Herrold's Egg Yolk Medium Research Articles

Grass silage contaminated with Mycobacterium avium subspecies paratuberculosis (MAP): a possible source of paratuberculosis infection in ruminants?

 Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis (Johne's disease) in ruminants. Paratuberculosis can cause severe economic losses and is acknowledged as one of the most important diseases of ruminants today. High amounts of MAP can be shed in the faeces of infected individuals and can survive for a long period in the environment. In the presented trial, baled grass silage was inoculated with a MAP-suspension, and the viability of MAP was studied over time. Samples from the bales were taken at increasing intervals and subsequently tested for the presence of MAP by solid culture on Herrold's Egg Yolk Media (HEYM), liquid culture and real time Polymerase Chain Reaction (PCR) for the IS900 and F57 fragments. No growth of MAP was observed at any time on solid or in liquid cultures, except at the time of inoculation; PCR detections were positive in the majority of the bales. From the results of the presented study, baled grass silage can be classed as a minor risk for the transmission of MAP.

Rapid Assessment of the Viability of Mycobacterium avium subsp. paratuberculosis Cells after Heat Treatment, Using an Optimized Phage Amplification Assay

Thermal inactivation experiments were carried out to assess the utility of a recently optimized phage amplification assay to accurately enumerate viable Mycobacterium avium subsp. paratuberculosis cells in milk. Ultra-heat-treated (UHT) whole milk was spiked with large numbers of M. avium subsp. paratuberculosis organisms (10(6) to 10(7) CFU/ml) and dispensed in 100-microl aliquots in thin-walled 200-microl PCR tubes. A Primus 96 advanced thermal cycler (Peqlab, Erlangen, Germany) was used to achieve the following time and temperature treatments: (i) 63 degrees C for 3, 6, and 9 min; (ii) 68 degrees C for 20, 40, and 60 s; and (iii) 72 degrees C for 5, 10, 15, and 25 s. After thermal stress, the number of surviving M. avium subsp. paratuberculosis cells was assessed by both phage amplification assay and culture on Herrold's egg yolk medium (HEYM). A high correlation between PFU/ml and CFU/ml counts was observed for both unheated (r(2) = 0.943) and heated (r(2) = 0.971) M. avium subsp. paratuberculosis cells. D and z values obtained using the two types of counts were not significantly different (P > 0.05). The D(68 degrees C), mean D(63 degrees C), and D(72 degrees C) for four M. avium subsp. paratuberculosis strains were 81.8, 9.8, and 4.2 s, respectively, yielding a mean z value of 6.9 degrees C. Complete inactivation of 10(6) to 10(7) CFU of M. avium subsp. paratuberculosis/ml milk was not observed for any of the time-temperature combinations studied; 5.2- to 6.6-log(10) reductions in numbers were achieved depending on the temperature and time. Nonlinear thermal inactivation kinetics were consistently observed for this bacterium. This study confirms that the optimized phage assay can be employed in place of conventional culture on HEYM to speed up the acquisition of results (48 h instead of a minimum of 6 weeks) for inactivation experiments involving M. avium subsp. paratuberculosis-spiked samples.

Inactivation ofMycobacterium aviumssp.paratuberculosisin milk by UV treatment

To determine the effect of UV radiation on the viability of two strains of Mycobacterium avium ssp. paratuberculosis (Map) inoculated into milk. Mycobacterium avium ssp. paratuberculosis in a ultra heat treated milk matrix was subjected to increasing doses of UV-C radiation from 0 to 1836 mJ ml(-1) using a pilot-scale UV reactor (20 l capacity). Survival of Map was monitored by culture on Herrold's egg yolk medium, Middlebrook 7H10 medium and the FASTPlaqueTB phage assay. Differences in sensitivity to UV treatment were observed between strains, however, at 1000 mJ ml(-1) a Map kill rate of 0.1-0.6 log(10) was achieved regardless of strain used or method employed to enumerate Map. Although the inactivation trend was similar on the culture and phage assay, the former gave a consistently higher viable count. The use of UV radiation alone does not represent an alternative to current pasteurization regimes for a large reduction in viable Map in milk. To the authors' knowledge the work here represents the first pilot-scale UV treatment process used to assess UV efficacy to inactivate Map in milk. The results are similar to those obtained with a laboratory-scale process indicating the difficulties associated with UV treatment of an opaque liquid and the recalcitrance of Map towards inimical treatments.

