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.

Complete mitochondrial genome analyses confirm that bat Polychromophilus and ungulate Plasmodium constitute a distinct clade independent of other Plasmodium species.

In recent phylogenetic studies, bat Polychromophilus and ungulate Plasmodium, two relatively understudied haemosporidian parasites within the Apicomplexa phylum, have often been overlooked. Instead, the focus has been primarily on haemosporidian parasites in primates, rodents, and birds. Several phylogenetic analyses of bat Polychromophilus have relied on limited datasets and short informative DNA sequences. As a result of these inherent limitations, the substantiation of their evolutionary stance has encountered a diminished degree of robust validation. This study successfully obtained complete mitochondrial genome sequences from 11 Polychromophilus parasites originating from Hipposideros gentilis and Myotis siligoensis bats for the first time. Additionally, the authors have sequenced the apicoplast caseinolytic protease C genes from Polychromophilus murinus and a potentially new Polychromophilus species. These mitochondrial genomes range in length from 5994 to 6001 bp and consist of three protein-coding genes (PCGs), seven small subunit ribosomal RNA genes (SSU rRNA), 12 large subunit ribosomal RNA genes (LSU rRNA), and seven miscellaneous RNA genes. Phylogenetic analyses using Bayesian Inference and Maximum Likelihood methods indicated robust support for the grouping of ungulate Plasmodium and bat Polychromophilus in a single clade separate from other Plasmodium spp., confirming previous reports, albeit with stronger evidence in this study. The divergence between Polychromophilus in bats and Plasmodium in ungulates occurred approximately 29.61 to 55.77 million years ago (Mya), with a node age estimated at 40.63 Mya. These findings highlight that the genus Plasmodium, which includes species found in ungulates, birds, reptiles, and other mammals, does not form a monophyletic group. By incorporating Polychromophilus in bats and Plasmodium in ungulates, this study contributes significantly to understanding the phylogenetic relationships within the Haemosporida order. It provides valuable insights into the evolutionary history and interconnections among these diverse parasites, thereby expanding knowledge in this field.

Molecular and morphological identification of Lernaea spp. in Cyprinid fishes from two districts in Yogyakarta, Indonesia.

Background and Aim: Parasitic infection commonly affects freshwater ornamental fishes. Parasites in fish may impede their growth and even cause death, resulting in a decline in fecundity. The prevalence of lernaeosis in aquaculture ponds in Indonesia requires attention because of missing data, especially from Yogyakarta. Therefore, this study aimed to identify the Lernaea species found in fish in Indonesia, particularly in Yogyakarta, molecularly and morphologically, as well as an overview of their distribution and the water condition they inhabit. Materials and Methods: Lernaea species were collected from three different fish species in two districts of Yogyakarta, Indonesia, for precise identification. Lernaea specimens were characterized morphologically and subjected to molecular identification based on 18S rRNA and 28S rRNA genes. Results: Lernaea in this study was morphologically and genetically confirmed as Lernaea cyprinacea, and the infection rate in each fish species was different. Water conditions might have contributed to the differences in infection levels. Conclusion: This study characterized L. cyprinacea isolated from Yogyakarta. Future research should focus on sequencing as much molecular information as possible and carrying out more experimental infections.

Detection of Babesia bovis using loop-mediated isothermal amplification (LAMP) with improved thermostability, sensitivity and alternative visualization methods.

