Multi-locus sequence analysis of Anaplasma bovis in goats and ticks from Thailand, with the initial identification of an uncultured Anaplasma species closely related to Anaplasma phagocytophilum-like 1.

Ticks and tick-borne pathogens (TTBP) pose a serious threat to animal and human health globally. Anaplasma bovis, an obligatory intracellular bacterium, is one of the more recent species of the Family Anaplasmaceae to be formally described. Owing to its diminutive size, microscopic detection presents a formidable challenge, leading to it being overlooked in laboratory settings lacking advanced equipment or resources, as observed in various regions, including Thailand. This study aimed to undertake a genetic analysis of A. bovis and determine its prevalence in goats and ticks utilizing three genetic markers (16S rRNA, gltA, groEL). A total of 601 goat blood and 118 tick samples were collected from 12 sampling sites throughout Thailand. Two tick species, Haemaphysalis bispinosa (n = 109), and Rhipicephalus microplus (n = 9) were identified. The results herein showed that 13.8 % (83/601) of goats at several farms and 5 % (1/20) of ticks were infected with A. bovis. Among infected ticks, A. bovis and an uncultured Anaplasma sp. which are closely related to A. phagocytophilum-like 1, were detected in each of H. bispinosa ticks. The remaining R. microplus ticks tested positive for the Anaplasma genus. A nucleotide sequence type network showed that A. bovis originated from Nan and Narathiwat were positioned within the same cluster and closely related to China isolates. This observation suggests the potential dispersal of A. bovis over considerable distances, likely facilitated by activities such as live animal trade or the transportation of infected ticks via migratory birds. The authors believe that the findings from this study will provide valuable information about TTBP in animals.

Seasonal dynamics and genetic characterization of bovine arthropod-borne parasites in Nan Province, Thailand with molecular identification of Anaplasma platys and Trypanosoma theileri.

Virulent species or strains of hematophagous borne pathogens such as Anaplasma spp., Babesia spp., Theileria spp., and Trypanosoma spp., are lethal to susceptible animals or reduce their productivity on a global scale. Nonetheless, efforts to diagnose the causative agents and assess the genotypic profiles as well as quantify the parasite burden of aforementioned parasites across seasons remain limited. Therefore, the present investigation sought to elucidate the genotypic composition of Anaplasma spp., Babesia spp., Theileria spp., and Trypanosoma spp. The findings revealed heightened infection rates during the summer, manifesting a correlation between Trypanosoma spp. infection and seasonal fluctuations. Among the identified pathogens, Anaplasma marginale emerged as the most dominant species, while the occurrence of Anaplasma platys in Thai cattle was confirmed via the sequencing of the groEL gene. Moreover, the study successfully identified two lineages of Trypanosoma theileri. The findings of this investigation offer valuable insights that can inform the development of preventive strategies for vector-borne diseases, such as considering the appropriate use of insect repellent, mosquito or insect nets, or eliminating breeding places for insects in each season.

Myzomyia and Pyretophorus series of Anopheles mosquitoes acting as probable vectors of the goat malaria parasite Plasmodium caprae in Thailand.

Unlike malaria parasites in humans, non-human primates, rodents, and birds, ungulate malaria parasites and their vectors have received little attention. As a result, understanding of the hosts, vectors, and biology of ungulate malaria parasites has remained limited. In this study, we aimed to identify the vectors of the goat malaria parasite Plasmodium caprae. A total of 1019 anopheline and 133 non-anopheline mosquitoes were collected from goat farms in Thailand, where P. caprae-infected goats were discovered. Anopheline mosquitoes were identified using molecular biological methods that target the cytochrome c oxidase subunit 1 (cox1), the cytochrome c oxidase subunit 2 (cox2) genes, and the internal transcribed spacer 2 (ITS2) region. Pool and individual mosquitoes were tested for P. caprae using the head-thorax parts that contain the salivary glands, with primers targeting three genetic markers including cytochrome b, cytochrome c oxidase subunit 1, and 18S small subunit ribosomal RNA genes. Additionally, goat blood samples were collected concurrently with mosquito surveys and screened to determine the status of malaria infection. This study revealed nine mosquito species belonging to six groups on goat farms, including Hyrcanus, Barbirostris, Subpictus, Funestus, Tessellatus, and Annularis. The DNA of P. caprae was detected in Anopheles subpictus and Anopheles aconitus. This is the first time An. subpictus and An. aconitus have been implicated as probable vectors of P. caprae.

Molecular characterization of anopheline mosquitoes from the goat malaria-endemic areas of Thailand.

Despite the fact that over a 100 anopheline mosquito species have been identified as human malaria vectors, little is known about ungulate malaria vectors. Consequently, we focused on investigating the bionomics and genetic characterizations of anopheline mosquitoes in goat malaria-endemic regions. We also attempted to screen for ungulate malaria potential vectors. A total of 1019 female anopheline mosquitoes were collected from six goat farms in four provinces of Thailand from 2020 to 2021. Mosquitoes were morphologically identified and subsequently confirmed using the mitochondrial DNA barcoding region-cytochrome oxidase c subunit I (MtDNA-COI), mitochondrial DNA-cytochrome c oxidase subunit II (MtDNA-COII), and ribosomal DNA internal transcribed spacer 2 (rDNA-ITS2) sequences. The current study reveals the genetic characteristics and distribution of nine mosquito species within the Anopheles and Cellia subgenera. Four dominant species, including Anopheles peditaeniatus, Anopheles subpictus, Anopheles vagus, and Anopheles aconitus exhibited significant intraspecific gene flow within their corresponding species. Although malaria parasites were not found in 126 mosquito pools, meaning more investigation is necessary, the current study adds to the existing DNA barcoding data collection and improves the current understanding of the genetic structure and distribution of anopheline mosquito species, which could be useful for effective control of mosquito-borne diseases.

