Anaplasma Capra Research Articles

The first molecular detection of Anaplasma capra in domestic ruminants in the central part of Turkey, with genetic diversity and genotyping of Anaplasma capra.

Tick-borne diseases have been an increasing threat to human and animal health all over the world. Anaplasmosis is one of the emerging tick-borne diseases and has zoonotic potential. A new novel species, which was detected in China in 2010-2012 and provisionally named Anaplasma capra in 2015, causes zoonotic infections and infects many different animal species. In this study, we investigated the presence of A. capra in domestic ruminants from Turkey. A total of 468 blood samples (cattle, sheep, and goat) were examined by the gltA gene-specific nested polymerase chain reaction, revealing the presence of A. capra in six samples (1.28%): one of them from cattle (0.41%) and the other five from sheep (3.22%). According to DNA sequences results of the gltA gene, A. capra isolates identified in the present study were shown high nucleotide similarity with A. capra isolates detected from different hosts. However, the nucleotide differences were detected in the same nucleotide positions between A. capra isolates. For this reason, we thought that at least two different A. capra genotypes could be circulating in the world. As a result, it is seen that A. capra, which was determined to be a new species with zoonotic potential, was revealed in European and Asian countries and in different hosts. In order to raise awareness about human anaplasmosis infections, it is important to reveal the prevalence of the species in the world. The emergence of A. capra in Turkey reveals the need for a re-evaluation of the human and animal health risk analysis in terms of anaplasmosis.

Anaplasma capra in sheep and goats on Corsica Island, France: A European lineage within A. capra clade II?

Anaplasmosis is a tick-transmitted disease due to several species of the genus Anaplasma. In 2019, we demonstrated the presence of Anaplasma capra in two deer species at a zoological park in mainland France. As we suspected its presence in Corsica, we surveyed 11 geographically distant sheep or goat farms. Using molecular tools such as nested PCR targeting 16S ribosomal RNA (rRNA), citrate synthase (gltA) and heat-shock protein (groEL) genes, we detected the presence of A. capra on 5/11 farms, in 26/108 blood samples (24%), in sheep as well as in goats. Genotyping and phylogenetic analysis of A. capra revealed that isolates from Corsica island grouped closely with A. capra isolates reported in red deer and swamp deer from a zoological reserve in mainland France, as well as in roe deer from Spain, in a separate and well supported clade within A. capra clade II. This third report of the tick-borne bacterium A. capra in Europe suggests a potentially larger presence of this pathogen on the European continent, on domestic, native as well as wild ruminants, a broad host range already described in Asian countries for this species.

Ticks and Tick-Borne Pathogens Abound in the Cattle Population of the Rabat-Sale Kenitra Region, Morocco.

Tick-borne pathogens cause the majority of diseases in the cattle population in Morocco. In this study, ticks were collected from cattle in the Rabat-Sale-Kenitra region of Morocco and identified morphologically, while tick-borne pathogens were detected in cattle blood samples via polymerase chain reaction assay and sequencing. A total of 3394 adult ixodid ticks were collected from cattle and identified as eight different tick species representing two genera, Hyalomma and Rhipicephalus. The collected ticks consisted of Hyalomma marginatum, Hyalomma anatolicum excavatum, Rhipicephalus sanguineus sensu lato, Rhipicephalus bursa, Hyalomma detritum, Hyalomma lusitanicum, Hyalomma dromedarii, and Hyalomma impeltatum. The overall prevalence of tick-borne pathogens in blood samples was 63.8%, with 29.3% positive for Babesia/Theileria spp., 51.2% for Anaplasma/Ehrlichia spp., and none of the samples positive for Rickettsia spp. Sequencing results revealed the presence of Theileria annulata, Babesia bovis, Anaplasma marginale, Theileria buffeli, Theileria orientalis, Babesia occultans, Anaplasma phagocytophilum, Anaplasma capra, Anaplasma platys, Anaplasma bovis, Ehrlichia minasensis, and one isolate of an unknown bovine Anaplasma sp. Crossbreeds, females, older age, and high tick infestation were the most important risk factors for the abundance of tick-borne pathogens, which occurred most frequently in Jorf El Melha, Sidi Yahya Zaer, Ait Ichou, and Arbaoua locations.

