vector responsible for the transmission of the studied agent. Additionally, these data provide information about the incidence of A. marginale among buffaloes in the northern region of Brazil and may guide future anaplasmosis control programs. Conflict of interest statement None of the authors of this work has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of this paper. Acknowledgements We thank the Coordination for the Improvement of Higher Level of Education Personnel (CAPES) foundation and the National Counsel of Technological and Scientific Development (CNPq) for financial support. References Bonizzi, L., Menandro, M.L., Pasotto, D., Lauzi, S., 2003. Transition cow: non-specific immune response. Vet. Res. Comm. 27, 137–142. Corrêa, F.N., 2011. Estudo Epidemiológico de Borrelia burgdorferi, Babesia bovis, Babesia bigemina e Anaplasma marginale em Búfalos (Bubalus bubalis) do Estado do Rio de Janeiro. Universidade Federal Rural do Rio de Janeiro, Seropédica, Rio de Janeiro (Tese de Doutorado), 99 pp. de Echaide, S.T., Knowles, D.P., Mcguire, T.C., Palmer, G.H., Suarez, C.E., Mcelwain, T.F., 1998. Detection of cattle naturally infected with Anaplasma marginale in a region of endemicity by nested PCR and a competitive enzyme linked immunosorbent assay using recombinant major surface protein 5. J. Clin. Microbiol. 36, 777–782. de la Fuente, J., Vicente, J., Höfle, U., Ruiz-Fons, F., Fernández de Mera, I.G., Van Den Bussche, R.A., Kocan, K.M., Gortazar, C., 2004. Anaplasma infection in free-ranging Iberian red deer in the region of Castilla-La Mancha, Spain. Vet. Microbiol. 100, 163–173. Dumler, J.S., Barbet, A.F., Bekker, C.P.J., Dasch, G.A., Palmer, G.H., Ray, S.C., Rikihisa, Y., Rurangirwa, F.R., 2001. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and ‘HGE agent’ as subjective synonyms of Ehrlichia phagocytophila. Int. J. Syst. Evol. Microbiol. 51, 2145–2165. Francis, J., 1966. Resistance of zebu and other cattle to tick infestation and babesiosis with special reference to Australia: an historical review. Brit. Vet. J. 122, 301–307. Guedes-Junior, D.S., Araújo, F.R., Silva, F.J.M., Rangel, C.P., Barbosa-Neto, J.D., Fonseca, A.H., 2008. Frequency of antibodies to Babesia bigemina, B, bovis, Anaplasma marginale, Trypanosoma vivax and Borrelia burgdorferi in cattle from the northeastern region of the state of Pará, Brazil. Rev. Bras. Parasitol. Vet. 17, 105–109. Instituto Brasileiro de Geografia e Estatística (IBGE) (2012). Disponível em: www.ibge.gov.br Acesso em: 05 jun 2011. Jonsson, N.N., Bock, R.E., Jorgensen, W.K., Morton, J.M., Stear, M.J., 2012. Is endemic stability of tick-borne disease in cattle a useful concept? Trends Parasitol. 28, 85–89.
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Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Original article
Molecular and serological prevalence of Anaplasma marginale in water buffaloes in northern Brazil Jenevaldo Barbosa da Silva a,∗ , Wagner Marcelo Sousa Vinhote b , Carlos Magno Chaves Oliveira b , Marcos Rogério André a , Rosangela Zacarias Machado a , Adivaldo Henrique da Fonseca c , José Diomedes Barbosa b a Laboratório de Imunoparasitologia, Departamento de Patologia Veterinária, Facudade de Ciências Agrárias e Veterinárias FCAV-UNESP, Via de Acesso Prof. Paulo Donato Castellane s/n, 14884-900 Jaboticabal, SP, Brazil b Instituto de Medicina Veterinária, Universidade Federal do Pará, Rodovia BR 316 Km 61, Bairro Saudade, 68740-970 Castanhal, PA, Brazil c Laboratório de Doenc¸as Parasitárias, Departamento de Epidemiologia e Saúde Pública, Universidade Federal Rural de Rio de Janeiro (UFRRJ), Br 465, Km 7, 23890-000, Seropédica, RJ, Brazil
a r t i c l e
i n f o
Article history: Received 25 May 2013 Received in revised form 5 September 2013 Accepted 11 September 2013 Available online 15 November 2013 Keywords: Anaplasmosis Brazil ELISA PCR Water buffaloes
a b s t r a c t Bovine anaplasmosis, caused by Anaplasma marginale, occurs in tropical and subtropical regions throughout the world and is a major constraint on cattle production in many countries. Approximately 60% of the buffalo herds in South America are located in northern Brazil. However, compared with the research on cattle, research on buffaloes has been neglected. Therefore, the present study was conducted to investigate the distribution of A. marginale in water buffaloes in northern Brazil. A total of 500 buffalo blood samples was randomly collected from 16 provinces and was analyzed using both nPCR assay and ELISA techniques. The percentage of animals that were seropositive for A. marginale according to ELISA was 49% (245/500). The main risk factors associated with seroprevalence were the region (p = 0.021; OR = 1.2) and the reproductive status (p = 0.0001; OR = 1.6). Anaplasma marginale DNA was detected in 5.4% (27/500) of the sampled buffaloes. Our data provide information about the incidence of A. marginale infection in water buffaloes and may guide future programs aimed at controlling the disease in the northern region of Brazil. Although these water buffaloes are exposed to A. marginale, a low rate of A. marginale PCR-positive animals was found, which could be explained by the habitat in which the sampled animals live because they exhibited a low rate of attached ticks on their skin. © 2013 Elsevier GmbH. All rights reserved.
