Effects of MAT1-2 Spore Ratios on Fruiting Body Formation and Degeneration in the Heterothallic Fungus Cordyceps militaris.

The medicinal mushroom Cordyceps militaris is widely exploited in traditional medicine and nutraceuticals in Asian countries. However, fruiting body production in C. militaris is facing degeneration through cultivation batches, and the molecular mechanism of this phenomenon remains unclear. This study showed that fruiting body formation in three different C. militaris strains, namely G12, B12, and HQ1, severely declined after three successive culturing generations using the spore isolation method. PCR analyses revealed that these strains exist as heterokaryons and possess both the mating-type loci, MAT1-1 and MAT1-2. Further, monokaryotic isolates carrying MAT1-1 or MAT1-2 were successfully separated from the fruiting bodies of all three heterokaryotic strains. A spore combination of the MAT1-1 monokaryotic isolate and the MAT1-2 monokaryotic isolate promoted fruiting body formation, while the single monokaryotic isolates could not do that themselves. Notably, we found that changes in ratios of the MAT1-2 spores strongly influenced fruiting body formation in these strains. When the ratios of the MAT1-2 spores increased to more than 15 times compared to the MAT1-1 spores, the fruiting body formation decreased sharply. In contrast, when MAT1-1 spores were increased proportionally, fruiting body formation was only slightly reduced. Our study also proposes a new solution to mitigate the degeneration in the heterokaryotic C. militaris strains caused by successive culturing generations.

First Molecular Evidence of Pathogens in Fleas Collected from Dogs in Northern Vietnam.

Fleas are considered as hosts for a wide range of pathogens that cause emerging and re-emerging zoonotic diseases worldwide. Data on fleas and flea-borne pathogens (FBPs) in the international literature are limited in Vietnam. This study aimed to investigate the species of fleas and the presence of pathogens of interest in fleas in northern Vietnam using PCR and sequence analysis. Out of 200 dogs enrolled in this study, 20% were infested by the flea species Ctenocephalides felis felis. In total, 62 fleas (35 females and 27 males) collected from domestic dogs were molecularly screened for the detection of pathogens. Out of the screened fleas, 39 were positive for Rickettsia felis (62.9%), 9 for Candidatus Mycoplasma hemobos (14.52%), and 6 for Mycoplasma wenyonii (9.68%). This study shows the first molecular detection of the above-mentioned pathogens in fleas collected from the studied areas and the potential risk of infection with examined FBPs in northern Vietnam.

Ancylostoma ceylanicum infections in humans in Vietnam.

Ancylostoma ceylanicum is recognized as the only zoonotic hookworm species that is able to mature into adult stage in the human intestine. While human infections caused by this hookworm species have been reported from neighboring countries and this hookworm is prevalent in dogs in Vietnam, human infection has never been reported in Vietnam. The present study, therefore, aimed to identify human infections with A. ceylanicum in Vietnam. A total of 526 fecal samples from the residents in Long An Province were collected and the presence of hookworm eggs was detected by the Kato-Katz method. The results indicated that the overall prevalence of human hookworm infection was 85/526 (16.2%). After filter paper culture, 3rd stage larvae were successfully obtained from 48 egg-positive samples. The larvae were identified for their species using semi-nested PCR-RLFP on the cox1 gene. As a result, two hookworm species were confirmed; single species infections with Necator americanus or A. ceylanicum, and mixed infections with both species were found in 47.9%, 31.3%, and 20.8% of the samples, respectively.

A case of Trypanosoma evansi in a German Shepherd dog in Vietnam.

