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 Herrold's 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.

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

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.

The effect of selenium supplementation before calving on early-lactation udder health in pastured dairy heifers.

Selenium (Se) deficiency has been associated with lowered resistance to mastitis in dairy cattle. However, little published data exists on the effect of Se supplementation before calving on udder health of pastured dairy heifers. Further, the relative efficacy of injectable barium selenate and oral organic Se for improving udder health in cows has not previously been tested. The objectives of this study were to determine the effects of precalving Se supplementation and type of supplementation on the blood activity of glutathione peroxidase and measures of udder health immediately after calving and during the first month of lactation in pastured dairy heifers. One hundred forty pregnant Chilean Holstein-Friesian heifers were fed a basal diet containing, on average, 0.15 mg of Se/kg of dry matter. One month before predicted calving, heifers were allocated to 1 of 3 groups. Group 1 (n=49) received no supplementary Se, group 2 (n=46) received a single subcutaneous injection of Se (1 mg/kg of live weight, as barium selenate), and group 3 (n=45) was fed Se yeast (3 mg/heifer/d until calving). Heifers supplemented with barium selenate had a higher glutathione peroxidase activity from 14 d in milk onwards. Selenium supplementation, irrespective of source, tended to reduce the prevalence of intramammary infection (IMI) and decrease the prevalence of quarters with high somatic cell count (SCC) at calving. Overall, Se supplementation did not result in a reduction of the incidence of new IMI or clinical mastitis or in decreased SCC during the balance of the first month of lactation. However, in pasture-based heifers injected with barium selenate before calving, and fed diets with 1.3 and 2.5 mg of Se/d precalving and during lactation, respectively, no cases of clinical mastitis were observed in the first month of lactation.

Barium selenate supplementation and its effect on intramammary infection in pasture-based dairy cows.

A significant proportion of cattle receive inadequate dietary Se because of its low content in soils and pastures of various regions of the world. Several economically important diseases in dairy cows, such as mastitis, have been associated with Se deficiency. The objective of this study was to evaluate the effect of a single injection of a long-acting form of Se at drying off on the risk and incidence rate of new intramammary infections and on milk somatic cell count in the subsequent lactation in pasture-based dairy cows. Forty-nine Chilean Holstein-Friesian cows were fed a diet containing <0.05 mg of Se/kg of ration dry matter. During the dry period, cows were allocated to 1 of 2 groups, a supplemented (n=24) group treated with a single subcutaneous injection of barium selenate 2 mo before calving and a control group (n=25) that remained unsupplemented. Duplicate foremilk samples were aseptically collected within 6 d after calving and every 2 wk until drying-off for bacteriological culture. Milk samples were also collected monthly for somatic cell count evaluation. Blood samples were collected before treatment and at 30, 90, 180, and 270 d after treatment for analysis of blood glutathione peroxidase (GPx) activity. The activity of glutathione peroxidase was higher in supplemented cows 30 d after the injection until the end of the study. The risk and incidence rate of new intramammary infections was not affected by supplementation. A progressive increase in somatic cell count was observed throughout lactation, but there was no effect of supplementation. In conclusion, a one-time injection of barium selenate 2 mo before calving in these pasture-based dairy cows did not affect udder health in the subsequent lactation, indicating that Se basal intake was adequate for preventing subclinical mastitis in pasture-based cows in southern Chile.

Antibody recognition to secreted proteins of Mycobacterium avium subsp. paratuberculosis in sera from infected ruminants.

