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Mycoplasma cynos and Mycoplasma felis are often associated with canine and feline infectious respiratory disease in dogs and cats, respectively. Mycoplasmas have a reduced genome and dearth of many biosynthetic pathways, making them dependent on rich medium for growth. Due to this fastidious nature, mycoplasmas have been historically underdiagnosed. The aim of this study was to develop a cost-effective and accurate sequencing workflow for genotypic characterization of clinical isolates of M. cynos and M. felis using a rapid long-read sequencing platform. We explored the following critical aspects of bacterial whole genome sequencing, including: (i) five solid and liquid-based culture approaches based on a specialized media formulation for Mycoplasma culture, (ii) three DNA extraction methods modified for long-read sequencing purposes, and (iii) two de novo assembly platforms, Flye and Canu, as key components of a bioinformatics pipeline. DNA extraction method 1, a solid-phase and column-based kit with enzymatic lysis, provided the best DNA quality and concentration followed by high coverage and sequencing contiguity. This was obtained with a culture volume of 45 ml in modified Hayflick’s broth incubated for 48 h. DNA extracted directly from colonies on agar or from small broth volumes (6 ml) did not meet the criteria required for long-read sequencing. Overall, Flye generated more contiguous assemblies than the Canu assembler and was more time efficient. This 4–5 day sample-to-sequence workflow provides the scientific and clinical communities with a more comprehensive tool than laborious conventional methods for complete genomic characterization of M. cynos and M. felis clinical isolates.

A mortality event among recently captured feral donkeys ( Equus asinus) occurred in south-central Utah in 2016. The deaths were sporadic, and clinical signs were indicative of respiratory disease, likely associated with an infectious etiology. Ten of 13 donkeys autopsied had moderate-to-severe interstitial fibrosing pneumonia, and one had pyogranulomatous pneumonia. Consensus PCRs directed toward the DNA polymerase and DNA packaging terminase subunit 1 for herpesviruses were performed followed by sequencing of the PCR amplicons and phylogenetic analysis. Asinine herpesvirus 4 (AsHV4) and 5 (AsHV5) were consistently identified in lung tissues of affected donkeys. No other herpesviruses were identified, and herpesviral DNA was not detected in lung tissues of 2 donkeys without evidence of respiratory disease. The detection of asinine gammaherpesviruses may have been associated with the lesions described. AsHV4 and AsHV5 have been reported in previous studies as novel gammaherpesviruses based on sequences obtained from donkeys with interstitial pneumonia and marked syncytial cell formation. Our findings suggest that the association of asinine gammaherpesviruses with respiratory conditions in equids deserves further attention.

Summary Widely distributed aquatic species such as terns are highly dependent on, and can serve as indicators of, the global health of marine and other aquatic environments. Documented mass mortality events in terns have been associated with anthropogenic, weather-related and, less commonly, infectious causes. This study describes a multispecies mortality event associated with brevetoxicosis and Bisgaard taxon 40-induced sepsis involving common (Sterna hirundo) and sandwich (Thalasseus sandvicensis) terns off the southwest coast of Florida, USA, in November and December 2018. During an approximately 6–8-week period, a large number of birds were found dead or displayed weakness, ataxia or other neurological signs. Many were admitted to a wildlife hospital for evaluation, but most died or were euthanized due to poor prognosis. Necropsy of 12 birds revealed minimal or non-specific gross lesions. Initial toxicology screening of tissues for brevetoxins revealed levels that could be consistent with brevetoxicosis. However, histology revealed multiorgan inflammation and necrosis associated with a gram-negative bacillus. A bacterium isolated on aerobic culture of liver and heart tissues was unidentifiable in the MALDI-TOF database. Subsequently, 16 S rRNA gene sequencing revealed that the isolate shared 99.33% homology with Bisgaard taxon 40 from the Pasteurellaceae family. While the source of the bacterium and potential association with brevetoxin exposure are unclear, histopathology suggests that the bacterium was the proximate cause of clinical signs and mortality in all birds examined as well as the scale of the mortality event. This report highlights the need to conduct detailed investigations into wildlife mortality events and expands on the current, limited knowledge of the effects of novel Pasteurellaceae bacteria on avian health.

