The GEA pipeline for characterizing Escherichia coli and Salmonella genomes.

Salmonella enterica and Escherichia coli are major food-borne human pathogens, and their genomes are routinely sequenced for clinical surveillance. Computational pipelines designed for analyzing pathogen genomes should both utilize the most current information from annotation databases and increase the coverage of these databases over time. We report the development of the GEA pipeline to analyze large batches of E. coli and S. enterica genomes. The GEA pipeline takes as input paired Illumina raw reads files which are then assembled followed by annotation. Alternatively, assemblies can be provided as input and directly annotated. The pipeline provides predictive genome annotations for E. coli and S. enterica with a focus on the Center for Genomic Epidemiology tools. Annotation results are provided as a tab delimited text file. The GEA pipeline is designed for large-scale E. coli and S. enterica genome assembly and characterization using the Center for Genomic Epidemiology command-line tools and high-performance computing. Large scale annotation is demonstrated by an analysis of more than 14,000 Salmonella genome assemblies. Testing the GEA pipeline on E. coli raw reads demonstrates reproducibility across multiple compute environments and computational usage is optimized on high performance computers.

Distribution of Extremely Heat-Resistant Escherichia coli in the Beef Production and Processing Continuum.

Understanding the dynamics of stress-resistant Escherichia coli (E. coli) across the meat production and processing continuum is important for tracking sources of such microbes and devising effective modes of control. The Locus of Heat Resistance (LHR) is a ∼14-19 Kb genetic element imparting extreme heat resistance (XHR) in Enterobacteriaceae. It has been hypothesized that thermal and antimicrobial interventions applied during meat processing may select for LHR+E. coli. Thus, our goal was to study the prevalence and molecular biology of LHR+E. coli among lots of beef cattle (n = 3) from production through processing. Two hundred thirty-two generic E. coli isolated from the same animals through seven stages of the beef processing continuum (cattle in feedyards to packaged strip loins) were examined. LHR+E. coli were rare (0.6%; 1 of 180) among the early stages of the beef continuum (feces and hides at feedlot, feces and hides at harvest, and preevisceration carcasses), whereas the prevalence of LHR+E. coli on final carcasses and strip loins was remarkably higher. Half (14 of 28) of the final carcass E. coli possessed the LHR, while 79.2% (19 of 24) of the strip loin E. coli did. Eighty-five percent (29 of 34) of the LHR+E. coli presented with the XHR phenotype. The selection or enrichment of LHR+E. coli from harvest steps to the final products appeared unlikely as the LHR+E. coli isolates were effectively controlled by antimicrobial interventions typically used during beef processing. Further, whole-genome sequencing of the isolates suggested LHR+E. coli are persisting in the chilled processing environment and that horizontal LHR transfer among E. coli isolates may take place.

Probiotics in milk replacer affect the microbiome of the lung in neonatal dairy calves.

Introduction: Probiotics have been investigated for their many health benefits and impact on the microbiota of the gut. Recent data have also supported a gut-lung axis regarding the bacterial populations (microbiomes) of the two locations; however, little research has been performed to determine the effects of oral probiotics on the microbiome of the bovine respiratory tract. We hypothesized that probiotic treatment would result in changes in the lung microbiome as measured in lung lavage fluid. Our overall goal was to characterize bacterial populations in the lungs of calves fed probiotics in milk replacer and dry rations from birth to weaning. Methods: A group of 20 dairy calves was split into two treatment groups: probiotic (TRT; N = 10, milk replacer +5 g/d probiotics; Bovamine Dairy, Chr. Hansen, Inc., Milwaukee, WI) and control (CON; N = 10, milk replacer only). On day 0, birth weight was obtained, and calves were provided colostrum as per the dairy SOP. On day 2, probiotics were added to the milk replacer of the treated group and then included in their dry ration. Lung lavages were performed on day 52 on five random calves selected from each treatment group. DNA was extracted from lavage fluid, and 16S ribosomal RNA (rRNA) gene hypervariable regions 1-3 were amplified by PCR and sequenced using next-generation sequencing (Illumina MiSeq) for the identification of the bacterial taxa present. Taxa were classified into both operational taxonomic units (OTUs) and amplicon sequence variants (ASVs). Results: Overall, the evaluation of these samples revealed that the bacterial genera identified in the lung lavage samples of probiotic-fed calves as compared to the control calves were significantly different based on the OTU dataset (p < 0.05) and approached significance for the ASV dataset (p < 0.06). Additionally, when comparing the diversity of taxa in lung lavage samples to nasal and tonsil samples, taxa diversity of lung samples was significantly lower (p < 0.05). Discussion: In conclusion, analysis of the respiratory microbiome in lung lavage samples after probiotic treatment provides insight into the distribution of bacterial populations in response to oral probiotics and demonstrates that oral probiotics affect more than the gut microbiome.

