Isolation of vapB-positive Rhodococcus equi from submaxillary lymph nodes with or without granulomatous lesions in growing-finishing pigs in Japan.

To investigate the etiological role of vapB-positive Rhodococcus equi in pigs, R. equi was isolated from the submaxillary lymph nodes with or without macroscopically detectable lesions of apparently healthy growing-finishing pigs at a slaughterhouse in Toyama Prefecture, Japan. R. equi was isolated from 57 (24.6%) of 232 pigs with macroscopically detectable lymph node lesions, and 56 (98.2%) of the 57 isolates were vapB-positive. R. equi was isolated from 10 (2.4%) of 420 pigs without lymph node lesions, and six (60%) of the 10 isolates were vapB-positive. Plasmid DNA was isolated from the 62 vapB-positive isolates and digested with EcoRI and NsiI to obtain the plasmid profile. Fifty-two (83.9%), three (4.8%), and four (6.5%) isolates contained pVAPB subtypes 1, 2, and 3, respectively, while the remaining three isolates were of pVAPB subtypes 9, 13, and 14, respectively. Twelve specimens from lymph nodes with macroscopically detectable lesions were randomly selected for histopathological staining. Granulomatous lesions resembling tuberculosis were found in 11 of the 12 specimens, and the remaining specimen showed typical foci of malakoplakia in the lymph node. The isolation rates of R. equi and vapB-positive R. equi from lymph nodes with macroscopically detectable lesions were significantly higher (P<0.05) than those of lymph nodes without lesions, suggesting an etiologic association between vapB-positive R. equi and macroscopically detectable granulomatous lesions in porcine submaxillary lymph nodes. Previous reports on the prevalence of vapB-positive R. equi in pigs are reviewed and discussed.

First description of Rhodococcus equi infection in common bottlenose dolphin (Tursiops truncatus).

Rhodococcus equi is a terrestrial bacterium and a common pathogen in foals (Equus caballus), in which causes pneumonia. This report describes for the first time the infection caused by R. equi in a common bottlenose dolphin (Tursiops truncatus) stranded in the Calabrian coast, Italy. The post mortem examination of the animal revealed lesions in lung and colon. The animal was also positive to dolphin morbillivirus. The histological study showed lesions attributable to R. equi infection, such as pyogranulomatous bacterial pneumonia and chronic granulomatous colitis. Whole genome sequencing of the isolated strain confirmed its identification as R. equi.

Development of monoclonal antibodies against Rhodococcus equi virulence-associated protein N and their application to pathological diagnosis.

IMPORTANCE: Rhodococcus equi can cause infection in ruminants, and its pathogenicity is suggested to be associated with VapN. Despite its wide distribution, no immunological diagnostic method has been developed for VapN-producing R. equi. Against this background, we attempted to develop monoclonal antibodies targeting VapN and assess their application in immunostaining. In the study, mice were immunized with recombinant VapN, and cell fusion and cloning by limiting dilution permitted the generation of three antibody-producing hybridomas. The utility of the antibodies produced from the hybridomas in immunostaining was demonstrated using an infected mouse model, and the antibodies were further applied to previously reported cases of R. equi infection in goats and cattle. Although the 4H4 antibody induced the strongest reactions, the reactivity of two other antibodies was improved by antigen retrieval. Our monoclonal antibodies will be utilized to support the definitive diagnosis of suspected R. equi infection, including cases that were previously missed.

Rhodococcus equi-Derived Extracellular Vesicles Promoting Inflammatory Response in Macrophage through TLR2-NF-κB/MAPK Pathways.