Mycoplasmas are respiratory pathogens in humans and animals and due to their fastidious nature, they have been historically underdiagnosed. Lack of standardised diagnostic, typing and antimicrobial susceptibility testing methods makes clinical management and epidemiological studies challenging. The aim of this study was to develop a cost-effective and accurate sequencing workflow for genotypic characterization of clinical isolates of respiratory mycoplasmas using a rapid long-read sequencing platform. Critical aspects of bacterial whole genome sequencing were explored using fastidious respiratory Mycoplasma (M. felis and M. cynos) isolated from animals including: (i) four solid and liquid-based media based on a specialized formulation for Mycoplasma culture, (ii) three DNA extraction methods modified for sequencing purposes, and (iii) two de novo assembly platforms as key components of a bioinformatic pipeline including Flye and Canu assemblers. DNA quality and quantity compatible with long-read sequencing requirements were obtained with culture volumes of 160ml in modified Hayflick’s broth incubated for 96 hours. The other three culture approaches investigated did not meet the DNA quality criteria required for long-read sequencing. The use of bead-beating bacterial cell lysis in the extraction protocol resulted in smaller fragments and shorter reads compared to enzymatic lysis methods. Overall, Flye generated more contiguous assemblies than the Canu assembler. This novel study provides a step-by-step sequencing workflow including mycoplasma culture, DNA extraction and de novo assembly approaches for the characterization of highly fastidious respiratory mycoplasmas. This workflow will provide diagnosticians, epidemiologists, and researchers with a more comprehensive tool than the laborious conventional methods for a complete genomic characterization of respiratory mycoplasmas.

Investigations describing the ocular and lacrimal gland lesions associated with rabies are sparse. Here we characterize the pathological changes and distribution of rabies viral antigen in the eye, optic nerve, and lacrimal gland of 18 rabies cases from different mammalian species. Histology and immunohistochemistry for rabies virus, CD3, CD20, and Iba1 were performed on tissue sections of eye, optic nerve, and lacrimal gland. Polymerase chain reaction (PCR) for rabies was performed on all cases, including 7 formalin-fixed, paraffin-embedded (FFPE) and 11 frozen tissue samples of eye and lacrimal gland. Pathological changes in the eye consisted of retinal necrosis (12/18 cases) with occasional viral inclusions within ganglion cells (8/12 cases). Immunohistochemically, viral antigen was detected within the nerve fiber layer, ganglion cells, and inner plexiform layer in all 12 cases with retinal lesions and in 2 cases with no retinal lesions, as well as optic nerve (6/18 cases) and lacrimal gland epithelium (3/18 cases). CD3+ T lymphocytes were present in the retina (11/18 cases), optic nerve (2/18 cases), and lacrimal gland (11/18 cases). No CD20+ B lymphocytes or Iba1+ macrophages were detected. PCR for rabies virus was positive in 9 of 11 frozen samples but in only 2 of 7 FFPE samples. Five samples that were negative for rabies by PCR were positive by immunohistochemistry, and 2 samples were negative by both tests. These results provide evidence that rabies virus infection extends to the eye, likely via the ocular nerve, and that the lacrimal gland might be a source of viral infection.

The etiology of otitis in dogs and cats is multifactorial and complex, involving bacterial and fungal pathogens. As empiric antimicrobial prescription is a common practice when treating such cases, antimicrobial resistance may represent a complicating factor. The aim of this study was to describe microbiological features and susceptibility profiles of pathogens associated with 142 cases of external otitis, comprising 138 dogs and 4 cats.. The specimens were processed to identify bacterial and fungal etiologies following standard microbiological methods. Antimicrobial susceptibility was determined in vitro against 15 antibiotics and 3 antifungals. Further, Staphylococcus spp. isolates were screened for the detection of β-lactamase enzymes using cefinase paper discs. Pseudomonas spp. and isolates from Enterobacteriaceae family were screened for colistin (Polymyxin E) resistance and for the mcr-1-mediated colistin resistance gene by PCR. The presence of mixed cultures of Enterobacteriaceae, Pseudomonas spp. and Staphylococcus spp., and co-infections with Malassezia spp., emphasizes the polymicrobial etiology of external otitis in small animals. Emerging rates of multidrug resistance observed in almost 50% of the isolates may alert for a near future of challenging veterinary cases unresponsive to first-line antimicrobials. In addition, these results highlight a potential public health concern of multidrug resistant bacteria, given the proximity of pets and their owners. This study addressed central aspects of external otitis, providing microbiologists and clinicians updated information on the etiology and treatment of challenging cases of multidrug resistant bacteria. It also provides priceless surveillance value in monitoring resistant bacteria in small animals.