A 2.5-year-old male German Shepherd was presented to a private veterinary clinic in Hanoi, Vietnam showing anorexia, weakness, lethargy, reluctant to go for walks with a recent history of intermittent fever. Clinical examination of the dog showed pale mucous membrane, impaired eyesight, edema of the back legs. Complete blood count revealed severe anemia; red blood cell 3.8 × 1012/l, hemoglobin 8.7 g/dl, hematocrit 26.4%, associated with thrombocytopenia 145 × 109/l. Biochemical analysis showed a moderate increase of alanine transaminase (150.7 UI/l) and alkaline phosphatase activities (266 UI/I) with mild hypoglycemia (71.46 mg/dl). Trypanosoma evansi was observed in Giemsa-stained blood smears under microscopic observation which was confirmed by PCR. This is the first report of canine trypanosomiasis caused by T. evansi in Vietnam.

Molecular detection of pathogens in ticks and fleas collected from companion dogs and cats in East and Southeast Asia.

BACKGROUND: Ticks and fleas are considered amongst the most important arthropod vectors of medical and veterinary concern due to their ability to transmit pathogens to a range of animal species including dogs, cats and humans. By sharing a common environment with humans, companion animal-associated parasitic arthropods may potentially transmit zoonotic vector-borne pathogens (VBPs). This study aimed to molecularly detect pathogens from ticks and fleas from companion dogs and cats in East and Southeast Asia. METHODS: A total of 392 ticks and 248 fleas were collected from 401 infested animals (i.e. 271 dogs and 130 cats) from China, Taiwan, Indonesia, Malaysia, Singapore, Thailand, the Philippines and Vietnam, and molecularly screened for the presence of pathogens. Ticks were tested for Rickettsia spp., Anaplasma spp., Ehrlichia spp., Babesia spp. and Hepatozoon spp. while fleas were screened for the presence of Rickettsia spp. and Bartonella spp. RESULT: Of the 392 ticks tested, 37 (9.4%) scored positive for at least one pathogen with Hepatozoon canis being the most prevalent (5.4%), followed by Ehrlichia canis (1.8%), Babesia vogeli (1%), Anaplasma platys (0.8%) and Rickettsia spp. (1%) [including Rickettsia sp. (0.5%), Rickettsia asembonensis (0.3%) and Rickettsia felis (0.3%)]. Out of 248 fleas tested, 106 (42.7%) were harboring at least one pathogen with R. felis being the most common (19.4%), followed by Bartonella spp. (16.5%), Rickettsia asembonensis (10.9%) and "Candidatus Rickettsia senegalensis" (0.4%). Furthermore, 35 Rhipicephalus sanguineus ticks were subjected to phylogenetic analysis, of which 34 ticks belonged to the tropical and only one belonged to the temperate lineage (Rh. sanguineus (sensu stricto)). CONCLUSION: Our data reveals the circulation of different VBPs in ticks and fleas of dogs and cats from Asia, including zoonotic agents, which may represent a potential risk to animal and human health.

Anaplasma marginale and A. platys Characterized from Dairy and Indigenous Cattle and Dogs in Northern Vietnam.

Anaplasma marginale and A. platys were detected and characterized (16S rDNA sequence analysis) from dairy and indigenous cattle, and the latter in domestic dogs in Vietnam. A phylogenetic tree was inferred from 26 representative strains/species of Anaplasma spp. including 10 new sequences from Vietnam. Seven of our Vietnamese sequences fell into the clade of A. marginale and 3 into A. platys, with strong nodal support of 99 and 90%, respectively. Low genetic distances (0.2-0.4%) within each species supported the identification. Anaplasma platys is able to infect humans. Our discovery of this species in cattle and domestic dogs raises considerable concern about zoonotic transmission in Vietnam. Further systematic investigations are needed to gain data for Anaplasma spp. and members of Anaplasmataceae in animal hosts, vectors and humans across Vietnam.

Ticks and associated pathogens from dogs in northern Vietnam.