Comparison of faecal culture and IS900 PCR assay for the detection of Mycobacterium avium subsp. paratuberculosis in bovine faecal samples

Comparative efficacy of faecal culture and IS900 Polymerase chain reaction (PCR) assay of faecal samples was investigated in 40 clinically suspected cases of Johne's disease in dairy cattle. The sensitivity of faecal culture and PCR assay in this study was 52.5% (21/40) and 90% (36/40) respectively. All isolates appeared only on the mycobactin J supplemented Herrold's egg yolk medium (HEYM) at 8-16 weeks post-inoculation, were acid-fast and were positive for IS900 PCR yielding a single amplicon of 217 bp. A total of 28 faecal samples out of 40 were positive by IS900 primary PCR assay for Mycobacterium avium subsp. paratuberculosis (Map) yielding an expected product of size 217 bp. Twelve faecal samples, which gave negative results in the primary PCR, were subjected to secondary PCR assay. Of the 12 samples, 8 gave positive results in the IS900 nested PCR (nPCR), which yielded a PCR product of 167 bp, proving better sensitivity of nPCR assay than single amplification PCR. PCR could detect additionally 15 samples as positive which were negative by faecal culture. The chi-square analysis showed a highly significant difference between the tests (P< 0.01). This study suggests that IS900-PCR-based detection of Map could be used as a potential diagnostic tool for rapid and effective Johne's disease surveillance.

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.

Strain‐Specific Sensitivity Estimates of Mycobacterium avium subsp. paratuberculosis Culture in Greek Sheep and Goats

The requirements for the isolation of Mycobacterium avium subsp. paratuberculosis (Map) may be related to the strain-type [sheep (S)- or cattle (C)-type] and not to the host. The objective of this paper was to estimate and compare strain- and biological sample (faeces or pooled-tissue)--specific sensitivities (Ses) of two solid culture media, Herrold's egg yolk medium (HEYM) and Lowenstein-Jensen (LJ) medium, for the isolation of Map from Greek dairy sheep and goats. From 400 faecal samples collected from sub-clinically infected sheep and goats of four flocks and from 214 pooled-tissue samples (142 from sheep and 72 from goats) collected, at the abattoir, from >1-year-old routinely slaughtered animals, with gross pathology suggestive of paratuberculosis, we isolated 34 Map strains. Of those, by the IS1311 PCR, 18 were categorized into the C-type and nine into the S-type; seven were not typed. We used a Bayesian approach to estimate the strain-specific Ses. SeHEYM-C-faecal=17% (95% credible interval: 7, 40) was higher than SeHEYM-S-faecal=2% (0.3, 11). Also, SeHEYM-C-faecal was higher than SeLJ-C-faecal=4% (1, 12). In pooled-tissue samples, the strain-specific Ses did not differ between the two media.

Detection of &lt;em&gt;Mycobacterium avium subspecies paratuberculosis&lt;/em&gt; in formula milk from Bogor using PCR IS 900

Crohn's disease (CD) that becomes a public health concern in developed countries shows similarities in clinical signs and pathological features with Johne's disease (JD) in ruminants infected by Mycobacterium avium subspecies paratuberculosis (MAP). Few researches conducted in Europe, the USA, and Australia showed relationships between MAP, CD, JD and dairy products. Indonesians consume milk and diary products from domestic and imported source. Adji in 2004 found some domestic dairy cows that were seropositive for MAP, and this could be a serious problem in dairy farm animals and human health in the future. The aim of this study was to detect MAP in the growing up formula milk. Fifty samples from five established factories were taken from supermarkets in Bogor. Polymerase chain reaction method (PCR) with insertion sequence (IS) 900 as primer and culture in Herrold's egg yolk media with mycobactin J (HEYM J) as a gold standard were used in this study. Neither MAP grew up in HEYM J medium after 20 weeks of culture period nor positive samples by PCR IS 900 were found. Although there were no positive samples found in this study, further extensive and comprehensive studies on MAP should be done with more and varied samples, as well as in human to provide data on MAP in Indonesia. (Med J Indones 2008; 17: 183-7)

Influence of Type of Culture Medium on Characterization ofMycobacterium aviumsubsp.paratuberculosisSubtypes

