Segmental Variation in a Duplicated msp2 Pseudogene Generates Anaplasma marginale Antigenic Variants.

Anaplasma marginale is a prototypical highly antigenically variant bacterial pathogen dependent on the sequential generation of major surface protein 2 (Msp2) outer membrane variants to establish persistent infection. Msp2 is encoded by a single expression site, and diversity is achieved by gene conversion of chromosomally encoded msp2 pseudogenes. Analysis of the full complement of msp2 pseudogenes in the St. Maries strain revealed identical sequences in different loci. The Florida strain shared the same locus structure, but in the loci where the St. Maries strain had two identical pseudogenes, the Florida strain had one whose sequence was identical to the St. Maries sequences, while the sequence of the second pseudogene differed. Consequently, we hypothesized that the msp2 pseudogene repertoire arose via gene duplication, allowing structural variation to occur in one copy but the utility of the other to be retained. Using comparative genomics, we first established that duplication of msp2 pseudogenes is common among A. marginale strains: all seven examined strains had at least one duplicate pair in which either the genes in the pair were maintained as identical copies or the genes contained segmental changes. We then demonstrated that a minimal segmental change in a duplicated pseudogene locus is sufficient for immune escape from the broad antibody response generated in a natural host, as is a completely divergent pseudogene sequence in an otherwise conserved locus. The results support a model in which a locus first duplicates, resulting in a second identical copy, and then progressively incorporates changes to generate an msp2 repertoire capable of generating sufficient antigenic variants to escape immunity and establish persistent infection.

Population Structure and Antimicrobial Resistance of Canine Uropathogenic Escherichia coli.

Escherichia coli is the most common cause of human and canine urinary tract infection (UTI). Clonal groups, often with high levels of antimicrobial resistance, are a major component of the E. coli population that causes human UTI. While little is known about the population structure of E. coli that causes UTI in dogs, there is evidence that dogs and humans can share fecal strains of E. coli and that human-associated strains can cause disease in dogs. In order to better characterize the E. coli strains that cause canine UTI, we analyzed 295 E. coli isolates obtained from canine urine samples from five veterinary diagnostic laboratories and analyzed their multilocus sequence types, phenotypic and genotypic antimicrobial resistance profiles, and virulence-associated gene repertoires. Sequence type 372 (ST372), an infrequent human pathogen, was the predominant sequence type in dogs at all locations. Extended-spectrum ß-lactamase-producing isolates with blaCTX-M genes were uncommon in canine isolates but when present were often associated with sequence types that have been described in human infections. This provides support for occasional cross-host-species sharing of strains that cause extraintestinal disease and highlights the importance of understanding the role of companion animals in the overall transmission patterns of extraintestinal pathogenic E. coli.

Evaluation of environmental sampling methods for detection of Salmonella enterica in a large animal veterinary hospital.

Environmental surveillance for Salmonella enterica can be used for early detection of contamination; thus routine sampling is an integral component of infection control programs in hospital environments. At the Purdue University Veterinary Teaching Hospital (PUVTH), the technique regularly employed in the large animal hospital for sample collection uses sterile gauze sponges for environmental sampling, which has proven labor-intensive and time-consuming. Alternative sampling methods use Swiffer brand electrostatic wipes for environmental sample collection, which are reportedly effective and efficient. It was hypothesized that use of Swiffer wipes for sample collection would be more efficient and less costly than the use of gauze sponges. A head-to-head comparison between the 2 sampling methods was conducted in the PUVTH large animal hospital and relative agreement, cost-effectiveness, and sampling efficiency were compared. There was fair agreement in culture results between the 2 sampling methods, but Swiffer wipes required less time and less physical effort to collect samples and were more cost-effective.

Évaluation des méthodes de prélèvements d'échantillons environnementaux pour la détection de Salmonella enterica dans une clinique vétérinaire pour grands animaux. La surveillance environnementale pour Salmonella enterica peut être utilisée pour la détection précoce de la contamination. Des prélèvements réguliers font donc partie intégrante des programmes de contrôle des infections dans les milieux hospitaliers. À l'Hôpital d'enseignement vétérinaire de l'Université Purdue (Purdue University Veterinary Teaching Hospital ou PUVTH), on emploie régulièrement la technique d'utilisation des éponges de gaze stérilisée dans l'hôpital pour grands animaux pour prélever des échantillons environnementaux, une méthode qui s'est avérée laborieuse et longue. Des méthodes d'échantillonnage de remplacement utilisent des linges électrostatiques de marque Swiffer pour le prélèvement des échantillons environnementaux et ces méthodes seraient efficaces et efficientes. On a émis l'hypothèse que l'utilisation des linges Swiffer pour le prélèvement d'échantillons serait plus efficace et moins coûteuse que l'utilisation des éponges de gaze. Une comparaison directe entre les deux méthodes d'échantillonnage a été réalisée dans l'hôpital pour grands animaux du PUVTH et la concordance relative, l'économie et l'efficacité d'échantillonnage ont été comparées. Il y avait une concordance modérée entre les deux méthodes d'échantillonnage, mais les linges Swiffer exigeaient moins de temps et moins d'efforts physiques pour prélever les échantillons et ils étaient plus économiques.(Traduit par Isabelle Vallières).

