Pathogen detection and gut bacteria identification in Apis cerana indica in Thailand

Abstract

Pathogen infection of honeybees can lead to economic losses in apiculture. The earlier the pathogen contamination can be found the better it will likely be for the treatment of the infected colony and prevention of the spread of the pathogen within and between colonies. A total of 50 colonies of Apis cerana were sampled in Samut Songkhram (five colonies) and Chumphon (45 colonies) provinces in the central and the south of Thailand, respectively. Diagnostic multiplex polymerase chain reaction (PCR) revealed that 20, 6, 4, 20 and 0% of the samples were infected by Paenibacillus larvae, Nosema ceranae, Nosema apis, Ascosphaera apis and Sacbrood virus (Morator aetatulus), respectively. Positive amplified PCR products of target genes were sequenced. A phylogenetic tree was constructed by the neighbor-joining (NJ) distance based, using the Phylogenetic Analysis Using Parsimony (PAUP 4.0b10) program. The phylogenetic relationship of each pathogen, based on the partial sequence of the 16S rRNA of P. larvae, N. ceranae and N. apis, and of the 5.8S rRNA of A. apis, revealed a significant difference from the non-Thai isolates of these pathogens, but no significant geographical isolation between the different Thai apiaries, although it separated some into closely related clusters. In order to reduce the use of antibiotics in an apiary, bacteria in the gut of healthy bees were focused. Interestingly, Bifidobacterium species, Lactobacillus species, Bacillus species, Lactobacillus spp. and other lactic acid bacteria, were isolated from larvae and adult workers, but gave conflicting preliminary identities based on their biochemistry-morphology versus sequence analysis of a partial fragment (1.4 kb) of their 16S rRNA.

Keywords: Apis cerana indica, bee pathogens, gut bacteria, multiplex polymerase chain reaction (PCR), 16S rRNA