Molecular detection of Anaplasma spp., Babesia spp. and Theileria spp. in yaks (Bos grunniens) and Tibetan sheep (Ovis aries) on the Qinghai-Tibetan Plateau, China

BackgroundAnaplasma, Babesia and Theileria are tick-borne pathogens (TBPs) that affect livestock worldwide. However, information on these pathogens in yaks (Bos grunniens) and Tibetan sheep (Ovis aries) on the Qinghai-Tibet Plateau (QTP), China, is limited. In this study, Anaplasma spp., Babesia spp. and Theileria spp. infections were assessed in yaks and Tibetan sheep from Qinghai Province.MethodsA total of 734 blood samples were collected from 425 yaks and 309 Tibetan sheep at nine sampling sites. Standard or nested polymerase chain reaction was employed to screen all the blood samples using species- or genus-specific primers.ResultsThe results showed that 14.1% (60/425) of yaks and 79.9% (247/309) of Tibetan sheep were infected with at least one pathogen. Anaplasma ovis, Anaplasma bovis, Anaplasma capra, Anaplasma phagocytophilum, Babesia bovis and Theileria spp. were detected in this study, with total infection rates for all the assessed animals of 22.1% (162/734), 16.3% (120/734), 23.6% (173/734), 8.2% (60/734), 2.7% (20/734) and 19.3% (142/734), respectively. For yaks, the infection rate of A. bovis was 6.4% (27/425), that of B. bovis was 4.7% (20/425) and that of Theileria spp. was 3.3% (14/425). Moreover, 52.4% (162/309) of the Tibetan sheep samples were infected with A. ovis, 30.1% (93/309) with A. bovis, 56.0% (173/309) with A. capra, 19.4% (60/309) with A. phagocytophilum and 41.4% (128/309) with Theileria spp.Conclusions This study revealed the prevalence of Anaplasma spp., Babesia spp. and Theileria spp. in yaks and Tibetan sheep in Qinghai Province, China, and provides new data for a better understanding of the epidemiology of TBPs in these animals in this area of the QTP, China.Graphical

First molecular detection of Anaplasma species in cattle from Kyrgyzstan; molecular identification of human pathogenic novel genotype Anaplasma capra and Anaplasma phagocytophilum related strain

Anaplasmosis is a rickettsial infection with significant effects on human and animal health, and the discovery of new species or genotypes with zoonotic potential in recent years has increased this importance. The aim of this study was to provide the first assessment of the molecular etiology and prevalence of bovine anaplasmosis in Kyrgyzstan (specifically in the Chuy, Talas, Djalal-Abad, Naryn, and Issyk-Kul regions). The prevalence of bovine anaplasmosis was determined as 1.7% (6/358). PCR and partial DNA sequencing results of the 16S ribosomal RNA (rRNA) gene revealed that Anaplasma centrale, A. phagocytophilum like-1, and the human pathogenic novel genotype A. capra are circulating in cattle herds in Kyrgyzstan. Six DNA nucleotide sequences obtained in this study were deposited in GenBank under the following accession numbers: A. centrale (MW672117, MW672118, MW672119, MW672120), A. phagocytophilum (MW672121), and A. capra (MW672115).

Diversity of Anaplasma species and importance of mixed infections in roe deer from Spain.

