A survey of gastrointestinal helminth infestation in smallholder backyard pigs and the first molecular identification of the two zoonotic helminths Ascaris suum and Trichuris suis in Myanmar.

BACKGROUND: Parasitic infestations have a substantial economic impact on pig production. This study aimed to investigate the gastrointestinal (GI) helminths in pigs and to molecularly characterise two important nematodes, Ascaris and Trichuris species. MATERIALS AND METHODS: A total of 500 pig faecal samples were collected from small holder backyard pig farms in five townships within Nay Pyi Taw, Myanmar. Microscopic examination was conducted to estimate the prevalence of GI helminth infestation in the pigs. DNA extraction and PCR were performed on faecal samples that were morphologically positive for Ascaris and Trichuris eggs. Molecular analysis was then conducted to characterise A. suum and T. suis, the most common and zoonotic helminths. RESULTS: According to microscopic examination, 69.2% (346/500) were positive for GI helminth eggs. The GI helminth species observed were A. suum, Strongyle, Strongyloides spp., T. suis, Metastrongylus spp., Hyostrongylus spp., Fasciolopsis spp., Paragonimus spp., and Schistosoma spp., with occurrences of 34.8%, 29.6%, 21.4%, 20.0%, 4.0%, 1.6%, 1.0%, 1.0%, and 0.4%, respectively. Mixed infections of GI helminths were noted in 31.0% of the samples. Overall, sampled pigs excreted mostly low levels (< 100 EPG) or moderate levels (> 100-500 EPG) of GI helminth eggs. The highest mean EPG for each parasite species was noted in A. suum. The presence of A. suum and T. suis was confirmed molecularly. The sequences of the internal transcribed spacer 1 (ITS1) region of A. suum showed high similarity with previously reported sequences. Likewise, the sequences of T. suis exhibited high similarity with the sequences reported from humans and pigs. Age was noted as an associated factor (P < 0.05) for GI helminth infection status. CONCLUSIONS: In this report, A. suum and T. suis were molecularly identified for the first time in Myanmar. It is important to extend the information among the farmers to be aware of the necessity of preventing zoonotic parasites by practicing regular deworming, proper use of anthelmintics and maintaining hygienic conditions in their pig farms.

First molecular detection of Theileria luwenshuni from goats in Myanmar.

Tick-borne intracellular protozoan parasites of the Theileria genus infect a wide range of both domestic and wild animals. In the present study, we describe the first PCR detection of Theileria luwenshuni in the blood of goats in Myanmar. Nested PCR targeting the Theileria 18S rRNA gene resulted in seven positive goats in central and northern Myanmar. Nucleotide sequencing of the PCR products revealed that all seven sequences were identical and showed 100% identity with T. luwenshuni sequences in GenBank from goats and sheep in China. Since T. luwenshuni parasites have recently been discovered and shown to have nationwide distribution in China, they might have been introduced into Myanmar via transboundary movement of infected domestic small ruminants and/or wild animals from China.

The strong influence of management factors on coccidian infections in smallholder pig farms and the first molecular identification of Cystoisospora suis in Myanmar.

A cross-sectional study was conducted to investigate coccidian infection and associated factors in smallholder pigs, and to identify Cystoisospora oocysts by PCR. A total of 500 pig faecal samples from 330 smallholder farms were collected in Nay Pyi Taw, Myanmar. The faecal flotation method was used to identify Eimeria and Cystoisospora species, and oocyst counts per gram (OPG) of faeces were recorded. Oocysts were differentiated after sporulation. Oocyst DNA was subjected to ITS1-targeted Cystoisospora-specific PCR. The overall coccidian oocyst detection rate by microscopic was 89.0% (445/500). Among the studied samples, 74.0% (370/500) and 70.6% (353/500), were found to be positive with Eimeria spp. and Cystoisospora suis oocysts, respectively. The sequences of C. suis detected were 100% identical to those of C. suis reported from Japan, and had 99.5% resemblance to sequences from Australia and China. Weaner pigs showed the significantly highest (p < 0.05) OPG when compared to other age groups. The highest intensity of coccidian infection (p < 0.05) was found in pigs fed local feed, pigs raised on earthen floors and pigs under poor hygienic conditions. Factors such as age, breed, feed type, and housing floors were found to be significantly associated with coccidian infection (p < 0.05). Age, as well as management factors including floor type, feed type, and hygiene practices on the farm, had a strong influence on the occurrence of coccidian infection in pigs. This is the first study in Myanmar on coccidian infection in pigs and molecular detection of C. suis.