Rhodococcus equi (R. equi) is a Gram-positive coccobacillus that causes pneumonia in foals of less than 3 months, which have the ability of replication in macrophages. The ability of R. equi persist in macrophages is dependent on the virulence plasmid pVAPA. Gram-positive extracellular vesicles (EVs) carry a variety of virulence factors and play an important role in pathogenic infection. There are few studies on R. equi-derived EVs (R. equi-EVs), and little knowledge regarding the mechanisms of how R. equi-EVs communicate with the host cell. In this study, we examine the properties of EVs produced by the virulence strain R. equi 103+ (103+-EVs) and avirulenct strain R. equi 103− (103−-EVs). We observed that 103+-EVs and 103−-EVs are similar to other Gram-positive extracellular vesicles, which range from 40 to 260 nm in diameter. The 103+-EVs or 103−-EVs could be taken up by mouse macrophage J774A.1 and cause macrophage cytotoxicity. Incubation of 103+-EVs or 103−-EVs with J774A.1 cells would result in increased expression levels of IL-1ß, IL-6, and TNF-α. Moreover, the expression of TLR2, p-NF-κB, p-p38, and p-ERK were significantly increased in J774A.1 cells stimulated with R. equi-EVs. In addition, we presented that the level of inflammatory factors and expression of TLR2, p-NF-κB, p-p38, and p-ERK in J774A.1 cells showed a significant decreased when incubation with proteinase K pretreated-R. equi-EVs. Overall, our data indicate that R. equi-derived EVs are capable of mediating inflammatory responses in macrophages via TLR2-NF-κB/MAPK pathways, and R. equi-EVs proteins were responsible for TLR2-NF-κB/MAPK mediated inflammatory responses in macrophage. Our study is the first to reveal potential roles for R. equi-EVs in immune response in R. equi-host interactions and to compare the differences in macrophage inflammatory responses mediated by EVs derived from virulent strain R. equi and avirulent strain R. equi. The results of this study have improved our knowledge of the pathogenicity of R. equi.

An Uncommon Case of Trueperella pyogenes Infection in an Adult Backyard Rooster and a Retrospective Study; 2000-20.

Trueperella pyogenes is an opportunistic Gram-positive bacterium that induces purulent lesions and abscesses in cattle, small ruminants, and swine. In birds, T. pyogenes infections have been linked to lameness and osteomyelitis in turkeys (Phasianidae) and hepatic fibriscess in turkeys and pigeons (Columbidae). An 18-mo-old backyard rooster with a history of progressive emaciation was submitted to the California Animal Health and Food Safety (CAHFS) laboratory system. At necropsy, unusual numerous miliary granulomas were identified, primarily in the spleen, but granulomas were also observed in air sacs and lungs. Microscopically, few to moderate numbers of granulomas with giant cells were observed in the spleen, lung, air sacs, and crop composed of necrosis and mixed inflammatory cell inflammation including multinucleated giant cells, fibrin deposition, and fibrosis. Trueperella pyogenes was isolated from the air sacs and trachea. Avibacterium paragallinarum PCR was positive from the tracheal swab. A retrospective analysis of CAHFS data on T. pyogenes between 2000 and 2020 identified 24 cases in avian species: chickens (Gallus gallus domesticus; 16/24), turkeys (5/24), Pekin duck (Anas platyrhynchos domesticus; 1/24), parrot (Psittaciformes; 1/24), and pheasant (Phasianidae; 1/24). Although T. pyogenes infection in birds is rare, the clinical signs and gross lesions might be indistinguishable from avian mycobacteriosis in some cases and should be considered in the differential diagnosis.

Reporte de caso­Un caso no común de infección por Trueperella pyogenes en un gallo adulto de traspatio y un estudio retrospectivo; entre los años 2000-20. Trueperella pyogenes es una bacteria grampositiva oportunista que induce lesiones purulentas y abscesos en bovinos, pequeños rumiantes y porcinos. En las aves, las infecciones por T. pyogenes se han relacionado con cojera y osteomielitis en pavos (Phasianidae) y fibrosis hepática en pavos y palomas (Columbidae). Un gallo de traspatio de 18 meses de edad con antecedentes de emaciación progresiva fue enviado al sistema de Laboratorios de Salud Animal y Seguridad Alimentaria de California (CAHFS). En la necropsia, se identificaron numerosos granulomas miliares inusuales, principalmente en el bazo, pero también se observaron granulomas en los sacos aéreos y los pulmones. Microscópicamente, se observaron pocos a moderados granulomas con células gigantes en el bazo, pulmón, sacos aéreos y buche compuesto por necrosis e inflamación celular inflamatoria mixta, incluidas células gigantes multinucleadas, depósito de fibrina y fibrosis. Trueperella pyogenes se aisló de los sacos aéreos y la tráquea. Un método de PCR para Avibacterium paragallinarum fue positivo realizado a partir de hisopos traqueales. Un análisis retrospectivo de los datos de CAHFS sobre T. pyogenes entre los años 2000 y 2020 identificó 24 casos en especies aviares: pollos (Gallus gallus domesticus; 16/24), pavos (5/24), pato Pekín (Anas platyrhynchos domesticus; 1/24), loro (Psittaciformes; 1/24) y faisán (Phasianidae; 1/24). Aunque la infección por T. pyogenes en aves es poco común, los signos clínicos y las lesiones macroscópicas pueden ser indistinguibles de micobacteriosis aviar en algunos casos y debe considerarse como diagnóstico diferencial.

