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.