J. Kruze

Diagnosis of paratuberculosis by fecal culture and ELISA on milk and serum samples in two types of Chilean dairy goat herds

Fecal culture has been the primary method used to diagnose paratuberculosis in goats. It is laborious, slow, and expensive. Validation of enzyme-linked immunosorbent assays (ELISAs) on milk samples could make paratuberculosis testing more widely available for goat farmers. The aim of this study was to determine the accuracy of serum and milk ELISAs for paratuberculosis, relative to fecal culture, in Chilean dairy goats. Eight dairy goat herds were selected. Feces, blood, and milk samples were collected from all female goats >2 years old. Fecal samples were cultured using Herrold egg yolk medium with mycobactin J and antibiotics. Serum and milk samples were tested using a commercial ELISA kit for Mycobacterium avium subsp. paratuberculosis antibody detection. A total of 383 goats were tested by ELISA and fecal culture. The sensitivity of ELISA on serum and milk relative to fecal culture was 74.3% (95% CI: 59.8-88.8) and 60% (95% CI: 43.8-76.2), respectively. The corresponding values for ELISA specificity based on the percentage of non-M. avium subsp. paratuberculosis-infected goats testing ELISA-negative were 98.6% (95% CI: 96.6-100) and 99.3% (95% CI: 97.9-100) on serum and milk, respectively. Proportions of positive results for serum and fecal samples were significantly different, whereas the proportions of positive results for milk and fecal samples were not significantly different. The milk ELISA had a moderate level of agreement with fecal culture results (Kappa = 0.57). The paratuberculosis ELISA on goat milk samples may be a cost-effective, accurate alternative to fecal culture.

FIRST ISOLATION OF MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS FROM WILD GUANACOS (LAMA GUANICOE) ON TIERRA DEL FUEGO ISLAND

The aim of this study was to search for Mycobacterium avium subsp. paratuberculosis (Map) infection in a free-ranging wild animal species in a region where Johness disease has yet to be reported and to classify Map isolates using a genomic typing method. Fecal samples were obtained from 501 wild guanacos (Lama guanicoe) from Tierra del Fuego Island, Chile, in August 2006. Samples were cultured using Herrolds egg yolk medium with and without mycobactin J. After 9 mo of incubation, suspected Map colonies showing mycobactin dependence were confirmed by real-time polymerase chain reaction (PCR) based on IS900 and F57. Isolates were further tested using IS1311 PCR with restriction endonuclease analysis in order to type the guanaco Map strains. Twenty-one of 501 (4.2%) animals were fecal culture–positive for Map; identity was confirmed by real-time PCR and isolates were classified as cattle-type. Most culture-positive animals were located in four contiguous geographic areas, and the infection was most commonly found among adult animals. Prevalence was higher in females (5.9%) than males (3.1%) but the difference was not statistically significant. This represents the first isolation of Map from a free-ranging wildlife species in Chile. It expands the geographic range of paratuberculosis and the diversity of wildlife species that can become infected with Map.

Goat paratuberculosis in Chile: first isolation and confirmation of Mycobacterium avium subspecies paratuberculosis infection in a dairy goat

In October 2004, 41 goats >2 years old from a Saanen dairy goat herd located in Purranque County, 10th Region, Chile, were sampled and tested for paratuberculosis. While collecting samples it was observed that several goats were thin and emaciated. One goat was sufficiently debilitated to warrant humane euthanasia. This animal was brought to the Veterinary School at the Universidad Austral de Chile for necropsy. The goat selected for necropsy was a 12-year-old doe. The animal showed classical clinical signs of caprine paratuberculosis: emaciation despite willingness to eat, dry and rough hair coat, and no evidence of diarrhea. Gross pathology and histopathology of the necropsied goat were consistent with paucibacillary paratuberculosis. Bacteriology, serology, and PCR confirmed the diagnosis. This is the first published report of goat paratuberculosis in Chile confirming a case of caprine paucibacillary paratuberculosis.