Although wildlife can act as reservoirs of some Anaplasma species, studies on the presence and distribution of Anaplasma spp. in wild cervids are mainly limited and focused on zoonotic species. In order to identify the Anaplasma species in roe deer from Spain and to detect co-infections, 224 spleen samples were tested for Anaplasma spp. using a commercial qPCR; positive samples were further characterized using generic 16S rRNA primers and species-specific primers targeting the msp2 and groEL genes. Anaplasma DNA was detected in the 50.9% of samples, and four Anaplasma species were identified. Anaplasma phagocytophilum (43.8%) was predominant, followed by Anaplasma bovis (13.8%), Anaplasma capra (5.8%) and Anaplasma ovis (2.2%). In addition, strains similar to Anaplasma platys were found in nine animals. Most positive roe deer (71.9%) were infected with a single Anaplasma species, whereas co-infections with two (19.3%) or three (8.8%) Anaplasma species were also found. This study confirms the widespread occurrence of Anaplasma spp. in roe deer from Spain, being the first report of A. platys-like strains and A. capra in this cervid; it is also the first report of A. capra in Spain. The detection of Anaplasma species pathogenic for humans and/or domestic animals in roe deer suggests that this cervid may play a role in the sylvatic cycle of these bacteria contributing to the appearance of clinical anaplasmosis cases. In addition, co-infections are common in roe deer revealing that Anaplasma species specific PCR assays are essential for a reliable identification as well as for determining their real prevalence.

The Novel Zoonotic Pathogen, Anaplasma capra, Infects Human Erythrocytes, HL-60, and TF-1 Cells In Vitro.

Anaplasma capra, a species of the family Anaplasmataceae, is zoonotic tick-borne obligate intracellular bacteria. There have been no reports of human infection with this pathogen since 2015. Therefore, the zoonotic characteristics of A. capra need to be further studied. To verify the ability of A. capra to infect human cells, A. capra were inoculated in human erythrocytes, HL-60, and TF-1 cell lines in vitro. Cell smears were taken after inoculation, using Giemsa staining, transmission electron microscope (TEM), chromogenic in situ hybridization and immunocytochemistry for detection. In the Giemsa staining, many dark colored corpuscles or purple granules were seen in the inoculated erythrocytes, HL-60, and TF-1 cells. The results of chromogenic in situ hybridization show that there were brown precipitates on the surface of most erythrocytes. Immunocytochemistry results show many dark brown vacuolar structures or corpuscles in the cytoplasm of erythrocytes, HL-60, and TF-1 cell lines. The A. capra morulae were seen in the cytoplasm of both HL-60 and TF-1 in TEM, and their diameter was about 295–518 nm. Both dense-cored (DC) and reticulate cell (RC) form morulae could be seen. This study confirmed the ability of A. capra to infect human erythrocytes, HL-60, and TF-1. This study is of profound significance in further verifying the zoonotic characteristics of the pathogen and for establishing an in vitro cultivation model.

Molecular detection of Rickettsia raoultii, Rickettsia tamurae, and associated pathogens from ticks parasitizing water deer (Hydropotes inermis argyropus) in South Korea

Most defined Rickettsiales, which can be transmitted by ticks, are known to be important zoonotic pathogens. Some of these pathogens can cause severe diseases in humans, including anaplasmosis, rickettsioses, and ehrlichiosis. Previous studies in South Korea have investigated tick-borne pathogens (TBPs) residing in ticks found on grassy vegetation and animals. However, there is limited phylogenetic information on TBPs in ticks parasitizing Korean water deer (KWD; Hydropotes inermis argyropus). This study evaluated the prevalence, risk factors (regions, tick stages, and tick species), and coinfections of TBPs in ticks parasitizing KWD. Were collected a total of 283 hard ticks, including Haemaphysalis longicornis, Haemaphysalis flava, and Ixodes nipponensis from KWD in South Korea from 2013 to 2017. In 173 tested tick pools, genes for seven TBPs, namely Rickettsia raoultii (20 %), Rickettsia tamurae (1 %), Candidatus Rickettsia longicornii (31 %), Ehrlichia canis (3 %), Anaplasma capra (3 %), Anaplasma bovis (2 %), and Anaplasma sp. (1 %), were detected. The unidentified Anaplasma sp. isolates revealed a 98.4 %–99.3 % sequence identity with Anaplasma sp. in GenBank sequences obtained from ticks. To the best of our knowledge, this is the first study to report the presence of the emerging human pathogen R. tamurae in South Korea. These results should increase awareness about the need for continued development of epidemiological control measures, and medical and veterinary communities must be informed of their high infection potential and clinical complexity in humans.