Severe nasal Prototheca cutis infection was diagnosed postmortem for an immunocompetent cat with respiratory signs. Pathologic examination and whole-genome sequencing identified this species of algae, and susceptibility testing determined antimicrobial resistance patterns. P. cutis infection should be a differential diagnosis for soft tissue infections of mammals.

Listeria monocytogenes is an important foodborne pathogen in human and veterinary health, causing significant morbidity and mortality including abortion. It has a particular tropism for the gravid uterus, however, the route of infection in reproductive tissues of ruminants (i.e. placentome), is much less clear. In this study, we aimed to investigate a bovine caruncular epithelial cell (BCEC) line as a model for L. monocytogenes infection of the bovine reproductive tract. The BCEC infection model was used to assess the ability of 14 different L. monocytogenes isolates to infect these cells. Lysozyme sensitivity and bacterial survival in 580 μg lysozyme/ml correlated with attenuated ability to proliferate in BCEC (p = 0.004 and p = 0.02, respectively). Four isolates were significantly attenuated compared to the control strain 10403S. One of these strains (AR008) showed evidence of compromised cell wall leading to increased sensitivity to ß-lactam antibiotics, and another (7644) had compromised cell membrane integrity leading to increased sensitivity to cationic peptides. Whole genome sequencing followed by Multi Locus Sequence Type analysis identified that five invasive isolates had the same sequence type, ST59, despite originating from three different clinical conditions. Virulence gene analysis showed that the attenuated isolate LM4 was lacking two virulence genes (uhpT, virR) known to be involved in intracellular growth and virulence. In conclusion, the BCEC model was able to differentiate between the infective potential of different isolates. Moreover, resistance to lysozyme correlated with the ability to invade and replicate within BCEC, suggesting co-selection for surviving challenging environments as the abomasum., Bioinformatics; Genetics; Microbiology; Molecular biology; Listeria monocytogenes; infection model; BCEC; MLST, sequence typing.

Antimicrobial resistance has been declared by the World Health Organization as one of the biggest threats to public health and Acinetobacter baumannii is a notable example. A. baumannii is an important human nosocomial pathogen, being along with other multidrug resistant (MDR) bacteria, one of the biggest public health concerns worldwide. In Veterinary Medicine, resistance patterns of Acinetobacter species other than A. baumanii are unclear, and the scarce information available is limited and fragmented. We applied a statistical modeling approach to investigate the occurrence, clinical relevance and antimicrobial resistant phenotypes of Acinetobacter spp. originated from animals. Seven Acinetobacter species were identified in clinical specimens of more than 15 different domestic, zoo and exotic animal species. We found a high rate of MDR A. baumannii of canine origin with some of these isolates originating from serious systemic or wound infections, which highlights their potential pathogenic profile and spread in the human environment. Data also revealed different antimicrobial resistance patterns of animal-origin Acinetobacter species, emphasizing the necessity to implement specific antimicrobial susceptibility recommendations for animal isolates as there are no such clinical breakpoints currently in place. This study provides substantial advancing in our understanding of Acinetobacter spp. in animal clinical specimens, and highlights the role of animals in the dynamics of multidrug resistance in bacteria. The data presented here is a valuable source of information for further establishment of clinical breakpoints for susceptibility testing of animal-associated Acinetobacter isolates.

Listeria monocytogenesis an important foodborne pathogen in human and veterinary health, causing significant morbidity and mortality including abortion. It has a particular tropism for the gravid uterus, however, the route of infection in reproductive tissues of ruminants (i.e. placentome), is much less clear. In this study, we aimed to investigate a bovine caruncular epithelial cell (BCEC) line as a model forL. monocytogenesinfection of the bovine reproductive tract. The BCEC infection model was used to assess the ability of 14 differentL. monocytogenesisolates to infect these cells. Lysozyme sensitivity and bacterial survival in 580 µg lysozyme/ml correlated with attenuated ability to proliferate in BCEC (p=0.004 and p=0.02, respectively). Four isolates were significantly attenuated compared to the control strain 10403S. One of these strains (AR008) showed evidence of compromised cell wall leading to increased sensitivity to ß-lactam antibiotics, and another (7644) had compromised cell membrane integrity leading to increased sensitivity to cationic peptides. Whole genome sequencing followed by Multi Locus Sequence Type analysis identified that five invasive isolates had the same sequence type, ST59, despite originating from three different clinical conditions. Virulence gene analysis showed that the attenuated isolate LM4 was lacking two virulence genes (uhpT,virR) known to be involved in intracellular growth and virulence.In conclusion, the BCEC model was able to differentiate between the infective potential of different isolates. Moreover, resistance to lysozyme correlated with the ability to invade and replicate within BCEC, suggesting co-selection for surviving challenging environments as the abomasum.