Genetic characterization of genes encoding the major surface proteins of Anaplasma marginale from cattle isolates in Thailand reveals multiple novel variants.

Bovine anaplasmosis is a serious tick-borne disease that is responsible for economic loss worldwide. The major surface proteins (MSPs), encoded by msp1 to msp5 genes of Anaplasma marginale, play an important role in host-pathogen and tick-pathogen interactions. These markers have been used for genetic characterization and phylogenetic studies. Despite domestic reports concerning suspected outbreaks of anaplasmosis in Thailand, genetic analysis of A. marginale in the country remains largely limited. Therefore, we aim to investigate the infection rate of the rickettsia organism in the Anaplasmataceae family throughout five regions of Thailand and to further characterize the key genetic markers: msp1a, msp2, and msp5 of A. marginale. From 2016 to 2021, we collected a total of 384 cattle blood samples across 18 provinces. Overall, the infection rate of the rickettsia organism in the Anaplasmataceae family was 46.1%. Over 65% of the positive samples were confirmed as A. marginale. We successfully obtained a total of 138 A. marginale msp1a (38), msp2 (79), and msp5 (21) sequences from all regions of the country. The msp1a and msp2 genes exhibit a high degree of genetic diversity, while the msp5 gene is highly conserved among the Thai isolates. Our findings regarding msp1a corroborated the genetic heterogeneity of A. marginale strains in endemic regions worldwide. Additionally, we found multiple novel variants for the first time in the current nationwide survey. We found 45 tandem repeat characters of the msp1a sequence. Among them, 24 characters were not shared with other countries. Collectively, we expanded the extent of genetic diversity in key markers; msp1a and msp2 genes, and further confirmed the previous finding that msp5 was highly conserved. The msp1a and msp2 genes could be useful for the surveillance of newly introduced strains. The current data may also be useful in designing a vaccine containing potential epitopes of different antigens in the future.

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.

Development and application of a novel multiplex PCR assay for the differentiation of four haemosporidian parasites in the chicken Gallus gallus domesticus.

Haemosporidian infections in domestic chickens (Gallus gallus domesticus) are not only widely prevalent but also cause economic loss. Diagnosis is usually made by microscopic examination; however, the method has several drawbacks such as requiring an experienced microscopist, being unreliable when parasitemia is low and being unable to accurately differentiate between co-infections from multiple parasite species. Therefore, the current extent of haemosporidian infections might be underestimated and neglected. We have developed a novel multiplex PCR assay to simultaneously detect and differentiate between four haemosporidian parasites: Leucocytozoon caulleryi, Leucocytozoon sabrazesi, Plasmodium juxtanucleare and Plasmodium gallinaceum. Primers in the present study specifically amplified the corresponding targets with no cross-species amplification detected. The multiplex PCR exhibited a significantly greater detection rate when compared with microscopic examination (p = 0.0001). The results demonstrate that the detection rate of multiplex PCR for L. sabrazesi, P. juxtanucleare, and P. gallinaceum are all greater than that of microscopic examination with p = 0.002, 0.0001 and 0.004, respectively. Co-infections were also detected more effectively by multiplex PCR. We applied the current method to field samples originating from Nan, Prachinburi, and Chachoengsao Provinces. The current study revealed that positive rates of haemosporidian parasites in chickens in the three study sites ranging from 39.5%-93.8%. The present assay offers a timesaving option for molecular diagnosis instead of using singleplex PCRs for detecting the parasites individually. Within a single reaction, this assay would be a useful tool for the detection of avian haemosporidian parasites either single or under co-infection conditions and for large-scale epidemiology studies.

Author Correction: Genetic homogeneity of goat malaria parasites in Asia and Africa suggests their expansion with domestic goat host.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Genetic homogeneity of goat malaria parasites in Asia and Africa suggests their expansion with domestic goat host.

Plasmodium was first identified in a goat in Angola in 1923, and only recently characterized by DNA isolation from a goat blood sample in Zambia. Goats were first domesticated in the Fertile Crescent approximately 10,000 years ago, and are now globally distributed. It is not known if the Plasmodium identified in African goats originated from parasites circulating in the local ungulates, or if it co-evolved in the goat before its domestication. To address this question, we performed PCR-based surveillance using a total of 1,299 goat blood samples collected from Sudan and Kenya in Africa, Iran in west Asia, and Myanmar and Thailand in southeast Asia. Plasmodium DNA was detected from all locations, suggesting that the parasite is not limited to Africa, but widely distributed. Whole mitochondrial DNA sequences revealed that there was only one nucleotide substitution between Zambian/Kenyan samples and others, supporting the existence of a goat-specific Plasmodium species, presumably Plasmodium caprae, rather than infection of goats by local ungulate malaria parasites. We also present the first photographic images of P. caprae, from one Kenyan goat sample.