Two liquid culture media to obtain secreted proteins of Mycobacterium avium subsp. paratuberculosis at different incubation periods were evaluated. Middlebrook 7H9-OADC (7H9) and Watson-Reid (WR) broths were inoculated with a field strain of M. paratuberculosis and growth curves determined using nonlinear regression analysis. Most culture filtrate (CF) proteins were of low molecular weight and reacted strongly against sera from cultured-positive cases of paratuberculosis. CF proteins obtained in WR yielded a higher number of bands and were detected earlier than those obtained from 7H9. A high degree of variability in CF protein immunoreactivity was seen among infected animals. Sera from cattle with clinical paratuberculosis or heavy fecal shedders of M. paratuberculosis reacted more intensively and to more CF proteins than did sera from other infected cattle. Immunoblots showed differences in antibody binding to CF proteins when sera were absorbed with M. avium but not with others environmental mycobacteria. Immunoblots with sera from infected goats and a sheep showed reactivity with proteins of 32, 33 and 46kDa both before and after the sera were absorbed with M. phlei. Antibodies found in serum of infected deer reacted with CF proteins in a similar way as did for cattle. These results suggest that a pool of CF proteins of M. paratuberculosis could be good candidates as antigens for serodiagnosis of paratuberculosis.

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 Johnes's 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 Herrold's 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.

Somatic cell count in milk of selenium-supplemented dairy cows after an intramammary challenge with Staphylococcus aureus.

The objective of this study was to evaluate the effect of selenium (Se) supplementation on milk somatic cell count (SCC) in dairy cows. Twelve multiparous Holstein-Friesian cows were fed a diet containing a suboptimal Se concentration (<0.05 ppm, dry basis) starting 2 months before calving. Supplemented cows (n=6) received a single s.c. injection of barium selenate (1 ml/50 kg BW) 45 days prior to calving, whereas control group was kept unsupplemented. Twenty weeks after calving, two mammary quarters (right side) of each cow were challenged with 205,000 cfu/ml of Staphylococcus aureus (strain Newbould 305). Blood was collected bi-weekly until day 150 of lactation for the analysis of blood glutathione peroxidase (GPx1; EC 1.11.1.9) activity. To re-isolate the challenging pathogen and to evaluate SCC, aseptic milk samples were collected daily starting on the day of challenge, and finishing 7 days after inoculation. Unsupplemented cows had a lower activity of GPx1 through the experiment (P<0.001). Natural log SCC (lnSCC) was higher in unsupplemented than Se-supplemented cows (P=0.04), showing evidence of significance after 5 days. Selenium supplementation of dairy cows fed a diet containing a suboptimal Se concentration, resulted in higher blood activity of GPx1, and lower mean lnSCC after an intramammary challenge with Staph. aureus.

Bayesian analysis to validate a commercial ELISA to detect paratuberculosis in dairy herds of southern Chile.

In Chile, Mycobacterium avium subsp. paratuberculosis (Map) has been isolated on several occasions and clinical cases have been reported. Nevertheless, diagnostic tests have not yet been validated for this agent in the Chilean setting. The objective of the study was to validate a commercial ELISA to detect Map shedding dairy cows in management conditions, prevalence and stages of infection existing in Southern Chile, utilising different statistical approaches. Blood and faeces were collected from 1333 lactating cows in 27 dairy herds (both large commercial and smallholder dairy farms) between September 2003 and August 2004. Within the herds up to a maximum of 100 dairy cows were selected based on age (>or=3 years old) and, if present, clinical signs of a Map infection. In herds with less than 100 cows, all cows >or=3 years old were sampled. Blood samples were tested using a commercial ELISA kit (IDEXX Laboratories, Inc.). Faecal samples were cultured on Herrold's Egg Yolk Medium (HEYM). Latent class models (i.e. maximum likelihood (ML) methods and Bayesian inference) were used to determine the validity of the ELISA. Map was cultured from 54 (4.1%) cows and 10 (37.0%) herds, which were all large, commercial dairy herds. As a result of empty cells in the cross-tabulations, the ML model provided the same results as the validation with faecal culture as the gold-standard. In the Bayesian model, the Se and Sp of the ELISA were estimated to be 26% (95% CI: 18-35%) and 98.5% (95% CI: 97.4-99.4%), respectively. For faecal culture, the Se was 54% (95% CI: 46-62%) and the Sp was 100% (95% CI: 99.9-100%). Interestingly, the prevalence in the smallholder dairy farms was estimated to be 8% even though there were no faecal culture positive cows detected in those herds. There was no significant correlation between the two tests. The advantage of Bayesian inference is that the Se and Sp of both tests are obtained in one model relative to the (latent) true disease status, the model can handle small datasets and empty cells and the estimates can be corrected for the correlation between tests when the tests are not conditionally independent. Therefore, Bayesian analysis was the preferred method for Map that lacks a gold-standard and usually has low cow-level prevalence.