Introduction Anaplasma marginale, a tick-borne obligate intracellular ␣ proteobacterium belonging to the order Rickettsiales, family Anaplasmataceae, is the agent of bovine anaplasmosis (Dumler et al., 2001). Anaplasma marginale is the most prevalent tick-borne pathogen of cattle worldwide, with an endemic occurrence in several regions of the Americas, Africa, Asia, and Australia (Kocan et al., 2010). Brazil is a developing agricultural country located in South America, where the development of the livestock industry has been hampered by the high occurrence of tick-borne diseases, particularly bovine anaplasmosis (Vidotto et al., 1998). Water buffaloes are located predominantly in the northern part of the country, where
they meet many important human needs by providing meat, milk, and leather, plowing the land and transporting people and crops (IBGE, 2012). Although water buffaloes are raised with cattle and are potential carriers of Anaplasma, only Corrêa (2011) and Silva et al. (2013) have reported the occurrence of A. marginale among the water buffaloes in Brazil. Therefore, epidemiological surveys of A. marginale infections in water buffaloes are expected to be beneficial in reducing the economic losses of the Brazilian livestock industry. For this reason, we investigated the molecular and serological prevalence of A. marginale infection in water buffaloes in the northern region of Brazil.
Materials and methods ∗ Corresponding author. Tel.: +55 21 2682 29 40. E-mail addresses: [email protected] (J. Barbosa da Silva), marcos [email protected] (M.R. André), [email protected] (R.Z. Machado), [email protected] (A.H. da Fonseca), [email protected] (J.D. Barbosa). 1877-959X/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ttbdis.2013.09.007
Design and population studies A cross-sectional serological and molecular study was conducted from January to December 2011 in buffalo herds in
J. Barbosa da Silva et al. / Ticks and Tick-borne Diseases 5 (2014) 100–104
16 provinces located in the state of Pará in the northeastern region of Brazil. We randomly selected a total of 7 provinces on Marajó Island (Soure, Salvaterra, Muaná, Chaves, Ponta de Pedras, Cachoeira do Arari, and Santa Cruz do Arari) and 9 provinces on the continent (Abaetetuba, Ipixuna do Pará, Marapanim, Moju, Nova Timboteua, Tailândia, Paragominas, Santa Izabel do Pará, and São Caetano de Odivelas). On the continent, the vegetation is predominantly provided by the Amazon rainforest (tropical rainforest). The buffaloes are raised on land and are managed predominantly with beef cattle (Nelore). The buffaloes are vaccinated against brucellosis and foot-and-mouth disease, and they periodically receive endectocidal drugs (ivermectin). Furthermore, these animals are handled in pens using a production system that aims to profit by marketing them. In contrast, large areas of bog and grassland along the floodplains of rivers are found on Marajó Island. In this area, buffaloes are reared in the wetlands. Although they are vaccinated against the same agents as those reared on the continent, endo- and ectoparasite control is rarely used. These animals are reared using an extensive subsistence system. Sampling The number of samples required to assess the prevalence of A. marginale in the water buffaloes in the northern region of Brazil was determined using the formula recommended by the Pan American Zoonosis Center (CEPANZO, 1979) for the study of chronic diseases, as follows: N=
p(100 − p)Z 2 (dp/100)
2
where N is the number of samples; p is the expected prevalence; Z is the confidence level, and d is the error margin. The estimated prevalence of A. marginale-infected buffaloes was 40%. A confidence level of 95.0% and an error margin of 5.0% were established. We estimated that 500 samples should be analyzed. Thus, we randomly selected all of the female water buffaloes on at least 3 farms to provinces, which included 260 buffaloes from Marajó Island and 240 buffaloes from the continent, with 255 belonging to the Murrah breed and 245 to the Mediterranean breed. Regarding their reproductive status, 252 females were pregnant and 248 were not. Whole blood samples were collected from the caudal or jugular veins of individual water buffaloes. To prepare the serum samples, blood