Background Mycoleptodiscus indicus is a dematiaceous hyphomycete fungus found on plant leaves. It has been rarely reported as a cause of human or animal disease, possibly because it is difficult to culture and identify from clinical specimens. Infections are presumably acquired by traumatic implantation. Case presentation An 8-year-old non-immunosuppressed cat from Georgia, USA, presented with a left front leg swelling without lameness. Cytology from a fine needle aspirate revealed pyogranulomatous inflammation with both cytoplasmic and extracellular fungal elements. There were septate hyphae with irregularly sized segments, non-staining uneven walls, and rounded yeast-like forms from which longer hyphae arose in a hub-and-spoke pattern. A mold was isolated on agar from a fine needle aspirate collected 1 week later and identified as M. indicus by morphology, DNA sequencing and phylogenetic analysis. The cat recovered completely and uneventfully with antifungal treatment. Conclusions We report a previously undescribed presentation of M. indicus causing a subcutaneous infection in a cat with successful antifungal treatment. In this study we highlight the potential of M. indicus to infect immunocompetent animals, and the veterinary medical community should be aware of its unusual but characteristic clinical, microbiological and cytologic presentation.

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.

Neurologic manifestations other than cerebellar hypoplasia are rarely associated with feline panleukopenia virus (FPV) infection in cats. Here the authors describe lymphoplasmacytic meningoencephalitis and neuronal necrosis in 2 cats autopsied after exhibiting ataxia and nystagmus. Gross changes consisted of cerebellar herniation through the foramen magnum, with flattening of cerebrocortical gyri and narrowing of sulci. Histologically, lymphoplasmacytic meningoencephalitis, extensive neuronal necrosis, and neuroaxonal degeneration with digestion chambers were present in the telencephalon and brain stem in both cats. Frozen brain tissue of both cats was positive for parvoviral antigen via fluorescent antibody testing, and formalin-fixed, paraffin-embedded tissue sections of brain were immunoreactive for parvovirus antigen and positive for parvoviral DNA on in situ hybridization. Frozen brain tissue from 1 case was positive for parvovirus NS1 and VP2 genes using conventional polymerase chain reaction, and subsequent DNA sequencing and phylogenetic analysis revealed that the viral strain was a FPV. Reverse transcription quantitative polymerase chain reaction on formalin-fixed, paraffin-embedded brain tissue revealed high levels of parvovirus in both cases, supporting an acute and active viral infection. Although rare, FPV infection should be considered in cases of lymphoplasmacytic meningoencephalitis and neuronal necrosis in cats.

Supplemental Material, DS1_VET_10.1177_0300985819837723 for Lymphoplasmacytic Meningoencephalitis and Neuronal Necrosis Associated With Parvoviral Infection in Cats by Anna Kokosinska, Grazieli Maboni, Kathleen M. Kelly, Alex Molesan, Susan Sanchez, Jeremiah T. Saliki, and Daniel R. Rissi in Veterinary Pathology

We describe lipid pneumonia in 5 of 24 Arctic foxes ( Vulpes lagopus) in association with morbillivirus infection, and lymphoid depletion in 3 of these 5 foxes. Canine distemper virus (CDV) immunohistochemistry yielded positive staining in lung, lymph nodes, spleen, adipose tissue, and renal pelvic urothelial cells in 5 cases. Liver and bone marrow samples collected from these cases tested positive for morbillivirus by reverse-transcription PCR assay. Strains belonged to the CDV Arctic lineage based on sequencing of the hemagglutinin gene followed by phylogenetic analysis. Phylogenetic analysis of the phosphoprotein gene showed that the identified CDV strains were not closely related to any previously documented strains responsible for outbreaks in different animals in other parts of the world.

