Original article
Low genetic diversity of Anaplasma marginale in calves in an endemic area for bovine anaplasmosis in the state of São Paulo, Brazil

https://doi.org/10.1016/j.ttbdis.2015.07.018Get rights and content

Abstract

Major surface protein 1a has been used as a marker for genetic stability in identifying geographical isolates of Anaplasma marginale. We conducted a longitudinal study to ascertain the genetic diversity of A. marginale in a dairy cattle herd with a history of clinical anaplasmosis. A total of 20 calves were evaluated every 3 months, from birth to 1 year of age. They were evaluated using blood smears, IFAT, ELISA, and qPCR. Additionally, samples positive for the msp1a gene using nPCR were sequenced. The detection of A. marginale ranged from 20 to 90% using blood smears, 20–80% using ELISA/IFAT, and 15–100% using qPCR. We found evidence that suggested transplacental transmission of A. marginale in 15% (3/20) of the calves based on qPCR and 20% (4/20) based on blood smears. Additionally, these four animals were A. marginale ELISA/IFAT-positive. The A. marginale strains found were α-β3-Γ (n = 7), α-β2 (n = 1), and α-β2-190-Γ (n = 1). The new MSP1a tandem repeat 190 was described. The results showed that the genetic diversity of A. marginale in a group of calves up to 1 year of age from Taiaçu (SP) was low, with only three different strains identified, showing the microsatellite genotype E. Eighty percent of the animals evaluated had clinical signs of bovine anaplasmosis and were treated using oxytetracycline and imidocarb dipropionate. Additionally, it was found that 30% (6/20) and 10% (2/20) of the animals required a second and third treatment, respectively, based on clinical signs. Four animals infected with the α-β3-Γ strain died, showing that this strain is also involved in the clinical anaplasmosis cases in Brazil.

Introduction

Anaplasma marginale (Rickettsiales: Anaplasmataceae) is the most prevalent tick-borne pathogen in the world. It is distributed on six continents and is responsible for a high morbidity, and mortality among cattle in temperate, subtropical, and tropical regions (Kocan et al., 2010). Anaplasma marginale is an obligate intracellular pathogen that may be transmitted biologically by ticks, mechanically by blood sucking flies or infected blood in fomites, and also transplacentally (Aubry and Geale, 2011).

Brazil can be classified as an area of endemic stability for the occurrence of bovine anaplasmosis, as previously established for the Babesia bovis-Rhipicephalus microplus system in Australia by Mahoney and Ross in 1972. The prevalence of bovine anaplasmosis in Brazil ranges from 16.3% in the semi-arid zone of the state of Sergipe (Oliveira et al., 1992) to close to 100% in the states of Rio de Janeiro (Silva et al., 2014). In the state of São Paulo, a serological survey showed that 98% of the cattle were positive for A. marginale (Andrade et al., 2004).

Major surface protein 1 alpha (MSP1a) has been used in identifying geographical strains according to differences in molecular weight and amino acid sequences (de la Fuente et al., 2007). While the C-terminus of the protein is highly conserved, the N-terminus is highly variable, with the number and sequence of amino acids constant for a given strain of A. marginale (de la Fuente et al., 2001, Palmer et al., 2001). The genetic diversity of A. marginale has been described for cattle herds in Europe (de la Fuente et al., 2005), Africa (Mtshali et al., 2007; Mutshembele et al., 2014), the Middle East (Molad et al., 2009), North America (de la Fuente et al., 2001, Palmer et al., 2001; Alamzán et al., 2008), South America (Garcia-Garcia et al., 2004, Ruybal et al., 2009), and, most recently, in Asia (Ybañez et al., 2014). In Brazil, significant variations in the MSP1a tandem repeats of strains of A. marginale have been found in the states of Paraná (Ferreira et al., 2001; Vidotto et al., 2006), Minas Gerais (de la Fuente et al., 2005, Pohl et al., 2013), and Rio de Janeiro (Silva et al., 2015). In the MSP1a tandem repeats of strains of A. marginale described in Brazil, the most frequent sequences are 16, α, and τ in Paraná (Vidotto et al., 2006), 72 and α in Minas Gerais (Pohl et al.,2013), and 4 and τ in Rio de Janeiro (Silva et al., 2015). Additionally, the analysis of MSP1a microsatellite sequences has resulted in five (B, C, D, E, and G) different genotypes amongst A. marginale strains from Brazil (Vidotto et al., 2006, Pohl et al., 2013, Silva et al., 2015).