Resistencia bacteriana en cepas patógenas aisladas de mastitis en vacas lecheras de la V Región, Región Metropolitana y Xª Región, Chile

3Cooprinsem, Casilla 827, Osorno, Chile. SUMMARY Antimicrobial chemotherapy in human and veterinary medicine is one of the most important therapeutic tool against pathogenic agents causing infectious diseases; nevertheless, the development of multiple resistant strains during the last years has been reported. Some of the measures adopted to control this problem have been the veterinary prescription of antimicrobials for animal use, the permanent rotation of drugs, and the implementation of continuous monitoring programs for bacterial resistance. In the present paper the sensitivity of pathogenic bacteria isolated from dairy cows suffering mastitis in different regions of Chile against antimicrobials most frequently used in dairy herds is reported. The Plate Dilution Method and the Minimum Inhibitory Concentration (MIC) were used to evaluate the bacterial resistance of each isolated strain. A total of 449 bacterial strains were isolated from 963 aseptically collected milk samples in the 5th and Metropolitan Regions of Chile, E. coli being the most frequent mastitis pathogen recovered. In the 10th Region, however, S. aureus was the main pathogen among 1012 bacterial strains isolated from 2000 milk samples. S. aureus, Streptococcus spp and coagulase-negative Staphilococcus (CNS) were shown to be highly resistant to amoxicilin, ampicillin, penicillin, streptomycin and lincomicyn. Resistance to cloxacilin of S. aureus strains isolated in the 5th-Metropolitan and 10th Regions were 6.2% and 3.7% respectively. On the other hand, a high rate of sensitivity was observed in E. coli with resistance values below 25%. According to these results it is possible to conclude that the mastitis pathogens present in the geographical regions under study are resistant to more than one antimicrobial drug and, therefore, usage of these drugs under medical prescription and implementation of permanent monitoring programs for bacterial resistance are strongly recommended.

European Hares in Chile: A Different Lagomorph Reservoir for Mycobacterium avium subsp. paratuberculosis?

Ruminants are the principal host for infection by Mycobacterium avium subsp. paratuberculosis (Map), the cause of Johnes disease. Based on studies of a Map-infected population of European rabbits (Oryctolagus cuniculus) in Scotland, lagomorphs as a broad taxonomic order were proposed as potential nonruminant reservoirs for Map. To determine whether a different lagomorph species may serve as a wildlife reservoir, we investigated Map infection in European hares (Lepus europaeus) sharing habitat with known Map-infected dairy cattle in southern Chile. Fecal, mesenteric lymph node, and ileal samples were aseptically collected from 385 wild hares for liquid culture and real-time polymerase chain reaction identification of acid-fast isolates. All tissue samples were also acid-fast stained and examined microscopically. We isolated Map from at least one tissue from 48 hares (12.6%) and fecal samples from 16 hares (4.2%). No Map was found in tissues of eight of the fecal-culture–positive hares. Histologically, all tissues from all hares were within normal limits, and no acid-fast organisms were observed in any sample. Active infection, implying amplification of the organism secondary to resultant disease, was not evident. With this report Map isolations on a population versus incidental detection have now been made from two lagomorph species. However, although the rabbit population studied in Scotland appears to function as a Map reservoir, the hares studied in Chile appear to be a dead-end host, serving only as potential mechanical vectors for the organism.

Herd-level prevalence of Map infection in dairy herds of southern Chile determined by culture of environmental fecal samples and bulk-tank milk qPCR.

Paratuberculosis, an infectious disease of domestic and wild ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map), is an economically important disease in dairy herds worldwide. In Chile the disease has been reported in domestic and wildlife animals. However, accurate and updated estimations of the herd-prevalence in cattle at national or regional level are not available. The objectives of this study were to determine the herd-level prevalence of dairy herds with Map infected animals of Southern Chile, based on two diagnostic tests: culture of environmental fecal samples and bulk-tank milk qPCR. Two composite environmental fecal samples and one bulk-tank milk sample were collected during September 2010 and September 2011 from 150 dairy farms in Southern Chile. Isolation of Map from environmental fecal samples was done by culture of decontaminated samples on a commercial Herrolds Egg Yolk Medium (HEYM) with and without mycobactin J. Suspicious colonies were confirmed to be Map by conventional IS900 PCR. Map detection in bulk-tank milk samples was done by real time IS900 PCR assay. PCR-confirmed Map was isolated from 58 (19.3%) of 300 environmental fecal samples. Holding pens and manure storage lagoons were the two more frequent sites found positive for Map, representing 35% and 33% of total positive samples, respectively. However, parlor exits and cow alleyways were the two sites with the highest proportion of positive samples (40% and 32%, respectively). Herd prevalence based on environmental fecal culture was 27% (true prevalence 44%) compared to 49% (true prevalence 87%) based on bulk-tank milk real time IS900 PC. In both cases herd prevalence was higher in large herds (>200 cows). These results confirm that Map infection is wide spread in dairy herds in Southern Chile with a rough herd-level prevalence of 28-100% depending on the herd size, and that IS900 PCR on bulk-tank milk samples is more sensitive than environmental fecal culture to detect Map-infected dairy herds.

