Circulation of four species of Anaplasmataceae bacteria in ticks in Harbin,northeastern China

Ticks play an important role in the evolution and transmission of Anaplasmataceae bacteria which are agents of emerging infectious diseases. In this study, a total of 1286 adult ticks belonging to five species were collected from cattle, goats, horses and vegetation in Harbin area, Heilongjiang province, northeastern China. The tick-borne Anaplasmataceae bacteria were identified by amplifying and sequencing the 16S rRNA (rrs) and heat shock protein-60 encoding (groEL) genes. The results showed that Ixodes persulcatus was dominant (38.8%, 499/1283) among the five tick species, and Anaplasmataceae bacteria were detected in all tick species with an overall prevalence of 7.4%. Four species of Anaplasmataceae bacteria (Anaplasma phagocytophilum, Anaplasma ovis, Anaplasma bovis, and “Candidatus Neoehrlichia mikurensis”), which are pathogenic to humans and/or animals, were identified from tick samples by phylogenetic analyzes of the rrs and groEL gene sequences. Interestingly, the cluster 1 strains were first identified in Asian, and a novel cluster was also detected in this study. These data revealed the genetic diversity of Anaplasmataceae bacteria circulating in ticks in Harbin area, highlighting the need to investigate these tick-borne pathogens and their risks to human and animal health.     

Molecular detection of Rickettsia,Anaplasma, and Bartonella in ticks from free-ranging sheep in Gansu Province,China

Ticks pose a serious threat to public health as carriers and often vectors of zoonotic pathogens. There are few systematic studies on the prevalence and genetic diversity of tick-borne bacterial pathogens in Western China. In this study, 465 ticks were collected from free-ranging sheep in Gansu Province in China. Ticks were divided into 113 pools and tick DNA was extracted from these ticks. PCR assays were performed using specific primers to screen for tick-borne pathogens as well as sequence analysis based on the 16S rRNA (rrs), ompB, gltA, ompA genes for Rickettsia, rrs, groEL genes for Anaplasma, and ssrA and rpoB genes for Bartonella. The PCR results showed that the minimum infection rates with Rickettsia, Anaplasma, and Bartonella were 16.8% (78/465), 18.9% (88/465), and 0.9% (4/465), respectively. Sequence analysis based on the concatenated sequences of rrs-ompB-gltA-ompA indicated that the Rickettsia species identified in the ticks belonged to Rickettsia raoultii, Rickettsia slovaca, and Rickettsia sibirica, respectively; phylogenetic analysis based on the groEL gene showed that all Anaplasma strains identified were Anaplasma ovis; and phylogenetic analysis based on the ssrA and rpoB genes indicated that all Bartonella strains in the ticks belonged to Bartonella melophagi. The results of this study showed that ticks in Gansu Province harbored multiple pathogens that may cause rickettsial diseases and bartonellosis. These diseases were neglected in the area and physicians and public health workers need to pay attention to their diagnoses to prevent human infection.     

Genetic diversity and antigenicity variation of Babesia bovis merozoite surface antigen-1 (MSA-1) in Thailand

Babesia bovis, an intraerythrocytic protozoan parasite, causes severe clinical disease in cattle worldwide. The genetic diversity of parasite antigens often results in different immune profiles in infected animals, hindering efforts to develop immune control methodologies against the B. bovis infection. In this study, we analyzed the genetic diversity of the merozoite surface antigen-1 (msa-1) gene using 162 B. bovis-positive blood DNA samples sourced from cattle populations reared in different geographical regions of Thailand. The identity scores shared among 93 msa-1 gene sequences isolated by PCR amplification were 43.5–100%, and the similarity values among the translated amino acid sequences were 42.8–100%. Of 23 total clades detected in our phylogenetic analysis, Thai msa-1 gene sequences occurred in 18 clades; seven among them were composed of sequences exclusively from Thailand. To investigate differential antigenicity of isolated MSA-1 proteins, we expressed and purified eight recombinant MSA-1 (rMSA-1) proteins, including an rMSA-1 from B. bovis Texas (T2Bo) strain and seven rMSA-1 proteins based on the Thai msa-1 sequences. When these antigens were analyzed in a western blot assay, anti-T2Bo cattle serum strongly reacted with the rMSA-1 from T2Bo, as well as with three other rMSA-1 proteins that shared 54.9–68.4% sequence similarity with T2Bo MSA-1. In contrast, no or weak reactivity was observed for the remaining rMSA-1 proteins, which shared low sequence similarity (35.0–39.7%) with T2Bo MSA-1. While demonstrating the high genetic diversity of the B. bovis msa-1 gene in Thailand, the present findings suggest that the genetic diversity results in antigenicity variations among the MSA-1 antigens of B. bovis in Thailand.     