The first detection of Anaplasma capra, an emerging zoonotic Anaplasma sp., in erythrocytes

ABSTRACT An emerging infectious disease caused by “Anaplasma capra” was reported in a 2015 survey of 477 hospital patients with a tick-bite history in China. However, the morphological characteristics and parasitic location of this pathogen are still unclear, and the pathogen has not been officially classified as a member of the genus Anaplasma. Anaplasma capra-positive blood samples were collected, blood cells separated, and DNA of whole blood cells, erythrocytes, and leukocytes extracted. Multiplex PCR detection assay was used to detect whole blood cell, erythrocytes and leukocytes, DNA samples, and PCR identification, nucleic acid sequencing, and phylogenetic analyses based on A. capra groEL, 16S rRNA, gltA, and msp4 genes. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), Wright–Giemsa staining, chromogenic in situ hybridization (CISH), immunocytochemistry, and indirect immunofluorescence assay (IFA) were used to identify the location and morphological characteristics of A. capra. Multiple gene loci results demonstrated that erythrocyte DNA samples were A. capra-positive, while leukocyte DNA samples were A. capra-negative. Phylogenetic analysis showed that A. capra is in the same clade with the A. capra sequence reported previously. SEM and TEM showed one or more pathogens internally or on the outer surface of erythrocytes. Giemsa staining, CISH, immunocytochemistry, and IFA indicated that erythrocytes were A. capra-positive. This study is the first to identify the novel zoonotic tick-borne Anaplasma sp., “Anaplasma capra,” in host erythrocytes. Based on our results, we suggest revision of Genus Anaplasma and formally name “A. capra” as Anaplasma capra sp. nov.

Novel Anaplasma Variants in Small Ruminants From Central China.

Anaplasma capra is an emerging zoonotic pathogen that pose a risk to the health of human and veterinary animal. Numerous variants in a variety of domestic and wild animals had been reported since its discovery and confirmation in humans in 2015 and its first detection from goat blood during 2012–2013. In order to find out more A. capra variants data of A. capra in central China, 16S rRNA, gltA, groEL, and msp4 genes of this pathogen were amplified from sheep and goat samples collected during 2011–2015 and phylogenetic analysis of these sequences were conducted. The results of 16S rRNA and gltA manifested that partial sequences obtained in this study were 100% identical with A. capra isolates, while phylogenetic analysis results of groEL and msp4 showed that the obtained sequences were independent with all other Anaplasmas, formed separate branches on the evolutionary trees. What needed to be emphasized was that the 16S rRNA and gltA gene sequences of X51 (KX505302 and KX450269), a sample from Shandong in 2011, were found to be 100% identical with A. capra. Therefore, we could speculate that the occurrence of A. capra may be earlier than its first discovery and report. And the A. capra isolates in central China were novel variants which were different from known genotypes.

Anaplasma platys-Like Infection in Goats, Beijing, China.

As one of the important tick-borne zoonotic pathogens, Anaplasma has both veterinary and public health significance. Here, we performed a survey of Anaplasma infection in the goats from a farm in Beijing, China, and found 44.6% (41/92) were infected with Anaplasma capra, and 22.8% (21/92) were infected with Anaplasma sp. This Anaplasma sp. bacterium was close to a recently emerging Anaplasma platys strain based on gltA and groEL gene phylogenetic analysis. As to further understand the characteristics of Anaplasma sp., we raised a couple of positive goats (n = 2) in the laboratory with tick-free settings. We observed inappetence, vomiting, high fever, and weakness of limbs in the goat's offspring (n = 3). In addition, the blood samples from all offspring were all positive of this Anaplasma spp. We did not see any intracellular morulae in neutrophils, monocytes, and erythrocytes, but we identified some in the platelets of the blood smears from the positive goats by light microscopy. We named it A. platys-like and suggested it may infect platelets and be transmitted vertically through the placenta of goats. These findings deserve further evaluation.

