Lawsonia intracellularis in Pigs: Progression of Lesions and Involvement of Apoptosis.

The purpose of this study was to follow the progression of gross and histologic lesions and apoptosis events in Lawsonia intracellularis-infected enterocytes through the course of the disease, proliferative enteropathy (PE). Thirty 5-week-old pigs were divided into 2 groups: 20 challenged and 10 control animals. Groups of 3 pigs, 2 challenged and 1 control, were euthanized at 1, 3, 5, 8, 11, 15, 19, 24, 29, and 35 days after inoculation. Complete necropsies were performed with gross evaluation. Tissue samples from different sites of the gastrointestinal tract and other visceral organs were collected for routine histologic staining and for immunohistochemistry (IHC) for L. intracellularis. In addition, caspase-3, terminal deoxyuridine nick-end labeling assay, and electron microscopy were performed in ileum samples. Macroscopic and histologic lesions suggestive of PE were first detected 11 days after infection and continued through day 24. L. intracellularis antigen was first detected in the intestine by IHC on day 5 after inoculation, and the bacterium was first detected by transmission electron microscopy on day 15. Positive IHC staining for [L. intracellularis] and enterocyte proliferation, but no gross lesion, were detected on day 29. All 3 pigs euthanized on day 35 were grossly and histologically normal and IHC negative. Hyperplastic crypts in challenge pigs had more apoptotic cells on days 15, 19, and 24 postinfection ( P < .05) compared to control pigs. Our results demonstrated the progression of lesions and infection by L. intracellularis and that inhibition of enterocyte apoptosis is not involved in the pathogenesis of proliferative enteropathy.

Pathogenesis of Senecavirus A infection in finishing pigs.

Senecavirus A (SVA) is an emerging picornavirus that has been associated with vesicular disease and neonatal mortality in swine. Many aspects of SVA infection biology and pathogenesis, however, remain unknown. Here the pathogenesis of SVA was investigated in finishing pigs. Animals were inoculated via the oronasal route with SVA strain SD15-26 and monitored for clinical signs and lesions associated with SVA infection. Viraemia was assessed in serum and virus shedding monitored in oral and nasal secretions and faeces by real-time reverse transcriptase quantitative PCR (RT-qPCR) and/or virus isolation. Additionally, viral load and tissue distribution were assessed during acute infection and following convalescence from disease. Clinical signs characterized by lethargy and lameness were first observed on day 4 post-inoculation (pi) and persisted for approximately 2-10 days. Vesicular lesions were first observed on day 4 pi on the snout and/or feet, affecting the coronary bands, dewclaws, interdigital space and heel/sole of SVA-infected animals. A short-term viraemia was observed between days 3 and 10 pi, whereas virus shedding was detected between days 1 and 28 pi in oral and nasal secretions and faeces. Notably, RT-qPCR and in situ hybridization (ISH) performed on tissues collected on day 38 pi revealed the presence of SVA RNA in the tonsils of all SVA-infected animals. Serological responses to SVA were characterized by early neutralizing antibody responses (day 5 pi), which coincided with decreased levels of viraemia, virus shedding and viral load in tissues. This study provides significant insights into the pathogenesis and infectious dynamics of SVA in swine.

Swine conjunctivitis outbreaks associated with Mycoplasma hyorhinis.

Conjunctivitis is an uncommon finding in commercial swine herds, and the etiology of the disease is rarely studied. We investigated cases of conjunctivitis in 3 wean-to-finish swine farms. Eye swabs and tissues were obtained from clinically affected pigs (8-22 wk of age), from unaffected pigs in contact with affected pen-mates, and from age-matched pigs from an unaffected herd. Real-time PCR (rtPCR) testing for Mycoplasma hyorhinis demonstrated consistent detection and high bacterial load in samples from affected herds (clinically affected animals and non-clinical pen-mates). Ct values in affected pigs were 18.9-25.3; values were 36.4-38.6 in unaffected pigs from unaffected herds. Additionally, M. hyorhinis was identified within inflamed palpebral conjunctivae by in situ hybridization. The association of rtPCR and in situ detection of M. hyorhinis, along with the lack of detection of other potential pathogens and noninfectious causes, suggests the involvement of M. hyorhinis in the etiology and pathogenesis of the reported swine conjunctivitis.