Whole genome sequencing of Moraxella bovis strains from North America reveals two genotypes with different genetic determinants.

BACKGROUND: Moraxella bovis and Moraxella bovoculi both associate with infectious bovine keratoconjunctivitis (IBK), an economically significant and painful ocular disease that affects cattle worldwide. There are two genotypes of M. bovoculi (genotypes 1 and 2) that differ in their gene content and potential virulence factors, although neither have been experimentally shown to cause IBK. M. bovis is a causative IBK agent, however, not all strains carry a complete assortment of known virulence factors. The goals of this study were to determine the population structure and depth of M. bovis genomic diversity, and to compare core and accessory genes and predicted outer membrane protein profiles both within and between M. bovis and M. bovoculi. RESULTS: Phylogenetic trees and bioinformatic analyses of 36 M. bovis chromosomes sequenced in this study and additional available chromosomes of M. bovis and both genotype 1 and 2 M. bovoculi, showed there are two genotypes (1 and 2) of M. bovis. The two M. bovis genotypes share a core of 2015 genes, with 121 and 186 genes specific to genotype 1 and 2, respectively. The two genotypes differ by their chromosome size and prophage content, encoded protein variants of the virulence factor hemolysin, and by their affiliation with different plasmids. Eight plasmid types were identified in this study, with types 1 and 6 observed in 88 and 56% of genotype 2 strains, respectively, and absent from genotype 1 strains. Only type 1 plasmids contained one or two gene copies encoding filamentous haemagglutinin-like proteins potentially involved with adhesion. A core of 1403 genes was shared between the genotype 1 and 2 strains of both M. bovis and M. bovoculi, which encoded a total of nine predicted outer membrane proteins. CONCLUSIONS: There are two genotypes of M. bovis that differ in both chromosome content and plasmid profiles and thus may not equally associate with IBK. Immunological reagents specifically targeting select genotypes of M. bovis, or all genotypes of M. bovis and M. bovoculi together could be designed from the outer membrane proteins identified in this study.

Characterization of Escherichia coli harboring colibactin genes (clb) isolated from beef production and processing systems.

Certain strains of Escherichia coli possess and express the toxin colibactin (Clb) which induces host mutations identical to the signature mutations of colorectal cancer (CRC) that lead to tumorigenic lesions. Since cattle are a known reservoir of several Enterobacteriaceae including E. coli, this study screened for clb amongst E. coli isolated from colons of cattle-at-harvest (entering beef processing facility; n = 1430), across the beef processing continuum (feedlot to finished subprimal beef; n = 232), and in ground beef (n = 1074). Results demonstrated that clb+ E. coli were present in cattle and beef. Prevalence of clb+ E. coli from colonic contents of cattle and ground beef was 18.3% and 5.5%, respectively. clb+ E. coli were found susceptible to commonly used meat processing interventions. Whole genome sequencing of 54 bovine and beef clb+ isolates showed clb occurred in diverse genetic backgrounds, most frequently in phylogroup B1 (70.4%), MLST 1079 (42.6%), and serogroup O49 (40.7%).

Twenty-Four-Month Longitudinal Study Suggests Little to No Horizontal Gene Transfer In Situ between Third-Generation Cephalosporin-Resistant Salmonella and Third-Generation Cephalosporin-Resistant Escherichia coli in a Beef Cattle Feedyard.