The medical and veterinary significance of ticks and tick-borne pathogens (TBPs) in tropical and subtropical zones is well recognized. Although ticks and TBPs are known to occur in Southeast Asia, limited data is available in the international literature for some countries, such as Vietnam. The aim of this study was to investigate the species of ticks and TBPs associated with dogs in northern Vietnam. Out of 359 dogs enrolled in this study, 26.2% (n = 94) were infested by 466 ticks (i.e., 287 males, 139 females, 30 nymphs, and 10 larvae). All ticks were morphologically identified as Rhipicephalus sanguineus sensu lato, and some of them genetically characterized as belonging to the tropical lineage. A total of 302 ticks were molecularly screened for the detection of selected TBPs. Three ticks were positive for Hepatozoon canis, one for Ehrlichia canis, and one for Babesia vogeli, representing the first molecular characterization of these pathogens in Vietnam. In conclusion, the tropical lineage of R. sanguineus s.l. is the dominant tick taxon infesting dogs from northern Vietnam, where different TBPs are circulating.

Prevalence, genotyping and risk factors of Giardia duodenalis from dogs in Vietnam.

This study was carried out to determine the prevalence, genotypes/assemblages and possible risk factors associated with Giardia duodenalis infection in dogs in central Vietnam. A total of 209 dog fecal samples, randomly collected from private owned dogs (n=105) and dogs from stores (n=104), were examined for Giardia cysts by microscopy. Positive samples were genotyped by PCR-sequence analysis of ß-giardin and triosephosphate isomerase genes markers. Risk factors were studied using a structured questionnaire and collected data were analyzed by univariate and multivariate logistic regression analyses. Results indicated that the overall infection rate was 8.6% (18/209) with the detected parasites were belonging to the non-zoonotic assemblages C and D. Age, gender and origin of animals were the main risk factors associated with G. duodenalis infection in dogs under study. Occurrence of infection was more likely in young animals compared to old ones and in females compared to males. Dogs originated from stores were more prone to Giardia infection compared to private owned counterparts.

Updated molecular phylogenetic data for Opisthorchis spp. (Trematoda: Opisthorchioidea) from ducks in Vietnam.

BACKGROUND: An opisthorchiid liver fluke was recently reported from ducks (Anas platyrhynchos) in Binh Dinh Province of Central Vietnam, and referred to as "Opisthorchis viverrini-like". This species uses common cyprinoid fishes as second intermediate hosts as does Opisthorchis viverrini, with which it is sympatric in this province. In this study, we refer to the liver fluke from ducks as "Opisthorchis sp. BD2013", and provide new sequence data from the mitochondrial (mt) genome and the nuclear ribosomal transcription unit. A phylogenetic analysis was conducted to clarify the basal taxonomic position of this species from ducks within the genus Opisthorchis (Digenea: Opisthorchiidae). METHODS: Adults and eggs of liver flukes were collected from ducks, metacercariae from fishes (Puntius brevis, Rasbora aurotaenia, Esomus metallicus) and cercariae from snails (Bithynia funiculata) in different localities in Binh Dinh Province. From four developmental life stage samples (adults, eggs, metacercariae and cercariae), the complete cytochrome b (cob), nicotinamide dehydrogenase subunit 1 (nad1) and cytochrome c oxidase subunit 1 (cox1) genes, and near-complete 18S and partial 28S ribosomal DNA (rDNA) sequences were obtained by PCR-coupled sequencing. The alignments of nucleotide sequences of concatenated cob + nad1 + cox1, and of concatenated 18S + 28S were separately subjected to phylogenetic analyses. Homologous sequences from other trematode species were included in each alignment. RESULTS: Phylogenetic trees were inferred from concatenated (cob + nad1 + cox1) nucleotide sequences and combined 18S + 28S nucleotide sequences of five Opisthorchis sp. BD2013 samples and additional reference taxa. Both trees demonstrated the anticipated clustering of taxa within the superfamily Opisthorchioidea, the paraphyly of the genus Opisthorchis and the sister-species relationship of Opisthorchis sp. BD2013 with O. viverrini. CONCLUSIONS: While it is likely that Opisthorchis sp. BD2013 is distinct from O. viverrini, it is clearly a sister taxon of O. viverrini within the limited number of Opisthorchis species for which appropriate sequence data are available. The new sequences provided here will assist the diagnosis and the taxonomic clarification of the opisthorchiid species.