Association between the severity of histopathological lesions and Mycobacterium avium subspecies paratuberculosis (MAP) molecular diversity in cattle in southern Chile.

The objective was to evaluate the association between the severity of histopathological lesions caused by Mycobacterium avium subspecies paratuberculosis (MAP) infection and the molecular diversity of this pathogen. Blood, ileum, and mesenteric lymph node samples were collected at slaughter, from 1,352 adult cattle [source population 1 (SP1)]. In addition, 42 dairy herds (n = 4,963 cows) were followed for 2 years, and samples from compatible paratuberculosis clinical cases [source population 2 (SP2)] were collected. MAP infection was confirmed using an ELISA test, liquid media culture, and PCR. Isolates were genotyped using five MIRU-VNTR markers. Tissues from confirmed samples were subjected to a histopathological examination. A histopathological severity score (HSS) system was developed and used to grade (0 to 5) the magnitude of lesions caused by MAP. In general, the HSS system assesses the number of foci and degree of macrophage infiltration, together with the presence of multinucleated giant cells (MGCs) and acid-fast bacilli (AFB), in addition to the fusion of the intestinal villi and hyperplasia of the crypts. Despite the large sampling effort, only 79 MAP isolates were successfully genotyped, where 19 different haplotypes were described. A mixed-effect Poisson regression model was used to assess the relationship between haplotypes and HSS values. The model was controlled by animal age, and the farm was used as a random effect. Haplotypes were grouped based on their relative frequency: the most frequent haplotype (group i, 49.4%), the second most frequent haplotype (group ii, 12.7%), and all other haplotypes (group iii, 37.9%). Model outputs indicated that group i had significantly higher HSS values than group iii. In addition, group i was also associated with higher optical density (OD) values of the ELISA test. These results support the existence of differences in pathogenicity between MAP haplotypes. However, results were based on a relatively small sample size; thus, these should be taken with caution. Despite this, study findings suggest that haplotypes would be associated with differences in disease progression, where the dominant haplotype tends to generate more severe lesions, which could be linked to a greater shed of MAP cells than non-dominant haplotypes, increasing their chances of transmission.

Molecular Survey and Genetic Diversity of Bartonella spp. in Small Indian Mongooses (Urva auropunctata) and Their Fleas on Saint Kitts, West Indies.

This study aimed to molecularly survey and evaluate the genetic diversity of Bartonella spp. in mongooses and their fleas from St. Kitts. Spleen (n = 54), blood (n = 71), and pooled flea samples, all identified as Ctenocephalides felis (n = 53), were submitted to TaqMan real-time quantitative PCR (qPCR) targeting Bartonella-nuoG fragment (84 bp). Positive samples underwent further conventional PCR assays targeting five loci (gltA, rpoB, fstZ, nuoG, and ITS), subsequent sequencing, and phylogenetic and haplotype analyses. The overall occurrence of Bartonella spp. in mongooses and fleas was 51.2% (64/125 [95% CI (42.1-60.2%)]) and 62.3% (33/53) [95% CI (47.9-75.2%)]), respectively. From samples sequenced across the five loci, 50.8% (33/65) were identified as Bartonella henselae, 26.2% (17/65) were 96.74-99.01% similar by BLAST analysis to an unidentified Bartonella sp. previously reported in Japanese badgers (Meles anakuma), and 23.1% (15/65) were co-infected with both species. Nucleotide polymorphism analysis showed low diversity amongst haplotypes but did concur with phylogenetic analysis, placing the unidentified species in a separate clade from B. henselae by multiple mutational events. Our data confirms that mongooses and Ctenocephalides felis fleas collected from them are not only potential reservoirs for B. henselae but also a novel Bartonella sp. which we propose be called 'Candidatus Bartonella kittensis'.

Molecular survey of Bartonella spp. and haemoplasmas in American minks (Neovison vison).