The World Organization for Animal Health has proposed that enrofloxacin, imidocarb, and oxytetracycline should be used for eliminating persistent A. marginale infections in cattle. However, studies have shown that these antimicrobials are not capable of fully eliminating persistent A. marginale infections (Coetzee et al., 2005, Coetzee et al., 2006). Additionally, there are no studies evaluating the capacity of these chemical components to induce genetic diversity in A. marginale or the possibility that some strains may be more or less sensitive to the drugs.

Among the different strains of A. marginale, some have been associated with occurrences of disease outbreaks. The α-β3-Γ strain has already been reported in bovine anaplasmosis outbreaks in Mexico (Almazán et al., 2008) and Argentina (Ruybal et al., 2009). In Brazil, this strain was isolated from a calf with clinical anaplasmosis in the state of São Paulo and has been kept cryopreserved for over 20 years. It was shown to be highly pathogenic in successive experimental infections (Andrade et al., 2004, Silva et al., 2015). Thus, a longitudinal study was conducted to ascertain the genetic diversity of A. marginale in a dairy cattle herd with a history of clinical anaplasmosis over the last 10 years.

Section snippets

Materials and methods

Area and animals. The farm was selected based on its history of cases of clinical anaplasmosis in calves and heifers over the last 10 years (unpublished data). Calves aged from 0 to 4 months were kept in individual stalls, with access to a pasture area of Tifton 85 grass (Cynodon dactylon). In this phase, the animals received 2 kg of milk in the morning and 2 kg in the afternoon, and 1 kg of concentrate per day. When the calves reached 5 months of age, they were transferred to a rotation area

Results

Detection of A. marginale infections. Calves were diagnosed as positive for A. marginale by means of blood smears, ELISA, IFAT, nPCR, and qPCR (Table 1). We found evidence that suggested transplacental transmission of A. marginale in 15% (3/20) of the calves from qPCR and 20% (4/20) from blood smears. Additionally, these four animals were A. marginale ELISA/IFAT-positive (Table 2). The detection of A. marginale ranged from 20% (in newborns) to 90% (3- and 6-month olds) according blood smears;

Discussion

Our results showed that only three A. marginale strains were circulating in the animals of this study. Two possibilities can be considered for this low genetic diversity of A. marginale. First, in areas that are endemic for bovine anaplasmosis, the low genetic diversity of A. marginale has been correlated with low tick populations (Ruybal et al., 2009). The animals in the present study underwent spray treatment against R. microplus, with products based on pyrethroids, every 21 days during the

Conclusion

The α-β3-Γ strain of A. marginale was the most common strain detected among calves up to 1 year old showing clinical anaplasmosis in a farm in the state of São Paulo, southeastern Brazil. Although there was high molecular detection of A. marginale, low genetic diversity of MSP1a was found.

Conflict of interest statement

None of the authors of this work has a financial or personal relationship with other people or organizations that would inappropriately influence or bias the content of this paper.

Ethical statement

This research (Process # 017259/14) is according to the Committee on Animal Research and Ethics (CARE).

Acknowledgments

We thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for their financial support (Process #2012/21371-4) and the Coordination Office for Improvement of Higher-Education Staff (CAPES) for the J. B. Silva fellowship (Process #2012-2015). We thank the Germania Farm for access to the study animals.

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