Multilocus sequence typing of intercontinental bovine Staphylococcus aureus isolates.

A total of 258 bovine-associated Staphylococcus aureus isolates from the United States, Chile, and the United Kingdom, plus the reference isolate S. aureus Newbould 305 (NCIMB 702892), were analyzed by multilocus sequence typing (MLST). A collection of previously characterized United Kingdom isolates were also included in the analysis. The results demonstrated that MLST is suitable for the differentiation of bovine S. aureus isolates from various sites (milk, teat skin, milking machine unit liners, hands, and bedding) and countries. The theory of the host specificity of S. aureus is supported by the detection of a previously undescribed clonal complex that comprised 87.4% of the isolates studied, with representatives from all geographic locations investigated. This suggests that a single clonal group has achieved a widespread distribution and is responsible for the majority of infections. Some sequence types (STs; ST25, ST115, ST124, and ST126) demonstrated site specificity, as they were significantly (P < 0.05) associated with milk or teat skin.

Risk factors associated with BMSCC greater than 200 000 cells/ml in dairy herds in southern Chile.

A cross-sectional survey of 523 dairy farms in the south of Chile was carried out to quantify risk factors associated with bulk-milk somatic-cell count (BMSCC) >200 x 10(3)cells/ml. Questionnaires followed by one reminder were sent to 3710 dairy farms via the 11 milk-processing plants that they supplied in October 1998. The response proportion was 14.1%. The median BMSCC was 289 x 10(3) cells/ml (range: 74 x 10(3) to 1800 x 10(3)cells/ml). The median herd size was 70 cows (range: 7-616); herd size was not associated with BMSCC. The annual milk yield of 33.2% of the herds was <4000 l and 53.4% had an annual milk yield of 4 x 10(3) to 6 x 10(3) l. Clinical-mastitis records were kept by 55.3% of the farmers. Seventy-six percent of the farmers (377/499) reported <10 clinical cases of mastitis in the year prior to the questionnaire. Logistic multiple regression indicated that BMSCC >200 x 10(3)cells/ml was more likely when foremilking was practised, and when cows were collected in a yard before milking. BMSCC was less likely to be >200 x 10(3)cells/ml when teats were washed with water containing disinfectant compared with plain water; when the udder and teats were always checked before milking compared with, sometimes or never; when cows with mastitis were milked first compared with any other ordering, and when farmers recorded individual-cow somatic-cell count (ICSCC) compared with when ICSCC was not recorded.

[Virulence factors of coagulase-negative Staphylococcus of human and bovine origin]. / Factores de virulencia de Staphylococcus coagulasa negativo (SCN) de origen humano y bovino.

Virulence factors such as hydrophobicity of cell surface, capsule formation and slime production were investigated in 393 CNS strains, 190 isolated from bovine mastitis and 203 from human infections; in addition a selected number of CNS species were experimentally infused into the teat cistern of lactating rabbits for pathogenicity tests. Most human (95%) and bovine (82.6%) strains showed cell hydrophobicity properties, however only 11.3% and 7.9% were capsulated strains, respectively. Slime production was detected in 10.5% of bovine strains but only in 0.9% of human strains. Using the lactating rabbit model it was shown that S. chromogenes, S. epidermidis, S. saprophyticus and S. haemolyticus were the most pathogenic species.