Antibacterial activity and safety of commercial veterinary cationic steroid antibiotics and neutral superoxidized water.

Antibiotic resistance of bacteria common to the ocular surface is an evolving problem. Thus, novel treatment options with new modes of action are required. We investigated the antibacterial activity and safety of three commercially available topical veterinary ophthalmic products (cationic steroid antibiotics, products A and B, and a neutral superoxidized water, product C) to determine their potential use as antimicrobial alternatives. The minimum inhibitory concentrations (MIC) of the three products were determined against 17 antibiotic resistant bacterial clinical isolates from the ocular surface. Using a standard cytotoxicity assay, the products at varying concentrations were evaluated with a corneal fibroblast cell line and a macrophage-like cell line to determine their potential toxic effect in vitro. The commercial ophthalmic solutions, ofloxacin 0.3%, tobramycin 0.3% and gentamicin 0.3% were used as positive controls for the MIC and tobramycin 0.3% was used as positive control for the cytotoxicity assays. For the MIC, Product C showed no inhibition of growth for any organisms, while Products A and B showed inhibition of growth similar to slightly less than the positive controls. For the cytotoxicity assays, Product C exhibited minimal toxicity while Products A and B exhibited toxicity similar to the controls. In conclusion, Product C had no antibacterial activity in these assays, while Products A and B had antibacterial profiles similar to slightly less than common topical ophthalmic antibiotics and cytotoxicity profiles similar to common topical ophthalmic antibiotics. To our knowledge, this is the first report on the antibacterial activity and safety of the cationic steroid antibiotics and superoxidized water.

Detection of hemoplasma infection of goats by use of a quantitative polymerase chain reaction assay and risk factor analysis for infection.

OBJECTIVE To develop and validate a real-time quantitative PCR (qPCR) assay for the detection and quantification of Mycoplasma ovis in goats and investigate the prevalence and risk factors for hemoplasma infection of goats located in Indiana. ANIMALS 362 adult female goats on 61 farms. PROCEDURES Primers were designed for amplification of a fragment of the dnaK gene of M ovis by use of a qPCR assay. Blood samples were collected into EDTA-containing tubes for use in total DNA extraction, blood film evaluation, and determination of PCV. Limit of detection, intra-assay variability, interassay variability, and specificity of the assay were determined. RESULTS Reaction efficiency of the qPCR assay was 94.45% (R(2), 0.99; slope, -3.4623), and the assay consistently detected as few as 10 copies of plasmid/reaction. Prevalence of infection in goats on the basis of results for the qPCR assay was 18.0% (95% confidence interval, 14% to 22%), with infected goats ranging from 1 to 14 years old, whereby 61% (95% confidence interval, 47% to 73%) of the farms had at least 1 infected goat. Bacterial load in goats infected with M ovis ranged from 1.05 × 10(3) target copies/mL of blood to 1.85 × 10(5) target copies/mL of blood; however, no bacteria were observed on blood films. Production use of a goat was the only risk factor significantly associated with hemoplasma infection. CONCLUSIONS AND CLINICAL RELEVANCE The qPCR assay was more sensitive for detecting hemoplasma infection than was evaluation of a blood film, and production use of a goat was a risk factor for infection.

Antibacterial Characterization of Novel Synthetic Thiazole Compounds against Methicillin-Resistant Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius is a commensal organism of companion animals that is a significant source of opportunistic infections in dogs. With the emergence of clinical isolates of S. pseudintermedius (chiefly methicillin-resistant S. pseudintermedius (MRSP)) exhibiting increased resistance to nearly all antibiotic classes, new antimicrobials and therapeutic strategies are urgently needed. Thiazole compounds have been previously shown to possess potent antibacterial activity against multidrug-resistant strains of Staphylococcus aureus of human and animal concern. Given the genetic similarity between S. aureus and S. pseudintermedius, this study explores the potential use of thiazole compounds as novel antibacterial agents against methicillin-sensitive S. pseudintermedius (MSSP) and MRSP. A broth microdilution assay confirmed these compounds exhibit potent bactericidal activity (at sub-microgram/mL concentrations) against both MSSA and MRSP clinical isolates while the MTS assay confirmed three compounds (at 10 µg/mL) were not toxic to mammalian cells. A time-kill assay revealed two derivatives rapidly kill MRSP within two hours. However, this rapid bactericidal activity was not due to disruption of the bacterial cell membrane indicating an alternative mechanism of action for these compounds against MRSP. A multi-step resistance selection analysis revealed compounds 4 and 5 exhibited a modest (two-fold) shift in activity over ten passages. Furthermore, all six compounds (at a subinihibitory concentration) demonstrated the ability to re-sensitize MRSP to oxacillin, indicating these compounds have potential use for extending the therapeutic utility of ß-lactam antibiotics against MRSP. Metabolic stability analysis with dog liver microsomes revealed compound 3 exhibited an improved physicochemical profile compared to the lead compound. In addition to this, all six thiazole compounds possessed a long post-antibiotic effect (at least 8 hours) against MRSP. Collectively the present study demonstrates these synthetic thiazole compounds possess potent antibacterial activity against both MSSP and MRSP and warrant further investigation into their use as novel antimicrobial agents.

