Methods: The study was conducted in the Department of Medical Microbiology and Parasitology, University Putra Malaysia in 2014-2017. Saline extract protein from the infective larvae of S. ratti was used to immunize BALB/c mice and subsequent fusion of the B-cells with myeloma cells (SP2/0) using 50% PEG. The hybridomas were cultured in HAT medium and cloned by limiting dilutions. Positive hybrids were screened by indirect ELISA. The ascites fluid from the antibody-secreting hybridoma was purified and the MAb was characterized by western-blots and evaluated in sandwich ELISA for reactivity against the homologous and heterologous antigens.
Results: An IgG1 that recognizes a 30 and 34 kDa protein bands was obtained. The MAb was recognized by all S. ratti-related antigens and cross-reacted with only Toxocara canis antigens in both assays. The minimum antigen detection limit was found to be 5 ng/ml. All antibody-positive rat and dog sera evaluated have shown antigen-positive reactions in Sandwich-ELISA.
Conclusion: The MAb produced, was able to detect antigens in strongyloidiasis and toxocariasis in animal models and may also be useful for the serological detection of active strongyloidiasis and visceral toxocariasis in human sera.
RESULTS: Tumors with a variety of clinical and pathological characteristics were selected. Gene expression stability and the optimal number of reference genes for gene expression normalization were calculated. RPS5 and HNRNPH were highly stable among OS cell lines, while RPS5 and RPS19 were the best combination for primary tumors. Pairwise variation analysis recommended four and two reference genes for optimal normalization of the expression data of canine OS tumors and cell lines, respectively.
CONCLUSIONS: Appropriate combinations of reference genes are recommended to normalize mRNA levels in canine OS tumors and cell lines to facilitate standardized and reliable quantification of target gene expression, which is essential for investigating key genes involved in canine OS metastasis and for comparative biomarker discovery.
METHODS: For this study, seven Haemaphysalis sp. ticks were removed from dogs and collected from the vegetation. These showed morphological differences from congeneric species known to occur in Vietnam. In addition, three Ixodes sp. ticks were collected from pygmy slow lorises (Xanthonycticebus pygmaeus), and a Dermacentor female had been previously collected from the vegetation. After DNA extraction, these were molecularly or phylogenetically analyzed based on the cytochrome c oxidase subunit I (cox1) and 16S rRNA genes.
RESULTS: The three species were morphologically identified as (i) Ixodes granulatus, which had nearly or exactly 100% sequence identities to conspecific ticks reported from large (approximately 2000 km) geographical distances but was more different (having lower, only 94.2% cox1 and 96.7% 16S rRNA sequence identity) from samples collected within 1000 km of Vietnam in Southern China and Malaysia, respectively; (ii) Haemaphysalis bispinosa, which showed 100% sequence identity to samples reported within both narrow and broad geographical ranges; and (iii) a new species, Dermacentor pseudotamokensis Hornok sp. nov., described here morphologically and shown to be phylogenetically a sister species to Dermacentor tamokensis.
CONCLUSIONS: Haemaphysalis bispinosa shows genetic homogeneity in the whole of South and Southeast Asia, probably owing to its frequent association with domestic ruminants and dogs (i.e. frequently transported hosts). However, I. granulatus, the Asian rodent tick, has a mixed geographical pattern of haplotypes, probably because it may associate with either synanthropic or wild-living rodents as primary hosts. This tick species is recorded here, for the first time to our knowledge, as parasitizing lorises in Vietnam and its region. Based on phylogenetic analyses, D. pseudotamokensis Hornok sp. nov., recognized and described here for the first time, was almost certainly misidentified previously as Dermacentor steini, drawing attention to the need to barcode all Dermacentor spp. in Southern Asia.