ABSTRACT: Third-generation cephalosporins (3GCs) are preferred treatments for serious human Salmonella enterica infections. Beef cattle are suspected to contribute to human 3GC-resistant Salmonella infections. Commensal 3GC-resistant Escherichia coli are thought to act as reservoirs of 3GC resistance because these strains are isolated more frequently than are 3GC-resistant Salmonella strains at beef cattle feedyards. During each of 24 consecutive months, four samples of pen surface material were obtained from five pens (N = 480) at a Nebraska feedyard to determine to the contribution of 3GC-resistant E. coli to the occurrence of 3GC-resistant Salmonella. Illumina whole genome sequencing was performed, and susceptibility to 14 antimicrobial agents was determined for 121 3GC-susceptible Salmonella, 121 3GC-resistant Salmonella, and 203 3GC-resistant E. coli isolates. 3GC-susceptible Salmonella isolates were predominantly from serotypes Muenchen (70.2%) and Montevideo clade 1 (23.1%). 3GC-resistant Salmonella isolates were predominantly from serotypes Montevideo clade 2 (84.3%). One bla gene type (blaCMY-2) and the IncC plasmid replicon were present in 100 and 97.5% of the 3GC-resistant Salmonella, respectively. Eleven bla gene types were detected in the 3GC-resistant E. coli, which were distributed across 42 multilocus sequence types. The blaCMY-2 gene and IncC plasmid replicon were present in 37.9 and 9.9% of the 3GC-resistant E. coli, respectively. These results suggest that 3GC resistance in Salmonella was primarily due the persistence of Salmonella Montevideo clade 2 with very minimal or no contribution from 3GC-resistant E. coli via horizontal gene transfer and that 3GC-resistant E. coli may not be a useful indicator for 3GC-resistant Salmonella in beef cattle production environments.

MALDI-TOF MS Biomarker Detection Models to Distinguish RTX Toxin Phenotypes of Moraxella bovoculi Strains Are Enhanced Using Calcium Chloride Supplemented Agar.

Moraxella bovoculi is the bacterium most often cultured from ocular lesions of cattle with infectious bovine keratoconjunctivitis, also known as bovine pinkeye. Some strains of M. bovoculi contain operons encoding for a repeats-in-toxin (RTX) toxin, which is a known virulence factor of multiple veterinary pathogens. We explored the utility of MALDI-TOF MS and biomarker detection models to classify the presence or absence of an RTX phenotype in M. bovoculi. Ninety strains that had undergone whole genome sequencing were classified by the presence or absence of complete RTX operons and confirmed with a visual assessment of hemolysis on blood agar. Strains were grown on Tryptic Soy Agar (TSA) with 5% sheep blood, TSA with 5% bovine blood that was supplemented with 10% fetal bovine serum, 10 mmol/LCaCl2, or both. The formulations were designed to determine the influence of growth media on toxin production or activity, as calcium ions are required for toxin secretion and activity. Mass spectra were obtained for strains grown on each agar formulation and biomarker models were developed using ClinProTools 3.0 software. The most accurate model was developed using spectra from strains grown on TSA with 5% bovine blood and supplemented with CaCl2, which had a sensitivity and specificity of 93.3% and 73.3%, respectively, regarding RTX phenotype classification. The same biomarker model algorithm developed from strains grown on TSA with 5% sheep blood had a substantially lower sensitivity and specificity of 68.0% and 52.0%, respectively. Our results indicate that MALDI-TOF MS biomarker models can accurately classify strains of M. bovoculi regarding the presence or absence of RTX toxin operons and that agar media modifications improve the accuracy of these models.

Differences between predicted outer membrane proteins of genotype 1 and 2 Mannheimia haemolytica.