The aim of this study was to perform a molecular survey and characterize Bartonella spp. and haemotropic Mycoplasma (haemoplasmas) in invasive American minks (Neovison vison) from Southern Chile. Additionally, we addressed risk factors for positivity in both groups of agents. Blood and/or tissue samples from 246 minks were analysed by qPCR targeting the nuoG gene for Bartonella spp. and conventional (c)PCR for 16S rRNA for haemotropic Mycoplasma spp. nuoG qPCR-positive Bartonella spp. samples were submitted to cPCR assays (ITS, ribC, gltA, rpoB, pap-31 and ftsZ genes) to perform phylogenetic inferences. Haemotropic Mycoplasma spp. 16S-positive samples were further amplified by cPCR targeting RNaseP gene (160-210 bp) and by two overlapping 16S rRNA cPCR assays to amplify a larger portion of the gene (1,200bp) for phylogenetics. Bartonella DNA was detected in 8.9% of minks (22/246). Out of 22 nuoG qPCR-positive samples, one and two showed positive results in cPCR assays based on ITS and ribC, respectively. Consistent sequencing results were obtained for only one ITS sample (464 bp sequence), which shared 99.6% identity with B. clarridgeiae. Two per cent of minks (5/246) were positive for 16S rRNA haemotropic Mycoplasma-cPCR assay. Two concatenated sequences of 16S rRNA (1,176 and 1,230 bp) were obtained: one sample shared 97.87% identity with haemotropic Mycoplasma sp. from a wild rodent, and the other 96.49% identity with 'Candidatus Mycoplasma haematoparvum' from a dog. All BLAST results were supported by phylogenetic analysis. One haemoplasma RNase P sequence shared 94.86% identity with Mycoplasma haemofelis from a cat. No risk factors for PCR positivity were identified. In a nutshell, Bartonella clarridgeiae and a potentially novel haemoplasma closely related to haemoplasmas previously reported in rodents, dogs, domestic and wild cats were described for the first time in American minks.

Antimicrobial resistance in E. coli isolated from dairy calves and bedding material.

INTRODUCTION: E. coli is a ubiquitous bacterium commonly used as a sentinel in antimicrobial resistance studies. Here, E. coli was isolated from three groups (sick calves, healthy calves and bedding material), to assess the presence of antimicrobial resistance, describe resistance profiles, and compare these resistances among groups. MATERIAL AND METHODS: Samples were collected from calves and calving pens from 20 dairy farms. Using the disc diffusion method, E. coli isolates were screened for antimicrobial resistance against seven antimicrobials: Amoxicillin, Ceftiofur, Gentamicin, Enrofloxacin, Trimethoprim-sulfamethoxazole, Florfenicol and Oxytetracycline. Isolates resistant to all these seven antimicrobials were tested again against an extended 19 antimicrobial drug panel and for the presence of the most common E. coli pathogenicity genes through PCR. RESULTS & DISCUSSION: Three hundred forty-nine E. coli isolates were obtained; most isolates were resistant to a single antimicrobial, but 2.3% (8) were resistant to 16 to 19 of the antimicrobials tested. The group with the highest percentage of multiresistant isolates was the calves with diarrhea group. Younger calves provided samples with higher antimicrobial resistance levels. CONCLUSIONS: There is a high rate of antimicrobial resistance in dairy farms calving pens. These bacteria could not only be a resistance gene reservoir, but also could have the potential to spread these determinants through horizontal gene transfer to other susceptible bacteria. Measures should be taken to protect colonization of younger calves, based on hygienic measures and proper management.

Sarcocystis spp. in red deer (Cervus elaphus), fallow deer (Dama dama), and pudu (Pudu pudu) in southern Chile / Sarcocystis spp. em veado (Cervus elaphus), Gamo (Dama dama) e Pudu (Pudu pudu) no sul do Chile

Worldwinde, cervids are considered an important source of infection and dissemination of a wide variety of pathogens, both for farm animals and humans. Among this diseases is sarcosporidiosis, which is a parasitic disease caused by Sarcocystis spp. (Protozoa: Apicomplexa). Most frequent clinical signs are hemolytic anemia, weakness, weight loss and decrease of growth and some species of Sarcocystis might cause abortions. The clinical disease in ruminants is fairly rare but the infection is very frequent. Infections are accumulative and the parasite does not generate immunity in any of the hosts. Ovine sarcosporidiosis is a serious issue in the some regions of Chile due to the macrocysts located in the muscle which means condemnation of the whole carcass. Sarcocystis spp. has been widely reported in red deer and other cervid species but in Chile the situation remains unknown. Nowadays there is little to no evidence of Sarcocystis in foreign deer in Chile and there is only one report of the parasite on pudu. The main goal of this study is to demonstrate the presence of Sarcocystis spp. in myocardium of red deer and fallow deer in Chile, and confirm the presence of Sarcocystis spp. in pudu. All cervid cases from 1994 to 2013 of the Institute of Animal Pathology of the Universidad Austral de Chile were reviewed. The animals selected were those in which a myocardium sample was taken. From the histopathological samples observed, it was found that 5 of the 9 red deer, 1 of the 4 fallow deer and in 11 of the 23 pudu there were Sarcocystis cysts in the myocardium. This study represents the first record for Chile of Sarcocystis spp. in myocardium of red deer and fallow deer. Stablishing the red deer, fallow deer and pudu as hosts of Sarcocystis aids to have a better understanding of the parasite epidemiology in Chile and the role of wild and captive cervids in the maintenance and spread of these parasites.(AU)