Identification of Zoonotic Tick-Borne Pathogens from Korean Water Deer (Hydropotes inermis argyropus).

Korean water deer (Hydropotes inermis argyropus) are widespread in the Republic of Korea (ROK). Mostly, Korean water deer are essential hosts for maintaining ticks and tick-borne diseases (TBDs). Here, we investigated the prevalence of tick-borne pathogens (TBPs) among rescued Korean water deer. Anaplasma phagocytophilum (21.4%, 6/28), Anaplasma capra (14.3%, 4/28), Babesia capreoli (3.6%, 1/28), and Coxiella burnetii (3.6%, 1/28) were detected, but Borrelia burgdorferi, Ehrlichia, Rickettsia, and Theileria infections were not found. A. phagocytophilum was the most commonly detected pathogen, and co-infection with A. capra and B. capreoli was also noted in one Korean water deer. To our knowledge, this is the first article of B. capreoli infection in Korean water deer in the ROK. The infecting isolate of A. phagocytophilum was genetically characterized by 16S ribosomal RNA (rRNA) gene and ankyrin-related protein (ankA) gene. Although the 16S rRNA gene alone may not be informative enough to delineate distinct host species, ankA-based phylogeny revealed a high identity of Korean water deer sequences with those of the causative agent of human granulocytic anaplasmosis. A. capra was detected by using citrate synthase gene (gltA), heat-shock protein (groEL), and major surface protein 4 (msp4) genes. Phylogenetic tree based on these gene markers revealed that there were at least two distinct variants within A. capra circulating in the ROK. One variant originated from different hosts including humans, ticks, goats, and sheep, whereas the other variant was reported recently in Korean water deer in the ROK. Consequently, these sequences were identified to belong to a zoonotic species. Sequencing analysis of the 18S rRNA gene revealed that our isolate belonged to B. capreoli and was distinct from Babesia divergens and Babesia venatorum. Moreover, our isolate showed 92.2% homology with B. capreoli sequences, indicating that these differences may be attributed to the different tick species that transmit B. capreoli or to different host species. Genotyping and phylogenetic analysis of C. burnetii based on 16S rRNA and IS1111 genes revealed that our isolate was grouped with several strains of C. burnetii and was genetically distant from Coxiella-like bacteria isolates. The present results highlight that Korean water deer act as potential reservoir hosts for zoonotic TBPs, and thus play an important role in the transmission of TBDs in humans, animals, and livestock.

Genotypic Analysis of Piroplasms and Associated Pathogens from Ticks Infesting Cattle in Korea.

Tick-borne pathogens (TBPs) impose an important limitation to livestock production worldwide, especially in subtropical and tropical areas. Earlier studies in Korea have examined TBPs residing in ticks and animals; however, information on multiple TBPs in ticks infesting cattle is lacking. This study assessed the prevalence of TBPs in ticks parasitizing cattle. A total of 576 ticks, including 340 adults and 236 nymphs, were collected from cattle in Korea between 2014 and 2018. All ticks collected were identified as Haemaphysalis longicornis based on their morphological and molecular characteristics. Among piroplasms and other tick-associated pathogens, seven TBP genes, namely Theileria orientalis (5.0%), Anaplasma bovis (2.3%), Anaplasma capra (4.7%), Anaplasma phagocytophilum-like Anaplasma spp. (APL) clades A (1.9%) and B (0.5%), Ehrlichia canis (1.6%), and Candidatus Rickettsia longicornii (17.5%), were detected. Bartonella spp. and severe fever with thrombocytopenia syndrome virus were not found. To our knowledge, this is the first study to report the presence of the pathogens T. orientalis major piroplasm surface protein genotypes 3 and 7, A. capra, and APL in ticks from Korea. Cattle ticks may be maintenance hosts for many TBPs, and veterinary and medical clinicians should be aware of their high probability of infection and clinical complexity in humans.

