Genotypic and Phenotypic Characterization of Treponema phagedenis from Bovine Digital Dermatitis

This study aimed to isolate and characterize Treponema spp. from bovine digital dermatitis (BDD)-infected dairy cattle. Seven isolates were characterized in this study. Isolates exhibited slow growth, and colonies penetrated the agar and exhibited weak β-hemolysis. Round bodies were observed in old and antibiotic-treated cultures. Cells ranged from 9–12 µm in length, 0.2–2.5 µm in width, and were moderately spiraled. The 16S rRNA analysis revealed the isolates as Treponema phagedenis with >99% sequence homology. Isolates had alkaline phosphatase, acid phosphatase, β-galactosidase, N-acetyl-β-glucosaminidase, esterase (C4), esterase lipase (C8), naphthol-AS-BI-phosphohydrolase, and β-glucuronidase activities. Low concentrations of ampicillin, erythromycin, and tetracycline were required to inhibit the growth of isolates. Formic, acetic, and butyric acids were produced, while propionic acid was significantly utilized, indicating its essentiality for treponemal growth. The isolates shared the same characteristics and, therefore, were considered as a single strain. Isolate HNL4 was deposited as a representative isolate (Treponema phagedenis KS1). The average nucleotide identity of strain KS1 showed a small difference with the human strain (99.14%) compared with bovine strain (99.72%). This study was the first to isolate and characterize Treponema phagedenis from BDD in Korea and, hence, it delivered pathogenicity-related insights and provided valuable information that can be used for the management of BDD.

The Inhibitory Activity of Anthraquinones against Pathogenic Protozoa, Bacteria, and Fungi and the Relationship to Structure

Plant-derived anthraquinones were evaluated in cell assays for their inhibitory activities against the parasitic protozoa Trichomonas vaginalis human strain G3 that causes the sexually transmitted disease trichomoniasis in women, Tritrichomonas foetus bovine strain D1 that causes sexually transmitted diseases in farm animals (bulls, cows, and pigs), Tritrichomonas foetus-like strain C1 that causes diarrhea in domestic animals (cats and dogs), and bacteria and fungi. The anthraquinones assessed for their inhibitory activity were anthraquinone, aloe-emodin (1,8-dihydroxy-3-hydroxymethylanthraquinone), anthrarufin (1,5-dihydroxyanthraquinone), chrysazin (1,8-dihydroxyanthraquinone), emodin (1,3,8-trihydroxy-6-methylanthraquinone), purpurin (1,2,4-trihydroxyanthraquinone), and rhein (1,8-dihydroxy-3-carboxyanthraquinone). Their activities were determined in terms of IC50 values, defined as the concentration that inhibits 50% of the cells under the test conditions and calculated from linear dose response plots for the parasitic protozoa, and zone of inhibition for bacteria and fungi, respectively. The results show that the different substituents on the anthraquinone ring seem to influence the relative potency. Analysis of the structure–activity relationships in protozoa indicates that the aloe-emodin and chrysazin with the highest biological activities merit further study for their potential to help treat the diseases in women and domestic and farm animals. Emodin also exhibited antifungal activity against Candida albicans. The suggested mechanism of action and the additional reported beneficial biological properties of anthraquinones suggest that they have the potential to ameliorate a broad spectrum of human diseases.

Eleven High-Quality Reference Genome Sequences and 360 Draft Assemblies of Shiga Toxin-Producing Escherichia coli Isolates from Human, Food, Animal, and Environmental Sources in Canada

We report high-quality closed reference genomes for 1 bovine strain and 10 human Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from serogroups O26, O45, O91, O103, O104, O111, O113, O121, O145, and O157. We also report draft assemblies, with standardized metadata, for 360 STEC strains isolated from watersheds, animals, farms, food, and human infections.

Within-Farm Changes in Dairy Farm-Associated Salmonella Subtypes and Comparison to Human Clinical Isolates in Michigan, 2000-2001 and 2009

ABSTRACTTemporal changes in the distribution ofSalmonellasubtypes in livestock populations may have important impacts on human health. The first objective of this research was to determine the within-farm changes in the population of subtypes ofSalmonellaon Michigan dairy farms that were sampled longitudinally in 2000-2001 and again in 2009. The second objective was to determine the yearly frequency (2001 through 2012) of reported human illnesses in Michigan associated with the same subtypes. Comparable sampling techniques were used to collect fecal and environmental samples from the same 18 Michigan dairy farms in 2000-2001 and 2009. Serotypes, multilocus sequence types (STs), and pulsed-field gel electrophoresis (PFGE) banding patterns were identified for isolates from 6 farms where >1Salmonellaisolate was recovered in both 2000-2001 and 2009. The distribution of STs was significantly different between time frames (P< 0.05); only two of 31 PFGE patterns were identified in both time frames, and each was recovered from the same farm in each time frame. Previously reported within-farm decreases in the frequency of multidrug-resistant (MDR)Salmonellawere due to recovery of MDR subtypes ofS. entericaserotypes Senftenberg and Typhimurium in 2000-2001 and genetically distinct, pansusceptible subtypes of the same serotypes in 2009. The annual frequency of human illnesses between 2001 and 2012 with a PFGE pattern matching a bovine strain decreased for patterns recovered from dairy farms in 2000-2001 and increased for patterns recovered in 2009. These data suggest important changes in the population ofSalmonellaon dairy farms and in the frequency of human illnesses associated with cattle-derived subtypes.

