Canine infectious respiratory disease (CIRD) is a syndrome where multiple viral and bacterial pathogens are involved sequentially or synergistically to cause illness. There is limited information regarding the prevalence of pathogens related to CIRD in the United States as well as the role of co-infections in the pathogenesis of the syndrome. We aimed to conduct a comprehensive etiologic and epidemiologic study of multiple CIRD agents in a diverse dog population using molecular methods and statistical modeling analyses. In addition, a novel probe-based multiplex real-time PCR was developed to simultaneously detect and differentiate two species of Mycoplasma ( M . canis and M . cynos ). Canine adenovirus, canine distemper virus, canine parainfluenza virus, coronavirus, influenza A virus (H3N2 and H3N8), Bordetella bronchiseptica , M . canis , M . cynos and Streptococcus equi subsp. zooepidemicus were investigated in specimens from clinically ill and asymptomatic dogs received at the Athens Veterinary Diagnostic Laboratory. Results showed low occurrence of classical CIRD agents such as B . bronchiseptica , canine adenovirus and distemper virus, while highlighting the potential role of emerging bacteria such as M . canis and M . cynos . Statistical modeling analyses of CIRD pathogens emphasized the impact of co-infections on the severity of clinical presentation, and showed that host factors, such as animal age, are the most important predictors of disease severity. This study provides new insights into the current understanding of the prevalence and role of co-infections with selected viruses and bacteria in the etiology of CIRD, while underscoring the importance of molecular diagnosis and vaccination against this disease.

Ovine footrot is a highly prevalent bacterial disease caused by Dichelobacter nodosus and characterised by the separation of the hoof horn from the underlying skin. The role of innate immune molecules and other bacterial communities in the development of footrot lesions remains unclear. This study shows a significant association between the high expression of IL1β and high D. nodosus load in footrot samples. Investigation of the microbial population identified distinct bacterial populations in the different disease stages and also depending on the level of inflammation. Treponema (34%), Mycoplasma (29%) and Porphyromonas (15%) were the most abundant genera associated with high levels of inflammation in footrot. In contrast, Acinetobacter (25%), Corynebacteria (17%) and Flavobacterium (17%) were the most abundant genera associated with high levels of inflammation in healthy feet. This demonstrates for the first time there is a distinct microbial community associated with footrot and high cytokine expression.

In this text we evaluated the in vitro antifungal activities of terbinafine combined with caspofungin, miconazole, ketoconazole, and fluconazole against 17 Pythium insidiosum strains by using the microdilution checkerboard method. Synergistic interactions were observed with terbinafine combined with caspofungin (41.2% of the strains), fluconazole (41.2%), ketoconazole (29.4%), and miconazole (11.8%). No antagonistic effects were observed. The combination of terbinafine plus caspofungin or terbinafine plus fluconazole may have significant therapeutic potential for treatment of pythiosis.Pythiosis is a life-threatening infectious disease in humans and animals that is caused by the aquatic oomycete Pythium insidiosum (9). Horses are the most frequently infected animals, and equine pythiosis typically involves ulcerative granulomas (8). In humans, the infection occurs as ophthalmic, subcutaneous, and systemic forms, which are frequently associated with ␣-and ␤-thalassemia (5, 7). Pythiosis therapy, which is based on amphotericin B or azoles, has been ineffective or controversial, and the associated prognosis for human and equine pythiosis is poor (5,7,8,9,12). Therefore, surgical procedures, including amputation, are often effective, but disease reoccurrence rates are unfortunately high (7).Combinations of antifungal agents against pythiosis have not been thoroughly studied, and therefore, such in vitro combinatory activities against P. insidiosum require attention (1, 6).The purpose of this study was to investigate the in vitro activity of terbinafine (TRB) combined with caspofungin (CAS), miconazole (MNZ), ketoconazole, and fluconazole (FLC) against 17 strains of Pythium insidiosum isolated from animals.A total of 15 Brazilian P. insidiosum strains isolated from equines with pythiosis and two standard strains (ATCC 58637 and CBS 101555) were tested. All strains were maintained in cornmeal agar, and strain identification was confirmed by a PCR-based assay (4).The susceptibility of the P. insidiosum strains to the antifungal agents was tested by microdilution, based on protocol M38-A2 (2). The inoculum consisted of P. insidiosum zoospores obtained following zoosporogenesis. Cell numbers of zoospores were counted on a hemocytometer; zoospores were diluted in RPMI 1640 containing L-glutamine and buffered to pH 7.0 with 0.165 M MOPS (morpholinepropanesulfonic acid) to obtain a final concentration range of 2 ϫ 10 3 to 3 ϫ 10 3 zoospores/ml (10). The combinations of TRB (Novartis) plus CAS (Merck), TRB plus MNZ (Labware), TRB plus ketoconazole (Janssen), and TRB plus FLC (Pfizer) were evaluated using the checkerboard technique, according to the broth microdilution design (2,14). In the individual tests, 100 l of each drug concentration was plated in microplate wells and an equal volume of the inoculum was added to each well. In the combination tests, the antifungals were plated at a 4ϫ concentrate of 50 l of drug A plus 50 l of drug B and 100 l of the inoculum, resulting in a final 1ϫ drug concentration of each compound....

