Gut transcriptome of replete adult female cattle ticks, Rhipicephalus (Boophilus) microplus, feeding upon a Babesia bovis-infected bovine host.

As it feeds upon cattle, Rhipicephalus (Boophilus) microplus is capable of transmitting a number of pathogenic organisms, including the apicomplexan hemoparasite Babesia bovis, a causative agent of bovine babesiosis. The R. microplus female gut transcriptome was studied for two cohorts: adult females feeding on a bovine host infected with B. bovis and adult females feeding on an uninfected bovine. RNA was purified and used to generate a subtracted cDNA library from B. bovis-infected female gut, and 4,077 expressed sequence tags (ESTs) were sequenced. Gene expression was also measured by a microarray designed from the publicly available R. microplus gene index: BmiGI Version 2. We compared gene expression in the tick gut from females feeding upon an uninfected bovine to gene expression in tick gut from females feeding upon a splenectomized bovine infected with B. bovis. Thirty-three ESTs represented on the microarray were expressed at a higher level in female gut samples from the ticks feeding upon a B. bovis-infected calf compared to expression levels in female gut samples from ticks feeding on an uninfected calf. Forty-three transcripts were expressed at a lower level in the ticks feeding upon B. bovis-infected female guts compared with expression in female gut samples from ticks feeding on the uninfected calf. These array data were used as initial characterization of gene expression associated with the infection of R. microplus by B. bovis.

Choosing Wisely clinical decision support adherence and associated inpatient outcomes.

OBJECTIVES: To determine whether utilization of clinical decision support (CDS) is correlated with improved patient clinical and financial outcomes. STUDY DESIGN: Observational study of 26,424 patient encounters. In the treatment group, the provider adhered to all CDS recommendations. In the control group, the provider did not adhere to CDS recommendations. METHODS: An observational study of provider adherence to a CDS system was conducted using inpatient encounters spanning 3 years. Data comprised alert status (adherence), provider type (resident, attending), patient demographics, clinical outcomes, Medicare status, and diagnosis information. We assessed the associations between alert adherence and 4 outcome measures: encounter length of stay, odds of 30-day readmission, odds of complications of care, and total direct costs. The associations between alert adherence and the outcome measures were estimated using 4 generalized linear models that adjusted for potential confounders, such as illness severity and case complexity. RESULTS: The total encounter cost increased 7.3% (95% CI, 3.5%-11%) for nonadherent encounters versus adherent encounters. We found a 6.2% (95% CI, 3.0%-9.4%) increase in length of stay for nonadherent versus adherent encounters. The odds ratio for readmission within 30 days increased by 1.14 (95% CI, 0.998-1.31) for nonadherent versus adherent encounters. The odds ratio for complications increased by 1.29 (95% CI, 1.04-1.61) for nonadherent versus adherent encounters. CONCLUSIONS: Consistent improvements in measured outcomes were seen in the treatment group versus the control group. We recommend that provider organizations consider the introduction of real-time CDS to support adherence to evidence-based guidelines, but because we cannot determine the cause of the associations between CDS interventions and improved clinical and financial outcomes, further study is required.

Bioinformatic prediction of G protein-coupled receptor encoding sequences from the transcriptome of the foreleg, including the Haller's organ, of the cattle tick, Rhipicephalus australis.

The cattle tick of Australia, Rhipicephalus australis, is a vector for microbial parasites that cause serious bovine diseases. The Haller's organ, located in the tick's forelegs, is crucial for host detection and mating. To facilitate the development of new technologies for better control of this agricultural pest, we aimed to sequence and annotate the transcriptome of the R. australis forelegs and associated tissues, including the Haller's organ. As G protein-coupled receptors (GPCRs) are an important family of eukaryotic proteins studied as pharmaceutical targets in humans, we prioritized the identification and classification of the GPCRs expressed in the foreleg tissues. The two forelegs from adult R. australis were excised, RNA extracted, and pyrosequenced with 454 technology. Reads were assembled into unigenes and annotated by sequence similarity. Python scripts were written to find open reading frames (ORFs) from each unigene. These ORFs were analyzed by different GPCR prediction approaches based on sequence alignments, support vector machines, hidden Markov models, and principal component analysis. GPCRs consistently predicted by multiple methods were further studied by phylogenetic analysis and 3D homology modeling. From 4,782 assembled unigenes, 40,907 possible ORFs were predicted. Using Blastp, Pfam, GPCRpred, TMHMM, and PCA-GPCR, a basic set of 46 GPCR candidates were compiled and a phylogenetic tree was constructed. With further screening of tertiary structures predicted by RaptorX, 6 likely GPCRs emerged and the strongest candidate was classified by PCA-GPCR to be a GABAB receptor.