In situ hybridization detection and subtyping of rotaviruses in swine samples.

Rotavirus groups A, B, and C (RVA, RVB, and RVC, respectively) have been the most prevalent and pathogenic in pigs. To date, immunohistochemistry is only available for RVA because of the lack of commercial antibodies for RVB and RVC. We developed a novel in situ hybridization RNA-based chromogenic technique (ISH-RNA) to detect and subtype RVA, RVB, and RVC. We evaluated 33 samples that were reverse-transcription PCR positive for RVA, RVB, and/or RVC. ISH-RNA was able to detect as few as 103 RV RNA copies/mL. The new ISH-RNA test can be useful for routine investigation of rotavirus enteritis in order to guide strategies for control of the infection in pigs, but a full validation study needs to be completed. Pathogenesis studies may be conducted using ISH-RNA based on the identification of replicating virus.

Next-Generation Sequencing Coupled With in situ Hybridization: A Novel Diagnostic Platform to Investigate Swine Emerging Pathogens and New Variants of Endemic Viruses.

Next generation sequencing (NGS) can be applied to identify and characterize the entire set of microbes within a sample. However, this platform does not provide a morphological context or specific association between the viral or bacterial sequences detected and the histological lesions. This limitation has generated uncertainty whether the sequences identified by NGS are actually contributing or not for the clinical outcome. Although in situ hybridization (ISH) and immunohistochemistry (IHC) can be used to detect pathogens in tissue samples, only ISH has the advantage of being rapidly developed in a context of an emerging disease, especially because it does not require development of specific primary antibodies against the target pathogen. Based on the sequence information provided by NGS, ISH is able to check the presence of a certain pathogen within histological lesions, by targeting its specific messenger RNA, helping to build the relationship between the pathogen and the clinical outcome. In this mini review we have compiled results of the application of NGS-ISH to the investigation of challenging diagnostic cases or emerging pathogens in pigs, that resulted in the detection of porcine circovirus type 3, porcine parvovirus type 2, Senecavirus A, and Mycoplasma hyorhinis.

A novel RNA-based in situ hybridization to detect Seneca Valley virus in neonatal piglets and sows affected with vesicular disease.

Seneca Valley virus (SVV) is the causative agent of an emerging vesicular disease in swine, which is clinically indistinguishable from other vesicular diseases such as foot-and-mouth disease. In addition, SVV has been associated with neonatal mortality in piglets. While a commercial SVV qRT-PCR is available, commercial antibodies are lacking to diagnose SVV infections by immunohistochemistry (IHC). Thus, a novel in situ hybridization technique-RNAscope (ISH) was developed to detect SVVRNA in infected tissues. From a total of 78 samples evaluated, 30 were positive by qRT-PCR and ISH-RNA, including vesicular lesions of affected sows, ulcerative lesions in the tongue of piglets and various other tissues with no evidence of histological lesions. Nineteen samples were negative for SVV by qRT-PCR and ISH-RNA. The Ct values of the qRT-PCR from ISH-RNA positive tissues varied from 12.0 to 32.6 (5.12 x 106 to 5.31 RNA copies/g, respectively). The ISH-RNA technique is an important tool in diagnosing and investigating the pathogenesis of SVV and other emerging pathogens.

Malignant peripheral nerve sheath tumour in a sow.

Nodular lung lesions in swine are frequently due to abscesses or granulomatous pneumonia. Although tumours are rarely reported in modern pig farming, they should be considered as a differential diagnosis when nodular lung lesions are found. A first-parity sow exhibiting respiratory signs was euthanized. Several whitish firm nodules, not encapsulated, ranging in diameter from 0.5 to 5 cm were present in all lung lobes. Microscopically, the nodules were composed of dense neoplastic cells, mainly in Antoni types A and B patterns, infiltrative and with development of emboli. All neoplastic cells stained positively by immunohistochemistry for vimentin and S-100 protein, with variable immunostaining for glial fibrillary acidic protein and stained negative for cytokeratin. Based on the gross, histological and immunohistochemical features, the tumor was diagnosed as malignant peripheral nerve sheath tumour.

