Elsevier

Veterinary Microbiology

Volume 261, October 2021, 109199
Veterinary Microbiology

Genetic characterization and phylogenetic of Anaplasma capra in Persian onagers (Equus hemionus onager)

https://doi.org/10.1016/j.vetmic.2021.109199Get rights and content

Highlights

  • This study identifies the occurrence of A. capra in blood samples of wild onagers in Iran.

  • Also, this study is the first report of A. capra in onagers and Equidae family, worldwide.

  • Phylogenetic characteristics suggest that this pathogen could be a potentially health threat to animals and human.

  • A. capra has not been reported from Iran previously in any other host and this is the first report from Iran.

Abstract

Anaplasma spp. are among the most recognized arthropod-borne infectious agents. Although the novel A. capra has been isolated from wildlife, livestock, and hard ticks from many parts of the world, there is no report regarding the identification of this pathogen from equines and little is known about the epidemiology of A. capra in Equidae. In this study, A. capra was identified in two out of ten blood specimens of wild onagers (Equus hemionus onager) during a routine health check-up in Semnan, Iran by light microscopy and molecular analyses while other pathogens were not detected. First, inclusions on RBC's were observed in two blood smears by light microscopy. Then, the blood specimens of both animals were analyzed by realtime-PCR for Anaplasma, Ehrlichia, and Theileria infections. A 1400 bp sequence of 16S rRNA belonging to Anaplasmataceae and 874 bp fragment for groEL gene for A. capra were amplified in Anaplasma positive samples and sequenced. Preliminary BLAST analysis of sequenced fragments showed high homology to A. capra strains in GenBank database. Finally, nested PCR and restriction enzyme fragment length polymorphism techniques confirmed the pathogen as A. capra. To the best of our knowledge, this study has reported the occurrence of A. capra in wild onagers for the first time and suggests that equines could be infected with this pathogen and act as reservoirs for A. capra.

Introduction

The Persian onager (Equus hemionus onager; Persian zebra) is one of the Asiatic wild ass subspecies native to the desert of Iran. This animal belongs to the family Equidae and the genus Equus (Weinstock et al., 2005; Askari et al., 2018a). Persian onagers are endangered animals according to the IUCN (International Union for Conservation of Nature) Red List with about 400 individuals reported in their natural environment (Thompson et al., 2020). Although these animals have been mainly found in Asia and Middle East (from Mongolia to Saudi Arabia), three subpopulations of wild Persian onagers are localized in Iran. Also, seventeen international institutions are protecting ex situ populations of onagers worldwide in Europe, United States and Australia (Askari et al., 2018b). The Equidae are hosts to a considerable variety of infectious agents, some of which can cause significant morbidity or mortality if infected animals remain untreated.

Anaplasma (Order: Rikettsiales) are classified as obligatory intracellular arthropod-borne organisms, which infect mammals (Qin et al., 2018). Major reservoirs for Anaplasma spp. are livestock and ticks (Ixodidae) but there is no considerably data regarding their epidemiology within wildlife (Williams et al., 2002; Lee et al., 2018). In vertebrates A. phagocytophilum, A. platys, A. capra, A. marginale, A. ovis, and A. bovis are considered as hematological pathogens (Liu et al., 2012; Li et al., 2015; Qin et al., 2018). Likewise, species with zoonotic potential have been reported as well as A. phagocytophilum, A. capra, and A. ovis which are transmitted most often through the bite of a hard tick (Acari: Ixodidae) to human (Chochlakis et al., 2010; Truchan et al., 2013). Depending on the Anaplasma spp., various cells in the reticuloendothelial system as well as granulocytes, platelets, erythrocytes, and bone marrow precursor cells or endothelial cells may be infected (Rymaszewska Anna, 2008) resulting a variety of clinical signs from mild to life-threatening illness including anemia, gastrointestinal and pulmonary disorders, and joint pain and lameness (Nicholson, 2018).

Anaplasmosis in equine mainly known as equine granulocytic anaplasmosis (EGA) is a seasonal infection and seen in many parts of the world. A. phagocytophilum is considered as the main causative agent of anaplasmosis in horses (Pusterla and Madigan, 2013). Moreover, A. bovis has been detected in a horse with anaplasmosis (Seo et al., 2019). Further, a high seroprevalence of Anaplasma antibodies (72.7 %) in plain zebras has been reported (Ngeranwa et al., 2008) but the identification of Anaplasma spp. in wild Equidae has not been reported previously. In equine, granulocytic anaplasmosis (EGA) typically has been found by mild anemia (a decrease of PCV, Hb, and RBC), thrombocytopenia, and leukopenia with neutropenia and lymphopenia in many clinical reports (Butler et al., 2008; Jahn et al., 2010). Elevated bilirubin, creatinine, blood urea nitrogen and proteinuria have been reported in horses with granulocytic anaplasmosis (Saleem et al., 2018a, 2018b). The most frequent biochemistry characteristic of EGA are mild to moderate hyperbilirubinemia created by elevated unconjugated bilirubin and a slight decrease in albumin (Li et al., 2011; Siska et al., 2013). The severity of hematological and biochemical disorders depends on factors such as animal species, age, sex, physiological statues, and co-infection for instance with Theileria spp. (Saleem et al., 2018a).

16S rRNA and groEL gene sequencing are powerful tools that have been used to trace phylogenetic relationships between microorganisms, and to identify pathogens from various hosts (Guo et al., 2019; Zobba et al., 2020). Gene sequencing is used today in clinical laboratories for routine identifications, especially for fastidious or unusual bacteria for instance Anaplasma spp. (Zobba et al., 2014; Staji et al., 2021). The present investigation deals with the finding and genetic characterization of A. capra in the blood samples of two cases of Persian onager from Khar Turan National Park in the north central Iran.

Section snippets

Ethics approval

Department of Environmental Protection (DEP), located in Semnan, Iran, aimed to transfer 10 wild onagers from Khar Turan National Park (36.5000 ° N 55.50000 °E) to Kavir National Park (34.6347 ° N 52.4333 °E) located in Semnan Province (north central Iran). This study, conducted in October 2018, did not receive approval from the related animal care committee of DEP because the analyzed findings were collected during a routine health check-up. Blood specimens were obtained by expert

Hematological, biochemical, and microbiology analyses of blood specimens

Based on the blood smears observation, RBC infecting organisms were noticed in two animals out of ten (Fig. 1). Considering that the reference intervals of hematology and serum biochemistry parameters of Persian onagers have not been determined in any source so far, therefore, in the present study hematology and serum biochemistry parameters of infected and non-infected onagers were compared. A slight decrease of RBC (1.20 %), HCT (1.24 %), and HGB (1.05 %), leukopenia (2.05 %), lymphopenia

Discussion

Wild animals can be infected and act as reservoirs for a variety of pathogens and no active surveillance programs regarding infectious diseases are present for wildlife as well as onagers in Iran. Unfortunately, infectious diseases of wildlife are often not included in epidemiological considerations, as the main focus stands on infections in farm animals and their potential zoonosis. Besides, predisposing factors such as environment and shared feed/water resources increase the risk of

Conclusion

This is the first report of the occurrence of A. capra in wild onagers. Although this pathogen has been identified in some wildlife species, our findings demonstrate that wild onagers as highly endangered animals with low papulations may also be infected by the pathogenic A. capra. Therefore, further epidemiological and experimental investigations should shed light on whether these were accidentally identified animals, or if A. capra can effectively infect equines and cause disease.

Declaration of Competing Interest

None.

Acknowledgments

We thank Mr. Rasoul Rostami Lima for his assistance in experimental procedures.

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