Prediction of G protein-coupled receptor encoding sequences from the synganglion transcriptome of the cattle tick, Rhipicephalus microplus.

The cattle tick, Rhipicephalus (Boophilus) microplus, is a pest which causes multiple health complications in cattle. The G protein-coupled receptor (GPCR) super-family presents a candidate target for developing novel tick control methods. However, GPCRs share limited sequence similarity among orthologous family members, and there is no reference genome available for R. microplus. This limits the effectiveness of alignment-dependent methods such as BLAST and Pfam for identifying GPCRs from R. microplus. However, GPCRs share a common structure consisting of seven transmembrane helices. We present an analysis of the R. microplus synganglion transcriptome using a combination of structurally-based and alignment-free methods which supplement the identification of GPCRs by sequence similarity. TMHMM predicts the number of transmembrane helices in a protein sequence. GPCRpred is a support vector machine-based method developed to predict and classify GPCRs using the dipeptide composition of a query amino acid sequence. These two bioinformatic tools were applied to our transcriptome assembly of the cattle tick synganglion. Together, BLAST and Pfam identified 85 unique contigs as encoding partial or full length candidate cattle tick GPCRs. Collectively, TMHMM and GPCRpred identified 27 additional GPCR candidates that BLAST and Pfam missed. This demonstrates that the addition of structurally-based and alignment-free bioinformatic approaches to transcriptome annotation and analysis produces a greater collection of prospective GPCRs than an analysis based solely upon methodologies dependent upon sequence alignment and similarity.

The ovarian transcriptome of the cattle tick, Rhipicephalus (Boophilus) microplus, feeding upon a bovine host infected with Babesia bovis.

BACKGROUND: Cattle babesiosis is a tick-borne disease of cattle with the most severe form of the disease caused by the apicomplexan, Babesia bovis. Babesiosis is transmitted to cattle through the bite of infected cattle ticks of the genus Rhipicephalus. The most prevalent species is Rhipicephalus (Boophilus) microplus, which is distributed throughout the tropical and subtropical countries of the world. The transmission of B. bovis is transovarian and a previous study of the R. microplus ovarian proteome identified several R. microplus proteins that were differentially expressed in response to infection. Through various approaches, we studied the reaction of the R. microplus ovarian transcriptome in response to infection by B. bovis. METHODS: A group of ticks were allowed to feed on a B. bovis-infected splenectomized calf while a second group fed on an uninfected splenectomized control calf. RNA was purified from dissected adult female ovaries of both infected and uninfected ticks and a subtracted B. bovis-infected cDNA library was synthesized, subtracting with the uninfected ovarian RNA. Four thousand ESTs were sequenced from the ovary subtracted library and annotated. RESULTS: The subtracted library dataset assembled into 727 unique contigs and 2,161 singletons for a total of 2,888 unigenes, Microarray experiments designed to detect B. bovis-induced gene expression changes indicated at least 15 transcripts were expressed at a higher level in ovaries from ticks feeding upon the B. bovis-infected calf as compared with ovaries from ticks feeding on an uninfected calf. We did not detect any transcripts from these microarray experiments that were expressed at a lower level in the infected ovaries compared with the uninfected ovaries. Using the technique called serial analysis of gene expression, 41 ovarian transcripts from infected ticks were differentially expressed when compared with transcripts of controls. CONCLUSION: Collectively, our experimental approaches provide the first comprehensive profile of the R. microplus ovarian transcriptome responding to infection by B. bovis. This dataset should prove useful in molecular studies of host-pathogen interactions between this tick and its apicomplexan parasite.