BACKGROUND: Mannheimia haemolytica strains isolated from North American cattle have been classified into two genotypes (1 and 2). Although members of both genotypes have been isolated from the upper and lower respiratory tracts of cattle with or without bovine respiratory disease (BRD), genotype 2 strains are much more frequently isolated from diseased lungs than genotype 1 strains. The mechanisms behind the increased association of genotype 2 M. haemolytica with BRD are not fully understood. To address that, and to search for interventions against genotype 2 M. haemolytica, complete, closed chromosome assemblies for 35 genotype 1 and 34 genotype 2 strains were generated and compared. Searches were conducted for the pan genome, core genes shared between the genotypes, and for genes specific to either genotype. Additionally, genes encoding outer membrane proteins (OMPs) specific to genotype 2 M. haemolytica were identified, and the diversity of their protein isoforms was characterized with predominantly unassembled, short-read genomic sequences for up to 1075 additional strains. RESULTS: The pan genome of the 69 sequenced M. haemolytica strains consisted of 3111 genes, of which 1880 comprised a shared core between the genotypes. A core of 112 and 179 genes or gene variants were specific to genotype 1 and 2, respectively. Seven genes encoding predicted OMPs; a peptidase S6, a ligand-gated channel, an autotransporter outer membrane beta-barrel domain-containing protein (AOMB-BD-CP), a porin, and three different trimeric autotransporter adhesins were specific to genotype 2 as their genotype 1 homologs were either pseudogenes, or not detected. The AOMB-BD-CP gene, however, appeared to be truncated across all examined genotype 2 strains and to likely encode dysfunctional protein. Homologous gene sequences from additional M. haemolytica strains confirmed the specificity of the remaining six genotype 2 OMP genes and revealed they encoded low isoform diversity at the population level. CONCLUSION: Genotype 2 M. haemolytica possess genes encoding conserved OMPs not found intact in more commensally prone genotype 1 strains. Some of the genotype 2 specific genes identified in this study are likely to have important biological roles in the pathogenicity of genotype 2 M. haemolytica, which is the primary bacterial cause of BRD.

Rapid differentiation of Moraxella bovoculi genotypes 1 and 2 using MALDI-TOF mass spectrometry profiles.

Moraxella bovoculi is the most frequently isolated bacteria from the eyes of cattle with Infectious Bovine Keratoconjunctivitis (IBK), also known as bovine pinkeye. Two distinct genotypes of M. bovoculi, genotype 1 and genotype 2, were characterized after whole genome sequencing showed a large degree of single nucleotide polymorphism (SNP) diversity within the species. To date, both genotypes have been isolated from the eyes of cattle without clinical signs of IBK while only genotype 1 strains have been isolated from the eyes of cattle with clinical signs of IBK. We used 38 known genotype 1 strains and 26 known genotype 2 strains to assess the ability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to accurately genotype M. bovoculi strains using mass spectrum biomarkers. Mass spectrum data was analyzed with ClinProTools 3.0 software and six models were developed that classify strain genotypes with accuracies ranging from 90.6% - 100%. Finally, using four of the most genotype-specific peaks that also exhibited high peak intensities from the six automated models, we developed a customized model (UNL assisted model) that had recognition capability, validation, and classification accuracies of 100% for genotype classification. Our results indicate that MALDI-TOF MS biomarkers can be used to accurately discriminate genotypes of M. bovoculi without the need for additional methods.

Digestive tract microbiota of beef cattle that differed in feed efficiency.

We hypothesized cattle that differed in BW gain had different digestive tract microbiota. Two experiments were conducted. In both experiments, steers received a diet that consisted of 8.0% chopped alfalfa hay, 20% wet distillers grain with solubles, 67.75% dry-rolled corn, and 4.25% vitamin/mineral mix (including monensin) on a dry matter basis. Steers had ad libitum access to feed and water. In experiment 1, 144 steers (age = 310 ± 1.5 d; BW = 503 ± 37.2 kg) were individually fed for 105 d. Ruminal digesta samples were collected from eight steers with the greatest (1.96 ± 0.02 kg/d) and eight steers with the least ADG (1.57 ± 0.02 kg/d) that were within ±0.32 SD of the mean (10.1 ± 0.05 kg/d) dry matter. In experiment 2, 66 steers (age = 396 ± 1 d; BW = 456 ± 5 kg) were individually fed for 84 d. Rumen, duodenum, jejunum, ileum, cecum, and colon digesta samples were collected from eight steers with the greatest (2.39 ± 0.06 kg/d) and eight steers with the least ADG (1.85 ± 0.06 kg/d) that were within ±0.55 SD of the mean dry matter intake (11.9 ± 0.1 kg/d). In both studies, DNA was isolated and the V1 to V3 regions of the 16S rRNA gene were sequenced. Operational taxonomic units were classified using 0.03 dissimilarity and identified using the Greengenes 16S rRNA gene database. In experiment 1, there were no differences in the Chao1, Shannon, Simpson, and InvSimpson diversity indexes or the permutation multivariate analysis of variance (PERMANOVA; P = 0.57). The hierarchical test returned six clades as being differentially abundant between steer classifications (P < 0.05). In experiment 2, Chao1, Shannon, Simpson, and InvSimpson diversity indexes and PERMANOVA between steer classified as less or greater ADG did not differ (P > 0.05) for the rumen, duodenum, ileum, cecum, and colon. In the jejunum, there tended to be a difference in the Chao1 (P = 0.09) and Simpson diversity (P = 0.09) indexes between steer classifications, but there was no difference in the Shannon (P = 0.14) and InvSimpson (P = 0.14) diversity indexes. Classification groups for the jejunum differed (P = 0.006) in the PERMANOVA. The hierarchical dependence false discovery rate procedure returned 11 clades as being differentially abundant between steer classifications in the jejunum (P < 0.05). The majority of the OTU were in the Families Corynebacteriaceae and Coriobacteriaceae. This study suggests that intestinal differences in the microbiota of ruminants may be associated with animal performance.