Genetic variations in merozoite surface antigen genes of Babesia bovis detected in Vietnamese cattle and water buffaloes

The genes that encode merozoite surface antigens (MSAs) in Babesia bovis are genetically diverse. In this study, we analyzed the genetic diversity of B. bovis MSA-1, MSA-2b, and MSA-2c genes in Vietnamese cattle and water buffaloes. Blood DNA samples from 258 cattle and 49 water buffaloes reared in the Thua Thien Hue province of Vietnam were screened with a B. bovis-specific diagnostic PCR assay. The B. bovis-positive DNA samples (23 cattle and 16 water buffaloes) were then subjected to PCR assays to amplify the MSA-1, MSA-2b, and MSA-2c genes. Sequencing analyses showed that the Vietnamese MSA-1 and MSA-2b sequences are genetically diverse, whereas MSA-2c is relatively conserved. The nucleotide identity values for these MSA gene sequences were similar in the cattle and water buffaloes. Consistent with the sequencing data, the Vietnamese MSA-1 and MSA-2b sequences were dispersed across several clades in the corresponding phylogenetic trees, whereas the MSA-2c sequences occurred in a single clade. Cattle- and water-buffalo-derived sequences also often clustered together on the phylogenetic trees. The Vietnamese MSA-1, MSA-2b, and MSA-2c sequences were then screened for recombination with automated methods. Of the seven recombination events detected, five and two were associated with the MSA-2b and MSA-2c recombinant sequences, respectively, whereas no MSA-1 recombinants were detected among the sequences analyzed. Recombination between the sequences derived from cattle and water buffaloes was very common, and the resultant recombinant sequences were found in both host animals. These data indicate that the genetic diversity of the MSA sequences does not differ between cattle and water buffaloes in Vietnam. They also suggest that recombination between the B. bovis MSA sequences in both cattle and water buffaloes might contribute to the genetic variation in these genes in Vietnam.     

Type-specific PCR assays for Babesia bovis msa-1 genotypes in Asia: Revisiting the genetic diversity in Sri Lanka,Mongolia, and Vietnam

Babesia bovis is the most virulent Babesia organism, resulting in a high mortality rate in cattle. The genetic diversity of B. bovis merozoite surface antigens (MSAs), such as MSA-1, MSA-2b, and MSA-2c, might be linked to altered immune profiles in the host animals. The present study aimed to develop type-specific PCR assays for Asian msa-1 genotypes, thereby re-analyzing the genetic diversity of msa-1 in Sri Lanka, Mongolia, and Vietnam. Specific primers were designed for nine Asian msa-1 genotypes, which had been detected based on the phylogeny constructed using msa-1 gene sequences retrieved from the GenBank database. Specificity of the type-specific PCR assays was confirmed using plasmids containing the inserts of msa-1 gene fragments that represent Asian genotypes. Furthermore, no amplicons were observed by these PCR assays when DNA samples of Babesia bigemina, Babesia ovata, Theileria annulata, Theileria orientalis, Trypanosoma evansi, Trypanosoma theileri, Anaplasma marginale, and Anaplasma bovis, and non-infected bovine blood were analyzed. In total, 109 B. bovis-positive blood DNA samples sourced from Sri Lanka (44 cattle), Mongolia (26 cattle), and Vietnam (23 cattle and 16 water buffaloes) were then screened by the type-specific PCR assays. The sequences derived from all of the PCR amplicons were phylogenetically analyzed. Out of 109 DNA samples, 23 (20 from cattle and 3 from water buffaloes) were positive for at least one genotype. In agreement with previous studies, five and four different genotypes were detected among the DNA samples from Sri Lanka and Vietnam, respectively. In contrast, four genotypes, including three novel genotypes, were detected from Mongolia. Five DNA samples were found to be co-infected with multiple genotypes. The sequences of the PCR amplicons clustered phylogenetically within the corresponding clades. These findings indicated that the type-specific PCR assays described herein are useful for the determination of genotypic diversity of the B. bovis msa-1 gene in Asia.     