Pathogenicity and genomic features of vapN-harboring Rhodococcus equi isolated from human patients.

Rhodococcus equi is a saprophytic soil bacterium and intracellular pathogen that causes refractory suppurative pneumonia in foals and has emerged as a pathogenic cause of zoonotic disease. Several studies have reported human infections caused by R. equi harboring a recently described third type of virulence plasmid, the ruminant-associated pVAPN, which carries the vapN virulence determinant. Herein, we analyzed pathogenicity and genomic features of nine vapN-harboring R. equi isolated from human patients with and without HIV/AIDS. Four of these strains showed significant VapN production and proliferation in cultured macrophages. These strains were lethally pathogenic after inoculation with 1.0 × 108 CFU in mice and reproduced a necrotizing granulomatous inflammation in the liver and spleen similar to that observed in humans. Additionally, we determined entire genome sequences of all nine strains. Lengths of sequences were 5.0-5.3 Mbp, and GC contents were 68.7 %-68.8 %. All strains harbored a 120- or 125-kbp linear plasmid carrying vapN (Type I or Type II pVAPN) classified on the basis of differences in the distal sequences on the 3' side. Interestingly, VapN production differed significantly among strains harboring nearly identical types of pVAPN with variation limited to several SNPs and short base pair indels. The pVAPN sequences possessed by the VapN-producing strains did not retain any common genetic characteristics, and more detailed analyses, including chromosomal genes, are needed to further elucidate the VapN expression mechanism.

A case report: Actinobaculum suis infection associated with formation of pyogranuloma, epididymitis and azoospermia in a boar.

BACKGROUND: Actinobaculum suis is a bacterium known to cause infections of the urogenital tract of sows. Infection can occur through close contact to boars, who frequently carry the pathogen in their preputial diverticulum but do not become clinically diseased themselves. In the current case, Actinobaculum suis was isolated from pyogranuloma of inflamed epididymis in a boar with poor fertility. CASE PRESENTATION: Increased return to oestrus rate, which worsened after the purchase of a new boar, was reported in an organic farm in Switzerland. During herd examination, azoospermia of the boar was diagnosed, and slaughter, followed by examination of its urogenital tract, was carried out. Pathologically, pyogranuloma formation and epididymitis were diagnosed. Bacteriology of the pyogranulomas showed growth of Actinobaculum suis and mixed flora. After the boar was replaced, the return to oestrus rate improved tremendously. CONCLUSION: A close relative of Actinobaculum suis, namely Actinotignum schaalii, has already been associated with epididymitis in humans. Considering the present case and the parallels in human medicine, Actinobaculum suis should be included in the list of differentials of boars with poor fertility.

Opsonization but not pretreatment of equine macrophages with hyperimmune plasma nonspecifically enhances phagocytosis and intracellular killing of Rhodococcus equi.

BACKGROUND: Evidence regarding the efficacy of equine hyperimmune plasma to prevent pneumonia in foals caused by Rhodococcus equi is limited and conflicting. HYPOTHESIS: Opsonization with R. equi-specific hyperimmune plasma (HIP) will significantly increase phagocytosis and decrease intracellular replication of R. equi by alveolar macrophages (AMs) compared to normal plasma (NP). ANIMALS: Fifteen adult Quarter Horses were used to collect bronchoalveolar lavage cells. METHODS: In the first experiment, AMs from 9 horses were pretreated (incubated) with either HIP, NP, or media only (control) and then infected with nonopsonized R. equi. In a second experiment, AMs from 6 horses were infected with R. equi either opsonized with HIP or opsonized with NP. For both experiments, AMs were lysed at 0 and 48 hours and the number of viable R. equi quantified by culture were compared among groups using linear mixed-effects modeling with significance set at P < .05. RESULTS: Opsonization with either HIP or NP increased phagocytosis by AMs (P < .0001) and decreased intracellular survival of organisms in AMs (P < .0001). Pretreating AMs with either HIP or NP without opsonizing R. equi had no effects on phagocytosis or intracellular replication. CONCLUSIONS AND CLINICAL IMPORTANCE: Opsonizing R. equi with either NP or HIP decreases intracellular survival of organisms in AMs, but the effect does not appear to be enhanced by using HIP. Mechanisms other than effects on AMs must explain any clinical benefits of using HIP over NP to decrease the incidence of R. equi pneumonia in foals.