samples collected without the anticoagulant EDTA were incubated at room temperature for 1 h and then centrifuged at 1000 × g; then, the sera were collected and stored at −20 ◦ C until use. EDTA-containing blood samples were also stored at −20 ◦ C until they were used for DNA extraction for the PCR assays. Enzyme-linked immunosorbent assay (ELISA) An Anaplasma marginale isolate from a calf in Jaboticabal in the state of São Paulo, Brazil, generously supplied by Professor Rosangela Zacarias Machado (Unesp-Jaboticabal), was used to infect a calf for crude ELISA and IFAT antigen production. For this purpose, a 3-month-old splenectomized calf was inoculated with 200 mL of A. marginale-infected blood (107 infected erythrocytes/mL). The rickettsemia peak (107 A. marginale-infected erythrocytes/mL) was observed 7 days after the experimental infection. After the blood was collected and processed for crude ELISA antigen production (Machado et al., 1997), the experimentally infected animal was treated with oxytetracycline (200 mg/kg). Briefly, 100 l of antigen diluted in 0.05 M carbonate/bicarbonate buffer, pH 9.6, was added to each well of a
101
micro-ELISA plate (Immulon; Dynatech Laboratories Inc., Alexandria, VA), and the protein concentration was adjusted to 10 g/mL. The plates were sealed and incubated overnight at 4 ◦ C. The plates were blocked with 3% ovalbumin in carbonate/bicarbonate buffer for l h at 37 ◦ C in a humid chamber. After 5 washes with PBS-Tween (phosphate-buffered saline, pH 7.2, containing 0.05% Tween 20), 100 l of diluted bovine sera (1:400) in PBS-Tween plus 5% normal rabbit serum was added in duplicate to the ELISA plate. The plates were incubated at 37 ◦ C in a humid chamber for 90 min and then washed 5 times with PBS-Tween. A 100-l aliquot of a 1:10,000 dilution of alkaline phosphatase-conjugated anti-bovine IgG (Sigma Chemical Co.) was added to each well, and the plates were incubated at 37 ◦ C under the same conditions for 90 min. The plates were washed 5 times with PBS-Tween. The appropriate substrate (p-nitrophenyl phosphate) was added, and the plates were sealed and incubated for 40 min at room temperature. Finally, the absorbance at a wavelength of 405 nm was read using a micro-ELISA reader (B.T.-100; Embrabio, São Paulo, Brazil). The cut-off values were calculated using receiver operating characteristic (ROC) analysis of the values for 30 non-Anaplasma-infected water buffaloes’ sera, using MedCalc statistical software (Version 11.4; http://www.medcalc.be) (Terkawi et al., 2011). Thirty sera samples obtained from foals before they suckled colostrum were used as negative controls. A positive reference group consisting of serum samples from 30 buffaloes that tested positive for A. marginale (titers of 1280 by IFAT) were used as positive controls for the serological assays. Semi-nested PCR DNA was extracted from 200 L of each of the 500 EDTA wholeblood samples using the QIAamp DNA Blood Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Initially, the presence of A. marginale DNA was detected using 1.0 l each (20 pmol) of primers targeting part of the MSP-5 gene, namely Amar msp-5 eF (5 GCA TAG CCT CCG CGT CTT TC 3 ) and Amar msp5 eR (5 TCC TCG CCT TGG CCC TCA GA 3 ), previously described by de Echaide et al. (1998) and optimized by Singh et al. (2012). The PCR protocol was at 94 ◦ C for 5 min, followed by 34 repetitive cycles of 94 ◦ C for 1 min, 58 ◦ C for 1 min, and 72 ◦ C for 1 min, followed by a final extension at 72 ◦ C for 10 min (Singh et al., 2012). Each sample of extracted DNA was used as a template in 25L reaction mixtures containing 10× PCR buffer, 1.5 mM MgCl2 , 0.5 mM deoxynucleotide triphosphate (dNTPs) mixture, and 1.0 U of Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA). The amplified products of the first reaction were used for another PCR using the primers Amar msp5 eR (5 TCC TCG CCT TGG CCC TCA GA 3 ) and Amar msp5 iF (5 TAC ACG TGC CCT ACC GAG TTA 3 ) (Singh et al., 2012). The cycling conditions were the same as for the PCR using the Amar msp-5 eF and Amar msp5 eR primers. Ultra-pure sterile water was used as the negative control. To prevent PCR contamination, the DNA extraction, reaction setup, PCR amplification, and electrophoresis were performed in separate