First molecular detection and genetic analysis of Anaplasma phagocytophilum in shelter cats in Seoul,Korea

Here, we report the molecular detection of Anaplasma phagocytophilum in shelter cats in Korea and the relationships between A. phagocytophilum gene sequences and the pathogenicity, region, and host specificity of this bacterium. Two (0.9%) out of 222 shelter cats from Seoul, Korea, yielded positive results for the A. phagocytophilum 16S rRNA, groEL, and msp2 genes. Phylogenetic analysis divided groEL gene sequences into two groups (alanine and serine), based on their nucleotide and amino acid sequences. A. phagocytophilum msp2 gene sequences were grouped per the region of isolation (Europe vs. USA, including Korea). Some nucleotide and amino acid sequences of groEL and msp2 showed distinctive patterns according to the region of isolation, which helped in distinguishing A. phagocytophilum gene sequences detected in Korea from those detected in the USA and Europe. Although the limited number of clinical anaplasmosis cases caused by A. phagocytophilum belonging to the alanine group prevents any firm conclusions, the results of the present study tend to refute the previous view that the pathogenicity of A. phagocytophilum is associated with the serine group. Moreover, our results suggest that genetic analyses of groEL and msp2 can be used to obtain a regional fingerprint of A. phagocytophilum.     

Molecular detection and genetic characterization of Anaplasma marginale and Anaplasma platys in cattle in Nigeria

Bovine anaplasmosis poses serious challenge to profitable livestock production in the tropics. Accurate information on the prevalence, distribution and genetic characteristics of Anaplasma spp. infections of cattle is invaluable for the design of cost-effective control measures. Blood samples from 275 cattle in Nigeria were screened for the DNA of Anaplasma spp. using species-specific primers and nucleotide sequence analysis. The DNA of Anaplasmataceae was detected based on 16S rRNA gene in 135 out of the 275 (49.1%) individuals examined, with 31 (23.0%) and 21(15.6%) being positive for Anaplasma marginale based on msp4 and msp2 genes, respectively. DNA of Anaplasma platys was detected in 62 (45.9%) based on groEL gene and in 27 (20.0%) using the A. platys species-specific primers. Presence of Anaplasma spp. DNA was significantly associated (p = 0.011) with the breed of the animals. Anaplasma nucleotide sequences of one group of the infected samples showed high identities of 99.0 to 100% (16S rRNA gene) and 99.6% (groEL gene) with reference sequences of A. platys, while those of another group matched to A. marginale references (msp2 with 98.9% and msp4 with 99.1%). Furthermore, phylogenetic analysis clustered the nucleotide sequences in this study with A. platys and A. marginale sequences in GenBank, confirming these relationships. For the first time, this study revealed the presence of mixed haplotypes in both A. platys and A. marginale in cattle in Nigeria. More studies are needed to elucidate the epidemiology and veterinary and public health significance of Anaplasma spp. infections in cattle in Nigeria.     