Clinicopathological and radiographic features in 40 cats diagnosed with pulmonary and cutaneous Rhodococcus equi infection (2012-2018).

OBJECTIVES: This retrospective study aimed to describe clinical manifestations, diagnostic options, radiological features, therapeutic plans and outcomes for cats infected with Rhodococcus equi. METHODS: Forty cats aged between 2 months and 11 years old (median 6 months) that were definitively diagnosed with rhodococcosis between 2012 and 2018 were recruited in this study. Medical records were reviewed for information on signalment, history, clinical presentation, diagnostic testing, treatment plans and clinical outcomes. RESULTS: Of the 40 cats, 36 showed the pulmonary form of the disease, with 35 (87.5%) presenting with dyspnoea, while four cats presented with only cutaneous lesions. Mean body temperature was 38.7 ± 0.2°C. Dyspnoea was noted in 87.5% of the cats. Leukocytosis (58.3%) with band neutrophilia (83.3%), monocytosis (58.3%) and thrombocytopenia (55.5%) were prominent findings in the haematology reports. Hyperproteinaemia (61.1%) with hypoalbuminaemia (22.2%) and hyperglobulinaemia (63.8%) with a low albumin:globulin ratio (38.9%) were prominent features of blood biochemistry reports. An alveolar-interstitial pattern was noted in 75% of pre-thoracocentesis radiographs. Pleural effusion, hepatomegaly, thoracic lymphadenopathy and atelectasis of any lung lobe were seen in 88.9%, 75%, 41.7% and 36.1% of cats, respectively. Overall, the mortality rate was 67.5% in both forms. CONCLUSIONS AND RELEVANCE: Clinicians should be aware that feline rhodococcosis manifests as a pulmonary disease at a much higher rate than previously reported. Further studies are required to address the epidemiology, pathophysiology, disease management and prognosis of feline rhodococcosis. The role of immunosuppression as a predisposing factor in feline rhodococcosis requires further investigation.

The extracellular thioredoxin Etrx3 is required for macrophage infection in Rhodococcus equi.

Rhodococcus equi is an intracellular veterinary pathogen that is becoming resistant to current antibiotherapy. Genes involved in preserving redox homeostasis could be promising targets for the development of novel anti-infectives. Here, we studied the role of an extracellular thioredoxin (Etrx3/REQ_13520) in the resistance to phagocytosis. An etrx3-null mutant strain was unable to survive within macrophages, whereas the complementation with the etrx3 gene restored its intracellular survival rate. In addition, the deletion of etrx3 conferred to R. equi a high susceptibility to sodium hypochlorite. Our results suggest that Etrx3 is essential for the resistance of R. equi to specific oxidative agents.

Clinical and pathological findings of Rhodococcus equi infection in foals / Achados clínicos e patológicos da infecção por Rhodococcus equi em potros

Infection by Rhodococcus equi is considered one of the major health concerns for foals worldwide. In order to better understand the disease's clinical and pathological features, we studied twenty cases of natural infection by R. equi in foals. These cases are characterized according to their clinical and pathological findings and immunohistochemical aspects. Necropsy, histologic examination, bacterial culture, R. equi and Pneumocystis spp. immunohistochemistry were performed. The foals had a mean age of 60 days and presented respiratory signs (11/20), hyperthermia (10/20), articular swelling (6/20), prostration (4/20), locomotor impairment (3/20) and diarrhea (3/20), among others. The main lesions were of pyogranulomatous pneumonia, seen in 19 foals, accompanied or not by pyogranulomatous lymphadenitis (10/20) and pyogranulomatous and ulcerative enterocolitis (5/20). Pyogranulomatous osteomyelitis was seen in 3 foals, one of which did not have pulmonary involvement. There was lymphoplasmacytic (4/20), lymphoplasmacytic and neutrophilic (1/20) or pyogranulomatous arthritis (1/20), affecting multiple or singular joints. Immunohistochemistry revealed to be a valuable tool for the detection of R. equi, confirming the diagnosis in all cases. Furthermore, pulmonary immunostaining for Pneumocystis spp. demonstrates that a coinfection with R. equi and this fungal agent is a common event in foals, seen in 13 cases.(AU)