rooms. Statistical analysis An individual animal served as the unit of analysis. The serological and molecular results (positive or negative) were the response variables for A. marginale in this study. Crude odds ratios (OR) were calculated for each risk factor. The exposure variables were the locations (island and continent), provinces (the 16 provinces studied), breed (Murrah or Mediterranean), and reproductive status (pregnant or nonpregnant). The confidence intervals of the overall and stratified individual buffalo’s seroprevalence for each variable evaluated were calculated as the proportion of ELISA-positive animals divided by the
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total number of buffaloes tested and adjusted for clustering within the herds and provinces. An ANOVA with a Bonferroni adjustment for multiple pairwise comparisons was used to compare the mean prevalence among the 4 variables evaluated in individual buffaloes. The kappa coefficient was calculated to evaluate the agreement among the results of the nPCR assay and the ELISA. The chi-squared test was used to evaluate the significance of the differences (p < 0.05) in the infection rates of animals from different breeds, reproductive status, and locations. The analytical procedures were performed using the R version 2.2.1 statistical package (R Development Core Team, 2005).
A
Percentage of Anaplasma marginale PCR-positive buffaloes
102
10.0
5.0
2.5
0.0
Island
Correlation between the serological and molecular assay results The number of samples that were positive for A. marginale using nPCR or ELISA was 50.4% (252/500; Table 2). A low correlation between serological tests and molecular assay was found in the present study (kappa index of 22.3%). Discussion Few studies have examined the occurrence of A. marginale among buffaloes, which are economically highly important in the northern region of Brazil, particularly on Marajó Island. Although previous studies reported the occurrence of Babesia sp. among buffaloes in China and Thailand (Liu et al., 1997; Terkawi et al., 2011), studies regarding the prevalence of A. marginale are scarce. In Pakistan, an examination of buffaloes’ Giemsa-stained blood smears revealed 22% prevalence for A. marginale (Rajput et al., 2005). In another survey using the complement-fixation test, only one out of 10 buffaloes (10%) sampled in Kenya were seropositive for A. marginale (Kimber et al., 2002). In Brazil, although a high seroprevalence (90%) for A. marginale was found among the buffaloes in Rio de Janeiro, only 1% of 295 sampled animals were positive according to PCR assays (Corrêa, 2011).
Percentage of Anaplasma marginale PCR-positive buffaloes
10.0
Mediterranean Murrah 7.5
ns (p=0.843)
5.0
2.5
0.0
Mediterranean
Murrah
C Percentage of Anaplasma marginale PCR-positive buffaloes
ELISA results indicated that IgG antibodies against A. marginale were found in 49% (245/500) of the buffaloes (Table 1). The percentage of buffaloes seropositive for A. marginale in the island localities (53.5%) was higher (p = 0.021; OR = 1.2) than that of the animals from the continent (44.2%). The seropositivity rate of the Murrah and Mediterranean breeds were not significantly different (p = 0.712; OR = 0.9; Table 1). Pregnant female buffaloes were more likely (p = 0.0001; OR = 1.6) to exhibit antibodies to A. marginale compared with nonpregnant female buffaloes.
Continent
B
Detection of Anaplasma marginale genomic DNA in water buffalo blood samples
IgG antibodies to Anaplasma marginale
Continent
7.5
Results
The prevalence of A. marginale-positive buffaloes according to the PCR assays was 5.4% (27/500). The number of animals found to be positive for A. marginale using the PCR assays was correlated with the sampling area, breed, and reproductive status (Fig. 1). The number of A. marginale PCR-positive buffaloes (6.4%; 16/250) on Marajó Island was significantly higher (p = 0.031) than that found on the continent (4.4%; 11/250; Fig. 1a). There was no difference between the number of A. marginale PCR-positive buffaloes of the Murrah and Mediterranean breeds (p = 0.843) (Fig. 1b). Pregnant female buffaloes were more likely (p = 0.0421) to have A. marginalepositive PCR results compared with nonpregnant females (3.0%; 9/302; Fig. 1c).
