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.

Morphological and molecular identification of trematode cercariae related with humans and animal health in freshwater snails from a lake and a dam in Myanmar.

Freshwater snails play an essential role in the transmission of trematode parasitic flatworms that can infect wild and domestic animals, as well as humans. This study aimed to investigate the rate of cercarial infections in freshwater snails collected from two study areas, Inlay Lake and Yezin Dam, in Myanmar. A total of 4,740 snail samples were collected from Inlay Lake (n = 3,837) and Yezin Dam (n = 903), and infection rate by cercarial emergence was examined. Cercarial DNA samples were analysed by PCR. Based on morphological characteristics, eleven snail species and eight cercarial types were identified. Snails of Melanoides tuberculata in the family Thiaridae were found as the most abundant, followed by Indoplanorbis exustus of the family Planorbidae, in both study areas. The infection rate by cercarial emergence in snails in Inlay Lake and Yezin Dam was 5.8% (224/3,837) and 48.6% (439/903), respectively. Echinostome cercariae showed the highest infection rate in both study areas. Phylogenetic analysis of cercarial internal transcribed spacer 2 (ITS2) sequences revealed that at least seven cercaria types belonged to five digenean trematode families, two of which were zoonotic trematodes in the families of Opisthorchiidae/Heterophyidae and Schistosomatidae. Furthermore, cercarial 28S ribosomal RNA gene analysis showed that the furcocercous cercariae in Yezin Dam were identified as Schistosoma spindale, a causative agent of ruminant schistosomiasis. This is the first report on zoonotic trematode cercariae in snails in Myanmar. The findings indicate that various snail species act as intermediate host for trematode species that infect aquatic animals, mammals and humans in the country.

Occurrence of gastrointestinal helminths and the first molecular detection of Ancylostoma ceylanicum, Trichuris trichiura, and Trichuris vulpis in dogs in Myanmar.

Dogs may serve as hosts for a variety of zoonotic or potentially zoonotic helminths, including Ancylostoma ceylanicum and Trichuris species. Cross-sectional study design was used to collect 210 faecal samples of dogs from Nay Pyi Taw area, Myanmar. According to microscopic examination, 180 samples (85.7%) were positive for eight species of gastrointestinal helminths. Among them, positive rates of Ancylostoma species and Trichuris species eggs were observed as 79.0% (166/210) and 11.9% (15/210), respectively. Molecular identification of A. ceylanicum and Trichuris species was confirmed by COX1 gene- and SSU rRNA gene-targeted PCR. Partial sequences of COX1 and SSU rRNA showed 100% identity with A. ceylanicum, Trichuris trichiura, and Trichuris vulpis deposited in GenBank.

Molecular detection and genetic characterization of infectious laryngotracheitis virus in poultry in Myanmar.

BACKGROUND: Avian infectious laryngotracheitis (ILT) is a highly contagious viral disease that causes severe economic losses to the poultry industry worldwide. In Southeast Asian countries, including Myanmar, poultry farming is a major industry. Although it is known that infectious respiratory pathogens, including infectious laryngotracheitis virus (ILTV), are a major threat to poultry farms, there are no data currently available on the epidemiology of ILTV in Myanmar. Therefore, in this study, we conducted a molecular detection of ILTV in 20 poultry farms in Myanmar. RESULTS: Of the 57 tested oropharyngeal swabs, 10 were positive for ILTV by polymerase chain reaction of a 647 bp region of the thymidine kinase (TK) gene, giving a prevalence of ILTV of 17.5% (10/57). Further sequencing analysis of infected cell protein 4 (ICP4) gene and glycoprotein B, G, and J (gB, gG, and gJ) genes indicated that these isolates were field strains. Phylogenetic analysis revealed that the Myanmar strains clustered together in a single branch and were closely related to other reference strains isolated from Asian countries. CONCLUSIONS: This study demonstrated the presence of ILTV in poultry farms in Myanmar. The genetic characterization analysis performed provides the fundamental data for epidemiological studies that monitor circulating strains of ILTV in Myanmar.

Molecular detection and genetic characterization of Mycoplasma gallisepticum, Mycoplama synoviae, and infectious bronchitis virus in poultry in Myanmar.

BACKGROUND: In Southeast Asian countries, including Myanmar, poultry farming is a major industry. In order to manage and maintain stable productivity, it is important to establish policies for biosecurity. Infectious respiratory diseases are a major threat to poultry farming. Avian influenza and Newcastle disease have been reported in Myanmar, but no scientific information is available for other respiratory pathogens, such as mycoplasmas and infectious bronchitis virus (IBV). Identifying the genotypes and serotypes of IBVs is especially important to inform vaccination programs. In this study, we detected Mycoplasma gallisepticum (MG), M. synoviae (MS), and IBV in several poultry farms in Myanmar. RESULTS: Samples were collected from 20 farms in three major poultry farming areas in Myanmar, and MG, MS, and IBV were detected on two, four, and eight farms, respectively, by polymerase chain reaction. Phylogenetic analysis revealed that the observed MG and MS isolates were not identical to vaccine strains. Three different genotypes of IBV were detected, but none was an unknown variant. CONCLUSIONS: Mycoplasmas and IBV were detected on poultry farms in Myanmar. Periodic surveillance is required to establish the distribution of each pathogen, and to institute better vaccine protocols.

Molecular detection of Dirofilaria immitis and its Wolbachia endosymbionts in dogs from Myanmar.