TITLE: Forte influence des facteurs de gestion sur les infections à coccidies dans les petites exploitations porcines et première identification moléculaire de Cystoisospora suis au Myanmar. ABSTRACT: Une étude transversale a été menée pour étudier l'infection coccidienne et les facteurs associés chez les porcs dans des petites exploitations, et pour identifier les oocystes de Cystoisospora par PCR. Au total, 500 échantillons de matières fécales de porcs provenant de 330 petites exploitations agricoles ont été collectés dans la région de Nay Pyi Taw, au Myanmar. La méthode de flottation fécale a été utilisée pour identifier les espèces d'Eimeria et de Cystoisospora, et le nombre d'oocystes par gramme (OPG) de matières fécales a été déterminé. Les oocystes ont été différenciés après sporulation. L'ADN des oocystes a été soumis à une PCR spécifique à Cystoisospora, ciblée sur ITS1. Le taux global de détection d'oocystes de coccidies au microscope était de 89,0 % (445/500). Parmi les échantillons étudiés, respectivement 74,0 % (370/500) et 70,6 % (353/500) ont été trouvés positifs pour Eimeria spp. et les oocystes de Cystoisospora suis. Les séquences de C. suis détectées étaient identiques à 100 % à celles de C. suis signalées au Japon, et avaient 99,5 % de ressemblance avec des séquences d'Australie et de Chine. Les porcs sevrés ont montré un OPG significativement plus élevé (p < 0,05) par rapport aux autres groupes d'âge. L'intensité la plus élevée de l'infection coccidienne (p < 0,05) a été observée chez les porcs nourris avec des aliments locaux, les porcs élevés sur des sols en terre battue et les porcs dans de mauvaises conditions d'hygiène. Des facteurs tels que l'âge, la race, le type d'alimentation et les étages se sont avérés être significativement (p < 0,05) associés à l'infection coccidienne. L'âge, ainsi que les facteurs de gestion, notamment le type de sol, le type d'alimentation et les pratiques d'hygiène dans la ferme, ont eu une forte influence sur la survenue d'une infection coccidienne chez les porcs. Il s'agit de la première étude au Myanmar sur l'infection coccidienne chez le porc et la détection moléculaire de C. suis.

PCR detection and genetic characterization of piroplasms from dogs in Myanmar, and a possible role of dogs as reservoirs for Theileria parasites infecting cattle, water buffaloes, and goats.

Canine vector-borne pathogens can act as zoonotic agents in humans; however, it poorly understood whether dogs play a role as reservoirs of vector-borne parasites in livestock animals. Here, we report the unexpected detection of 18S rRNA gene (rDNA) sequences of five ruminant Theileria species from the peripheral blood of dogs in Myanmar, in addition to those of two canine Babesia species. Using novel BTH primers capable of amplifying the 18S rDNA of Babesia, Theileria, and Hepatozoon spp., approximately 1,500 bp nested PCR products were detected in 19% (17/91) of local or imported dog breeds in different regions of Myanmar. Among the sequences of the 17 PCR products, ten were determined as Theileria 18S rDNA, including three as Theileria orientalis, three as Theileria buffeli, two as Theileria cf. velifera, one as Theileria luwenshuni, and one as Theileria sp. Most of these sequences showed higher identities with Theileria sequences determined in previous studies of cattle, water buffaloes, and goats in Myanmar. Six PCR products were identified as Babesia vogeli and one sample was determined as Babesia gibsoni. Furthermore, we obtained approximately 900 bp thrombospondin-related adhesive protein (TRAP) gene fragments from three dog blood DNA samples. Phylogenetic analysis of the TRAP gene showed that B. gibsoni parasites in Myanmar were considerably related to isolates from China, Korea, Japan, and Taiwan, but clearly separated from those from Bangladesh and India. These results provide new insights into a possible role of dogs in maintaining and spreading tick-borne pathogens among livestock and canine populations in Myanmar.