Island
*
(p=0.031)
10.0
Pregnant No pregnant 7.5
* (p=0.0421)
5.0
2.5
0.0
Pregnant
No pregnant
Fig. 1. Comparisons of the overall nPCR-based prevalence of Anaplasma marginale infection according to the area (A), breed (B), and reproductive status (C) of 500 female water buffaloes in the northern region of Brazil, 2012.
In the present study, the percentage of buffaloes positive for A. marginale was 43.4% according to ELISA and 5.4% according to PCR. Silva et al. (2013) found a molecular and serological prevalence of 2% and 25%, respectively, among buffaloes from the northern region of Brazil. In contrast, Guedes et al. (2008) found a seroprevalence greater than 90% among cattle sampled in the same region as the present study. According to de la Fuente et al. (2004), buffaloes are more resistant than cattle to infection upon exposure to A. marginale. Moreover, most of the sampled buffaloes were raised on submerged wetlands, thus avoiding tick attachment (Somparn et al., 2004; Terkawi et al., 2011) and precluding the biological transmission of A. marginale. However, it is worth mentioning the role of blood-sucking flies in the transmission of anaplasmosis (de la Fuente et al., 2004). Although no study has highlighted the importance of such flies in the transmission of A. marginale among buffaloes in the northern region of Brazil, we truly believe that those insects participate in the epidemiology of exposure to A. marginale among the sampled animals. Kessler (2001)
J. Barbosa da Silva et al. / Ticks and Tick-borne Diseases 5 (2014) 100–104 Table 1 ELISA-based serological prevalence of A. marginale infection among 500 female water buffaloes in the northern region of Brazil, 2012. Risk factors
N
P (%)
2
OR
p-Value
– 1.40 3.09 1.35 2.50 0.31 0.66
– 1.2 1.3 1.6 1.0 0.9 1.4
– 0.221 0.037 0.021 0.655 0.757 0.042
40.0% 53.3% 36.7% 46.7% 53.3% 36.0% 44.0% 50.0% 35.0%
– 3.34 0.87 0.23 5.21 1.68 1.21 5.76 1.26
– 0.8 1.1 0.9 0.8 1.1 0.9 0.8 1.1
– 0.046 0.235 0.549 0.032 0.234 0.639 0.029 0.654
260 240
53.5% 44.2%
– 5.38
– 1.2
– 0.041
Breed (1) Mediterraneana (2) Murrah
245 255
47.8% 50.2%
– 0.21
– 0.9
– 0.561
Pregnant (1) Positivea (2) Negative
252 248
60.3% 37.5%
– 30.92
– 1.6
– 0.001
Marajó Island (1) Sourcea (2) Salvaterra (3) Muaná (4) Chaves (5) Ponta de Pedra (6) Cachoeira do Arari (7) Santa Cruz do Arari
40 40 45 30 30 30 40
62.5% 52.5% 46.7% 40.0% 60.0% 73.3% 44.4%
Continent (1) Abaetetubaa (2) Ipixuna (3) Marapanim (4) Mojú (5) Nova Timboteua (6) Tailândia (7) Paragominas (8) Santa Izabel (9) São C. de Odivela
30 30 30 30 30 25 25 20 20
Localization (1) Continenta (2) Island
a Reference value. N, number of animals evaluated; P (%), prevalence of seropositivity to A. marginale; 2 = chi-square; OR, Odds ratio.
Table 2 Summary of the molecular and serological detection of A. marginale infection in female water buffaloes using an nPCR assay and ELISA techniques. The results of nPCR were cross-tabulated with those of ELISA. Anaplasma marginale PCRa (+) (−)
ELISAb (+)
(–)
20 225
7 248
a
The frequencies of positive and negative buffaloes in nPCR. The frequencies of positive and negative buffaloes as results of ELISA crosstabulated with nPCR results. b
experimentally demonstrated the mechanical transmission of A. marginale to cattle by stable flies (Stomoxys calcitrans) in Brazil. The prevalence of A. marginale in buffaloes sampled on Marajó Island was higher than that found among buffaloes from the continent. This finding could be explained by the use of tick and fly control associated with the nutritional management of herds on the continent. In that region, buffaloes are treated with avermectin 4–6 times annually and receive mineral and protein supplements during the dry season because they are raised with cattle. In contrast, on Marajó Island, buffaloes are not subjected to ectoparasite control and do not receive nutritional supplements. The slightly higher rate of infection detected in pregnant females compared with nonpregnant females most likely results from the physiology of pregnancy and the lactation period, which are associated with hormonal and immunological changes (Khansari et al., 1990; Mallard et al., 1998) and result in nonspecific immunosuppression. The magnitude and timing of immunosuppression also depends on many others factors, such as (in)appropriate hygienic and sanitary management, (in)appropriate feed and housing and genetic differences (Bonizzi et al., 2003).