Aislamiento de Mycobacterium avium subsp. Paratuberculosis de fecas en rebaños lecheros infectados mediante el Método de Cornell modificado

Con la finalidad de aumentar la tasa de aislamiento de Mycobacterium avium subsp. Paratuberculosis (Map), a partir de muestras de fecas bovinas, se evaluo un nuevo procedimiento de descontaminacion y cultivo de muestras fecales en 250 animales clinicamente sanos, provenientes de 14 rebanos infectados del sur de Chile. Para la descontaminacion de las muestras previo al cultivo se utilizo una solucion al 0.9% de cloruro de hexadecilpiridinio (RPC) y una solucion antibiotica con amfotericina B, vancomicina y acido nalidixico. Para el aislamiento del agente se utilizo el medio de Rerrold con yema de huevo y micobactina J adicionado de una solucion antibiotica similar a la empleada para la descontaminacion de las muestras. En el 16% (40) de las muestras analizadas fue posible aislar Map con un 7.6% de contaminacion con hongos solo a partir de la octava semana de incubacion. La identidad de las cepas aisladas fue confirmada en el 100% de los casos mediante PCR, utilizando partidores especificos para este agente (P90 y P91). La alta tasa de aislamiento, la especificidad del medio de cultivo y la baja tasa de contaminacion de los cultivos, durante el prolongado periodo de incubacion, hacen de este procedimiento una buena alternativa de diagnostico de Paratuberculosis bovina.

Concentraciones de inmunoglobulinas séricas y calostrales de vacas selenio-deficientes y en el suero sanguíneo de sus terneros

The colostrum immunoglobulin concentrations of selenium-deficient cows and in the blood of their calves were studied in twelve Friesian cows of 4-9 year old....

Paratuberculosis en rebaños caprinos chilenos

Resumen es: La paratuberculosis caprina esta ampliamente distribuida a nivel mundial y recientemente la enfermedad ha sido oficialmente reportada en Chile. El objeti...

Comparación de tres métodos de diagnóstico de Paratuberculosis bovina en rebaños lecheros infectados

Se comparo la sensibilidad y especifidad del examen microscopico directo de fecas (tincion de Ziehl Neelsen), cultivo de fecas (Medio de Herrold Modificado) y un metodo serologico (ELISA) para el diagnostico de Paratuberculosis bovina en 250 animales clinicamente sanos provenientes de 14 rebanos infectados. Las muestras de fecas fueron examinadas simultaneamente mediante cultivo y baciloscopia y las cepas aisladas fueron identificadas mediante la tecnica de PCR. Para el diagnostico serologico se utilizo un kit comercial de ELISA (IDEXX), utilizando un lector fotometrico a 620 nm con un punto de corte de DO 0.25. El 71.4% (10) de los rebanos y 16.0% (40) de las muestras resultaron positivos al cultivo. Al examen microscopico directo solo se obtuvieron resultados sospechosos en 35 muestras (14.0%), de las cuales solo 6 (17.0%) correspondieron a muestras positivas al cultivo (kappa: 0.013). El examen serologico detecto un 8.0% (20) de animales reaccionantes, con una sensibilidad de 32.5% y especificidad de 96.7% (kappa: 0.366). Los resultados obtenidos demuestran la conveniencia de utilizar simultaneamente cultivo de fecas y serologia para la deteccion de animales infectados subclinicamente y la inconveniencia de usar el examen microscopico directo como metodo de diagnostico de Paratuberculosis en animales asintomaticos.