Dynamic evolution of canine parvovirus in Thailand.

BACKGROUND AND AIM: According to the previous study, the circulating canine parvovirus (CPV) in Thailand is 2a and 2b. Nowadays, CPV mutants, including CPV-2c, have been identified in many parts of the world. This study aimed to investigate the genetic diversity of the circulating CPV in Thailand. MATERIALS AND METHODS: Eighty-five CPV-positive fecal samples were obtained from dogs with either acute hemorrhagic diarrhea or diarrhea. The complete VP2 gene of these samples was amplified using VP2 specific primers and polymerase chain reaction (PCR). The obtained full-length VP2 sequences were analyzed and a phylogenetic tree was constructed. RESULTS: Sixty and 25 CPV-positive fecal samples were collected in 2010 and 2018, respectively. Thirty-four samples were new CPV-2a and 31 samples were new CPV-2b due to amino acids substitution at position 297 (Ser-Ala). In 2018, 5 new CPV-2a, 19 CPV-2c, and 1 feline panleukopenia virus (FPV) were found, but no new CPV-2b was detected. Moreover, most of the CPV in this study had amino acids mutations at positions 324 and 440. The phylogenetic construction demonstrated the close relationship between the current new CPV-2a with the previous CPV-2a reported from Thailand, China, Uruguay, Vietnam, Singapore, and India. Interestingly, the current new CPV-2b in this study was not closely related to the previous CPV-2b reported in Thailand. The CPV-2c in this study was closer to Asian CPV-2c and further from either European or South America CPV-2c. Interestingly, FPV was identified in a diarrhea dog. CONCLUSION: The evolution of CPV in Thailand is very dynamic. Thus, it is important to monitor for CPV mutants and especially the clinical signs relating to these mutants to conduct surveillance for the emergence of new highly pathogenic CPV in the future.

Identification of uncultured bacteria from abscesses of exotic pet animals using broad-range nested 16S rRNA polymerase chain reaction and Sanger sequencing.

BACKGROUND: The Sanger sequencing technique has been questioned and challenged by advanced high-throughput sequencing approaches. Sanger sequencing seems to be an obsolete technology. However, there are still research problems that could be answered using the Sanger sequencing technology. Fastidious obligate anaerobic bacteria are mostly associated with abscesses in animals. These bacteria are difficult to isolate from abscesses and are frequently excluded due to the bias of conventional bacterial culturing. AIM: This study demonstrated the usefulness of a broad-range polymerase chain reaction (PCR) with Sanger sequencing to identify the majority population of bacteria in abscesses from exotic pet animals. MATERIALS AND METHODS: This study performed a pilot investigation of abscesses from 20 clinical cases (17 rabbits, 2 hedgehogs, and 1 sugar glider) using standard culture methods for both aerobes and anaerobes and broad-range nested PCR targeting the 16S rRNA gene followed by the Sanger sequencing technique. RESULTS: The standard culture and PCR techniques detected bacteria in 9 and 17 of 20 samples, respectively. From the 17 sequencings of the 16S rRNA, 10 PCR products were found to be closely related with obligate anaerobes including Bacteroides spp., Fusobacterium spp., Prevotella spp. Phylogenetic analysis using the rpoB gene revealed that the species for the Bacteroides was thetaiotaomicron and for the Fusobacterium was varium and nucleatum. However, the amplification of the rpoB gene for the Prevotella spp. was unsuccessful. Correlations between the standard culture and PCR techniques were found in 9 (6 positive and 3 negative samples) of 20 samples. Eleven samples were discordant between the standard culture and PCR techniques which were composed of eight samples negative by culture but positive by PCR and three samples had different bacteria by the culture and PCR techniques. CONCLUSION: According to this study, broad-range PCR combined with Sanger sequencing might be useful for the detection of dominant anaerobic bacteria in abscesses that were overlooked based on conventional bacterial culture.

Variation among Bm86 sequences in Rhipicephalus (Boophilus) microplus ticks collected from cattle across Thailand.

Anti-tick vaccines based on recombinant homologues Bm86 and Bm95 have become a more cost-effective and sustainable alternative to chemical pesticides commonly used to control the cattle tick, Rhipicephalus (Boophilus) microplus. However, Bm86 polymorphism among geographically separate ticks is reportedly associated with reduced effectiveness of these vaccines. The purpose of this study was to investigate the variation of Bm86 among cattle ticks collected from Northern, Northeastern, Central and Southern areas across Thailand. Bm86 cDNA and deduced amino acid sequences representing 29 female tick midgut samples were 95.6-97.0 and 91.5-93.5 % identical to the nucleotide and amino acid reference sequences, respectively, of the Australian Yeerongpilly vaccine strain. Multiple sequence analyses of these Bm86 variants indicated geographical relationships and polymorphism among Thai cattle ticks. Two larger groups of cattle tick strains were discernable based on this phylogenetic analysis of Bm86, a Thai group and a Latin American group. Thai female and male cattle ticks (50 pairs) were also subjected to detailed morphological characterization to confirm their identity. The majority of female ticks had morphological features consistent with those described for R. (B.) microplus, whereas, curiously, the majority of male ticks were more consistent with the recently re-instated R. (B.) australis. A number of these ticks had features consistent with both species. Further investigations are warranted to test the efficacies of rBm86-based vaccines to homologous and heterologous challenge infestations with Thai tick strains and for in-depth study of the phylogeny of Thai cattle ticks.