Infecção por Rhodococcus equi é considerado um dos maiores problemas sanitários para potros em todo o mundo. Para melhor compreender a apresentação clínica e patológica da enfermidade, foram avaliados vinte casos de infecção natural por R. equi em potros. Os casos são caracterizados de acordo com seus achados clínicos e patológicos e aspectos imuno-histoquímicos. Foram realizados exames de necropsia, histologia, bacteriologia e imuno-histoquímica para R. equi e Pneumocystis spp. Os potros tinham idade media de 60 dias e apresentaram sinais respiratórios (11/20), hipertermia (10/20), aumento de volume articular (6/20), prostração (4/20), distúrbios locomotores (3/20) e diarreia (3/20), entre outros. As lesões mais importantes eram pneumonia piogranulomatosa, vista em 19 potros, acompanhada ou não por linfadenite piogranulomatosa (10/20) e enterocolite ulcerativa (5/20). Osteomielite piogranulomatosa foi constatada em três potros, um dos quais não apresentava envolvimento pulmonar. Artrites afetando uma ou múltiplas articulações eram caracterizadas por infiltrado linfoplasmocítico (4/20), linfoplasmocítico e neutrofílico (1/20) e piogranulomatoso (1/20). A imuno-histoquímica demonstrou ser uma ferramenta valiosa na detecção de R. equi, permitindo confirmar o diagnóstico em todos os casos avaliados. Além disso, a imuno-histoquímica para Pneumocystis spp. demonstra que a coinfecção por R. equi e o agente fúngico é um evento frequente em potros, constatado em 13 casos.(AU)

Pyogranulomatous enteritis and mesenteric lymphadenitis in an adult llama caused by Rhodococcus equi carrying virulence-associated protein A gene.

We report herein Rhodococcus equi infection in an 11-y-old, male llama with a history of diarrhea and endoparasitism. Postmortem examination revealed granulomatous and ulcerative enteritis, pyogranulomatous mesenteric lymphadenitis, fibrinosuppurative peritonitis, and granulomatous hepatitis. Intralesional macrophages were laden with gram-positive cocci. Bacteriology identified R. equi, and cultures tested positive for R. equi choE and vapA genes by PCR. This case expands the reported spectrum of lesions associated with R. equi infections in llamas from pyogranulomatous bronchopneumonia and peripheral lymphadenitis to pyogranulomatous mesenteric lymphadenitis and enteritis. We also link a R. equi that is carrying the virulent-associated protein gene VapA to clinical disease in New World camelids.

Ulcerative, granulomatous glossitis and enteritis caused by Rhodococcus equi in a heifer.

Rhodococcus equi infection in horses is common and is characterized by pyogranulomatous pneumonia and ulcerative enterocolitis. R. equi clinical disease in cattle, however, is rare and typically manifests as granulomatous lymphadenitis discovered in the abattoir. A 19-mo-old female Santa Gertrudis had a history of intermittent inappetence and weight loss for a 3-mo period before euthanasia. Gross and histologic examination revealed severe, chronic, ulcerative, and granulomatous inflammation in the tongue, pharynx, and small intestine. Also, the heifer had severe, granulomatous pharyngeal and mesenteric lymphadenitis. Bacterial cultures from the ileum, tongue, and liver yielded numerous-to-moderate numbers of R. equi. PCR analysis of the isolate detected the linear virulence plasmid vapN, which is often identified in bovine isolates (traA- and vapN-positive). The bacteria also lack the circular plasmids vapA and vapB that are associated with virulence in horses and swine, respectively. We report herein an atypical and unusual clinical presentation of R. equi infection in cattle, which has zoonotic potential.

Renibacterium salmoninarum iron-acquisition mechanisms and ASK cell line infection: Virulence and immune response.

