Comparative genomics of Lactobacillaceae from the gut of honey bees, Apis mellifera, from the Eastern United States.

Lactobacillaceae are an important family of lactic acid bacteria that play key roles in the gut microbiome of many animal species. In the honey bee (Apis mellifera) gut microbiome, many species of Lactobacillaceae are found, and there is functionally important strain-level variation in the bacteria. In this study, we completed whole-genome sequencing of 3 unique Lactobacillaceae isolates collected from hives in Virginia, USA. Using 107 genomes of known bee-associated Lactobacillaceae and Limosilactobacillus reuteri as an outgroup, the phylogenetics of the 3 isolates was assessed, and these isolates were identified as novel strains of Apilactobacillus kunkeei, Lactobacillus kullabergensis, and Bombilactobacillus mellis. Genome rearrangements, conserved orthologous genes (COG) categories and potential prophage regions were identified across the 3 novel strains. The new A. kunkeei strain was enriched in genes related to replication, recombination and repair, the L. kullabergensis strain was enriched for carbohydrate transport, and the B. mellis strain was enriched in transcription or transcriptional regulation and in some genes with unknown functions. Prophage regions were identified in the A. kunkeei and L. kullabergensis isolates. These new bee-associated strains add to our growing knowledge of the honey bee gut microbiome, and to Lactobacillaceae genomics more broadly.

A real-time PCR method for quantification of the total and major variant strains of the deformed wing virus.

European honey bees (Apis mellifera) are critically important to global food production by virtue of their pollination services but are severely threatened by deformed wing virus (DWV) especially in the presence of the external parasite Varroa destructor. DWV exists as many viral strains with the two major variants (DWV-A and DWV-B) varying in virulence. A single plasmid standard was constructed containing three sections for the specific determination of DWV-A (VP2 capsid region), DWV-B (IRES) and a conserved region suitable for total DWV (helicase region). The assays were confirmed as specific and discriminatory with limits of detections of 25, 25 and 50 genome equivalents for DWV-A, DWV-B and total-DWV, respectively. The methods were successfully tested on Apis mellifera and V. destructor samples with varying DWV profiles. The new method determined a more accurate total DWV titre in samples with substantial DWV-B than the method currently described in the COLOSS Beebook. The proposed assays could be utilized for the screening of large quantities of bee material for both a total DWV load overview along with more detailed investigations into DWV-A and DWV-B profiles.

High rates of infection by blood parasites during the nestling phase in UK Columbids with notes on ecological associations.

Studies of blood parasite infection in nestling birds rarely find a high prevalence of infection. This is likely due to a combination of short nestling periods (limiting the age at which nestlings can be sampled) and long parasite prepatent periods before gametocytes can be detected in peripheral blood. Here we examine rates of blood parasite infection in nestlings from three Columbid species in the UK. We use this system to address two key hypotheses in the epidemiology of avian haemoparasites: first, that nestlings in open nests have a higher prevalence of infection; and second, that nestlings sampled at 14 days old have a higher apparent infection rate than those sampled at 7 days old. Open-nesting individuals had a 54% infection rate compared with 25% for box-nesters, probably due to an increased exposure of open-nesting species to dipteran vectors. Nestlings sampled at 14 days had a 68% infection rate compared with 32% in nestlings sampled at 7 days, suggesting that rates of infection in the nest are high. Further work should examine nestlings post-fledging to identify rates of successful parasite infection (as opposed to abortive development within a dead-end host) as well as impacts on host post-fledging survival and behaviour.

Propagation of the Israeli vaccine strain of Anaplasma centrale in tick cell lines.

Anaplasma centrale has been used in cattle as a live blood vaccine against the more pathogenic Anaplasma marginale for over 100 years. While A. marginale can be propagated in vitro in tick cell lines, facilitating studies on antigen production, immunisation and vector-pathogen interaction, to date there has been no in vitro culture system for A. centrale. In the present study, 25 cell lines derived from 13 ixodid tick species were inoculated with the Israeli vaccine strain of A. centrale and monitored for at least 12 weeks by microscopic examination of Giemsa-stained cytocentrifuge smears. Infection of 19 tick cell lines was subsequently attempted by transfer of cell-free supernate from vaccine-inoculated tick cells. In two separate experiments, rickettsial inclusions were detected in cultures of the Rhipicephalus appendiculatus cell line RAE25 28-32 days following inoculation with the vaccine. Presence of A. centrale in the RAE25 cells was confirmed by PCR assays targeting the 16S rRNA, groEL and msp4 genes; sequenced PCR products were 100% identical to published sequences of the respective genes in the Israeli vaccine strain of A. centrale. A. centrale was taken through three subcultures in RAE25 cells over a 30 week period. In a single experiment, the Dermacentor variabilis cell line DVE1 was also detectably infected with A. centrale 11 weeks after inoculation with the vaccine. Availability of an in vitro culture system for A. centrale in tick cells opens up the possibility of generating a safer and more ethical vaccine for bovine anaplasmosis.