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Although the use of Bos indicus cattle to control cattle ticks and babesiosis has been advocated since 1912 (Francis, 1966), the evidence of the relative susceptibility of B. taurus and B. indicus cattle to infection by A. marginale is contradictory (Jonsson et al., 2012). We did not observe any difference in the susceptibility of Murrah and Mediterranean buffaloes to parasitism by Rhipicephalus (Boophilus) microplus or to infection with A. marginale. Moreover, the significant differences in the prevalence of anaplasmosis among the different provinces suggest the necessity of further studies of the geographic distribution of tick and blood-sucking fly vectors in the surveyed areas. Conclusion This is one of the first epidemiological studies to investigate the occurrence of A. marginale in Brazilian water buffaloes using both molecular and serologic tests. Our results show that even on Marajó Island, where buffaloes are raised separate from cattle and exhibited a low density of ticks, a high percentage of the animals were seropositive for A. marginale, suggesting that in these areas, bloodsucking flies are the main vector responsible for the transmission of the studied agent. Additionally, these data provide information about the incidence of A. marginale among buffaloes in the northern region of Brazil and may guide future anaplasmosis control programs. Conflict of interest statement None of the authors of this work has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of this paper. Acknowledgements We thank the Coordination for the Improvement of Higher Level of Education Personnel (CAPES) foundation and the National Counsel of Technological and Scientific Development (CNPq) for financial support. References Bonizzi, L., Menandro, M.L., Pasotto, D., Lauzi, S., 2003. Transition cow: non-specific immune response. Vet. Res. Comm. 27, 137–142. Corrêa, F.N., 2011. Estudo Epidemiológico de Borrelia burgdorferi, Babesia bovis, Babesia bigemina e Anaplasma marginale em Búfalos (Bubalus bubalis) do Estado do Rio de Janeiro. Universidade Federal Rural do Rio de Janeiro, Seropédica, Rio de Janeiro (Tese de Doutorado), 99 pp. de Echaide, S.T., Knowles, D.P., Mcguire, T.C., Palmer, G.H., Suarez, C.E., Mcelwain, T.F., 1998. Detection of cattle naturally infected with Anaplasma marginale in a region of endemicity by nested PCR and a competitive enzyme linked immunosorbent assay using recombinant major surface protein 5. J. Clin. Microbiol. 36, 777–782. de la Fuente, J., Vicente, J., Höfle, U., Ruiz-Fons, F., Fernández de Mera, I.G., Van Den Bussche, R.A., Kocan, K.M., Gortazar, C., 2004. Anaplasma infection in free-ranging Iberian red deer in the region of Castilla-La Mancha, Spain. Vet. Microbiol. 100, 163–173. Dumler, J.S., Barbet, A.F., Bekker, C.P.J., Dasch, G.A., Palmer, G.H., Ray, S.C., Rikihisa, Y., Rurangirwa, F.R., 2001. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and ‘HGE agent’ as subjective synonyms of Ehrlichia phagocytophila. Int. J. Syst. Evol. Microbiol. 51, 2145–2165. Francis, J., 1966. Resistance of zebu and other cattle to tick infestation and babesiosis with special reference to Australia: an historical review. Brit. Vet. J. 122, 301–307. Guedes-Junior, D.S., Araújo, F.R., Silva, F.J.M., Rangel, C.P., Barbosa-Neto, J.D., Fonseca, A.H., 2008. Frequency of antibodies to Babesia bigemina, B, bovis, Anaplasma marginale, Trypanosoma vivax and Borrelia burgdorferi in cattle from the northeastern region of the state of Pará, Brazil. Rev. Bras. Parasitol. Vet. 17, 105–109. Instituto Brasileiro de Geografia e Estatística (IBGE) (2012). Disponível em: www.ibge.gov.br Acesso em: 05 jun 2011. Jonsson, N.N., Bock, R.E., Jorgensen, W.K., Morton, J.M., Stear, M.J., 2012. Is endemic stability of tick-borne disease in cattle a useful concept? Trends Parasitol. 28, 85–89.
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