Miguel Salgado

Fate of Mycobacterium avium subsp. paratuberculosis after application of contaminated dairy cattle manure to agricultural soils

ABSTRACT Details regarding the fate of Mycobacterium avium subsp. paratuberculosis (basonym, Mycobacterium paratuberculosis) after manure application on grassland are unknown. To evaluate this, intact soil columns were collected in plastic pipes (lysimeters) and placed under controlled conditions to test the effect of a loamy or sandy soil composition and the amount of rainfall on the fate of M. paratuberculosis applied to the soil surface with manure slurry. The experiment was organized as a randomized design with two factors and three replicates. M. paratuberculosis-contaminated manure was spread on the top of the 90-cm soil columns. After weekly simulated rainfall applications, water drainage samples (leachates) were collected from the base of each lysimeter and cultured for M. paratuberculosis using Bactec MGIT ParaTB medium and supplements. Grass was harvested, quantified, and tested from each lysimeter soil surface. The identity of all probable M. paratuberculosis isolates was confirmed by PCR for IS900 and F57 genetic elements. There was a lag time of 2 months after each treatment before M. paratuberculosis was found in leachates. The greatest proportions of M. paratuberculosis-positive leachates were from sandy-soil lysimeters in the manure-treated group receiving the equivalent of 1,000 mm annual rainfall. Under the higher rainfall regimen (2,000 mm/year), M. paratuberculosis was detected more often from lysimeters with loamy soil than sandy soil. Among all lysimeters, M. paratuberculosis was detected more often in grass clippings than in lysimeter leachates. At the end of the trial, lysimeters were disassembled and soil cultured at different depths, and we found that M. paratuberculosis was recovered only from the uppermost levels of the soil columns in the treated group. Factors associated with M. paratuberculosis presence in leachates were soil type and soil pH (P < 0.05). For M. paratuberculosis presence in grass clippings, only manure application showed a significant association (P < 0.05). From these findings we conclude that this pathogen tends to move slowly through soils (faster through sandy soil) and tends to remain on grass and in the upper layers of pasture soil, representing a clear infection hazard for grazing livestock and a potential for the contamination of runoff after heavy rains.

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.

A cross sectional observational study to estimate herd level risk factors for Leptospira spp. serovars in small holder dairy cattle farms in southern Chile

BackgroundThe south of Chile constitutes the main cattle milk producing area of the country. Regarding leptospirosis control in Chile, there is neither an official program nor an epidemiological characterization of smallholder dairy farms. This study was carried out to determine Leptospira seroprevalence and to evaluate risk factors associated with seropositivity at herd level in smallholder bovine dairy herds in southern Chile.A cross-sectional study was conducted, and a convenient sample of 1,537 apparently healthy dairy cows was included in the study. Individual blood samples were taken and examined for six selected reference Leptospira serovars by the Microscopic Agglutination Test (MAT).ResultsOf the included herds 75% (52/69) showed serological titers against one or more Leptospira serovar. Leptospira borgpetersenii serovar Hardjo was the serovar most frequently (81%) reported from animals with positive results. The variables considered risk factors for Leptospira seropositivity were calve natural breeding system, using a specific calving area and vaccination against Leptospira. Adult cows in contact with calves weaned, proved to be a protective factor against infection.ConclusionsHerds neglecting the management practices mentioned in this study could represent an important source of Leptospira infection for other herds in the same geographic area, as well as for other animal species.

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.

Performance of a Johne's disease enzyme-linked immunosorbent assay adapted for milk samples from goats

Antibody detection–based tests for paratuberculosis offer speed and economy, 2 diagnostic test attributes important to animal industries with narrow profit margins. Application of such tests to individual milk samples instead of serum samples can further improve testing efficiency and decrease testing cost. Accuracy of a commercial bovine paratuberculosis enzyme-linked immunosorbent assay (ELISA) adapted for use on goat serum and milk samples was determined. Fecal, blood, and milk samples were collected from 159 goats belonging to 2 Wisconsin goat herds with a prior history of paratuberculosis and 1 herd of 50 goats from a paratuberculosis-free Wisconsin herd. Fecal samples were cultured using the modified BACTEC 12B media. Sera were tested according to the manufacturers instructions for bovine samples. Milk samples were centrifuged and mixed with the ELISA kits Mycobacterium phlei–containing diluent at a ratio of 1:2. Using fecal culture as the “gold standard,” the sensitivity of the ELISA on goat serum was 64% and the sensitivity of the ELISA on goat milk was 48%. The milk ELISA had higher agreement with fecal culture results (kappa = 0.525) than the serum ELISA (kappa = 0.425). ELISA specificity was 100% on both serum and milk. Regression analysis also showed good correlation between serum and milk S/P values (r 2 = 0.67). Although less sensitive, the ELISA on goat milk samples appears to offer a useful, low-cost alternative for detection of goats with paratuberculosis that have progressed to the stage of shedding M. paratuberculosis in their feces.

Effect of Soil Slope on the Appearance of Mycobacterium avium subsp. paratuberculosis in Water Running off Grassland Soil after Application of Contaminated Slurry

ABSTRACT The study assessed the effect of soil slope on Mycobacterium avium subsp. paratuberculosis transport into rainwater runoff from agricultural soil after application of M. avium subsp. paratuberculosis-contaminated slurry. Under field conditions, 24 plots of undisturbed loamy soil 1 by 2 m2 were placed on platforms. Twelve plots were used for water runoff: 6 plots at a 3% slope and 6 plots at a 15% slope. Half of the plots of each slope were treated with M. avium subsp. paratuberculosis-contaminated slurry, and half were not treated. Using the same experimental design, 12 plots were established for soil sampling on a monthly basis using the same spiked slurry application and soil slopes. Runoff following natural rainfall was collected and analyzed for M. avium subsp. paratuberculosis, coliforms, and turbidity. M. avium subsp. paratuberculosis was detected in runoff from all plots treated with contaminated slurry and one control plot. A higher slope (15%) increased the likelihood of M. avium subsp. paratuberculosis detection but did not affect the likelihood of finding coliforms. Daily rainfall increased the likelihood that runoff would have coliforms and the coliform concentration, but it decreased the M. avium subsp. paratuberculosis concentration in the runoff. When there was no runoff, rain was associated with increased M. avium subsp. paratuberculosis concentrations. Coliform counts in runoff were related to runoff turbidity. M. avium subsp. paratuberculosis presence/absence, however, was related to turbidity. Study duration decreased bacterial detection and concentration. These findings demonstrate the high likelihood that M. avium subsp. paratuberculosis in slurry spread on pastures will contaminate water runoff, particularly during seasons with high rainfall. M. avium subsp. paratuberculosis contamination of water has potential consequences for both animal and human health.

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.

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...

Association between cattle herd Mycobacterium avium subsp. paratuberculosis (MAP) infection and infection of a hare population

Paratuberculosis has long been considered a disease of domestic and wild ruminants only. The known host range of Mycobacterium avium subsp. paratuberculosis (MAP) was recently extended to include non-ruminant wildlife species believed to be exposed to spillover of MAP from infected domestic cattle herds. The aim of the present study was to assess the association between cattle herd MAP infection pressure level and the infection level of a hare population in two dairy farms of southern Chile. Fifty hares from a herd A and 42 hares from herd B were captured and sampled for MAP culture. The results showed a statistically significant association between the cattle herds’ infection prevalence and the hare infection prevalence.