Identification, genetic variation, and structural analysis of 18S rRNA of Theileria orientalis and Theileria velifera-like isolates from Myanmar.

Ribosomal RNA genes have been widely used for the identification and phylogenetic analysis of various organisms, including parasitic protozoa. Here, we report nine near full-length Theileria orientalis 18S rRNA gene sequences from cattle from different areas of Myanmar. Phylogenetic analysis of the 18S rRNA genes revealed a considerably close genetic relationship among T. orientalis isolates from Australia, China, Japan, Korea, Myanmar, and Pakistan. We also obtained four Theileria velifera-like (Theileria cf. velifera) 18S rRNA gene sequences from two cattle and two water buffaloes from the northernmost area of Myanmar. The phylogenetic analysis of T. cf. velifera isolates from Myanmar along with T. velifera and T. cf. velifera isolates from African countries suggested an evolutionary lineage of greater complexity in T. velifera-related parasites. DNA alignment analysis indicated the presence of 51 and 55 nucleotide variation positions within the 18S rRNA genes from 15 T. orientalis and 11 T. velifera-related isolates, respectively. Alignment entropy analysis of the 18S rRNA sequences indicated that both T. orientalis and T. velifera-related isolates had three hyper variable regions, corresponding to V2, V4, and V7 regions in eukaryotes. The degree of variation was prominent in the V2 in T. orientalis and V4 in T. velifera-related isolates. The secondary structure analysis of the 18S rRNA predicted using minimum free energy algorism revealed that the structure of V4 region differed most significantly between T. orientalis and T. velifera. These results provide novel insights into common structures, variations and functions of small subunit rRNA in Theileria species.

Anthropogenic interferences lead to gut microbiome dysbiosis in Asian elephants and may alter adaptation processes to surrounding environments.

Human activities interfere with wild animals and lead to the loss of many animal populations. Therefore, efforts have been made to understand how wildlife can rebound from anthropogenic disturbances. An essential mechanism to adapt to environmental and social changes is the fluctuations in the host gut microbiome. Here we give a comprehensive description of anthropogenically induced microbiome alterations in Asian elephants (n = 30). We detected gut microbial changes due to overseas translocation, captivity and deworming. We found that microbes belonging to Planococcaceae had the highest contribution in the microbiome alterations after translocation, while Clostridiaceae, Spirochaetaceae and Bacteroidia were the most affected after captivity. However, deworming significantly changed the abundance of Flavobacteriaceae, Sphingobacteriaceae, Xanthomonadaceae, Weeksellaceae and Burkholderiaceae. These findings may provide fundamental ideas to help guide the preservation tactics and probiotic replacement therapies of a dysbiosed gut microbiome in Asian elephants. More generally, these results show the severity of anthropogenic activities at the level of gut microbiome, altering the adaptation processes to new environments and the subsequent capability to maintain normal physiological processes in animals.

Detection and molecular identification of Leucocytozoon and Plasmodium species from village chickens in different areas of Myanmar.