Use of oral fluids to detect anti Lawsonia intracellularis antibodies in experimentally infected pigs / Utilização de fluidos orais para detecção de anticorpos anti Lawsonia intracellularis em suínos experimentalmente infectados

Several pathogens and antibodies derived from serum or produced in tissues associated with the oral cavity are present in the oral fluid (OF). Considering the applicability of this alternative sample, recent studies in veterinary medicine have tested OF as a replacement for serum in diagnostic assays. The aim of this study was to standardize the immunoperoxidase monolayer assay (IPMA) to detect anti-Lawsonia intracellularis immunoglobulin A (IgA) and immunoglobulin G (IgG) in OF samples from experimentally infected pigs. Sixty-two pigs were divided into two groups: control (T1, n=30) and inoculated with L. intracellularis (T2, n=32). Blood, OF and fecal samples were collected at 0, 7, 14, 21, 28 and 42 days post-inoculation (dpi). Some adaptations of the standard technique for serum were made to IPMA for the detection of IgA and IgG in OF. The IPMA showed high specificity and sensitivity for serum samples and high specificity and moderate sensitivity for the detection of IgA and IgG in OF. There was high agreement between the results of serum IgG and OF IgA and IgG. Based on our results, oral fluid samples may be used for the evaluation and determination of anti-L. intracellularis antibodies in pigs, but not for individual diagnosis of swine proliferative enteropathy.(AU)

Vários patógenos e anticorpos derivados do soro ou produzidos em tecidos associados a cavidade oral estão presentes no fluido oral (FO). Considerando a aplicabilidade dessa amostra alternativa, estudos recentes em medicina veterinária têm testado o FO como substituto do soro para testes diagnósticos. O objetivo desse estudo foi padronizar a imunoperoxidase em monocamada de célula (IPMC) para a detecção de imunoglobulina A e imunoglobulina G anti-Lawsonia intracellularis em amostras de FO de suínos experimentalmente infectados. Um total de 62 suínos foram divididos em dois grupos: controle (T1, n=30) e inoculados com L. intracellularis (T2, n=32). Sangue, FO e amostras de fezes foram coletados aos 0, 7,14, 21, 28 e 42 dias após a inoculação (dpi). Algumas adaptações da técnica foram realizadas na técnica padrão da IPMC para a detecção de IgA e IgG. A IPMC demostrou alta especificidade e sensibilidade para amostras de soro e alta especificidade de moderada sensibilidade para a detecção de IgA e IgG em FO. Houve alta concordância entre resultados de detecção de IgG em soro com a IgA e IgG em amostras de FO. Baseado em nossos resultados, amostras de fluido oral podem ser usadas em avaliações e detecção de anticorpos anti-L. intracellularis em suínos, porém não de forma individual.(AU)

Minimum inhibitory concentration of Brazilian Brachyspira hyodysenteriae strains / Concentração inibitória mínima de cepas Brachyspira hyodysenteriae brasileiras

The objectives of this study were to characterize Brachyspira hyodysenteriae isolates and to evaluate the antimicrobial susceptibility patterns of strains obtained from pigs in Brazil based on the minimal inhibitory concentration test (MIC). The MIC was performed for 22 B. hyodysenteriae isolates obtained from 2011 to 2013 using the following antimicrobial drugs: tylosin, tiamulin, valnemulin, doxycycline, lincomycin and tylvalosin. Outbreaks of swine dysentery were diagnosed based on clinical presentation, bacterial isolation, gross and microscopic lesions, duplex PCR for B. hyodysenteriae and B. pilosicoli and nox gene sequencing. All obtained MIC values were consistently higher or equal to the microbiological cut-off described in the literature. The MIC 90 values for the tested drugs were 8µg/ml for doxycycline, >4µg/ml for valnemulin, 8µg/ml for tiamulin, 32µg/ml for tylvalosin, >64µg/ml for lincomycin and >128µg/ml for tylosin. These results largely corroborate those reported in the literature. Tiamulin, doxycycline and tylvalosin showed the lowest MIC results. All of the samples subjected to phylogenetic analysis based on the nox gene sequence exhibited similar results, showing 100% identity to B. hyodysenteriae. This is the first study describing the MIC pattern of B. hyodysenteriae isolated in Brazil.(AU)