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.

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.

Moraxella bovis is historically known as the primary agent of infectious bovine keratoconjunctivitis (IBK). However, Moraxella bovoculi and Moraxella ovis are also reported to be involved in the pathogenesis of IBK, therefore, these three species should be included in the development of a new vaccine with a broad-spectrum protection against the disease natural challenge. In this study we investigated the antigenic properties of clinical isolates and reference strains of M. bovis, M. bovoculi and M. ovis using a novel in vitro approach for vaccine evaluation based on two techniques, flow cytometry and western blotting (WB). Here, we demonstrated that rabbit antisera produced against reference M. bovis strain and commercial bacterin showed low number of IgG with capacity to recognize a panel of heterologous strains composed by M. bovoculi and M. ovis. On the other hand, the antisera generated against two clinical isolates of M. ovis (Mov2 and Mov3) presented high cross-reactivity levels against all M. ovis and M. bovis strains evaluated. Similarly, the antisera against Mbv3 (clinical isolate of M. bovoculi) had high levels of IgG associated on the surface of all M. bovoculi strains and most of the M. ovis strains analyzed. The WB analysis demonstrated that Moraxella spp. has multiple immunogenic antigens and most of them are shared between the three species. Based on the cross-reactivity analysis and considering the relative number of IgGs associated on the bacterial surface, we suggest that a multivalent vaccine including Mbv3, Mov2 and Mov3 strains may provide a strong and broad protection against all strains involved in IBK outbreaks.

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.

Foram obtidas 154 amostras de lesões cutâneas de cães com pioderma superficial atendidos no Serviço de Dermatologia Veterinária do Hospital Veterinário Universitário (HVU) da Universidade Federal de Santa Maria (UFSM), com o objetivo de determinar a suscetibilidade antimicrobiana de isolados de Staphylococcus spp. e avaliar a presença de multirresistência. Após isolamento e identificação, as cepas foram submetidas ao teste de sensibilidade aos antimicrobianos, cujos resultados evidenciaram menores percentuais de resistência à associação amoxicilina e ácido clavulânico (1,9%), cefadroxil (1,9%), cefalexina (1,9%) e vancomicina (0,6%). Os maiores percentuais de resistência foram frente à amoxicilina (60,4%) e penicilina G (60,4%). A multirresistência foi detectada em 23,4% e a resistência à meticilina em 5,8% das amostras. Pode-se concluir que os isolados de Staphylococcus spp. apresentam elevada suscetibilidade aos antimicrobianos comumente utilizados no tratamento dos piodermas superficiais em cães atendidos no HVU-UFSM, como a cefalexina e a amoxicilina associada ao ácido clavulânico, confirmando a eleição desses fármacos para o tratamento de cães com esta afecção. A suscetibilidade diminuída das cepas frente às fluoroquinolonas, também recomendadas pela literatura para o tratamento de pioderma, permite sugerir que estes fármacos não devem mais ser considerados na seleção empírica. A identificação de Staphylococcus spp. multirresistentes na população canina estudada justifica análises bacteriológicas periódicas e regionais de lesões cutâneas de cães com pioderma superficial, a fim de minimizar a seleção de bactérias resistentes, possíveis falhas terapêuticas e também motiva a antimicrobianoterapia prudente. A total of 154 samples of skin lesions were obtained from dogs with superficial pyoderma that were assisted by the Veterinary Dermatology Service at the University Veterinary Hospital (HVU), Universidade Federal de Santa Maria (UFSM), aiming to determine the antimicrobial susceptibility of Staphylococcus spp. isolates and evaluate the presence of multidrug resistance profile. After bacterial isolation and identification, the strains were tested for antimicrobial susceptibility, and the results showed lower percentages of resistance to the amoxicillin and clavulanic acid association (1.9%), cefadroxil (1.9%), cephalexin (1.9%) and vancomycin (0.6%). The highest percentages were towards amoxicillin (60.4%) and penicillin G (60.4%). The multidrug resistance was detected in 23.4% and the methicillin resistance in 5.8% of the samples. It may be concluded that the Staphylococcus spp. isolates present high susceptibility to key antimicrobials used in the treatment of superficial pyodermas in dogs at the HVU-UFSM, such as cephalexin and the amoxicillin and clavulanic acid association, confirming the preference for these drugs when treating dogs with this disorder. The susceptibility of the strains to fluoroquinolones, also recommended in the literature for the treatment of pyodermas, allows suggesting that such drugs should not be considered in the empirical selection. The identification of multidrug-resistant Staphylococcus spp. in the studied canine population justifies periodic and regional bacteriological tests of skin lesions in dogs with superficial pyoderma, possible therapeutic failures and also motivates wise use of antimicrobial therapy.