Bovine babesiosis is one of the most economically important tick-borne diseases in tropical and subtropical countries. A conventional microscopic diagnosis is typically used because it is inexpensive and expeditious. However, it is highly dependent on well-trained microscopists and tends to be incapable of detecting subpatent and chronic infections. Here, we developed a novel nucleic acid-based amplification method using loop-mediated isothermal amplification (LAMP) in conjunction with a colori-fluorometric dual indicator for the rapid and accurate detection of Babesia bovis based on the mitochondrial cytochrome b gene. We aimed to improve the thermostability, sensitivity, specificity, and alternative visualization of LAMP-based methods. We assessed its diagnostic performance compared to two conventional PCR agarose gel electrophoresis (PCR-AGE) methods. The thermostability of LAMP reaction mixtures and DNA templates in variable conditions was also assessed. In addition, we evaluated alternative visualization methods using different light sources including neon, LED, and UV lights. We found that the LAMP-neon was ten times more sensitive than the PCR-AGE, while the LAMP-LED and LAMP-UV were 1,000 times more sensitive. The current LAMP method showed no cross-amplification with uninfected cattle DNA or other common blood parasites in cattle, including Babesia bigemina, Theileria orientalis, Anaplasma marginale, and Trypanosoma evansi. In addition, the developed LAMP method has good thermostability and the potential for on-site utility as B. bovis DNA could still be detected up to 72 h after initial preparation. Our findings suggested that the developed LAMP method provides an alternative approach for B. bovis detection with sensitivity higher than PCR-AGE diagnostics, high specificity, and the flexibility to use neon, LED, and UV light sources for positive signal observations.

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 identification of cercaria Fasciola gigantica in lymnaeid snails in Kulon Progo, Yogyakarta.

Lymnaeid snails play an essential role in transmitting fasciolosis as intermediate hosts. Therefore, this study aims to use the molecular method to identify liver fluke in lymnaeid snails. A total of 320 lymnaeid snails were collected from a rice field. The samples were dissected to collect cercaria and identified using polymerase chain reaction. Furthermore, the internal transcribed spacer 2 (ITS2) was used as the target gene to identify the species of cercaria. The result showed that 3.75% (12/320) of the snails were infected by Fasciola gigantica, while the phylogenetic tree based on ITS2 showed that the cercaria in this study was monophyletic and similar to species from several countries in Southeast Asia, including China. Furthermore, the haplotype network showed that all four cercaria samples were similar with sequences from several countries. This study suggests that the F. gigantica cercaria isolated from lymnaeid snails in Kulon Progo, Yogyakarta, Indonesia, has a sequence similar to that of other species in Southeast Asian countries, although no hybrid type was detected in these sequences. This is the first report on the molecular identification of cercaria F. gigantica isolated from lymnaeid snails in Yogyakarta, Indonesia.

Myzorhynchus series of Anopheles mosquitoes as potential vectors of Plasmodium bubalis in Thailand.

Ungulate malaria parasites and their vectors are among the least studied when compared to other medically important species. As a result, a thorough understanding of ungulate malaria parasites, hosts, and mosquito vectors has been lacking, necessitating additional research efforts. This study aimed to identify the vector(s) of Plasmodium bubalis. A total of 187 female mosquitoes (133 Anopheles spp., 24 Culex spp., 24 Aedes spp., and 6 Mansonia spp. collected from a buffalo farm in Thailand where concurrently collected water buffalo samples were examined and we found only Anopheles spp. samples were P. bubalis positive. Molecular identification of anopheline mosquito species was conducted by sequencing of the PCR products targeting cytochrome c oxidase subunit 1 (cox1), cytochrome c oxidase subunit 2 (cox2), and internal transcribed spacer 2 (ITS2) markers. We observed 5 distinct groups of anopheline mosquitoes: Barbirostris, Hyrcanus, Ludlowae, Funestus, and Jamesii groups. The Barbirostris group (Anopheles wejchoochotei or Anopheles campestris) and the Hyrcanus group (Anopheles peditaeniatus) were positive for P. bubalis. Thus, for the first time, our study implicated these anopheline mosquito species as probable vectors of P. bubalis in Thailand.

Molecular identification and genetic diversity of Bartonella spp. in 24 bat species from Thailand.