We evaluated the effects of culture and/or enrichment methods on the selection of Mycobacterium avium subsp. paratuberculosis subtypes. M. avium subsp. paratuberculosis isolates from bovine fecal samples processed using a centrifugation protocol were investigated in both liquid (MGIT ParaTb tubes) and solid (Herrold's egg yolk medium) culture media. For this evaluation, M. avium subsp. paratuberculosis subtyping was based on the sequence variation in two of the most discriminatory short sequence repeat loci, i.e., mononucleotide G and trinucleotide GGT, in isolates from liquid and solid cultures. This study identified the existence of one major predominating fingerprint (>13G-5GGT) in bovine fecal samples, regardless of the type of medium used for isolation. Matched-pair analysis of subtypes showed that 69% of samples presented unique subtypes in the two culture media used, while 31% shared the same G-GGT allele. Furthermore, the liquid culture method appeared to select for a more genotypically diverse subtype population than the solid culture method. The variety of subtypes observed between liquid and solid cultures obtained from the same fecal samples suggests that the culture method could provide a "microbiological" bias and lead to a discrepancy in the growth of M. avium subsp. paratuberculosis strains. In conclusion, this study identified that these two types of culture media determined differential growth of M. avium subsp. paratuberculosis strains and that this should be considered in evaluating detection capabilities of diagnostic tests or interpreting data from molecular epidemiological studies performed using conventional solid fecal culture or automated liquid medium culture methods.

Presence and characterization of Mycobacterium avium subspecies paratuberculosis from clinical and suspected cases of Crohn's disease and in the healthy human population in India

To investigate and characterize Mycobacterium avium subspecies paratuberculosis (MAP) in patients with Crohn's disease, attendants of animals with suspected infection, and healthy humans, using multiple diagnostic tests. A total of 119 samples (35 stool, 76 serum, three blood clots, and five biopsies) were collected from five patients with Crohn's disease, eight attendants of animals with Johne's disease, and 93 apparently normal control subjects (Agra region) from North India. Samples were screened for the presence of MAP by smear examination, culture of stool, blood clot and biopsies, and ELISA. Colonies obtained by culture were further characterized using polymerase chain reaction (PCR) with IS900 MAP-specific primers. Using all diagnostic modalities, MAP and/or MAP antibodies were identified in 100% (5/5) of subjects with Crohn's disease; 75.0% (6/8) of attendants of MAP infected animals were positive and 38.0% (27/71) of apparently normal controls were also positive. Most sensitive test was ELISA (100%, 5/5), followed by culture (80.0%, 4/5), and acid-fast staining. Ziehl-Neelsen staining was positive in 37.5% (3/8) of subjects with active animal husbandry practices. In 71 serum samples from control subjects, seroprevalence of MAP was 38.0% using indigenous protoplasmic antigens (PPA) and 36.6% using commercial PPA. Of the serum samples from the Crohn's disease patients, 100% (5/5) were positive by ELISA using indigenous PPA and 40.0% (2/5) were positive by ELISA using commercial PPA. IS900 PCR was used to characterize tiny colonies of MAP that grew extremely slowly on Herrold's egg yolk medium, and of 15 (42.8%) cultures, 14 (93.3%) were typed as MAP. Paper documented the presence of MAP in all patients with Crohn's disease, in some animal attendants who had the history of working with goat herds infected with Johne's disease and in few normal healthy individuals. Presence of Ziehl Neelsen positive MAP. In the stool of attendants working with MAP-infected animals was unique to humans. ELISA based on antigens derived from indigenous MAP 'bison type' genotype of goat origin was most sensitive modality for screening Crohn's disease patients.

An inter-laboratory ring trial for the detection and isolation of Mycobacterium avium subsp. paratuberculosis from raw milk artificially contaminated with naturally infected faeces

There is a need for standardised, robust, reproducible molecular and culture methods to achieve clarification of the inactivation of Mycobacterium avium subsp. paratuberculosis (Map), the causative microbial agent of Johne's disease, in (faecally) contaminated milk and other food products such as meat. This study assessed the performance of a commercially available Map DNA extraction kit for milk (Adiapure ®) and accompanying PCR detection kit (Adiavet ®) alongside ‘in-house’ molecular and culture methods in an inter-laboratory ring trial using raw milk spiked with Map-infected faeces. The combined Adiapure-Adiavet ® Map DNA extraction and detection kit consistently detected 30 copies of IS900 (equivalent to approximately 2 cells) ml −1 raw milk, when used in four different laboratories. Improvements in sensitivity and ease of use for ‘in-house’ Map detection were observed when the Adiapure extraction kit was combined with ‘in-house’ detection assays. Detection by real-time PCR methods, using the commercial extraction and detection systems, resulted in an overall detection rate of 100%, 90%, 85% and 25% for respective Map concentrations of 300, 30, 3 and 0.3 copies of IS900 ml −1 raw milk. Map, at 300 copies of IS900 (equivalent to approximately 20 Map cells) ml −1 raw milk, was recovered from all samples cultured in mycobacteria growth indicator tube (MGIT) medium, from 10 of 12 samples on Herrold's egg yolk medium (HEYM) and not recovered from any samples using BACTEC medium. In conclusion, the Adiapure ® DNA extraction kit allows for sensitive and easy detection of Map in raw milk. The extraction method can form a candidate part of essential methodology and real-time PCR can further increase the sensitivity of the detection method. Moreover, MGIT medium is promising for culture-dependent detection of Map from raw milk.