Anaplasma platys-like strains in ruminants from Tunisia

Molecular diagnosis of Anaplasma platys and related strains (A. platys–like) in carnivores and ruminants is challenging due to co-infections with cross-reacting strains, and require post-amplification sequencing of the hemi-nested PCR products traditionally generated by targeting the groEL gene. In this study, a Restriction Enzyme Fragment Length Polymorphism (RFLP) assay coupled to hemi-nested groEL PCR was developed to discriminate among A. platys and genetically related strains. This novel approach was used for investigating A. platys-like infection in 963 domesticated ruminants (241 goats, 355 sheep, and 367 cattle) from 22 delegations located in North Tunisia. Overall prevalence rates of A. platys-like were 22.8, 11, and 3.5% in goats, sheep, and cattle, respectively. Alignment, identity comparison, and phylogenetic analysis of the groEL sequence variants obtained in this study confirmed RFLP data suggesting that Tunisian ruminants are infected by novel unclassified Anaplasma strains genetically related to A. platys. Compared to sequencing, RFLP assay allows fast detection of A. platys and A. platys-like pathogens in the same sample and has a potential value especially when screening ticks, cats and ruminants, which can be a common host for these two bacteria. This newly developed molecular technique would provide valuable molecular tool for epidemiological studies related to A. platys as well as remove concern over specificity of serological and molecular methods routinely used to identify diverse Anaplasma strains and species in wild and domestic ruminants.     

Molecular detection and characterization of tick-borne parasites in goats and ticks from Thailand

Ticks and tick-borne pathogens (TTBPs) pose a serious economic threat to ruminant production worldwide. Despite this, investigations focused on goats remain limited compared to those for pathogens infecting cattle. We carried out PCR-based surveys and phylogenetic analyses to examine TTBPs from 6 provinces in Thailand between January 2016 and June 2020. A total of 93 tick samples were collected as well as 969 blood samples from goats. All ticks were morphologically identified as Rhipicephalus microplus and confirmed for species based on 16S rRNA and cox1 gene sequences. The mitochondrial cox1 sequences in the present study were clustered into clades A and C. The overall infection rates of Anaplasma spp., piroplasmids, and co-infections of both parasites in goats were 13.5% (131/969), 2.7% (24/880), and 0.7% (7/969), respectively. We observed no statistically significant association between TTBP infections and age or sex. However, TTBP infections and the rainy season were linked (p < 0.05). Anaplasma bovis, Anaplasma marginale, and Anaplasma ovis were detected for the first time in goats in the country using primers targeting the chaperonin GroEL (groEL), major surface protein 2 (msp2), and major surface protein 4 (msp4) genes, while Anaplasma capra and Anaplasma phagocytophilum were not detected. Anaplasma bovis, A. marginale, and A. ovis isolates were clustered in a subclade that differed from the strains found in other countries. Among piroplasmids, only Theileria luwenshuni was detected in the current investigation. This work will add to the current understanding regarding the prevalence, genetic diversity, and genetic relationships of A. bovis, A. marginale, A. ovis, and T. luwenshuni among global isolates and those in Thailand.     

Multi-locus typing scheme for Babesia bovis and Babesia bigemina reveals high levels of genetic variability in strains from Northern Argentina

Bovine babesiosis, caused by the protozoa Babesia bovis and Babesia bigemina, is a tick-borne disease distributed in tropical regions worldwide. Current control measures are based on the use of acaricides and live attenuated vaccines. The major economic impact of babesiosis lies in the cattle industry.In order to gain insight into the extent of genetic diversity in populations of parasites in the field, we developed two MLST schemes for the molecular genotyping of B. bigemina and B. bovis. We have also developed a custom-designed bioinformatic pipeline to facilitate the automated processing of raw sequences and further diversity and phylogenetic analysis.The overall MLST scheme exhibited the maximum discriminatory power (Simpson Index = 1) for B. bovis and a high level of discrimination for B. bigemina (Simpson Index = 0.9545). Genetic diversity was very high and infections with multiple genotypes were frequently found for both parasites in outbreak samples from the Northeast and Northwest of Argentina. Recombination events, which could have arisen from these multiple infections, were suggested by intra-loci linkage disequilibrium analysis and the lack of congruence in phylogenetic trees from individual genes.The two MLST schemes developed here are a robust, objective and easily adoptable technology to analyze the genetic diversity and population structure of parasites of the genus Babesia.     