No mundo, os cervos são considerados uma fonte de infecção e propagação de uma grande variedade de patógenos para animais de criação e para os seres humanos. Entre estas doenças está a sarcosporidiosis, que é uma doença parasitária causada por Sarcocystis spp. (Protozoa: Apicomplexa). Os sinais clínicos mais comuns são anemia hemolítica, fraqueza, perda de peso e diminuição do crescimento e em algumas espécies de Sarcocystis podem causar abortos. A doença clínica em ruminantes é bastante rara, mas a infecção é muito comum. As infecções são cumulativos e o parasita não gera imunidade em nenhum dos seus hospedeiros. A Sarcosporidiosis ovina é um problema grave em algumas regiões do Chile devido a microcistos localizados no músculo provocando a reprovação total da carcaça. Sarcocystis spp. tem sido amplamente relatado em cervos vermelhos e outras espécies de cervídeos, mas no Chile a sua situação permanece desconhecida. Atualmente há pouca ou nenhuma evidência de Sarcocystis em cervos introduzidos no Chile e há apenas um relatório do parasita em pudú. O principal objetivo deste estudo é demonstrar a presença de Sarcocystis spp. no miocárdio no veado vermelho e cervo gamo no Chile e confirmar a presença de Sarcocystis spp. em pudus. Revisaram-se todos os casos de cervos desde 1994-2013 do Instituto de Patologia Animal da Universidad Austral de Chile. Os animais selecionados para o estudo foram aqueles em que se tomou amostra de miocárdio. Das amostras histopatológicas observadas, verificou-se que em cinco dos nove cervos vermelhos, em um dos quatro veados gamo e 11 dos 23 pudus tinham cistos de Sarcocystis no miocárdio. Este estudo representa o primeiro relatório para o Chile de Sarcocystis spp. no miocárdio de veados vermelhos e cervo gamo. Definir o veado vermelho, o cervo gamo e os pudú como anfitriões de Sarcocystis ajuda a uma melhor compreensão da epidemiologia deste parasita no Chile e o papel de cervos selvagens e em cativeiro para a manutenção e divulgação deste parasita.(AU)

Paratuberculosis: new histopathological findings in red deer (Cervus elaphus) and fallow deer (Dama dama) in Chile / Paratuberculosis: novos achados histopatológicos em Veado Vermelho (Cervus elaphus) e Gamo (Dama dama) no Chile

Paratuberculosis is a disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) that affects domestic and wild ruminants. The most common gross lesions are emaciation and corrugation and thickening of the mucosa of the small intestine. Mesenteric lymph nodes might be enlarged. For the present study, 14 red deer and 9 fallow deer from game reserves or venison farms were analyzed. The lesions found correspond to those found by other authors in other geographic locations, except for some differences in histopathological examinations. Among these differences, stands out that intestinal lesions were concentrated mostly in the ileum and granulomas were shown to be more frequent in this section of the intestine than in the corresponding lymph node. Furthermore, in multibacillary lesions the inflammatory infiltrate in the lymph nodes was mainly composed of macrophages. These differences may be due to individual variations of the animals, the stage of disease or a different strain of the pathogen. This study allowed to obtain basic information about the disease and to describe patterns of lesions found in red deer and fallow deer with prediagnosis of clinical paratuberculosis which were not described in the literature before.(AU)