Analysis of Babesia bovis infection-induced gene expression changes in larvae from the cattle tick, Rhipicephalus (Boophilus) microplus.

BACKGROUND: Cattle babesiosis is a tick-borne disease of cattle that has severe economic impact on cattle producers throughout the world's tropical and subtropical countries. The most severe form of the disease is caused by the apicomplexan, Babesia bovis, and transmitted to cattle through the bite of infected cattle ticks of the genus Rhipicephalus, with the most prevalent species being Rhipicephalus (Boophilus) microplus. We studied the reaction of the R. microplus larval transcriptome in response to infection by B. bovis. METHODS: Total RNA was isolated for both uninfected and Babesia bovis-infected larval samples. Subtracted libraries were prepared by subtracting the B. bovis-infected material with the uninfected material, thus enriching for expressed genes in the B. bovis-infected sample. Expressed sequence tags from the subtracted library were generated, assembled, and sequenced. To complement the subtracted library method, differential transcript expression between samples was also measured using custom high-density microarrays. The microarray probes were fabricated using oligonucleotides derived from the Bmi Gene Index database (Version 2). Array results were verified for three target genes by real-time PCR. RESULTS: Ticks were allowed to feed on a B. bovis-infected splenectomized calf and on an uninfected control calf. RNA was purified in duplicate from whole larvae and subtracted cDNA libraries were synthesized from Babesia-infected larval RNA, subtracting with the corresponding uninfected larval RNA. One thousand ESTs were sequenced from the larval library and the transcripts were annotated. We used a R. microplus microarray designed from a R. microplus gene index, BmiGI Version 2, to look for changes in gene expression that were associated with infection of R. microplus larvae. We found 24 transcripts were expressed at a statistically significant higher level in ticks feeding upon a B. bovis-infected calf contrasted to ticks feeding on an uninfected calf. Six transcripts were expressed at a statistically significant lower level in ticks feeding upon a B. bovis-infected calf contrasted to ticks feeding on an uninfected calf. CONCLUSION: Our experimental approaches yielded specific differential gene expression associated with the infection of R. microplus by B. bovis. Overall, an unexpectedly low number of transcripts were found to be differentially expressed in response to B. bovis infection. Although the BmiGI Version 2 gene index (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=b_microplus) was a useful database to help assign putative function to some transcripts, a majority of the differentially expressed transcripts did not have annotation that was useful for assignment of function and specialized bioinformatic approaches were necessary to increase the information from these transcriptome experiments.

Pyrosequencing-based analysis of the microbiome associated with the horn fly, Haematobia irritans.

The horn fly, Haematobia irritans, is one of the most economically important pests of cattle. Insecticides have been a major element of horn fly management programs. Growing concerns with insecticide resistance, insecticide residues on farm products, and non-availability of new generation insecticides, are serious issues for the livestock industry. Alternative horn fly control methods offer the promise to decrease the use of insecticides and reduce the amount of insecticide residues on livestock products and give an impetus to the organic livestock farming segment. The horn fly, an obligatory blood feeder, requires the help of microflora to supply additional nutrients and metabolize the blood meal. Recent advancements in DNA sequencing methodologies enable researchers to examine the microflora diversity independent of culture methods. We used the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) method to carry out the classification analysis of bacterial flora in adult female and male horn flies and horn fly eggs. The bTEFAP method identified 16S rDNA sequences in our samples which allowed the identification of various prokaryotic taxa associated with the life stage examined. This is the first comprehensive report of bacterial flora associated with the horn fly using a culture-independent method. Several rumen, environmental, symbiotic and pathogenic bacteria associated with the horn fly were identified and quantified. This is the first report of the presence of Wolbachia in horn flies of USA origin and is the first report of the presence of Rikenella in an obligatory blood feeding insect.