A Multiplex PCR Detection Assay for the Identification of Clinically Relevant Anaplasma Species in Field Blood Samples.

The genus Anaplasma (Rickettsiales: Anaplasmataceae), which includes the species Anaplasma capra, Anaplasma bovis, Anaplasma ovis, and Anaplasma phagocytophilum, is responsible for a wide variety of infections in both human and veterinary health worldwide. Multiple infections with these four Anaplasma pathogens have been reported in many cases. We introduce a novel multiplex PCR for the simultaneous detection of A. capra, A. bovis, A. ovis, and A. phagocytophilum, based on species-specific primers against the groEL (A. capra and A. bovis), msp4 (A. ovis), and 16S rRNA (A. phagocytophilum) genes. To verify the specificity of the PCR reactions, we evaluated four sets of primers to analyze samples containing different blood pathogens. The sensitivity of the multiplex PCR was evaluated by amplifying 10-fold dilutions of total genomic DNA extracted from sheep blood infected with A. capra, A. bovis, A. ovis, or A. phagocytophilum. The reproducibility of the assay was evaluated by testing 10-fold dilutions of total genomic DNA extracted from sheep blood infected with these pathogens from 100 to 10–3 ng/μL per reaction in triplicate on three different days. A total of 175 field blood DNA samples were used to evaluate the reproducibility of multiplex PCR compared with the simplex PCRs. PCR primers used in this study were confirmed to be 100% species-specific using blood pathogens previously identified by other methods. The lower limit of detection of the multiplex PCR with good repeatability enabled the detection of A. capra, A. bovis, A. ovis and A. phagocytophilum at concentrations of 3 × 10–5, 5 × 10–7, 2 × 10–5, and 7 × 10–7 ng/μL, respectively. There was no significant difference between conventional and multiplex PCR protocols used to detect the four Anaplasma species (P > 0.05). The results of the multiplex PCR revealed that the A. capra groEL gene, the A. bovis groEL gene, the A. ovis msp4 gene, and the A. phagocytophilum 16S rRNA gene were reliable target genes for species identification in clinical isolates, being specific for each of the four target Anaplasma species. Our study provides an effective, sensitive, specific, and accurate tool for the rapid differential clinical diagnosis and epidemiological surveillance of Anaplasma pathogens in sheep and goats.

Molecular investigation of Anaplasma species in sheep from Heilongjiang Province, northeast China identified four Anaplasma species and a novel genotype of Anaplasma capra

Anaplasmosis poses a great threat to the livestock industry and human health in most tropical and subtropical regions of the world. This study investigated the presence of Anaplasma in sheep from Heilongjiang Province, northeastern China. A total of 341 blood samples were detected by PCR with species-specific primers based on the msp4 gene of Anaplasma ovis, 16S rRNA gene of Anaplasma phagocytophilum and Anaplasma bovis and gltA gene of Anaplasma capra. The results showed that Anaplasma infection was found in 103 (30.2%) of 341 sheep. The infection rates were 2.6%, 8.8%, 15.8% and 10.0% for A. ovis, A. phagocytophilum, A. bovis and A. capra in sheep, respectively. Co-infection involving two Anaplasma species was found in 25 sheep (8.0%), which were usually A. phagocytophilum and A. bovis (72.0%). Co-infection involving A. phagocytophilum, A. capra, A. ovis with zoonotic potential, was found in one sheep. Sequence analysis revealed that the isolates of A. ovis, A. bovis and A. phagocytophilum identified in sheep were closely related to those previously reported in ticks and other animal hosts. Phylogenetic analysis showed that A. capra could be classified into two distinct clusters based on the gltA gene and the isolates identified in sheep from this study were clustered in the A. capra genotype II, which was clearly distinct with the human isolates. The findings in this study report four Anaplasma species and a novel A. capra genotype in sheep from northeastern China, and improve our knowledge of Anaplasma, contributing to the control of ovine anaplasmosis.

