Assessment of bacterial diversity in the cattle tick Rhipicephalus (Boophilus) microplus through tag-encoded pyrosequencing

Abstract

Background: Ticks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, Rhipicephalus (Boophilus) microplus, hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored. The objective of this study was to explore the R. microplus microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of R. microplus collected during outbreaks in southern Texas.

Results: Raw data from bTEFAP were screened and trimmed based upon quality scores and binned into individual sample collections. Bacteria identified to the species level include Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, Staphylococcus sciuri, Serratia marcescens, Corynebacterium glutamicum, and Finegoldia magna. One hundred twenty-one bacterial genera were detected in all the life stages and tissues sampled. The total number of genera identified by tick sample comprised: 53 in adult males, 61 in adult females, 11 in gut tissue, 7 in ovarian tissue, and 54 in the eggs. Notable genera detected in the cattle tick include Wolbachia, Coxiella, and Borrelia. The molecular approach applied in this study allowed us to assess the relative abundance of the microbiota associated with R. microplus.

Conclusions: This report represents the first survey of the bacteriome in the cattle tick using non-culture based molecular approaches. Comparisons of our results with previous bacterial surveys provide an indication of geographic variation in the assemblages of bacteria associated with R. microplus. Additional reports on the identification of new bacterial species maintained in nature by R. microplus that may be pathogenic to its vertebrate hosts are expected as our understanding of its microbiota expands. Increased awareness of the role R. microplus can play in the transmission of pathogenic bacteria will enhance our ability to mitigate its economic impact on animal agriculture globally. This recognition should be included as part of analyses to assess the risk for re-invasion of areas like the United States of America where R. microplus was eradicated.

Figure 1

Heat map depicting bacterial diversity and relative abundance in life stages and tissue samples from R. microplus. * Letters (A-C) used to identify individual life stage samples where applicable. Double hierarchical dendogram shows different bacteria distribution between taxonomic levels based on complete linkage clustering, and Manhattan distance methods with no scaling. Dendrogram linkages and distance of the bacterial taxa or traceback groups are not phylogenetic, but based upon relative abundance of the taxa within the samples. Traceback means bacterial classifications were based upon the percent identity of the sample sequence to known sequences, the percent divergence was then used to adjust identifications to the taxonomic level with the highest degree of confidence (e.g. a percent divergence < 3% can be expected to provide confidence at the species level, > 3% but < 5% at the genera level, etc.). Classifications were compiled after traceback. Legend and scale shown in upper left corner of the figure represent colors in heat map associated with the relative percentage of each traceback grouping of bacteria (cluster of variables in Y-axis) within each tick sample (X-axis clustering). Tick samples along the X-axis with Manhattan distances are indicated by branch length and associated with the scale located at the upper right corner of the figure. Bacterial traceback groups along the Y-axis are also clustered according to Manhattan distances; the respective scale is indicated in the figure's lower left corner.

Figure 2

Relative abundance of bacterial genera in life stages and tissue samples from R. microplus as detected by bTEFAP pyrosequencing. a) Adult female cattle tick. Mean percentages (n = 2). Values below 1% were grouped as "Other" with total value of 9.5%. "Other" group includes: Staphylococcus (0.7%), Bacillus (0.5%), Streptococcus (0.7%), Vagococcus (0.3%), Pseudobutyrivibrio (0.7%), Nocardioides (0.2%), Asteroleplasma (0.9%), Ruminococcus (0.4%), Escherichia (0.9%), Acetivibrio (0.3%), Erwinia (0.1%), Pedobacter (0.2%), Dermabacter (0.1%), Ornithinicoccus (0.2%), Oribacterium (0.7%), Alkaliflexus (0.2%), Paludibacter (0.5%), Pantoea (0.2%), Cytophaga (0.1%), Mitsuokella (0.1%), Enterobacter (0.1%), Paucisalibacillus (0.4%), Lachnobacterium (0.1%), Caldithrix (0.2%), Shigella (0.1%), Solirubrobacter (0.1%), Rhodobacter (0.1%), Desulfosporosinus (0.1%). b) Adult male cattle tick. Mean percentages (n = 2). Values below 1% were grouped as "Other" with total value of 3.8%. "Other" group includes: Coxiella (0.1%), Prevotella (0.3%), Rikenella (0.1%), Pseudomonas (0.2%), Escherichia (0.3%), Hallella (0.3%), Pantoea (0.1%), Moraxella (0.7%), Arthrobacter (0.1%), Enhydrobacter (0.1%), Mogibacterium (0.1%), Kocuria (0.5%), Enterobacter (0.1%), Exiguobacterium (0.2%), Lysinibacillus (0.1%), Belnapia (0.1%). c) Cattle tick egg. Mean percentages (n = 3). Values below 1% were grouped as "Other" with total value of 6.9%. "Other" group includes: Achromobacter (0.3%), Enterococcus (0.1%), Clostridium (0.1%), Serratia (0.7%), Ruminococcus (0.3%), Propionibacterium (0.4%), Klebsiella (0.2%), Acetivibrio (0.9%), Pedobacter (0.6%), Alkaliflexus (0.4%), Centipeda (0.5%), Pantoea (0.1%), Brevibacterium (0.2%), Rubrivivax (0.4%), Enhydrobacter (0.2%), Rhodoferax (0.3%), Sporocytophaga (0.1%), Alkanindiges (0.2%), Sphingopyxis (0.1%), Caulobacter (0.1%), Trichococcus (0.1%), Comamonas (0.1%), Anaerotruncus (0.1%), Akkermansia (0.1%), Legionella (0.1%). d) Adult female cattle tick gut. Pool of tissue from five ticks tested. Values below 1% were grouped as "Other" with total value of 0.3%. "Other" group includes: Corynebacterium (0.3%). e) Adult cattle tick ovary. Pool of tissue from five ticks tested. Values below 1% were grouped as "Other" with total value of 1.8%. "Other" group includes: Borrelia (0.9%), Cryobacterium (0.9%).