The study of bacterial zoonoses has been under-pursued despite the fact that bacteria cause the majority of zoonotic diseases, of which 70% have a wildlife origin. More Bartonella species are being identified as the cause of human diseases, and several of them have been linked to domestic and wild animals. Bats are outstanding reservoirs for Bartonella species because of their wide distribution, mobility, roosting behaviour, and long life span. Here, we carried out a PCR-based survey on bats that were collected from 19 sampling sites in eight provinces of Thailand from February 2018 to April 2021. Bartonella infection was investigated in a total of 459 bats that belong to 24 different bat species (21 species of which had never been previously studied in Thailand). PCR diagnostics revealed that 115 out of 459 (25.5%) blood samples tested positive for Bartonella. The nucleotide identities of the Bartonella 16S rRNA sequences in this study were between 95.78-99.66% identical to those of known zoonotic species (Bartonella ancashensis, Bartonella henselae, Bartonella bacilliformis and Bartonella australis) as well as to an unidentified Bartonella spp. In addition, the citrate synthase (gltA) and RNA polymerase-beta subunit (rpoB) genes of Bartonella were sequenced and analyzed in positive samples. The gltA and rpoB gene sequences from Hipposideros gentilis and Rhinolophus coelophyllus bat samples showed low nucleotide identity (<95%) compared to those of the currently deposited sequences in the GenBank database, indicating the possibility of new Bartonella species. The phylogenetic inference and genetic diversity were generated and indicated a close relationship with other Bartonella species previously discovered in Asian bats. Overall, the current study demonstrates the primary evidence pointing to a potential novel Bartonella species in bats. This discovery also contributes to our current understanding of the geographical distribution, genetic diversity, and host ranges of bat-related Bartonella.

Development of a novel multiplex PCR assay for the detection and differentiation of Plasmodium caprae from Theileria luwenshuni and Babesia spp. in goats.

Intraerythrocytic parasites are traditionally identified by the microscopic examination of Giemsa-stained blood smears. However, this method does not always allow for the identification of individual species in goat's RBCs. Moreover, its unreliability in detecting low levels of parasitemia makes it unsuitable for epidemiological investigations and leaves goat farms vulnerable to potential outbreaks. In the present study, a novel multiplex PCR (mPCR) targeting the cytochrome c oxidase subunit I (COI) gene was developed to detect and subsequently differentiate Plasmodium caprae, Theileria luwenshuni, and Babesia spp. The specificity of each primer set was assessed both in silico and with a panel of DNA samples from the hosts themselves and other goat hemoparasites. Amplicons generated from each pair of primers were 664, 555, and 320-bp for P. caprae, Babesia spp., and T. luwenshuni, respectively. These products were further confirmed by sequencing. Our novel mPCR reactions successfully demonstrated the accurate and simultaneous amplification of the three parasites' DNA samples. The current mPCR method showed no cross-amplification with unintended targets. The detection limit of the mPCR in this study was 108 parasites' DNA copies per reaction. The current mPCR was able to detect the minimum parasitemia of approximately 0.001%, 0.000005%, 0.00001% for P. caprae, Babesia spp. and T. luwenshuni, respectively. The diagnostic specificity in the detection of P. caprae and T. luwenshuni ranged from 94.9 to 100 %. The mPCR was further applied to a collection of field blood samples from five provinces in Thailand to validate its reliability and applicability. The results demonstrated the successful detection of P. caprae, Babesia spp. and T. luwenshuni in goat samples with the same sensitivity levels as conventional PCR methods. This study also confirmed the presence of T. luwenshuni and Babesia spp. in Thai goats. The current mPCR method offers an alternative for the diagnosis of P. caprae, T. luwenshuni, and Babesia spp., either single or under co-infection conditions, and for large-scale surveillance.

Trematodiasis occurrence in cattle along the Progo River, Yogyakarta, Indonesia.