Background Toll like receptors are one of the major innate immune system pathogen recognition systems. There is little data on the expression of the TLR10 member of this family in the horse. Results This paper describes the genetic structure of the Equine TLR10 gene and its RNA expression in a range of horse tissues. It describes the phylogenetic analysis of the Equine TLR1,6,10,2 annotations in the horse genome, firmly identifying them in their corresponding gene clades compared to other species and firmly placing the horse gene with other TLR10 genes from odd-toed ungulates. Additional 3’ transcript extensions to that annotated for TLR10 in the horse genome have been identified by analysis of RNAseq data. RNA expression of the equine TLR10 gene was highest in peripheral blood mononucleocytes and lymphoid tissue (lymph nodes and spleen), however some expression was detected in all tissues tested (jejunum, caudal mesenteric lymph nodes, bronchial lymph node, spleen, lung, colon, kidney and liver). Additional data on RNAseq expression of all equine TLR genes (1–4 and 6–10) demonstrate higher expression of TLR4 than other equine TLRs in all tissues. Conclusion The equine TLR10 gene displays significant homology to other mammalian TLR10 genes and could be reasonably assumed to have similar fuctions. Its RNA level expression is higher in resting state PBMCs in horses than in other tissues. Electronic supplementary material The online version of this article (doi:10.1186/s13104-016-2161-9) contains supplementary material, which is available to authorized users.

Ovine footrot is characterised by interdigital dermatitis (ID) and by the separation of the skin and hoof horn (under-running 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 species in biopsies from the ovine interdigital skin of healthy, ID and footrot-affected feet and (ii) to characterise the virulence of D nodosus strains in those biopsies. Postslaughter 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.

Additional file 1. Additional tables.

A discutida questão da substituição do uso de antibacterianos em rações (promotores de crescimento) requer urgentes alternativas. Face às necessidades de inibidores microbianos nesses alimentos, os óleos essenciais (OES) se constituem em alternativa, sob avaliação. Neste estudo, avaliou-se a atividade antimicrobiana dos OES de Origanum vulgare (orégano), Thymus vulgaris (tomilho), Cinnamomum zeylanicum (canela), Lippia graveolens (orégano mexicano), Zingiber officinale (gengibre), Salvia officinalis (sálvia), Rosmarinus officinalis (alecrim) e Ocimum basilicum (manjericão) frente a amostras de Escherichia coli isoladas de fezes de aves (n=43) e de bovinos (n=36). A concentração inibitória mínima (CIM) e a concentração bactericida mínima (CBM) foram determinadas para cada isolado através da técnica de microdiluição em caldo, a partir da máxima concentração de 6400µg mL-1 de cada OE testado. Observou-se atividade antimicrobiana para os OES de orégano, orégano mexicano, tomilho, canela. Para todas as amostras testadas, independente de sua origem, os OES mais e menos efetivos quanto à atividade antimicrobiana foram o orégano e a canela, respectivamente. Esses resultados confirmaram o potencial antibacteriano de alguns OES, os quais merecem novas investigações abordando sua adição na alimentação de aves e bovinos. The discussed issue about replacing the use of antibiotics in animal feed (growth promoters) requires emerging alternatives. To meet the needs of microbial inhibitors in these foods, the essentials oils (EOS) constitute potential alternatives under evaluation. In this study it was evaluated the antimicrobial activities of EOs from Oreganum vulgare (oregano), Thymus vulgaris (thyme), Lippia graveolens (Mexican oregano), Cinnamomum zeylanicum (cinnamon), Zingiber officinale (ginger), Salvia officinalis (sage), Rosmarinus officinalis (rosemary) and Ocimum basilicum (basil) against Escherichia coli strains isolated from poultry (n=43) and cattle faeces (n=36). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for each isolate by using the broth microdilution technique, from the maximum concentration of 6400µg mL-1 of each OE tested. Antimicrobial activity was observed on the essential oils of oregano, mexican oregano, thymus and cinnamon. For all strains tested, regardless of their origin, the OES more and less effective as antimicrobial activity were oregano and cinnamon, respectively. These results confirm the antimicrobial potential of some EOs, which deserve further research, addressing the addition of essential oils in poultry and cattle feeding.