Novel Ehrlichia and Hepatozoon genotypes in white-eared opossums (Didelphis albiventris) and associated ticks from Brazil

White-eared opossums (Didelphis albiventris) are well adapted to anthropized areas. The increased contact with domestic animals and humans mediates the transmission of arthropod-borne pathogens. Despite the worldwide occurrence of tick-borne Anaplasmataceae and Hepatozoidae species in a variety of vertebrates, few studies reported serological evidence or molecular detection of theses agentes in marsupials. Up to now, while Ehrlichia/Anaplasma spp. have only been detected in marsupials from Brazil, Hepatozoon spp. have been reported in marsupials from Chile, Australia and Brazil. The present work aimed to investigate, using molecular techniques and blood smear analysis, the presence of Ehrlichia spp., Anaplasma spp., and Hepatozoon sp. in the blood and ticks collected from D. albiventris in urban forest fragments from midwestern Brazil. Between May and December 2017, 43 D. albiventris (27 males and 16 females) were captured for blood and tick collection in the city of Campo Grande, state of Mato Grosso do Sul, midwestern Brazil. Ticks (46 Amblyomma dubitatum nymphs and 24 Amblyomma spp. larvae) were collected from 14 out 43 (32.5%) of the white-eared opossums. Panoptic-stained blood smears were performed using peripheral blood (tail tip) of the captured opossums. DNA extracted from blood and tick samples were subjected to PCR/qPCR assays for Anaplasmataceae agents (rrs, gltA, groEL, sodB, and dsb genes, and 23S-5S intergenic region) and Hepatozoon spp. (18S rRNA gene), followed by Sanger sequencing, BLASTn and phylogenetic analyses. An inclusion resembling Ehrlichia morulae was found in a white-eared opossum's monocyte from a blood smear stained with Panoptic. Five (11.63% [5/43]) white-eared opossums’ blood samples and 7 (25% [7/28]) tick samples (2 pools of Amblyomma spp. larvae and 5 pools of A. dubitatum nymphs) were positive for Anaplasmataceae via a PCR assay targeting the conserved rrs gene. Phylogenetic analysis based on the rrs gene positioned three sequences obtained from opossums and ticks together as a subclade within the Ehrlichia canis clade. However, all samples were negative in a qPCR assay specific for E. canis based on the dsb gene. Phylogenetic analyses positioned the gltA and 23S-5S ITS sequences obtained from opossums’ blood samples in a separate clade from the other validated Ehrlichia species. One (2.3% [1/43]) opossum blood sample was positive for the 18S rRNA gene of Hepatozoon sp. The phylogenetic analysis positioned the Hepatozoon sp. sequence obtained from a D. albiventris specimen in a clade with a sequence previously detected in a black storm petrel (Oceanodroma melania) from Mexico. All the other sequences of Hepatozoon sp. previously detected in marsupials from Brazil were positioned in a separated clade. The present work showed the occurrence of putative novel genotypes of Ehrlichia sp. and Hepatozoon sp. in white-eared opossums and associated A. dubitatum ticks from midwestern Brazil.     

Using msa-2b as a molecular marker for genotyping Mexican isolates of Babesia bovis

Variable merozoite surface antigens of Babesia bovis are exposed glycoproteins having a role in erythrocyte invasion. Members of this gene family include msa-1 and msa-2 (msa-2c, msa-2a₁, msa-2a₂ and msa-2b). To determine the sequence variation among B. bovis Mexican isolates using msa-2b as a genetic marker, PCR amplicons corresponding to msa-2b were cloned and plasmids carrying the corresponding inserts were purified and sequenced. Comparative analysis of nucleotide and deduced amino acid sequences revealed distinct degrees of variability and identity among the coding gene sequences obtained from 16 geographically different Mexican B. bovis isolates and a reference strain. Clustal-W multiple alignments of the MSA-2b deduced amino acid sequences performed with the 17 B. bovis Mexican isolates, revealed the identification of three genotypes with a distinct set each of amino acid residues present at the variable region: Genotype I represented by the MO7 strain (in vitro culture-derived from the Mexico isolate) as well as RAD, Chiapas-1, Tabasco and Veracruz-3 isolates; Genotype II, represented by the Jalisco, Mexico and Veracruz-2 isolates; and Genotype III comprising the sequences from most of the isolates studied, Tamaulipas-1, Chiapas-2, Guerrero-1, Nayarit, Quintana Roo, Nuevo Leon, Tamaulipas-2, Yucatan and Guerrero-2. Moreover, these three genotypes could be discriminated against each other by using a PCR–RFLP approach. The results suggest that occurrence of indels within the variable region of msa-2b sequences can be useful markers for identifying a particular genotype present in field populations of B. bovis isolated from infected cattle in Mexico.     