Paratuberculosis é uma doença causada por Mycobacterium avium subsp. paratuberculosis (MAP) que afecta ruminantes selvagens e domésticos. As lesões macroscópicas mais comuns são ondulação e espessamento da mucosa do intestino delgado. Os linfonodos mesentéricos podem aparecer com volume aumentado. Para este estudo, foram analisados 14 veados vermelhos e 9 veados gamo de reservas de caça e fazendas de carne. As lesões encontradas correspondem à encontrada por outros autores em outras localizações geográficas, com exceção de algumas diferenças no exame histopatológico. Entre essas diferenças, sobressai que as lesões intestinais se concentraram principalmente no íleo, os granulomas ocorreram com maior frequência nesta seção do intestino que no seu correspondente linfonodo. Além disso, nas lesões bacterianas, o infiltrado inflamatório linfonodos linfáticos era composta principalmente por macrófagos. Estas diferenças podem ser devidas a variações individuais dos animais, o estádio da doença ou de uma estirpe diferente do agente patogénico. Este estudo permitiu obter informação básica sobre a doença e descrever padrões de lesões encontradas em veados e em gamos com pré-diagnóstico, de paratuberculosis clínica nunca antes descritas na literatura.(AU)

Seroprevalence and risk factors for Neospora caninum in small dairy farms in central Chile / Seroprevalencia y factores de riesgo de Neospora caninum en pequeñas granjas lecheras de la zona central de Chile

ABSTRACT Objective. To provide information about seroprevalence and risk factors of Neospora caninum infection in bovines of central Chile. Material and Methods. The study population are small dairy farms that are part of a Government Technical Support Service (SAT) in the O´Higgins region in central Chile. Sera samples were collected from milking cows and analyzed by a commercial ELISA kit. Additionally, a questionnaire was applied to farmers to identify risk factors through logistic regression. Results. The farm level prevalence was found to be 67%, and within farms seroprevalence 55%. Abortion history (p= 0.037, OR=5.09), dogs feed source (p= 0.0429, OR=6), cattle drinking water source (p=0.034, OR=4.5) and abortions management (p=0.017, OR=7.43) were found as significant risk factors for infection. Conclusion. There is a high N. caninum seroprevalence in small SAT´s dairy farms in O´Higgins region. These results highlight the need of improving N. caninum surveillance, and the development of preventive measures to avoid losses related with this disease.

RESUMEN Objetivos. Proveer información sobre la seroprevalencia y factores de riesgo asociados a la infección por Neospora caninum en bovinos en Chile central. Materiales y métodos. La población estudiada corresponde a pequeñas lecherías que son parte de un programa gubernamental de Asistencia Técnica (SAT), en la región de O´Higgins en Chile central. Muestras de suero fueron recolectadas desde vacas en lactancia y analizadas mediante un kit comercial ELISA. Además, se aplicó un cuestionario a los dueños de los animales para la identificación de factores de riesgo a través de regresión logística. Resultados. Se identificó una prevalencia a nivel de granja del 67%, mientras que dentro de las granjas positivas, esta fue en promedio de 55%. El historial de abortos (p= 0.037, OR=5.09), la fuente de alimentación de perros (p= 0.0429, OR=6), la fuente de agua de bebida de las vacas (p=0.034, OR=4.5) y el manejo de los abortos (p=0.017, OR=7.43) fueron identificados como factores de riesgo para la infección. Conclusiones. Se observa una alta seroprevalencia de N. caninum en pequeños productores lecheros pertenecientes al SAT en Chile central. Los resultados presentados resaltan la necesidad de mejorar las acciones de vigilancia de esta enfermedad y el desarrollo de medidas preventivas para evitar las pérdidas asociadas a esta enfermedad.

Transient peripheral immune response and central nervous system leaky compartmentalization in a viral model for multiple sclerosis.

Theiler's virus-induced demyelination represents an important animal model to study the chronic-progressive form of multiple sclerosis (MS). The aim of the present study was to identify specific genes and pathways in the deep cervical lymph node (cLN) and spleen of experimentally infected SJL-mice, using DNA microarrays. Analyses identified 387 genes in the deep cLN and only 6 genes in the spleen of infected animals. The lymph node presented 27.4% of genes with fold changes +/-1.5 at 14 days post infection (dpi) and a reduced transcription at later time points. K-means clustering analyses resulted in five clusters. Accordingly, functional annotation revealed that the B-cell immune response pathway was the most up-regulated cluster at the early phase. Additionally, an increase of CD68- and lysozyme-positive cells in the deep cLN was observed by immunohistochemistry. Polioencephalitis was most intense at 14 dpi, and the spinal cord demyelinating leukomyelitis started at 42 dpi. In summary, early gene expression is indicative of virus-trigged immune responses in the central nervous system (CNS)-draining lymph node. The decreased gene transcription in the deep cLN during the chronic phase and the low number of spleen genes supports the hypothesis of a compartmentalized inflammation within the CNS, as described in progressive MS.