Made available in DSpace on 2018-12-11T17:22:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-07-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Universidade de Aveiro The diseases caused by Salmonella Gallinarum and S. Pullorum in chickens known as fowl typhoid and pullorum disease, respectively, pose a great threat to the poultry industry mainly in developing countries, since they have already been controlled in the developed ones. These bacteria are very similar at the genomic level but develop distinct host-pathogen relationships with chickens. Therefore, a deep understanding of the molecular mechanisms whereby S. Gallinarum and S. Pullorum interact with the host could lead to the development of new approaches to control and, perhaps, eradicate both diseases from the chicken flocks worldwide. Based on our previous study, it was hypothesised that metabolism-related pseudogenes, fixed in S. Pullorum genomes, could play a role in the distinct host-pathogen interaction with susceptible chickens. To test this idea, three genes (idnT, idnO and ccmH) of S. Gallinarum str. 287/91, which are pseudogenes on the S. Pullorum chromosomes, were inactivated by mutations. These genetically engineered strains grew well on the solid media without any colony morphology difference. In addition, similar growth curves were obtained by cultivation in M9 minimal medium containing D-gluconate as the sole carbon source. Infection of chickens with idnTO mutants led to increased numbers of bacteria in the livers and spleens at 5 days post-infection, but with slightly decreased heterophil infiltration in the spleens when compared to the wild-type strain. On the other hand, no significant phenotypic change was caused by mutation to ccmH genes. Apart from the above-mentioned alterations, all S. Gallinarum strains provoked similar infections, since mortality, clinical signs, macroscopic alterations and immune response were similar to the infected chickens. Therefore, according to the model applied to this study, mutation to the idnTO and ccmH genes showed minor impact on the fowl typhoid pathogenesis and so they may be relics from the ancestor genome. Our data hints at a more complex mechanism driving the distinct host-pathogen interaction of S. Gallinarum/Pullorum with chickens than differential inactivation of a few genes. Post Graduate Program in Agricultural and Livestock Microbiology Department of Veterinary Pathology School of Agriculture and Veterinarian Sciences São Paulo State University (Unesp), Campus at Jaboticabal Department of Veterinary Sciences Federal University of Paraíba School of Veterinary Medicine and Science University of Nottingham, Sutton Bonington Department of Veterinary Clinic and Surgery School of Veterinary Federal University of Minas Gerais (UFMG) Post Graduate Program in Agricultural and Livestock Microbiology Department of Veterinary Pathology School of Agriculture and Veterinarian Sciences São Paulo State University (Unesp), Campus at Jaboticabal FAPESP: 2013/22920-4 FAPESP: 2013/26127-7

Ovine footrot is a degenerative disease of sheep feet leading to the separation of hoof-horn from the underlying skin and lameness. This study quantitatively examined histological features of the ovine interdigital skin as well as their relationship with pro-inflammatory cytokine (IL-1β) and virulent Dichelobacter nodosus in footrot. From 55 healthy and 30 footrot ovine feet, parallel biopsies (one fixed for histology) were collected post-slaughter and analysed for lesions and histopathological analysis using haematoxylin and eosin and Periodic Acid-Schiff. Histological lesions were similar in both conditions while inflammatory scores mirror IL-1β expression levels. Increased inflammatory score corresponded with high virulent D. nodosus load and was significant (p D. nodosus load.

Records of in vitro susceptibility tests performed between 1992 and 2011 were retrospectively reviewed in order to evaluate the dynamic profiles of possible changes in antimicrobial resistance of Staphylococcus spp. isolated from milk samples of cows with mastitis during two decades. The results of 2,430 isolates tested by disk diffusion technique for susceptibility to oxacillin, penicillin, ampicillin, cephalexin, norfloxacin, tetracycline, sulfazotrim, gentamicin, and neomycin were analysed. Comparisons were performed between the percentages of resistance to antimicrobials and their classes and also between the decades studied. Additionally, the possible tendency or changes in the behaviour of these pathogens against the major drugs used in the last two decades were evaluated using regression analysis. The highest rates of resistance (P