Os objetivos deste trabalho foram a caracterização de isolados de Brachyspira hyodysenteriae e avaliar os padrões de sensibilidade antimicrobiana de isolados obtidos a partir de suínos no Brasil com base no teste de concentração inibitória mínima (MIC). A MIC foi realizada em 22 isolados de B. hyodysenteriae obtidos entre 2011 a 2013 usando os seguintes antimicrobianos: tilosina, tiamulina, valnemulina, doxiciclina, lincomicina e tilvalosina. Surtos de disenteria suína foram diagnosticados com base na apresentação clínica, isolamento bacteriano, lesões macroscópicas e microscópicas, PCR duplex para B. hyodysenteriae e B. pilosicoli e sequenciamento do gene nox. Todos os valores de MIC obtidos foram consistentemente mais elevados ou igual ao ponto de corte microbiológica descrito na literatura. Os valores de MIC 90 para os fármacos testados foram de 8 µg / mL para a doxiciclina, > 4 µg/ml de valnemulina, 8 µg / mL para a tiamulina, 32 µg / ml para tilvalosina, > 64 µg / ml para a lincomicina e > 128 µg / ml de tilosina. Estes resultados corroboram em grande parte com os relatados na literatura. Tiamulina, doxiciclina e tilvalosina apresentaram os menores resultados de MIC. Todas as amostras submetidas à análise filogenética com base na sequência do gene nox exibiram resultados semelhantes, indicando 100% de identidade com B. hyodysenteriae. Este é o primeiro estudo que descreve o padrão MIC de B. hyodysenteriae isoladas no Brasil.(AU)

Equine proliferative enteropathy on a brazilian farm / Enteropatia proliferativa em um haras brasileiro

Lawsonia intracellularis infection on a horse farm in the Midwest region of Brazil is described. Thirty-nine foals a few days to months old from a herd with 300 horses, experienced diarrhea with variable characteristics and intensities, weight loss, hyperemic mucous membranes and dehydration. In foals 3 to 6 months of age, hypoproteinemia associated with submandibular edema were also common. Intestinal fragments of a 7-month-old foal were sent to an animal disease laboratory for diagnosis. The observed macroscopic lesions were hyperemic serosa, thickening of the intestinal wall with a corrugation, thickening of the mucosa folds and reduction of intestinal lumen. Histological analysis of the small and large intestine revealed enterocyte hyperplasia of the crypts associated with diffuse marked decrease in the number of goblet cells and positive L. intracellularis antigen labeling by immunohistochemistry. Three out of 11 animals of the same property were seropositive for L. intracellularis, demonstrating the circulation of the agent throughout the farm, but none were PCR positive in fecal samples. Based on clinical signs and pathological findings, the diagnosis of equine proliferative enteropathy was confirmed.

Descreve-se a infecção por Lawsonia intracellularisem uma propriedade na região Oeste do Brasil. Em um rebanho de 300 equinos, 39 potros com poucos dias de vida à 21 meses apresentaram diarreia de características e intensidades variáveis, com perda de peso e desidratação. Em potros com três a seis meses de idade, hipoproteinemia associada a edema submandibular também foram frequentes. Fragmentos intestinais de um potro de 7 meses foram enviados ao laboratório de patologia animal para diagnóstico. Na macroscopia foi observada hiperemia de serosa e moderado espessamento de parede intestinal. Na histologia do intestino delgado existia hiperplasia de enterócitos de criptas difusa intensa com redução marcante de células caliciformes e marcação positiva na imuno-histoquímica para L. intracellularis. Na sorologia de 11 animais da mesma propriedade, três foram positivos. Já a PCR foi negativa para todos os animais. Com base nos sinais clínicos e nos achados patológicos confirmou-se o diagnóstico de enteropatia proliferativa equina, associada a sorologia positiva que demonstrava circulação do agente na propriedade.