Babesia bovis infection in cattle in the southwestern Brazilian Amazon

Ticks and Tick-borne Diseases 4 (2013) 78–82

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Original article

Babesia bovis infection in cattle in the southwestern Brazilian Amazon Luciana G. Brito a,∗ , Rodrigo B. Rocha a , Fábio da S. Barbieri a , Elisana S. Ribeiro a , Fabiano B. Vendrami b , Gislaine C.R. Souza a , Rodrigo Giglioti c , Luciana C.A. Regitano d , Thaís O.R.S. Falcoski e , Polyana C. Tizioto f , Márcia C.S. Oliveira d a

Embrapa Rondônia, Rodovia BR 364, km 5,5, Caixa Postal 127, 76815-800, Porto Velho, RO, Brazil Agência de Defesa Sanitária Agrosilvopastoril do Estado de Rondônia, Porto Velho, RO, Brazil Universidade Estadual Júlio de Mesquita Filho, Jaboticabal, SP, Brazil d Embrapa Pecuária Sudeste, São Carlos, SP, Brazil e Universidade Estadual Júlio de Mesquita Filho, Araraquara, SP, Brazil f Universidade Federal de São Carlos, São Carlos, SP, Brazil b c

a r t i c l e

i n f o

Article history: Received 11 June 2012 Received in revised form 3 August 2012 Accepted 6 August 2012 Keywords: Babesia bovis Epidemiology Cattle Amazonia Brazil

a b s t r a c t The present study provides the first epidemiological data on infection with Babesia bovis in cattle raised in the southwestern Brazilian Amazon. Blood clot samples were filtered through nylon cloth before being submitted to DNA extraction. PCR and nested-PCR were applied to assess the frequency of infection with B. bovis in calves with ages from 4 to 12 months bred in 4 microregions each in the states of Rondônia and Acre. After the DNA was extracted from the samples, the infection in cattle was investigated by amplification of the “rap1” gene from B. bovis. The DNA amplification results revealed a frequency of infection with B. bovis of 95.1% (272/286) in the samples from Rondônia and 96.1% (195/203) in those from Acre. The high frequency of B. bovis infection in the animals with ages from 4 to 12 months indicates a situation of enzootic stability in the regions studied. The infection rates are comparable to those detected by immunodiagnostic techniques in other endemic regions of Brazil. © 2012 Elsevier GmbH. All rights reserved.

Introduction Babesia bovis and B. bigemina are the causative agents of bovine babesiosis in Latin America, where the only known vector is the one-host tick Rhipicephalus microplus (Guglielmone, 1995). Cattle suffering from babesiosis show massive destruction of erythrocytes because of the multiplication of hemoparasites inside these cells (Bock et al., 2004). The cattle tick, R. microplus, is widely distributed in the southwestern Brazilian Amazon region, infesting cattle throughout the year (Brito et al., 2011). The high infestation rates are mainly related to the extremely favorable climate conditions for development of R. microplus. The equatorial climate predominant in this region of the Amazon is characterized by an average annual rainfall of 2500 mm and a mean annual temperature of 24 ◦ C, with well defined dry and rainy seasons (Fisch et al., 1998). It is known that calves are protected against the various forms of babesiosis by non-specific immunity until around 7 months of age (Guglielmone, 1992; Brown et al., 2006) and that infection during this period induces long-term immunity, while primary infection of adult cattle can produce serious manifestations of

∗ Corresponding author. Tel.: +55 69 3901 2544; fax: +55 69 3222-0409. E-mail address: [email protected] (L.G. Brito). 1877-959X/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ttbdis.2012.08.001

the disease and epidemic characteristics (Mahoney et al., 1973). Therefore, detection of the proportion of calves infected with these 2 babesiosis agents can be used to determine the probability of outbreaks in a region (FAO, 1984). Techniques involving direct microscopic examination of blood smears are still important to diagnose acute babesiosis, but the low sensitivity of these methods does not permit their use in epidemiological studies, where it is necessary to identify asymptomatic carrier animals (Almeria et al., 2001). Serological methods have been used extensively in field epidemiological studies. However, the occurrence of cross-reactions between B. bovis and B. bigemina is a disadvantage (Passos et al., 1998), as is the lack of discrimination between prior exposure and current infection (Wagner et al., 1992). Nowadays, techniques based on polymerase chain reaction (PCR) are increasingly being used due to the high sensitivity and specificity of this method of detecting infection with hemoparasites, both in the vertebrate host and the tick (Figueroa et al., 1992; Smeenk et al., 2000; Almeria et al., 2001; Birkenheuer et al., 2003; Miyama et al., 2005; Oyamada et al., 2005; Oliveira et al., 2005, 2008; Oliveira-Sequeira et al., 2005; Guerrero et al., 2007; Heim et al., 2007). Previous serological studies have suggested the occurrence of B. bovis infections in the northeastern Brazilian Amazon (Trindade et al., 2010; Guedes Junior et al., 2008). However, the incidence and distribution of B. bovis infections among the 13.2 million cattle population currently in the southwestern Brazilian Amazon, the location of the states