Heartworm disease in dogs and cats caused by Dirofilaria immitis continues to be a major clinical issue globally. This study focused on dogs suspicious of having tick-borne diseases (TBD) brought to a clinic and a veterinary teaching hospital in Myanmar. Blood samples were collected and initially screened using SNAP® 4Dx® Plus test kit. All dog blood samples were subjected to conventional PCR to detect both Dirofilaria spp. (cox1 gene) and Wolbachia spp. (16S rDNA) infections. Infection with D. immitis was detected in 14 (28.0%) of 50 examined samples, while the detection rate of TBD causative agents, including Anaplasma phagocytophilum and Ehrlichia canis, was 26.0% (13/50) and 26.0% (13/50), respectively, as determined by ELISA rapid test. In this study, D. immitis infection was moderately but significantly correlated with TBD infections (Pearson's r = 0.397, P = 0.008). Comparative sequence and phylogenetic analyses provided molecular identification of D. immitis in Myanmar and confirmed the identity of its Wolbachia endosymbiont with Wolbachia endosymbionts isolated from D. immitis, Rhipicephalus sanguineus and Aedes aegypti. The present study contributes to our understanding of the coexistence of D. immitis and Wolbachia endosymbiosis in dogs, and the findings may benefit the future prevention and control of dirofilariasis in dogs.

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.

Microscopic and molecular detection of Eimeria maxima and Eimeria praecox naturally infected in free-range village chickens of Myanmar.

PURPOSE: In Myanmar, village chicken production is an important source of both income and food for rural households. The present study is aimed to conduct microscopic detection and molecular identification of Eimeria species in free-range village chickens in Myanmar. METHODS: Faecal samples were taken from a total of 122 apparently healthy village chickens from three rural regions in Myanmar. The faecal samples were subjected to flotation method using a saturated sugar solution. Oocysts of Eimeria sp. were isolated by saturated sugar solution onto coverslips and identified to species at 400 × by light microscopy. Molecular identification was conducted for Eimeria oocysts collected from faecal samples using 18S rRNA and internal transcribed spacer-1 (ITS-1). RESULTS: Eimeria oocysts were found in 41 samples (33.6%) by flotation method. Oocysts morphologically identified as E. maxima and E. praecox, were detected in 33 (27.0%) and 15 (12.3%) samples, respectively. Mixed infection of these two species was found in 7 (5.7%). Partial sequences of the 18S rRNA gene amplified from morphologically identified oocysts of E. maxima and E. praecox, revealed 99.9% and 100%, identities with the sequences of each species deposited in GenBank, respectively. Species-specific PCR of the ITS-1 region was also confirmed the presence of these two Eimeria species. CONCLUSION: The results demonstrated the presence of E. maxima and E. praecox in free-range village chickens 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.

Molecular Prevalence and Identification of Ehrlichia canis and Anaplasma platys from Dogs in Nay Pyi Taw Area, Myanmar.

Ticks are vectors of different types of viruses, protozoans, and other microorganisms, which include Gram-negative prokaryotes of the genera Rickettsiales, Ehrlichia, Anaplasma, and Borrelia. Canine monocytic ehrlichiosis caused by Ehrlichia canis and canine cyclic thrombocytopenia caused by Anaplasma platys are of veterinary importance worldwide. In Myanmar, there is limited information concerning tick-borne pathogens, Ehrlichia and Anaplasma spp., as well as genetic characterization of these species. We performed nested PCR for the gltA gene of the genus Ehrlichia spp. and the 16S rRNA gene of the genus Anaplasma spp. with blood samples from 400 apparently healthy dogs in Nay Pyi Taw area. These amplicon sequences were compared with other sequences from GenBank. Among the 400 blood samples from dogs, 3 (0.75%) were positive for E. canis and 1 (0.25%) was positive for A. platys. The partial sequences of the E. canis gltA and A. platys 16SrRNA genes obtained were highly similar to E. canis and A. platys isolated from different other countries.

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.

Occurrence of Gastrointestinal Parasites in Small Ruminants in the Central Part of Myanmar.

Gastrointestinal parasite infection in small ruminants remains one of the major economic losses caused by reduced productivity. A total of 380 faecal samples were taken from 280 sheeps in Magway and Pwintbyu Townships and 100 goats in Natmauk Township, Myanmar. Faecal flotation and sedimentation methods were carried out to detect the presence of parasitic infections. Faecal egg and oocyst counts were carried out using the McMaster technique. The overall occurrence of gastrointestinal parasites in small ruminants was 98.4% (374/380). The occurrence of gastrointestinal parasites in sheep (99.3%) was higher than that in goats (96%). The highest occurrence was found in Eimeria spp. (96%), followed by Trichostrongyle (77.1%), Trichuris spp. (35%), and Moniezia expansa (14%). The mixed infection rate was 84.8% (317/374), while a single infection was 15.2% (57/374). The mean eggs per gram (EPG) and oocysts per gram (OPG) of faeces were ranged from 50 to 600 and 50 to 29,800, respectively. Among the 4 nucleotide sequences isolated, one sequence was 94.10-94.47% similarity with Trichostrongylus colubriformis, reported from Laos, and three sequences showed 96.64-99.46% identity with Haemonchus contortus from Laos, China, India, and Mongolia. As gastrointestinal parasite infection in small ruminants was relatively high in the study area, the development of appropriate treatment and control measures should be provided to reduce production losses.

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.