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A note on versions:The version presented here may differ from the published version or from the version of record. If you wish to cite this item you are advised to consult the publisher's version. Please see the repository url above for details on accessing the published version and note that access may require a subscription. Ovine footrot is characterised by interdigital dermatitis (ID) and by the separation of the skin and hoof horn (underrunning footrot). Dichelobacter nodosus is the essential pathogen causing footrot; the role of other microorganisms in this disease remains unclear. The aims of this study were: (i) to investigate the colonisation of D. nodosus, Fusobacterium necrophorum and Treponema spp. in biopsies from the ovine interdigital skin of healthy, ID and footrot affected feet and (ii) to characterize the virulence of D. nodosus strains in those biopsies. Post-slaughter biopsy samples (n=241) were collected and analysed by real-time PCR to determine prevalence and load of the different bacterial species. The highest prevalence and load of D. nodosus were found on feet with ID. The vast majority of samples contained virulent D. nodosus and some samples contained both virulent and benign D. nodosus. Notably, the more pathogenic subspecies of F. necrophorum was found in samples from UK sheep. Our findings provide further insights into the role bacterial colonisation may play in the early stage of ID and in the progression towards footrot.https://mc.manuscriptcentral.com/vetrec Veterinary Record

Skin infection studies are often limited by financial and ethical constraints, and alternatives, such as monolayer cell culture, do not reflect many cellular processes limiting their application. For a more functional replacement, 3D skin culture models offer many advantages such as the maintenance of the tissue structure and the cell types present in the host environment. A 3D skin culture model can be set up using tissues acquired from surgical procedures or post slaughter, making it a cost effective and attractive alternative to animal experimentation. The majority of 3D culture models have been established for aerobic pathogens, but currently there are no models for anaerobic skin infections. Footrot is an anaerobic bacterial infection which affects the ovine interdigital skin causing a substantial animal welfare and financial impact worldwide. Dichelobacter nodosus is a Gram-negative anaerobic bacterium and the causative agent of footrot. The mechanism of infection and host immune response to D. nodosus is poorly understood. Here we present a novel 3D skin ex vivo model to study anaerobic bacterial infections using ovine skin explants infected with D. nodosus. Our results demonstrate that D. nodosus can invade the skin explant, and that altered expression of key inflammatory markers could be quantified in the culture media. The viability of explants was assessed by tissue integrity (histopathological features) and cell death (DNA fragmentation) over 76 h showing the model was stable for 28 h. D. nodosus was quantified in all infected skin explants by qPCR and the bacterium was visualized invading the epidermis by Fluorescent in situ Hybridization. Measurement of pro-inflammatory cytokines/chemokines in the culture media revealed that the explants released IL1β in response to bacteria. In contrast, levels of CXCL8 production were no different to mock-infected explants. The 3D skin model realistically simulates the interdigital skin and has demonstrated that D. nodosus invades the skin and triggered an early cellular inflammatory response to this bacterium. This novel model is the first of its kind for investigating an anaerobic bacterial infection.

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Listerial keratoconjunctivitis ('silage eye') is a wide spread problem in ruminants causing economic losses to farmers and impacts negatively on animal welfare. It results from direct entry of Listeria monocytogenes into the eye, often following consumption of contaminated silage. An isolation protocol for bovine conjunctival swabbing was developed and used to sample both infected and healthy eyes bovine eyes (n=46). L. monocytogenes was only isolated from one healthy eye sample, and suggests that this organism can be present without causing disease. To initiate a study of this disease, an infection model was developed using isolated conjunctiva explants obtained from cattle eyes post slaughter. Conjunctiva were cultured and infected for 20 h with a range of L. monocytogenes isolates (n=11), including the healthy bovine eye isolate and also strains isolated from other bovine sources, such as milk or clinical infections. Two L. monocytogenes isolates (one from a healthy eye and one from a cattle abortion) were markedly less able to invade conjunctiva explants, but one of those was able to efficiently infect Caco2 cells indicating that it was fully virulent. These two isolates were also significantly more sensitive to lysozyme compared to most other isolates tested, suggesting that lysozyme resistance is an important factor when infecting bovine conjunctiva. In conclusion, we present the first bovine conjunctiva explant model for infection studies and demonstrate that clinical L. monocytogenes isolates from cases of bovine keratoconjunctivitis are able to infect these tissues.