Transfection of exogenous rotavirus rearranged RNA segments in cells infected with a WT rotavirus results in subsequent gene rearrangements

Group A rotaviruses, members of the family Reoviridae, are a major cause of infantile acute gastroenteritis. The rotavirus genome consists of 11 dsRNA segments. In some cases, an RNA segment is replaced by a rearranged RNA segment, which is derived from its standard counterpart by partial sequence duplication. It has been shown that some rearranged segments are preferentially encapsidated into viral progenies after serial passages in cell culture. Based on this characteristic, a reverse genetics system was used previously to introduce exogenous segment 7 rearrangements into an infectious rotavirus. This study extends this reverse genetics system to RNA segments 5 and 11. Transfection of exogenous rotavirus rearranged RNA segment 5 or 11 into cells infected with a WT helper rotavirus (bovine strain RF) resulted in subsequent gene rearrangements in the viral progeny. Whilst recombinant viruses were rescued with an exogenous rearranged segment 11, the exogenous segment was modified by a secondary rearrangement. The occurrence of spontaneous rearrangements of WT or exogenous segments is a major hindrance to the use of this reverse genetics approach.

G8P[6] rotaviruses isolated from Amerindian children in Mato Grosso do Sul, Brazil, during 2009: close relationship of the G and P genes with those of bovine and bat strains

During the 2009 national group A rotavirus (RVA) surveillance, five unusual strains of the human G8P[6] genotype were detected in Brazilian indian children with acute gastroenteritis. The aim of this study was to carry out sequence analysis of the two outer capsid proteins (VP4 and VP7) and the inner capsid protein (VP6) of the G8P[6] strains detected in order to provide further information on the genetic relationship between human and animal RVA. A total of 68 stool samples, collected in Mato Grosso do Sul during 2009, were tested for RVA using ELISA, following by reverse transcriptase-PCR and sequencing. RVA infection was detected in 7.3 % of samples (5/68). The IAL-RN376 G8 sequence shares a clade with bovine and human strains, displaying highest nucleotide identity to African human strains DRC86 and DRC88, followed by African bovine strain NGRBg8. IAL-RN376 and IAL-RN377 P[6] sequences showed highest identity to human strain R330 from Ireland, and a close genetic relationship to African fruit bat RVA strain KE4852/07. Strains IAL-RN376 and IAL-RN377 display genogroup I VP6 specificity and the I2 genotype, and share high nucleotide identities with human strains B1711, 272-BF and 06-242, and moderate identities with bovine (RUBV81, 86 and KJ9-1) and porcine (HP140) strains. This study suggested that a reassortment between bovine and bat RVA strains could have occurred in animal host(s) preceding the transmission to humans. In the indigenous population, zoonotic transmission is probably fairly frequent as the inhabitants live in close contact with animals under conditions of poor hygiene.

Different Rotavirus Strains Enter MA104 Cells through Different Endocytic Pathways: the Role of Clathrin-Mediated Endocytosis

ABSTRACT Rotaviruses, the single most important agents of acute severe gastroenteritis in children, are nonenveloped viruses formed by a three-layered capsid that encloses a genome formed by 11 segments of double-stranded RNA. The mechanism of entry of these viruses into the host cell is not well understood. The best-studied strain, RRV, which is sensitive to neuraminidase (NA) treatment of the cells, uses integrins α2β1 and αvβ3 and the heat shock protein hsc70 as receptors and enters MA104 cells through a non-clathrin-, non-caveolin-mediated pathway that depends on a functional dynamin and on the presence of cholesterol on the cell surface. In this work, using a combination of pharmacological, biochemical, and genetic approaches, we compared the entry characteristics of four rotavirus strains known to have different receptor requirements. We chose four rotavirus strains that represent all phenotypic combinations of NA resistance or sensitivity and integrin dependence or independence. We found that even though all the strains share their requirements for hsc70, dynamin, and cholesterol, three of them differ from the simian strain RRV in the endocytic pathway used. The human strain Wa, porcine strain TFR-41, and bovine strain UK seem to enter the cell through clathrin-mediated endocytosis, since treatments that inhibit this pathway block their infectivity; consistent with this entry route, these strains were sensitive to changes in the endosomal pH. The inhibition of other endocytic mechanisms, such as macropinocytosis or caveola-mediated uptake, had no effect on the internalization of the rotavirus strains tested here.

Molecular characterization of full-length genomic segment 2 of a bovine picobirnavirus (PBV) strain: evidence for high genetic diversity with genogroup I PBVs

We report here the molecular characterization of a bovine genogroup I picobirnavirus strain RUBV-P detected from a 1-month-old diarrhoeic calf in eastern India. Sequence comparisons and phylogenetic analysis of a short stretch of gene segment 2 of RUBV-P revealed low nucleotide identities (51.2–64.9 %) with and distant genetic relatedness to other genogroup I picobirnaviruses. The complete gene segment 2 sequence of RUBV-P was obtained by the single primer amplification method with modifications. Gene segment 2 of RUBV-P was 1758 bp long, encoded a predicted protein of 554 aa and exhibited low nucleotide (58.1–58.8 %) and amino acid (51.3–55.4 %) identities with genogroup I human strains Hy005102 and 1-CHN-97. The 5′- and 3′-end nucleotide sequences, and the three motifs of RNA-dependent RNA polymerases of double-stranded RNA viruses, were conserved among these strains. Our findings suggested that bovine strain RUBV-P might be distinct from genogroup I picobirnaviruses of humans and other animals.