L.G. Brito et al. / Ticks and Tick-borne Diseases 4 (2013) 78–82

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of Rondônia and Acre, remains unknown. Therefore, systematic studies to determine the magnitude of babesiosis infections are needed for development of control strategies in this area. For this purpose, a molecular study for diagnosis of B. bovis was carried out in Rondônia and Acre states. The protocols used to extract DNA from cattle hemoparasites involved collecting fresh blood with an anticoagulant. As an alternative, we tested a different extraction method in an attempt to establish a standard and easily-to-perform protocol to extract DNA from clotted blood, taking advantage of the collections made by animal health authorities in programs to monitor animal health in Rondônia and Acre. In these programs, the health authorities use bovine serum for antibody research, and the blood clots are considered as residue of the samples collected. This paper also presents the first epidemiological data on infection by B. bovis in naturally infected cattle exposed to ticks in Brazil’s southwestern Amazon region, where cattle ranching activity has been expanding in recent years.

at −30 ◦ C until use. The blood clots were cut into fragments with a sterile scalpel to form aliquots, according to the method proposed by Tas (1990). These fragments were placed on circular pieces of nylon tissue measuring 10 cm in diameter, of 2 types, one with mesh of 200 ␮m (treatment 1, T1) and the other one with mesh of 1200 ␮m (treatment 2, T2), according to the method described by Garg et al. (1996). The tissues (one per sample) were affixed to 50 ml glass test tubes, attached with a rubber band and centrifuged at 7000 rpm for 15 min. The DNA was extracted from the centrifuged blood clot samples and also from the total blood samples (treatment 3, T3) using the GFXTM Genomic Blood DNA Purification kit (GE Healthcare), according to the manufacturer’s recommendations. The same volume, 300 ␮l, of filtered blood clot and total blood samples was used for DNA extractions. The DNA samples were suspended in 100 ␮l of TE buffer (TRIS-HCl10 mM, EDTA 1 mM, pH 7.4) and stored in a freezer at −80 ◦ C. DNA and protein concentrations were determined using a spectrophotometer (Nanodrop Technologies Inc., Wilmington, DE, USA).

Materials and methods

PCR assays

Determination of the sample size

The PCR and nested-PCR (n-PCR) techniques were used for the amplification of B. bovis DNA using the primer sequences described by Figueroa et al. (1993). Only PCR-negative blood samples were submitted to n-PCR. The 25-␮l PCR reaction mixture consisted of 5 ␮l of the template DNA, 12.5 ml of Ampliquon Taq DNA Polymerase Master Mix Red (Denmark) containing 4.0 mM MgCl2 , 150 mM, Tris–HCl pH 8.5, 40 mM (NH4 )2 SO4 , 0.2% Tween 20, 0.4 mM dNTPs, 0.05 units/␮l taq DNA polymerase; 5.5 ␮l of distilled water, and 1.0 ␮l (1.0 mM) of each specific primer (BoF: 5 -CAC GAG GAA GGA ACT ACC GAT GTT GA-3 and BoR: 5 -CCA AGG AGC TTC AAC GTA CGA GGT CA-3 ). The same buffer at the same concentrations and 2 ␮l of the previously amplified products were used for n-PCR using de primers BoFN 5 TCA ACA AGG TAC TCT ATA TGG CTA CC-3 and BoRN 5 -CTA CCG AGC AGA ACC TTC TTC ACC AT-3 . Samples of B. bovis from clinically affected calves raised at the Embrapa Research Center in Porto Velho were used as a positive control for the PCR. A negative control was used in each battery of 20 reactions, where the genomic DNA was replaced by an equal volume of ultra-pure water. The amplification products were submitted to electrophoresis in 1.5% agarose gel, stained with ethidium bromide, visualized under UV light, and photographed with the L-Pix Image software (Loccus Biotechnology). The size of the amplification products was estimated by the inclusion of a base-pair standard (GeneRulerTM 100 bp DNA Ladder). Samples that presented bands of about 350 (PCR) and 290 (n-PCR) bp were considered positive for B. bovis. For confirmation that the fragments visualized by agarose gel electrophoresis were really from B. bovis, 2 samples were randomly selected and sequenced in an ABI 3100 automated DNA sequencer (Applied Biosystems) as an additional control and were confirmed to correspond to the GenBank accession AF030061 by the maximum composite likelihood model (Tamura et al., 2004). A nucleotide sequence corresponding to the rap1 gene from B. bovis obtained from the extraction of DNA from a clotted blood sample from the Cacoal microregion was deposited in GenBank, receiving the access code JX090146.