Molecular detection and phylogeny of Anaplasma spp. closely related to Anaplasma phagocytophilum in small ruminants from China

The genus Anaplasma comprises eight bacterial species that are obligate intracellular pathogens that affect human and animal health. The zoonotic species A. phagocytophilum is the causative agent of tick-borne fever in ruminants, and of granulocytic anaplasmosis in horses, dogs, and humans. Recently, novel strains related to A. phagocytophilum (A. phagocytophilum-like 1/Japanese variant and A. phagocytophilum-like 2/Chinese variant) have been identified. The aim of this study was to reveal the prevalence and phylogeny of A. phagocytophilum and related stains in small ruminants and ticks in China based on sequences of the 16S rRNA combined restriction fragment length polymorphism (RFLP) and groEL genes. PCR–RFLP and phylogenetic analyses based on the 16S rRNA gene showed the presence of A. phagocytophilum-like 1 and 2 variants in sampled animals from China, with prevalence rates of 22.6% (303/1338) and 0.7% (10/1338), respectively. Only A. phagocytophilum-like 1 DNA was found in Haemaphysalis longicornis. The phylogeny based on the groEL gene showed inclusion of A. phagocytophilum-like 1 and some A. phagocytophilum-like 2 strains in two unique clades distinct from, but related to, Japanese and Chinese strains of related A. phagocytophilum, respectively. One noteworthy result was that the SSAP2f/SSAP2r primers detected Ehrlichia spp. strains. Moreover, the A. phagocytophilum-like 1 and 2 strains should be considered in the differential diagnosis of caprine and ovine anaplasmosis. Further investigations should be conducted to provide additional epidemiological information about A. phagocytophilum and A. phagocytophilum-like variants in animals and ticks.     

Rickettsiaceae in two reptile-associated tick species,Amblyomma exornatum and Africaniella transversale: First evidence of Occidentia massiliensis in hard ticks (Acari: Ixodidae)

All species of hard ticks associated with reptiles as hosts throughout their life cycle, are currently assigned to genera including Amblyomma and Africaniella. Among these species, based on literature data, Africaniella transversale has never been investigated for the presence of tick-borne pathogens. In this study, seven DNA extracts (two from A. transversale and five from Amblyomma exornatum) were screened for the presence of important tick-borne protozoa (piroplasms) and bacteria (Anaplasmataceae and Rickettsiaceae) with conventional PCRs and sequencing. A new heat shock protein chaperonin (groEL) gene-specific PCR was also developed to identify Occidentia spp. in these samples.In A. transversale, Occidentia massiliensis (previously detected in rodent-associated soft ticks) and Rickettsia hoogstraalii were present. While the latter was molecularly identical with formerly reported sequences of this rickettsia, the genotype of O. massiliensis was new based on sequence and phylogenetic analyses of its groEL gene. In A. exornatum, a Rickettsia genotype closely related to R. tamurae and R. monacensis, was detected. The ompA sequence of this genotype was identical to that of Rickettsia sp. Ae-8 reported from A. exornatum in a reptile breeding facility in the USA.These results show that A. transversale might carry O. massiliensis which (unless having a symbiotic nature in ticks) may originate either from the reptile host of this hard tick species or the rodent prey of reptiles. This is also the first detection of the reptile tick-associated Rickettsia sp. Ae-8 (phylogenetically aligning with R. tamurae, R. monacensis) in Africa, i.e. within the original geographical range of A. exornatum.     