Classification of small ruminant lentivirus subtype A2, subgroups 1 and 2 based on whole genome comparisons and complex recombination patterns.

Background: Small ruminant lentiviruses (SRLVs) cause a multisystemic chronic wasting disease in sheep across much of the world. SRLV subtype A2 is prevalent in North America and further classified into multiple subgroups based on variation in the group antigens gene (gag) and envelope (env) genes. In sheep, the ovine transmembrane protein 154 (TMEM154) gene is associated with SRLV susceptibility. Ewes with at least one copy of TMEM154 encoding a full-length protein with glutamate at position 35 (E35; haplotypes 2 and 3), are highly susceptible to SRLV infection while ewes with any combination of TMEM154 haplotypes which encodes lysine (K35; haplotype 1), or truncated proteins (haplotypes 4 and 6) are several times less so. A2 subgroups 1 and 2 are associated with host TMEM154 genotypes; subgroup 1 with the K35/K35 genotype and subgroup 2 with the E35/E35 genotype. Methods:  Sequence variation within and among full-length assemblies of SRLV subtype A2 subgroups 1 and 2 was analyzed to identify genome-scale recombination patterns and subgroup-specific variants. Results:  Consensus viral genomes were assembled from 23 infected sheep, including animals of assorted TMEM154 genotypes comprised of haplotypes 1, 2, or 3. Viral genome analysis identified viral subgroups 1 and 2 among the samples, and revealed additional sub-structure within subgroup 2 based on models predicting complex patterns of recombination between the two subgroups in several genomes. Animals with evidence of dual subgroup infection also possessed the most diverse quasi-species and the most highly recombined consensus genomes. After accounting for recombination, 413 subgroup diagnostic single nucleotide polymorphisms (SNPs) were identified. Conclusions:  The viral subgroup framework developed to classify SRLV consensus genomes along a continuum of recombination suggests that animals with the TMEM154 E35/K35 genotype may represent a reservoir for producing viral genomes representing recombination between A2 subgroups 1 and 2.

Next-Generation Sequencing Identifies Polyunsaturated Fatty Acid Responsive Genes in the Juvenile Rat Cerebellum.