The number of samples to assess the prevalence of B. bovis in the southeastern Brazilian Amazon region was determined using the formula recommended by the Pan American Zoonosis Center (CEPANZO, 1979) for the study of chronic diseases: n=

p(100 − p)Z 2 (dp/100)

2

where n, number of samples; p, expected prevalence; Z, confidence level, and d, error margin. Based on an estimated prevalence of 90% of samples positive for B. bovis, ascertained in a pilot study with 100 DNA samples extracted from blood clots, where a confidence level of 95.0% and an error margin of 10.0% were established, we determined that 45 samples from each microregion should be analyzed. Animals and sample collection A total of 489 blood samples was collected from cross-bred dairy cattle (B. taurus × B. indicus) with ages ranging from 4 to 12 months with a vacuum system without the addition of an anticoagulant. These samples were collected by the official veterinary service for monitoring bovine diseases in the Amazon region. The clots were separated from the blood serum and sent to the Molecular Biology Laboratory at Embrapa Rondônia. The clotted calf blood samples were chosen at random in 4 microregions in the states of Rondônia (Alvorada do Oeste, Cacoal, Guajará-Mirim, and Ji-Paraná) and Acre (Cruzeiro do Sul, Rio Branco, Tarauacá, and Brasiléia). Of this total, 286 samples came from Rondônia and 203 from Acre. All the blood clot samples were kept at −30 ◦ C until the DNA extraction process. The municipalities making up the microregions in Rondônia and Acre are those defined by the Brazilian Institute of Geography and Statistics (IBGE, 2006). Preliminary assays

Estimated sensitivity of PCR and n-PCR To compare DNA quality from whole blood and blood clot samples, we used whole blood from 15 dairy calves reared in the Embrapa Research Center in Porto Velho, Rondônia. Five blood samples of 10 ml were aseptically withdrawn using EDTA as an anticoagulant, and 10 samples were collected without anticoagulant. All the samples were left still at room temperature for 20 min and then were maintained at 4 ◦ C for up to 2 days or maintained

PCR and n-PCR sensitivities were estimated considering a control sample with a mean of 6 × 106 erythrocytes/␮l of blood and an estimated parasitemia of 0.0017%, equivalent to 1 × 104 erythrocytes parasitized by B. bovis per microliter of blood. The control sample was diluted 15 times by 10-fold dilutions and submitted to amplification. Sensitivity was calculated according to

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the number of parasitized erythrocytes present in the last dilution in which it was possible to identify the diagnostic bands. Statistical analysis The mean, standard deviation, and intra-test coefficient of variation were calculated for all the DNA quantification results. Student’s t-test for paired data was used at 1% significance. The efficiency of the extraction methods was evaluated by plane dispersion to obtain the linear regression coefficient and correlation coefficient between the methods analyzed. The Chi-square and Fisher’s exact tests were used to compare the rates of infection with B. bovis between the states of Rondônia and Acre, considering their microregions. These analyses were performed by using the FREQ procedure of the SAS statistical program (SAS, 2002–2003).