Renibacterium salmoninarum is the aetiological agent of bacterial kidney disease (BKD) in salmonid farms. This pathogen possesses at least three iron-acquisition mechanisms, but the link between these mechanisms and virulence is unclear. Therefore, this study used RT-qPCR to assess the effects of normal and iron-limited conditions on iron-uptake genes controlled by IdeR and related to iron acquisition in Chilean R. salmoninarum strain H-2 and the type strain DSM20767T . Further evaluated was the in vitro immune-related response of the Atlantic Salmon Kidney (ASK) cell line, derived from the primary organ affected by BKD. R. salmoninarum grown under iron-limited conditions overexpressed genes involved in haemin uptake and siderophore transport, with overexpression significantly higher in H-2 than DSM20767T . These overexpressed genes resulted in higher cytotoxicity and an increased immune response (i.e., TNF-α, IL-1ß, TLR1 and INF-γ) in the ASK cell line. This response was significantly higher against bacteria grown under iron-limited conditions, especially H-2. These observations indicate that iron-acquisition mechanisms are possibly highly related to the virulence and pathogenic capacity of R. salmoninarum. In conclusion, treatments that block iron-uptake mechanisms or siderophore synthesis are attractive therapeutic approaches for treating R. salmoninarum, which causes significant aquaculture losses.

A model of clinical endometritis in Holstein heifers using pathogenic Escherichia coli and Trueperella pyogenes.

Bacterial infection of the uterus causes clinical endometritis in 15 to 20% of postpartum dairy cows and reduces fertility, even after the resolution of disease. However, it is difficult to disentangle the mechanisms linking reduced fertility with endometritis because cows have multiple confounding postpartum conditions. The aim of the present experiment was to develop an in vivo model of clinical endometritis in Holstein heifers using pathogenic Escherichia coli and Trueperella pyogenes. Estrous cycles of heifers were synchronized using a 5-d Co-Synch protocol, and subsequently received exogenous progesterone to elevate circulating progesterone at the time of uterine infusion. Endometrial scarification was performed before uterine infusion of live pathogenic Escherichia coli and Trueperella pyogenes, or sterile vehicle. Effects of infusion were evaluated by measuring rectal temperature, plasma haptoglobin, hematology, grading pus in the vaginal mucus, quantifying 16S rRNA in vaginal mucus, and transrectal ultrasonography. Bacterial infusion increased the median vaginal mucus to grade 2 by d 3 postinfusion, and to grade 3 from d 4 to 6 postinfusion. Control heifers maintained a median vaginal mucus grade ≤1 from d 1 to 6. Transrectal ultrasound revealed the accumulation of echogenic fluid in the uterus of heifers following bacterial infusion, which was absent in control heifers. Total 16S rRNA in vaginal mucus was elevated in bacteria-infused heifers compared with control heifers at d 5. Rectal temperature was increased in bacteria-infused heifers. Plasma haptoglobin, general health, and appetite did not differ between groups. As indicated by increased vaginal mucus grade after bacterial infusion and absence of systemic signs of illness, this model successfully induced symptoms resembling clinical endometritis in virgin Holstein heifers. The model allows the isolation of effects of uterine disease on fertility from confounding factors that can occur during the postpartum period in dairy cows.

Identification of a VapA virulence factor functional homolog in Rhodococcus equi isolates housing the pVAPB plasmid.

Rhodococcus equi is a facultative intracellular bacterium of macrophages and is an important pathogen of animals and immunocompromised people wherein disease results in abcessation of the lungs and other sites. Prior work has shown that the presence of the major virulence determinant, VapA, encoded on the pVAPA-type plasmid, disrupts normal phagosome development and is essential for bacterial replication within macrophages. pVAPA- type plasmids are typical of R. equi strains derived from foals while strains from pigs carry plasmids of the pVAPB-type, lacking vapA, and those from humans harbor various types of plasmids including pVAPA and pVAPB. Through the creation and analysis of a series of gene deletion mutants, we found that vapK1 or vapK2 is required for optimal intracellular replication of an R. equi isolate carrying a pVAPB plasmid type. Complementation analysis of a ΔvapA R. equi strain with vapK1 or vapK2 showed the VapK proteins of the pVAPB-type plasmid could restore replication capacity to the macrophage growth-attenuated ΔvapA strain. Additionally, in contrast to the intracellular growth capabilities displayed by an equine R. equi transconjugant strain carrying a pVAPB-type plasmid, a transconjugant strain carrying a pVAPB-type plasmid deleted of vapK1 and vapK2 proved incapable of replication in equine macrophages. Cumulatively, these data indicate that VapK1 and K2 are functionally equivalent to VapA.

Actinomadura vinacea isolated from a nonhealing cutaneous wound in a cat.