Dogs as New Hosts for the Emerging Zoonotic Pathogen Anaplasma capra in China.

Anaplasma capra is an emerging zoonotic tick-borne pathogen with a broad host range, including many mammals. Dogs have close physical interactions with humans and regular contact with the external environment. Moreover, they have been previously reported to be hosts of Anaplasma phagocytophilum, A. platys, A. ovis, and A. bovis. To confirm whether dogs are also hosts of A. capra, pathogen DNA was extracted from blood samples of 521 dogs, followed by PCR amplification of the citrate synthase (gltA) gene, heat shock protein (groEL) gene, and major surface protein 4 (msp4) gene of the A. capra. A total of 12.1% (63/521) of blood samples were shown to be A. capra-positive by PCR screening. No significant differences were observed between genders (P = 0.578) or types (P = 0.154) of dogs with A. capra infections. However, significantly higher A. capra infections occurred in dogs with regular contact with vegetation (P = 0.002), those aged over 10 years (P = 0.040), and during the summer season (P = 0.006). Phylogenetic analysis based on gltA, groEL, and msp4 sequences demonstrated that the isolates obtained in this study were clustered within the A. capra clade, and were distinct from other Anaplasma species. In conclusion, dogs were shown to be a host of the human pathogenic A. capra. Considering the affinity between dogs and humans and the zoonotic tick-borne nature of A. capra, dogs should be carefully monitored for the presence of A. capra.

Duplex TaqMan real-time PCR assay for simultaneous detection and quantification of Anaplasma capra and Anaplasma phagocytophilum infection

Anaplasma capra and A. phagocytophilum, two species of the family Anaplasmataceae, are zoonotic tick-borne obligate intracellular bacteria affecting wild and domestic ruminants, dogs, cats, horses and humans. A. capra and A. phagocytophilum infections have been steadily increasing in both number and geographic distribution, and the accurate diagnosis of these infections is challenging. This study aimed to develop a rapid, sensitive and reliable duplex real-time PCR assay for the specific detection and differentiation of these Anaplasma species.We designed primers and probes against the conserved regions of A. capra groEL and A. phagocytophilum 16S rRNA genes. A range of PCR-related parameters were evaluated such as the dosage of primers and probes, and annealing temperature. The specificity, sensitivity and repeatability of this assay were evaluated. Assay performance was further evaluated using samples collected from 124 goats in four regions of Henan, China. This set of samples was also tested using conventional PCR under conditions previously described.The developed duplex real-time PCR assay allowed the simultaneous detection of A. capra and A. phagocytophilum in a reasonably short time at levels as small as 102 copies/μL, respectively, with optimal specificity and reproducibility. In addition, this duplex real-time PCR assay is the first DNA-based method designed to detect A. capra and A. phagocytophilum, and will be valuable for timely diagnosis and treatment of these infections.

Molecular Detection and Phylogenetic Analysis of Anaplasma spp. in Korean Native Goats from Ulsan Metropolitan City, Korea.

Background: Tick-borne diseases (TBDs) can be fatal to humans as well as to animals causing severe economic losses globally to livestock industries. Many countries conduct regular surveillance of TBDs in livestock. Serological and molecular surveillance of TBDs in livestock and humans was carried out in the Republic of Korea. However, there are not a lot of data on analyses of anaplasmosis in Korean native goats and the correlation with rearing methods and seasons. Methods: In this study, goats in Ulsan city were tested for anaplasmosis by PCR and 16S rRNA sequencing. A total of 452 goat blood samples were collected from 20 farms in 2016. The goat farms in Ulsan city had three different types of rearing methods: conventional, confined, and mixed grazing-confined. Results: Forty-nine of the 452 goats (10.8%) were anaplasmosis positive. Sequence analysis of the PCR products from these 49 goats revealed that 39 of 452 goats (8.6%) were Anaplasma bovis positive, and 10 of 452 goats (2.2%) were infected with Anaplasma capra. The highest outbreaks of anaplasmosis occurred in mixed grazing-confined type of farms (27.1%, 33/122) (χ2 = 60.72, df = 2, p < 0.05), but there was no significant difference in the occurrence of anaplasmosis between spring, summer, and fall seasons. Conclusions: This study was the first detection of A. bovis in Korean native goats and its relationship with rearing methods and seasons. These findings suggested that Korean native goats were highly exposed to Anaplasma spp. during summers when the tick population is the highest and in farms employing mixed grazing-confined rearing methods.