DNA isolation The technique used to obtain DNA from blood clots shows that intraerythrocytic parasites are concentrated when a blood clot is formed, and similar DNA yields from equal volumes of blood and clots indicate that the extraction procedure from clots is not as efficient as from blood. However, the obtained material sufficed for the objectives of this study. The values observed for the DNA A260/A280 ratio indicate that the DNA samples extracted from the clotted blood samples had satisfactory purity levels in comparison with those extracted from whole blood. The concentrations obtained were similar in the 3 treatments studied, not differing according to Student’s t-test in comparison with the control method from whole blood. The coefficients of variation were also acceptable for routine laboratory work, according to the quality control standards for laboratory assays (Tibúrcio, 1995). Table 1 presents the DNA 260 nm/280 nm absorbance ratios obtained from the samples of total and clotted blood from the spectrophotometric analyses. As can be observed, the DNA samples were highly pure according to all the methods tested, with A260/280 = 1.23–1.90. Therefore, we chose to use protocol T2 for the DNA extraction from blood clots used in the present study. The sequences of the rap1 protein of B. bovis obtained from the blood clots (GenBank accession no. JX090146) revealed similarity of 99.97% with the published sequence of B. bovis from the Uruguay strain (rap 1 protein, GenBank accession no. AF030061). Table 1 Total DNA concentration and A260/280 ratio obtained from clotted blood centrifuged using filters with pore sizes of 200 ␮m (T1) and 1200 ␮m (T2) and from whole-blood samples (control, T3).

Blood clot (T1) Mean ± SD Range Coefficient of variation Blood clot (T2) Mean ± SD Range Coefficient of variation Whole blood (control, T3) Mean ± SD Range Coefficient of variation

Microregion

Total samples

Positive

Negative

Prevalence (%)

Alvorada do Oeste Cacoal Ji-Paraná Guajará Mirim Rondônia Cruzeiro do Sul Rio Branco Tarauacá Brasiléia Acre

68 101 63 54 286 45 58 45 55 203

67 93 61 51 272 42 56 44 53 195

1 8 2 3 14 3 2 1 2 8

98.53 92.1 96.8 94.4 95.1a 93.3 96.6 97.8 96.4 96.1a

Means followed by the same letter do not differ significantly from each other.

Estimated analytical sensitivity of PCR and n-PCR The estimated sensitivities of PCR and n-PCR for B. bovis corresponded to parasite rates of 0.00001% (1 × 102 parasitized erythrocytes per microliter of blood) and 0.0000001% (1 × 100 parasitized erythrocytes per microliter of blood), respectively.

Results

Samples

Table 2 Prevalence of infection with Babesia bovis in the different microregions of the states of Rondônia and Acre.

A260/280 ratio

DNA concentration (␮g/ml)

1.61a ± 0.068 1.23–1.90 0.04

33.39b ± 1.17 24.61–38.0 3.5

1.58a ± 0.035 1.40–1.83 2.21

31.76b ± 0.663 28.00–36.66 2.08

1.63a ± 0.028 1.40–1.87 1.71

32.35b ± 0.676 28.00–37.5 2.08

Means followed by the same letters do not differ from the mean of the control group by Student’s t-test for paired data at 1% significance.

Detection of B. bovis infection in cattle blood clot samples from Rondônia and Acre A ratio of 272/286 samples from Rondônia were positive for B. bovis (95.1%), and of the 203 samples from Acre, 195 were positive (96.1%). Cattle infected with B. bovis were observed in all the microregions evaluated, without a significant difference between regions in the states of Rondônia and Acre (Table 2). Discussion In this study, the amplification of B. bovis DNA from blood clot samples demonstrated that the use of this material to obtain template DNA for diagnosis of hemoparasites is feasible and inexpensive. This was the first epidemiological study of infection of cattle with a hemoparasite carried out in the southwestern Brazilian Amazon region. The polymerase chain reactions performed showed high specificity and high sensitivity for use in epidemiological studies of cattle hemoparasites, corroborating the observations of Figueroa et al. (1993), Oliveira-Sequeira et al. (2005), Silva et al. (2009) and Brito et al. (2010). The high infection rates found in all the microregions studied indicates that the overall area covered by these regions can be considered endemic for B. bovis. The level of positive samples in this study is above the 75% infection threshold established by Mahoney (1975) for an area to be considered stably endemic. This result was expected, because R. microplus parasitizes cattle in the majority of areas between 32◦ south latitude and 40◦ north latitude in nearly all months (Mahoney and Ross, 1972; Kuttler, 1988), and this tick is widely disseminated in all the microregions studied (Brito et al., 2011). According to Mahoney and Ross (1972) and Madruga et al. (1984), the prevalence of babesiosis in cattle at levels greater than or equal to 75% indicates that the animals become infected while younger than one year of age, with immunity to subsequent infections so that only a small number of adults present clinical babesiosis. Therefore, because of the absence of statistically significant differences in the rates of infection with B. bovis in the microregions studied, it can be inferred that the risks of babesiosis outbreaks are low, since the infection by this hemoparasite is similar among the microregions. However, the possibility of outbreaks does exist if animals are introduced from areas free of infection or