The aim of this study was to determine the differences in the antimicrobial susceptibility profiles of Moraxella bovis, M. bovoculi and M. ovis. Thirty-two strains of Moraxella spp. isolated from cattle and sheep with infectious keratoconjunctivitis were tested via broth microdilution method to determine their susceptibility to ampicillin, cefoperazone, ceftiofur, cloxacillin, enrofloxacin, florfenicol, gentamicin, neomycin, oxytetracycline and penicillin. The results demonstrated that Moraxella spp. strains could be considered sensitive for most of the antimicrobials tested in this study, but differences between the antimicrobial susceptibility profiles of these three Moraxella species were found. M. bovis might differ from other species due to the higher MIC and MBC values it presented.

The cytotoxin A (MbxA) is one of the main virulence factors of Moraxella bovis involved in the pathogenesis of infectious bovine keratoconjunctivitis (IBK). Moraxella ovis and Moraxella bovoculi, suspected to be associated with infectious keratitis in sheep and cattle respectively, also have a gene that encodes the cytotoxin A (movA and mbvA, respectively). The aim of this study was to determine the molecular sequence of the 3' region of the cytotoxin gene of Moraxella spp. strains isolated from clinical cases to establish phylogenetic and evolutionary comparisons. PCR amplification, nucleotide sequencing (nt) and amino acid (aa) sequence prediction were performed, followed by the sequences comparison, identity level calculation and selective pressure analysis. The phylogenetic reconstruction based on nt and aa sequences clearly differentiate M. bovis (n=15), M. bovoculi (n=11) and M. ovis (n=7) and their respective reference strains. An alignment of 843nt revealed high similarity within bacterial species (MbxA=99.9% nt and aa; MbvA=99.3% nt and 98.8% aa; MovA=99.5% nt and 99.3% aa). The similarity of partial sequences (nt 1807-2649) of MbxA in relation to MbvA and MovA ranged from 76.3 to 78.5%; similarity between MbvA and MovA ranged from 95.7 to 97.5%. A negative selection on mbvA and movA sequences was revealed by the molecular evolution analysis. The phylogenetic analysis of movA and mbvA allowed different strains of Moraxella spp. to be grouped according to the period of isolation. Sequence analysis of cytotoxin may provide insights into genetic and evolutionary relationships and into the genetic/molecular basis of Moraxella spp.

NTPDase (EC 3.6.1.5) occurs in lymphocytes and plays an important role in immune function, in that hydrolyzes extracellular nucleoside tri- and/or diphoshates to form AMP. Pythium insidiosum causes the disease pythiosis, a pyogranulomatous disease of horses, dogs, cattle, cats and humans. Most antifungal drugs are ineffective against this pathogen, and immunotherapy, a treatment approach that relies on the injection of P. insidiosum antigen, has been successfully used in humans and horses to manage this disease. In this study, we investigated NTPDase activity in lymphocytes from rabbits inoculated with zoospores of P. insidiosum. After immunotherapy, we investigated the relationship between enzymatic activity and the pattern of the immune response. One milliliter of zoospores was inoculated subcutaneously into the coastal region of each rabbits. An average of 17,500 viable mobile zoospores/mL of induction medium was administered. Inoculated rabbits were checked weekly, and the subcutaneous nodular area (cm 2 ) was measured 28 days after inoculation. Rabbits that developed lesions received eight doses of immunotherapy at intervals of 14 days. Blood samples were collected by heart puncture twice a month for the determination of NTPDase activity. The results demonstrated that NTPDase activity in lymphocytes was increased in relation to ATP hydrolysis (by about 100%) in pythiosis and returned to normal values after immunotherapy. The data demonstrating NTPDase activity before and after immunotherapy reinforce the previously elaborated hypothesis that the change from a Th2 to a Th1 immune response is responsible for the curative properties of immunotherapy.

Pythium insidiosum is a zoosporic organism which causes pythiosis in humans and animals. This study aimed to report the paradoxical growth of Brazilian P. insidiosum strains when submitted to in vitro susceptibility tests with caspofungin. The growth at concentrations above the minimal inhibitory concentration (MIC) ranged from 16 to 128 μg/ml and it was observed in 50% of the isolates tested. This paradoxical growth in the presence of caspofungin has been observed with Candida and Aspergillus strains, however, the phenomenon involving oomycetes was described here for the first time. © 2010.