Dietary n-3 polyunsaturated fatty acids (PUFA) influence postnatal brain growth and development. However, little data exist regarding the impacts of dietary n-3 PUFA in juvenile animals post weaning, which is a time of rapid growth. We tested the hypothesis that depleting dietary n-3 PUFA would result in modifications to the cerebellar transcriptome of juvenile rats. To test this hypothesis, three week old male rats (an age that roughly corresponds to an 11 month old child in brain development) were fed diets containing either soybean oil (SO) providing 1.1% energy from α-linolenic acid (ALA; 18:3n-3; ALA-sufficient) or corn oil (CO) providing 0.13% energy from ALA (ALA-deficient) for four weeks. Fatty acids (FAs) in the cerebellum were analyzed and revealed a 4-fold increase in n-6 docosapentaenoic acid (DPA; 22:5n-6), increases in arachidonic acid (AA; 20:4n-6) and docosatetraenoic acid (DTA; 22:4n-6), but no decrease in docosahexaenoic acid (DHA; 22:6n-3), in animals fed CO versus SO. Transcript abundance was then characterized to identify differentially expressed genes (DEGs) between the two diets. Upper quartile (UQ) scaling and transcripts per million (TPM) data normalization identified 100 and 107 DEGs, respectively. Comparison of DEGs from the two normalization methods identified 70 genes that overlapped, with 90% having abundance differences less than 2-fold. Nr4a3, a transcriptional activator that plays roles in neuroprotection and learning, was elevated over 2-fold from the CO diet. These data indicate that expression of Nr4a3 in the juvenile rat cerebellum is responsive to dietary n-3 PUFA, but additional studies are needed clarify the neurodevelopmental relationships between n-3 PUFA and Nr4a3 and the resulting impacts.

Whole genome sequencing of Moraxella bovoculi reveals high genetic diversity and evidence for interspecies recombination at multiple loci.

Moraxella bovoculi is frequently cultured from the ocular secretions and conjunctiva of cattle with Infectious Bovine Keratoconjunctivitis (IBK). Previous work has shown that single nucleotide polymorphism (SNP) diversity in this species is quite high with 81,284 SNPs identified in eight genomes representing two distinct genotypes isolated from IBK affected eyes (genotype 1) and the nasopharynx of cattle without clinical IBK signs (genotype 2), respectively. The goals of this study were to identify SNPs from a collection of geographically diverse and epidemiologically unlinked M. bovoculi strains from the eyes of IBK positive cattle (n = 183) and another from the eyes of cattle (most from a single population at a single time-point) without signs of IBK (n = 63) and to characterize the genetic diversity. Strains of both genotypes were identified from the eyes of cattle without IBK signs. Only genotype 1 strains were identified from IBK affected eyes, however, these strains were isolated before the discovery of genotype 2, and the protocol for their isolation would have preferentially selected genotype 1 M. bovoculi. The core genome comprised ~74% of the whole and contained >127,000 filtered SNPs. More than 80% of these characterize diversity within genotype 1 while 23,611 SNPs (~18%) delimit the two major genotypes. Genotype 2 strains lacked a repeats-in-toxin (RTX) putative pathogenesis factor and any of ten putative antibiotic resistance genes carried within a genomic island. Within genotype 1, prevalence of these elements was 0.85 and 0.12 respectively in strains from eyes that were IBK positive. Recombination appears to be an important source of genetic diversity for genotype 1 and undermines the utility of ribosomal-locus-based species identification. The extremely high genetic diversity in genotype 1 presents a challenge to the development of an efficacious vaccine directed against them, however, several low-diversity pilin-like genes were identified. Finally, the genotype-defining SNPs described in this study are a resource that can facilitate the development of more accurate M. bovoculi diagnostic tests.

First Complete Genome Sequence of a Genotype A2, Subgroup 4 Small Ruminant Lentivirus.

Genetic variation in the ovine TMEM154 gene associates with susceptibility to small ruminant lentivirus (SRLV) infection. We report here the first complete genome sequence for a genotype A2, subgroup 4 SRLV isolated from a Hampshire ewe with two copies of a TMEM154 frameshift mutation predicted to abolish protein function.

Complete Genome Sequence of Moraxella bovis Strain Epp-63 (300), an Etiologic Agent of Infectious Bovine Keratoconjunctivitis.

We report here the complete closed genome sequence of Moraxella bovis strain Epp-63 (300) (Epp63). This strain was isolated from an infectious bovine keratoconjunctivitis (IBK) case in 1963. Since then, Epp63 has been used extensively for IBK research. Consequently, the genome sequence of Epp63 should help elucidate IBK host-pathogen interactions.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of Moraxella bovoculi and Moraxella bovis isolates from cattle.