that are enzootically unstable. In this case, the imported animals would require previous immunization. Brazil has the world’s largest commercial cattle herd, with approximately 200 million animals (IBGE, 2009). Of this total, it is estimated that only a small portion are exposed to conditions of enzootic instability (Almeida et al., 2006), since the distribution of Babesia spp. is closely associated with the presence of the cattle tick R. microplus, which is highly prevalent in most of Brazil (Lima et al., 2000; Paiva Neto, 2004; Brito et al., 2011). However, even in regions with endemic stability for infection with B. bovis, this microorganism is still considered a cause of disease and mortality in calves (Madruga et al., 1984), mainly under conditions of heavy tick infestation. This often occurs in the Brazilian Amazon region, where the climate is highly favorable to the development of this vector in virtually all months of the year. The prevalence data of B. bovis in Brazil are mainly based on serological analysis. The rate of infection with B. bovis in the southwestern Amazon does not differ from that observed in most other regions of the country, where infection with this parasite is largely independent on age, sex, and breed. Two exceptions are the southern states of Rio Grande do Sul and Paraná, where the climate is more temperate. In the first case, Martins et al. (1994) found that of 101 cattle herds studied for the presence of anti-B. bovis antibodies in the region of Santana do Livramento, located in the southernmost part of the country, 53% were in a situation of enzootic instability. In the second case, Osaki et al. (2002) found that 64.2% of the animals studied were serologically positive for B. bovis in the Umuarama region. Published findings for other states are in line with those found here. In Goiás, Linhares et al. (1992) observed 98.72% prevalence of antibodies; in Bahia, Araújo et al. (1998) reported a figure of 95.30%; in the state of Rio de Janeiro, Soares et al. (2000) found 90.98% prevalence of anti-B. bovis antibodies; and in Pernambuco, Berto et al. (2008) detected the presence of IgG antibodies for B. bovis in 76.59% of the samples analyzed. Finally, in the western Brazilian Amazon, studies of the seroprevalence in the states of Tocantins and Pará revealed rates of anti-B. bovis antibodies of 91.7% and 98.8%, respectively, characterizing that region as endemic for B. bovis (Trindade et al., 2010; Guedes Junior et al., 2008). In conclusion, the molecular diagnosis of B. bovis utilizing clotted blood as a source to obtain template DNA proved efficient in detecting hemoparasites in asymptomatic cattle bred in the regions studied, also demonstrating that these regions can be classified as endemically stable. Acknowledgments We acknowledge funding for this work from Empresa Brasileira de Pesquisa Agropecuária – Embrapa (SGE/Embrapa, project number 030433600) and the Agência de Defesa Sanitária Agrosilvopastoril do Estado de Rondônia for the partnership in obtaining the samples. We also thank the laboratory technician Antônio Xavier do Nascimento (Embrapa Rondônia) for his dedication and contributions in the field activities. References Almeida, M.B., Tortelli, F.P., Riet-Correa, B., Ferreira, J.L.M., Soares, M.P., Farias, N.A.R., Riet-Correa, F., Schild, A.L., 2006. Tick fever in southern Brazil: a retrospective study of 1978–2005. Brazil J. Vet. Res. 26, 237–242. ˜ A., Estrada-Pena, ˜ A., Gutierrez, J.F., 2001. Almeria, S., Castella, J., Ferrer, D., Ortuno, Bovine piroplasmosis in Minorca (Balearic Islands, Spain): a comparison of PCRbased and light microscopy detection. Vet. Parasitol. 99, 249–259. Araújo, F.R., Madruga, C.R., Leal, C.R.B., Schenk, M.A.M., Kessler, R.H., Marques, A.P.C., Lemaire, D.C., 1998. Comparison between enzyme-linked immunosorbent assay, indirect fluorescent antibody and rapid conglutination tests in detecting antibodies against Babesia bovis. Vet. Parasitol. 74, 101–108. Berto, R.S., Faustino, M.A.G., Melo, L.E.H., Alves, L.C., Madruga, C.R., Almeida, M.A.O., Ramos, C.A.N., Tenório, T.G.S., Silva, F.F., 2008. Frequency of IgG antibodies

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