Village chicken production, a traditional, small-scale, and extensive backyard poultry industry, has been profitable for local farmers in Myanmar. However, there is scanty information available concerning the infection of these chickens with avian pathogens, including haemoprotozoan parasites. In the present study, we provide the first report of microscopic detection and molecular identification of Leucocytozoon and Plasmodium parasites from seven different areas of Myanmar. Leucocytozoon gametocytes were detected in 17.6% (81/461) of the blood smears from village chickens. The nested polymerase chain reaction (PCR) for targeting Leucocytozoon mitochondrial cytochrome b (cyt b) genes had a 17.6% positive rate. Although the positive rate of nested PCR targeting Plasmodium/Haemoproteus cyt b was 34.3%, the PCR protocol was observed to possibly amplify DNA of a certain species of Leucocytozoon. There were no obvious clinical signs in the infected birds. Statistical analysis of the microscopic detection and PCR detection rates using the age and sex of birds as internal factors revealed that the statistical significances differed according to the study area. The sequencing of 32 PCR products obtained from each study area revealed infection by Leucocytozoon caulleryi in three birds, Leucocytozoon sabrazesi in two birds, Leucocytozoon schoutedeni in two birds, Leucocytozoon sp. in eighteen birds, and Plasmodium juxtanucleare in seven birds; however, Haemoproteus infection was not detected. While L. sabrazesi was detected in chickens from the central region of Myanmar, the other haemosporidians were detected in those from different areas. In the haplotype analysis, we detected 17 haemosporidian cyt b haplotypes, including two for L. caulleryi, one for L. sabrazesi, two for L. schoutedeni, nine for Leucocytozoon sp., and three for P. juxtanucleare. Phylogenetic analysis of the cyt b haplotypes revealed a considerably close genetic relationship among chicken haemosporidians detected in Myanmar, Thailand, and Malaysia. These results indicate that well-recognized widespread species of chicken Leucocytozoon and Plasmodium are distributed nationwide in Myanmar, providing new insights into the ecosystem and control strategies of haemosporidian parasites in domesticated chickens in Myanmar.

Morphological and molecular identification of cyathostomine gastrointestinal nematodes of Murshidia and Quilonia species from Asian elephants in Myanmar.

Gastrointestinal nematode parasites have long been recognized in Asian elephants. The most common parasites belong to the subfamily Cyathostominae of the family Strongylidae, which are small to medium-sized with a cylindrical buccal capsule surrounded by coronal leaflets. Diagnostic keys of such parasites are provided from old illustrations in the form of line drawings. However, there very few photomicrographs and no genetic information of these parasites exist. In the present study we obtained adult worm specimens from faeces of Asian elephants after anthelmintic treatment in two elephant camps in Myanmar. Here, we provided photomicrographs for five cyathostomine parasites, Murshidia falcifera, Murshidia indica, Murshidia neveulemairei, Quilonia renniei, and Quilonia travancra almost 100 years after their original drawings. In addition, we determined the mitochondrial cytochrome c oxidase subunit I (COI) gene sequences of these species. Phylogenetic analysis of the COI genes of Murshidia and Quilonia species from Asian and African elephants revealed parasite speciation in each elephant host. The present study also indicated that several Murshidia and Quilonia species were widely distributed in Asian elephants in Myanmar, providing new insight into control strategies and evolution of cyathostomine gastrointestinal parasites in elephants.

Haematophagous mites on poultry farms in the Republic of the Union of Myanmar.

Haematophagous ectoparasites of poultry, such as Ornithonyssus sylviarum, northern fowl mites (NFMs), Dermanyssus gallinae, poultry red mites (PRMs), and Ornithonyssus bursa, tropical fowl mites (TFMs) are prevalent worldwide. Although poultry farming is a major industry in Southeast Asia, there are only a few reports concerning the prevalence of avian mites in this region. In this study, we sampled twenty farms in four major poultry farming areas in Myanmar. We detected the mites on six farms, and they showed morphological similarities to NFMs and TFMs. The nucleotide sequences of cytochrome c oxidase subunit I indicated that some mites were NFMs. This is the first report confirming the presence of NFMs and TFMs among the hematophagous mites infesting chickens on Myanmar poultry farms.

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.

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

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