First report of Anaplasma marginale infection in goats, Brazil

Anaplasma marginale, the causative agent of bovine anaplasmosis, is a tick-borne bacterium that causes significant economic losses for cattle industries and is increasingly being detected in other animal species. Rhipicephalus microplus is the main vector of this bacterium and may be found parasitizing small ruminants. In northeastern Brazil, multispecies grazing is a common family subsistence practice on smallholder farms possibly facilitating interspecies transmission of pathogens. Considering that A. marginale infection has been previously molecularly described in sheep, this study has aimed to estimate the prevalence of A. marginale and factors associated with the infection in goats from northeastern Brazil. A total of 403 goat blood samples were included in the study. An epidemiological questionnaire was applied to each farm owner addressing age, gender, presence of ticks and multispecies grazing. All samples were screened for A. marginale- and A. ovis-infection using primers targeting the Anaplasma spp. msp4 gene. The identity of A. marginale in the blood was confirmed by PCR amplification of msp5 followed by sequencing. Anaplasma spp. were differentiated by sequencing of the repeat region of the msp1α gene. For the statistical analysis the Chi-square or the Fisher’s exact test was used to verify association of the individual factors (age, gender, presence of ticks, and multispecies grazing) with Anaplasma spp. infection. We report the first molecular detection of A. marginale in goats from northeastern Brazil, based on msp1α, msp4 and msp5 gene sequencing analysis. Sequencing of the detected A. marginale msp1α gene revealed the F repeat. Amblyomma parvum and R. microplus were found feeding on animals.

Introduction Anaplasma marginale, the causative agent of bovine anaplasmosis, is a tick-borne bacterium that causes significant economic losses for cattle industries [1]. A. marginale infects cattle through tick transmission worldwide. The bacterium has been detected in non-bovine species, but the relevance of these other species in the ecology of bovine anaplasmosis is unknown. At least 20 ixodid tick species have been implicated in the transmission of A. marginale, including Dermacentor spp. and Rhipicephalus spp. [1].
Anaplasma marginale is found in regions where tick vectors are endemic. In tropical and subtropical regions, Rhipicephalus microplus is the vector of bovine anaplasmosis. Despite host specificity of R. microplus for cattle [2], this tick species may be found parasitizing small ruminants [3]. In Brazil, R. microplus is endemic [4] and hampers livestock production resulting in annual economic losses estimated at U$ 3.24 billion [5].
In northeastern Brazil, multispecies grazing is a common family subsistence practice on smallholder farms possibly facilitating interspecies transmission of pathogens. A study of cograzing ruminants has shown a single A. marginale strain infecting coexisting cattle, buffalo and ticks [6]. A. marginale infection has been previously molecularly described in sheep from Iran [7]. However, to the best of our knowledge, A. marginale has never been detected in goats. This study aimed to estimate the prevalence of A. marginale and factors associated with the infection in goats from the State of Paraíba, northeastern Brazil.

Material and methods
With the approval from the Ethics Committee for Animal Experimentation and Animal Welfare of the Universidade Federal da Paraíba (protocol 3305/14), a total of 403 blood samples from goats (368 females and 35 males), previously surveyed for other pathogens [8], were included in this study. All samples were collected in an anticoagulant tube containing ethylenediaminetetraacetic acid and stored at -80˚C. An epidemiological questionnaire was given to each farm owner addressing age, gender, presence of ticks and multispecies grazing. The age of goats was stratified into groups of one year and > one year.
Genomic DNA was extracted from 200 μL of whole blood using a commercial kit (GE Healthcare, Little Chalfont, UK), according to the manufacturer's instructions. Negative controls using ultra-pure water were performed in parallel to monitor cross-contamination in each batch of 30 samples.
PCR amplification of the caprine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene was done to verify successful DNA extraction, as previously described [9]. Samples were screened for A. marginale-and A. ovis-infection using previously described primers targeting the Anaplasma spp. msp4 gene (%870 bp) [10]. Amplified DNA fragments of the msp4 gene from two Anaplasma spp. isolates were directly sequenced using the Sanger method, and analyzed sequences compared by BLASTn with those present in the GenBank 1 database.
The identity of A. marginale in the blood was confirmed by PCR amplification of msp5 followed by sequencing (GenBank accession numbers: MH037254-MH037263). Anaplasma spp. were differentiated by amplifying, cloning and sequencing of the repeat region of the msp1α gene as previously described [11]. The msp1α gene sequences obtained herein have been submitted to GenBank under the following accession numbers: MH590661-MH590670.
Either the Chi-square or the Fisher's exact test was used to assess association of the individual factors such as age, gender, presence of ticks, and multispecies grazing including sheep and cattle with Anaplasma spp. infection. P-values were calculated and considered significantly The rate of A. marginale-infection and corresponding estimated parameters for each of the evaluated potential risk factor is shown in Table 1.
Tick-infested goats were six times more likely to be infected with A. marginale (p = 0.02788). The tick species feeding on the studied goats were identified as Amblyomma parvum (49/52, 94.23%) and R. microplus (3/52, 5.77%). All A. marginale-positive goats were found on farms with multispecies (sheep and cattle) grazing (p = 0.04300). To determine the A. marginale tandem repeats as previously described [12], we performed msp1α PCR (Fig 1A) and the amplicons were cloned and sequenced. The sequence results revealed MSP1a tandem repeat F in Brazilian goats (Fig 1B).

Discussion
Although some goat Anaplasma MSP4 and MSP5 sequences previously reported in GenBank database have greater similarity to A. marginale than A. ovis (e.g. JN572928 and NZ_PKOE 00000000), this study describes the first molecular report of A. marginale in goats based on msp1a gene sequence analysis. Anti-Anaplasma spp. antibodies have been identified in goats from northeastern Brazil [13]. However, direct molecular detection of A. marginale in small ruminants has been only reported in sheep from Iran [7]. R. microplus has been described as the main vector of A. marginale [1], yet no study has tested the vector competence of A. parvum for A. marginale. Previous studies have suggested that Amblyomma ticks may be involved in the transmission of A. marginale [6,14]. Further studies are necessary to evaluate if A. parvum is a competent vector of A. marginale in Brazil.
Co-grazing of goats, sheep and cattle is the most common practice of northeastern Brazil, since it allows a wider diversification of products for commercialization. A previous study in Rio de Janeiro State, southeastern Brazil, has shown that A. marginale strains identified in water buffaloes were closely related to A. marginale from cattle as determined by sequencing msp1α [6]. In the Brazilian goats, the detected A. marginale was also closely related to cattle species and all detected MSP1a displayed the F repeat. Due to the different number of tandemly repeated 29 amino acid units, the molecular weight of MSP1a varied between A. marginale isolates [15]. A previous report demonstrated individual animals had the same tandem repeat but different numbers of repeats ranging from 2 to 6 repeats [12]. In another study, molecular weight variation was used to differentiate and characterize geographic genotypes [15]. However, in both cases, sequences were used to determine A. marginale genotypes. In Brazil, six microsatellite genotypes have been reported, including repeats B, C, D, E, G and H, with genotype E being the most common in cattle [16,17]. Unfortunately, the nucleotide distance between the Shine-Dalgarno (SD) sequence and the translation initiation codon (ATG) could not be determined [18]. Thus, it is unknown whether other A. marginale genotypes infect goats. Additional studies are necessary to elucidate this question.

Conclusions
This study provides the first insight into A. marginale infection in goats from Paraíba State, northeastern Brazil and demonstrates that goats may play a role in the epidemiology of A. marginale as a yet unrecognized reservoir. Competent ticks feeding on goats and cattle may transfer the pathogen between the two livestock species.