Anaplasma phagocytophilum in European bison (Bison bonasus) and their ticks from Lithuania and Poland

The increasing population of European bison (Bison bonasus) can contribute to the prevalence of zoonotic pathogens. The aim of the present study was to assess the presence of A. phagocytophilum infection in European bison tissues as well as ticks removed from European bison in Lithuania and Poland. A further objective of this work was to compare the detected A. phagocytophilum strains. A total of 85 tissue samples (spleen) of European bison and 560 ticks belonging to two species, Ixodes ricinus (n = 408) and Dermacentor reticulatus (n = 152) were tested. DNA of A. phagocytophilum was detected based on RT-PCR in 40% of the European bison samples, 8.8% of the I. ricinus and 5.9% of the D. reticulatus ticks. Analysis of the obtained partial 16S rRNA gene sequences of A. phagocytophilum revealed the presence of three variants with two polymorphic sites. Furthermore, phylogenetic analysis with partial msp4 gene sequences grouped A. phagocytophilum variants into three clusters. This study revealed that the groEL gene sequences of A. phagocytophilum from European bison and their ticks grouped into ecotype I and only one sequence from Lithuanian European bison belonged to ecotype II. The results of the present study indicated that European bison may play a role as a natural reservoir of A. phagocytophilum.     

Molecular survey of Rickettsia,Ehrlichia, and Anaplasma infection of domestic cats in Japan

The prevalence of Rickettsia, Ehrlichia, and Anaplasma in 1764 DNA samples extracted from feline peripheral blood from all 47 prefectures in Japan was evaluated by screening real-time PCR, genus-specific PCR, and DNA nucleotide sequencing. The survey revealed that all cats were negative for Rickettsia infection. Two cats were positive for Ehrlichia or Anaplasma based on the screening PCR assay. Nucleotide sequence analysis of the partial 16S rRNA including the divergent region near the 3′-end revealed that the 2 positives were most similar to Anaplasma bovis with percent identities of 99.8% and 99.2%. This was the first detection of A. bovis DNA fragments in cats. Although these 2 cats showed stomatitis, both were also infected with feline immunodeficiency virus. The relationship between A. bovis carriage and clinical disease is not yet understood.     

Molecular detection and genetic characterization of Ehrlichia canis and Ehrlichia sp. in neotropical primates from Brazil

The Anaplasmataceae family includes obligate, arthropod-transmitted intracellular bacteria that can be zoonotic and potentially fatal. Studies focusing on the interaction between neotropical primates and the agents of this family are scarce. The present study aimed to identify agents of the Anaplasmataceae family in the whole blood of free-living and captive neotropical primates in the State of Mato Grosso, Central-West Brazil. Thirty-eight samples of six nonhuman primate (NHP) species were collected in seven municipalities and analysed through polymerase chain reaction (PCR), nucleotide sequencing, and phylogenetic analysis of the dsb, groEL, 16S rRNA, and gltA genes. DNA fragments similar to those of Ehrlichia canis were detected in Sapajus apella and Ehrlichia chaffeensis from Mico melanurus. The sequences generated in this study and homologous sequences retrieved from GenBank® were used for phylogenetic analyses to characterize the Ehrlichial agents detected in NHPs. The agents were then grouped into clades corresponding to different isolates from the NHP species. In addition, an Anaplasma sp. closely related to Anaplasma marginale was identified in two S. apella individuals. These findings shed light on the susceptibility of neotropical NHPs to Anaplasmataceae agents. These bacteria are known to be transmitted by ticks, which can also serve as possible sources of infection for other animals, including humans.     

First molecular survey and novel genetic variants’ identification of Anaplasma marginale,A. centrale and A. bovis in cattle from Tunisia