AIM: This study aimed to measure the occurrence of trematodiasis in cattle along the Progo River, a district of Yogyakarta, Indonesia. The findings help to establish the magnitude of the disease and encourage prevention and treatment of this condition. MATERIALS AND METHODS: Trematode eggs were extracted from 100 fecal samples collected from cattle. The eggs were examined using the sedimentation technique, and the method of Parfitt and Banks was used to differentiate Paramphistomum spp. eggs from Fasciola spp. eggs. RESULTS: The infection rate of trematode parasites was 50%. Cattle experienced multiple infections of both Paramphistomum spp. and Fasciola spp., as well as single infections of one species or the other. All breeds were vulnerable to infections of both trematode species, although different cattle breeds, including Peranakan Ongole crossbreeds, Simmental crossbreeds, and Limousin crossbreeds, showed differences in infection rate. The highest rate of infection with Paramphistomum spp. (15.78%) occurred in the Simmental crossbreeds. The highest rate of infection (31.57%) with Fasciola spp. was in the Peranakan Ongole crossbreeds. Multiple infections of both Paramphistomum spp. and Fasciola spp. were highest in Simmental crossbreed cattle (28.97%). CONCLUSION: The high infection rates of trematode parasites found in fecal samples, particularly of Fasciola spp., indicate that the cattle along the Progo River in Indonesia experience a high rate of trematodiasis disease.

Ticks (Acari: Ixodidae) infestation on cattle in various regions in Indonesia.

BACKGROUND AND AIM: Ticks (Ixodidae) not only cause blood loss in cattle but also serve as vectors for various diseases, thus causing direct and indirect losses. Moreover, tick infestation can cause significant economic losses. This study aimed to identify the diverse species of ticks infesting cattle in five different regions in Indonesia. MATERIALS AND METHODS: Tick specimens were obtained from local cattle in five different areas in Indonesia. The morphology of the specimens was macroscopically and microscopically evaluated, and the resulting data were descriptively and qualitatively analyzed. RESULTS: In total, 1575 ticks were successfully collected from 26 animals. In total, two genera and three species, namely, Rhipicephalus microplus, Haemaphysalis bispinosa, and Rhipicephalus pilans, were identified. The cattle in Yogyakarta and Riau were infested by H. bispinosa, while the cattle in Sukabumi, Bali, and Lombok were infested by R. microplus and R. pilans. The level of infestation varied among regions, with R. microplus being the most commonly found species. CONCLUSION: The results of this study revealed that cattle in different regions of Indonesia were infested by variable numbers of tick species. In particular, the cattle in Yogyakarta and Riau were solely infested by H. bispinosa; this is a new finding in terms of the distribution of tick species in the country. Increased tick infestation in cattle decreases productivity and causes health problems; therefore, it deserves serious attention. Our findings can help in the formulation of an effective strategy for controlling and preventing cattle tick infestation in the country.

Evaluation of effects of a novel probiotic feed supplement on the quality of broiler meat.

BACKGROUND AND AIM: A local microorganism-based probiotic has been developed as an alternative to using antibiotic growth promoter and its effect on broiler meat quality has been studied, when supplemented with poultry feed at different concentrations. This study aimed to understand the effect of local microorganism-based probiotic sourced from cattle rumen and chicken intestine and added as feed supplement at different concentrations on broiler meat quality. MATERIALS AND METHODS: The local microorganism-based probiotic made from cattle rumen and chicken intestine contained Lactobacillus spp., Bifidobacterium spp., Streptococcus spp., and Bacillus spp. The experiments followed a completely randomized design. Treatments in the study were: P0, i.e., control (without probiotic), P1 (probiotic administered at 5 ml/kg feed), P2 (probiotic administered at 10 ml/kg feed), and P3 (probiotic administered at 15 ml/kg feed). Each treatment was repeated 5 times. Parameters examined in this study were pH, meat tenderness, fat content, and meat protein content. RESULTS: Based on a total of 200 chickens, the percentage of meat protein content in treatments P1, P2, and P3 showed an increase of 19.34%, 19.42%, and 19.64%, respectively, when compared with P0 that showed a protein content increase of 19.14%. The fat content of meat for P1, P2, and P3 was 21.54%, 21.46%, and 21.30%, respectively, which was less than the value for P0 (21.69%). The treatments did not significantly affect pH or meat tenderness when compared with the control. The usage of this novel probiotic as a feed supplement resulted in an increase in meat protein content and a decrease in fat content. CONCLUSION: This study indicates that using the local microorganism-based probiotic sourced from cattle rumen and chicken intestine to supplement poultry feed did not have a significantly different effect (p>0.05) on meat pH; however, it had a significantly different (p<0.05) on protein and fat content of broiler meat.