Antibacterial activity of novel cationic peptides against clinical isolates of multi-drug resistant Staphylococcus pseudintermedius from infected dogs.

Staphylococcus pseudintermedius is a major cause of skin and soft tissue infections in companion animals and has zoonotic potential. Additionally, methicillin-resistant S. pseudintermedius (MRSP) has emerged with resistance to virtually all classes of antimicrobials. Thus, novel treatment options with new modes of action are required. Here, we investigated the antimicrobial activity of six synthetic short peptides against clinical isolates of methicillin-susceptible and MRSP isolated from infected dogs. All six peptides demonstrated potent anti-staphylococcal activity regardless of existing resistance phenotype. The most effective peptides were RRIKA (with modified C terminus to increase amphipathicity and hydrophobicity) and WR-12 (α-helical peptide consisting exclusively of arginine and tryptophan) with minimum inhibitory concentration50 (MIC50) of 1 µM and MIC90 of 2 µM. RR (short anti-inflammatory peptide) and IK8 "D isoform" demonstrated good antimicrobial activity with MIC50 of 4 µM and MIC90 of 8 µM. Penetratin and (KFF)3K (two cell penetrating peptides) were the least effective with MIC50 of 8 µM and MIC90 of 16 µM. Killing kinetics revealed a major advantage of peptides over conventional antibiotics, demonstrating potent bactericidal activity within minutes. Studies with propidium iodide and transmission electron microscopy revealed that peptides damaged the bacterial membrane leading to leakage of cytoplasmic contents and consequently, cell death. A potent synergistic increase in the antibacterial effect of the cell penetrating peptide (KFF)3K was noticed when combined with other peptides and with antibiotics. In addition, all peptides displayed synergistic interactions when combined together. Furthermore, peptides demonstrated good therapeutic indices with minimal toxicity toward mammalian cells. Resistance to peptides did not evolve after 10 passages of S. pseudintermedius at sub-inhibitory concentration. However, the MICs of amikacin and ciprofloxacin increased 32 and 8 fold, respectively; under similar conditions. Taken together, these results support designing of peptide-based therapeutics for combating MRSP infections, particularly for topical application.

Global transcriptional analysis reveals surface remodeling of Anaplasma marginale in the tick vector.

BACKGROUND: Pathogens dependent upon vectors for transmission to new hosts undergo environment specific changes in gene transcription dependent on whether they are replicating in the vector or the mammalian host. Differential gene transcription, especially of potential vaccine candidates, is of interest in Anaplasma marginale, the tick-borne causative agent of bovine anaplasmosis. METHODS: RNA-seq technology allowed a comprehensive analysis of the transcriptional status of A. marginale genes in two conditions: bovine host blood and tick derived cell culture, a model for the tick vector. Quantitative PCR was used to assess transcription of a set of genes in A. marginale infected tick midguts and salivary glands at two time points during the transmission cycle. RESULTS: Genes belonging to fourteen pathways or component groups were found to be differentially transcribed in A. marginale in the bovine host versus the tick vector. One of the most significantly altered groups was composed of surface proteins. Of the 56 genes included in the surface protein group, eight were up regulated and 26 were down regulated. The down regulated surface protein encoding genes include several that are well studied due to their immunogenicity and function. Quantitative PCR of a set of genes demonstrated that transcription in tick cell culture most closely approximates transcription in salivary glands of recently infected ticks. CONCLUSIONS: The ISE6 tick cell culture line is an acceptable model for early infection in tick salivary glands, and reveals disproportionate down regulation of surface protein genes in the tick. Transcriptional profiling in other cell lines may help us simulate additional microenvironments. Understanding vector-specific alteration of gene transcription, especially of surface protein encoding genes, may aid in the development of vaccines or transmission blocking therapies.

Protective immunity induced by immunization with a live, cultured Anaplasma marginale strain.

Despite significant economic losses resulting from infection with Anaplasma marginale, a tick-transmitted rickettsial pathogen of cattle, available vaccines provide, at best, only partial protection against clinical disease. The green-fluorescent protein expressing mutant of the A. marginale St. Maries strain is a live, marked vaccine candidate (AmStM-GFP). To test whether AmStM-GFP is safe and provides clinical protection, a group of calves was vaccinated, and clinical parameters, including percent parasitized erythrocytes (PPE), packed cell volume (PCV) and days required to reach peak bacteremia, were measured following inoculation and following tick challenge with wild type St. Maries strain (AmStM). These clinical parameters were compared to those obtained during infection with the A. marginale subsp. centrale vaccine strain (A. centrale) or wild type AmStM. AmStM-GFP resulted in similar clinical parameters to A. centrale, but had a lower maximum PPE, smaller drop in PCV and took longer to reach peak bacteremia than wild type AmStM. AmStM-GFP provided clinical protection, yielding a stable PCV and low bacteremia following challenge, whereas A. centrale only afforded partial clinical protection.