Few data are available about the presence and distribution of Anaplasma species in cattle in North African countries. In this study prevalence, co-infections, risk factors and genetic diversity of Anaplasma species were evaluated in bovines from Northern Tunisia. A total of 232 cattle from 36 randomly selected farms in three Tunisian localities were investigated for the presence of Anaplasma species in blood by Real-time PCR and/or nested PCR. Overall infection rates of Anaplasma spp., Anaplasma marginale, Anaplasma centrale and Anaplasma bovis were 34.9%, 25.4%, 15.1%, and 3.9%, respectively. Anaplasma phagocytophilum was not detected in cattle. The most common co-infection pattern was an association of A. marginale and A. centrale (11.2%). Five cattle (2.1%) all reared in the sub-humid bioclimatic area, were co-infected by the three Anaplasma species. Molecular prevalence of Anaplasma infection varied significantly according to locality, bioclimatic area, tick infestation and type of breeding. Animals of the Holstein breed were less infected by A. marginale and A. centrale than other breeds. Genetic analysis of A. marginale msp4 gene indicated a high sequence diversity of Tunisian strains, suggesting a multiple introduction of infected cattle from different origins. Phylogenetic studies based on the 16S rRNA gene showed that the most prevalent A. centrale strains were closely related to the A. centrale vaccine strain. Moreover, all A. bovis variants clustered with other A. bovis sequences obtained from domestic and wild ruminant strains. This is the first molecular investigation on Anaplasma species in Tunisian cattle providing pivotal background for designing epidemiological studies and to develop control strategies in the country.     

Phylogenetic analysis of Babesia species in China based on cytochrome b (COB) gene

In this study, a mitochondrial marker consisting of an approximately 550-bp region of the Cytochrome b genes (COB) was amplified by polymerase chain reaction (PCR) and sequenced from individual Babesia species. Sequence variation between Babesia species from China was 1.6–30.8%. The constructed phylogenetic tree based on the three unlinked gene sequences (partial COB gene, 18S rDNA and ITS) that evolve at different rates by the method of Neighbor-joining revealed the phylogenetic relationship of Babesia species in China compared with other published corresponding sequences from Babesia species. These data indicate that the 18S rDNA more reliably distinguish the deeper branches among some Babesia species than the partial COB gene and ITS, however, the partial COB gene sequence is better for recognizing close lineages among some Babesia species than the 18S rDNA and ITS sequences. So the combined phylogenetic analysis based on the multiple unlinked loci with different evolving rates can facilitate to establish the more reliable phylogenetic relationship of the Babesia genus. The data could be applicable for the survey of parasite dynamics, epidemiological studies as well as prevention and control of the disease.     

Genetic diversity of merozoite surface antigens in Babesia bovis detected from Sri Lankan cattle

Babesia bovis, the causative agent of severe bovine babesiosis, is endemic in Sri Lanka. The live attenuated vaccine (K-strain), which was introduced in the early 1990s, has been used to immunize cattle populations in endemic areas of the country. The present study was undertaken to determine the genetic diversity of merozoite surface antigens (MSAs) in B. bovis isolates from Sri Lankan cattle, and to compare the gene sequences obtained from such isolates against those of the K-strain. Forty-four bovine blood samples isolated from different geographical regions of Sri Lanka and judged to be B. bovis-positive by PCR screening were used to amplify MSAs (MSA-1, MSA-2c, MSA-2a1, MSA-2a2, and MSA-2b), AMA-1, and 12D3 genes from parasite DNA. Although the AMA-1 and 12D3 gene sequences were highly conserved among the Sri Lankan isolates, the MSA gene sequences from the same isolates were highly diverse. Sri Lankan MSA-1, MSA-2c, MSA-2a1, MSA-2a2, and MSA-2b sequences clustered within 5, 2, 4, 1, and 9 different clades in the gene phylograms, respectively, while the minimum similarity values among the deduced amino acid sequences of these genes were 36.8%, 68.7%, 80.3%, 100%, and 68.3%, respectively. In the phylograms, none of the Sri Lankan sequences fell within clades containing the respective K-strain sequences. Additionally, the similarity values for MSA-1 and MSA-2c were 40–61.8% and 90.9–93.2% between the Sri Lankan isolates and the K-strain, respectively, while the K-strain MSA-2a/b sequence shared 64.5–69.8%, 69.3%, and 70.5–80.3% similarities with the Sri Lankan MSA-2a1, MSA-2a2, and MSA-2b sequences, respectively. The present study has shown that genetic diversity among MSAs of Sri Lankan B. bovis isolates is very high, and that the sequences of field isolates diverged genetically from the K-strain.