Novel variants of the newly emerged Anaplasma capra from Korean water deer (Hydropotes inermis argyropus) in South Korea

BackgroundAnaplasma spp. are tick-borne Gram-negative obligate intracellular bacteria that infect humans and a wide range of animals. Anaplasma capra has emerged as a human pathogen; however, little is known about the occurrence and genetic identity of this agent in wildlife. The present study aimed to determine the infection rate and genetic profile of this pathogen in wild animals in the Republic of Korea.MethodsA total of 253 blood samples [198 from Korean water deer (Hydropotes inermis argyropus), 53 from raccoon dogs (Nyctereutes procyonoides) and one sample each from a leopard cat (Prionailurus bengalensis) and a roe deer (Capreolus pygargus)] were collected at Chungbuk Wildlife Center during the period 2015–2018. Genomic DNA was extracted from the samples and screened for presence of Anaplasma species by PCR/sequence analysis of 429 bp of the 16S rRNA gene marker. Anaplasma capra-positive isolates were genetically profiled by amplification of a longer fragment of 16S rRNA (rrs) as well as partial sequences of citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4). Generated sequences of each gene marker were aligned with homologous sequences in the database and phylogenetically analyzed.ResultsAnaplasma capra was detected in blood samples derived from Korean water deer, whereas samples from other animal species were negative. The overall infection rate in tested samples was 13.8% (35/253) and in the water deer the rate was 17.8% (35/198), distributed along the study period from 2015 to 2018. Genetic profiling and a phylogenetic analysis based on analyzed gene markers revealed the occurrence of two distinct strains, clustered in a single clade with counterpart sequences of A. capra in the database.ConclusionsAnaplasma capra infection were detected in Korean water deer in the Republic of Korea, providing insight into the role of wildlife as a potential reservoir for animal and human anaplasmosis. However, further work is needed in order to evaluate the role of Korean water deer as a host/reservoir host of A. capra.

First detection and molecular identification of the zoonotic Anaplasma capra in deer in France.

Cervids are known to be reservoirs of zoonotic bacteria transmitted by ticks. This study aimed to identify the Anaplasma species carried by captive red deer and swamp deer in a wild fauna reserve in France. Blood from 59 red deer and 7 swamp deer was collected and analyzed over a period of two years. A semi-nested PCR targeting the 23S rRNA was performed to detect and characterize Anaplasma spp. and determine the presence of zoonotic species. Anaplasma phagocytophilum was identified in 14/59 red deer (23.7%) but it was not identified in any of the swamp deer (7 animals). Three sequences could not be assigned to any particular species based on the 23S rRNA sequences. Complementary nested PCR targeting 16S rRNA, gltA and groEL genes and sequencing analysis then identified these sequences as a recently reported zoonotic species, Anaplasma capra; this species was found in 2 red deer (Cervus elaphus) and 1 swamp deer (Rucervus duvaucelii). This is the first report of the tick-borne zoonotic bacterium A. capra in France, a species otherwise described only in China, Japan, Malaysia and South Korea in goats, sheep, deer, cattle and Japanese serows (Capricornis crispus). While this bacterium may have been introduced into the reserve by infected imported animals, its local epidemiological cycle via tick transmission seems possible as locally born deer were found infected. Diagnostic methods, especially molecular ones, should take into account the potential infection of animals and humans with this species.