A 6-year-old, spayed female, domestic shorthair cat was presented to the University of Georgia Veterinary Teaching Hospital for a 3-year history of a nonhealing wound on the right tarsus. The wound temporarily resolved with medical management, but intermittently recurred when antimicrobials were discontinued. At presentation, the wound had become refractory to antimicrobial therapy. Physical examination revealed a 1 cm diameter crust along the medial aspect of the right tarsus. Proximal to the crust, were 2 non-painful, fluctuant swollen areas that were free of drainage. Cytologic evaluation revealed atypical granulated cells, and a mesenchymal neoplasm was interpreted as a top differential diagnosis. Histopathology revealed marked, chronic, multifocal, pyogranulomatous dermatitis with abundant intralesional colonies of gram-positive, acid-fast-negative, filamentous bacteria. PCR and sequencing confirmed the infection to be caused by Actinomadura vinacea.

Isoprenoids increase bovine endometrial stromal cell tolerance to the cholesterol-dependent cytolysin from Trueperella pyogenes.

Preventing postpartum uterine disease depends on the ability of endometrial cells to tolerate the presence of the bacteria that invade the uterus after parturition. Postpartum uterine disease and endometrial pathology in cattle are most associated with the pathogen Trueperella pyogenes. Trueperella pyogenes secretes a cholesterol-dependent cytolysin, pyolysin, which causes cytolysis by forming pores in the plasma membrane of endometrial stromal cells. The aim of the present study was to identify cell-intrinsic pathways that increase bovine endometrial stromal cell tolerance to pyolysin. Pyolysin caused dose-dependent cytolysis of bovine endometrial stromal cells and leakage of lactate dehydrogenase into supernatants. Cell tolerance to pyolysin was increased by inhibitors that target the mevalonate and cholesterol synthesis pathway, but not the mitogen-activated protein kinase, cell cycle, or metabolic pathways. Cellular cholesterol was reduced and cell tolerance to pyolysin was increased by supplying the mevalonate-derived isoprenoid farnesyl pyrophosphate, or by inhibiting farnesyl-diphosphate farnesyltransferase 1 or geranylgeranyl diphosphate synthase 1 to increase the abundance of farnesyl pyrophosphate. Supplying the mevalonate-derived isoprenoid geranylgeranyl pyrophosphate also increased cell tolerance to pyolysin, but independent of changes in cellular cholesterol. However, geranylgeranyl pyrophosphate inhibits nuclear receptor subfamily 1 group H receptors (NR1H, also known as liver X receptors), and reducing the expression of the genes encoding NR1H3 or NR1H2 increased stromal cell tolerance to pyolysin. In conclusion, mevalonate-derived isoprenoids increased bovine endometrial stromal cell tolerance to pyolysin, which was associated with reducing cellular cholesterol and inhibiting NR1H receptors.

A case report on disseminated Rhodococcus equi infection in a Japanese black heifer.

Rhodococcus equi was isolated from the granulomatous lesions of the lung, kidney, liver, and hepatic, mesenteric, and abomasum lymph nodes of a Japanese black heifer. R. equi isolates were analyzed by polymerase chain reaction for virulence-associated protein genes. The vapN gene was detected in all the isolates examined. This is the first report in which vapN-positive R. equi was isolated from cattle in Japan.

Identification of B-cell linear epitopes in domains 1-3 of pyolysin of Trueperella pyogenes using polyclonal antibodies.

Trueperella pyogenes is an important opportunistic pathogen. Pyolysin (PLO) makes important contributions to the pathogenicity of T. pyogenes. However, the structure and function of PLO has not been well documented. In the current study, epitopes in domain 1-3 of PLO have been mapped using rabbit anti-recombinant PLO (rPLO) polyclonal antibodies, and then the results were re-checked by using mouse and chicken anti-rPLO polyclonal antibodies, respectively. The results indicated that the region of aa 281-393 in PLO could not elicit antibodies against linear epitopes. A total of six B cell linear epitopes have been found in domain 1 of PLO. Two of the six epitopes (EP1 and EP2) were used to immunize mice and chicken. Chicken anti-EP1 and anti-EP2 serum and mouse anti-EP2 serum could react with rPLO and corresponding epitope polypeptide in western blot assay; however, only mouse anti-EP2 serum shows weak anti-hemolysis effect in the rPLO and sheep red blood system. Our results provide some new information to the research field of PLO structure and function.