Infectious bovine keratoconjunctivitis (IBK) is an economically significant disease caused by Moraxella bovis. Moraxella bovoculi, although not reported to cause IBK, has been isolated from the eyes of cattle diagnosed with IBK. Identification of M. bovis and M. bovoculi can be performed using biochemical or DNA-based approaches, both of which may be time consuming and inconsistent between laboratories. We conducted a comparative evaluation of M. bovoculi and M. bovis identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with a database provided by Bruker Daltonics (termed the BDAL database), the BDAL database supplemented with spectra generated in our study (termed the UNLVDC database), and with PCR-restriction-fragment length polymorphism (PCR-RFLP) typing. M. bovoculi ( n = 250) and M. bovis ( n = 18) isolates from cattle with or without IBK were used. MALDI-TOF MS using the UNLVDC database correctly identified 250 of 250 (100%) of M. bovoculi and 17 of 18 (94%) of M. bovis isolates. With the BDAL database, MALDI-TOF MS correctly identified 249 of 250 (99%) of M. bovoculi and 7 of 18 (39%) of M. bovis isolates. In comparison, the PCR-RFLP test correctly identified 210 of 250 (84%) of M. bovoculi and 12 of 18 (66%) of M. bovis isolates. Thus, MALDI-TOF MS with the UNLVDC database was the most effective identification methodology for M. bovis and M. bovoculi isolates from cattle.

Unexpected High Intragenomic Variation in Two of Three Major Pest Thrips Species Does Not Affect Ribosomal Internal Transcribed Spacer 2 (ITS2) Utility for Thrips Identification.

The mitochondrial cytochrome oxidase I gene (mtCO1) and the ribosomal internal transcribed spacer 2 region (ITS2) are among the most widely used molecular markers for insect taxonomic characterization. Three economically important species of thrips, Scirtothripsdorsalis, Thripspalmi, and Frankliniellaoccidentalis were selected to examine the extent of intragenomic variation within these two marker regions in the family Thripidae, and determine if this variation would affect the utility of markers in thrips molecular diagnostics. For each species, intragenomic (within individual) variation and intergenomic (among individuals) variation was assessed by cloning and sequencing PCR-amplified copies. Intergenomic variation was generally higher than intragenomic variation except in cases where intergenomic variation was very low, as in mtCO1 from S.dorsalis and F.occidentalis. Intragenomic variation was detected in both markers in all three of the thrips species, however, 2-3 times more intragenomic variation was observed for ITS2 than mtCO1 in both S.dorsalis and T.palmi. Furthermore, levels of intragenomic variation were low for both of the genes in F.occidentalis. In all of the three thrips species, no sex-based clustering of haplotypes was observed in either marker. Unexpected high intragenomic variation in ITS2 for two of three thrips species did not interfere with thrips diagnostics. However, caution should be taken in applying ITS2 to certain studies of S.dorsalis and T.palmi when high levels of intragenomic variation could be problematic or confounding. In such studies, mtCO1 may be a preferable marker. Possible reasons for discrepancies in intragenomic variation among genomic regions are discussed.

Rediscovering digitules in Aphidomorpha and the question of homology among Sternorrhyncha (Insecta, Hemiptera).

We explore and expand on the morphological term digitule. The term was originally proposed for toe-like setae on a species of Phylloxera Boyer de Fonscolombe, 1834 (Hemiptera, Sternorrhyncha, Aphidomorpha) by Henry Shimer, an American naturalist. While it is standard terminology in scale systematics (Hemiptera, Sternorrhyncha, Coccidomorpha), the term digitule was ignored by aphid specialists despite being the original taxon for which the term was described. Similar setae occur on many arthropod groups, so the homology is poorly understood even within any superfamily of Hemiptera. We provide the etymology of the term, a proposed explanation for why it was used among scale taxonomists and not aphid taxonomists, and discuss briefly options to progress beyond the confusion between terminology for morphology and homology in Sternorrhyncha.

Complete Genome Sequences of Two Genotype A2 Small Ruminant Lentiviruses Isolated from Infected U.S. Sheep.

Two distinct subgroups of genotype A2 small ruminant lentiviruses (SRLVs) have been identified in the United States that infect sheep with specific host transmembrane protein 154 (TMEM154) diplotypes. Here, we report the first two complete genome sequences of SRLV strains infecting U.S. sheep belonging to genotype A2, subgroups 1 and 2.