Effect of high pressure and pasteurization on Mycobacterium avium ssp. paratuberculosis in milk

To determine the effect of high pressures alone and in conjunction with pasteurization on the viability of two strains of Mycobacterium avium ssp. paratuberculosis (Map). Map in a milk matrix was subjected to 400, 500 and 600 MPa with and without pasteurization (72 degrees C for 15 s) and plated onto Herrold's egg yolk medium (HEYM) and Middlebrook 7H10 (7H10) agar, both containing antibiotic supplements. Medium 7H10 was found to give a significantly (P < 0.001) better recovery than HEYM. A significantly greater (P < 0.001) reduction in viable numbers was observed using 500 MPa (mean log reduction of 6.52) compared with 400 MPa (mean log reduction of 2.56) and between 400 MPa and control (no applied pressure) for 10 min treatments. A treatment time of 10 min resulted in significantly (P < 0.001) fewer survivors than 5 min. Low numbers of survivors were still detected when pressure treatment at 400 and 600 MPa was combined with subsequent pasteurization. The use of high-pressure was effective in reducing viable numbers of Map but even when combined with pasteurization there were still survivors, albeit when high inoculum levels of Map were used. To the authors' knowledge the work reported here represents the first study of the efficacy of high-pressure treatments alone and in combination with pasteurization to kill Map. The results indicate that further research is warranted before more commercial-scale studies are commissioned.

UV Light Inactivation of Mycobacterium avium subsp. paratuberculosis in Milk as Assessed by FASTPlaque TB Phage Assay and Culture

UV light inactivation of Mycobacterium avium subsp. paratuberculosis in Middlebrook 7H9 broth and whole and semiskim milk was investigated using a laboratory-scale UV machine that incorporated static mixers within UV-penetrable pipes. UV treatment proved to be less effective in killing M. avium subsp. paratuberculosis suspended in milk (0.5- to 1.0-log(10) reduction per 1,000 mJ/ml) than that suspended in Middlebrook 7H9 broth (2.5- to 3.3-log(10) reduction per 1,000 mJ/ml). The FASTPlaqueTB phage assay provided more rapid enumeration of surviving M. avium subsp. paratuberculosis (within 24 h) than culture on Herrold's egg yolk medium (6 to 8 weeks). Despite the fact that plaque counts were consistently 1 to 2 log(10) lower than colony counts throughout the study, UV inactivation rates for M. avium subsp. paratuberculosis derived using the phage assay and culture results were not significantly different (P = 0.077).

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.

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 (≥3 years old) and, if present, clinical signs of a Map infection. In herds with less than 100 cows, all cows ≥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.

Modified Culture Protocol for Isolation ofMycobacterium aviumsubsp.paratuberculosisfrom Raw Milk

A modified culture method using C18-carboxypropylbetaine (CB-18) and microscopic screening was evaluated for time to and limit of detection of Mycobacterium avium subsp. paratuberculosis (MAP) in raw milk. Bulk-tank milk samples were spiked with six different concentrations (10(1) to 10(6) CFU/mL) of MAP. Samples were processed using two different protocols. The first protocol involved specimen processing with the zwitterionic detergent C18-carboxypropylbetaine (CB-18) and lytic enzymes followed by culture on modified Middlebrook 7H10 agar plates with microscopic screening. The second protocol used 0.75% hexadecylpyridinium chloride (HPC) for specimen processing, followed by culture on Herrold's egg yolk medium (HEYM). Both protocols were repeated eight independent times, and the detection limit and time to detection were compared. The presence of MAP in spiked milk samples was detected between 14 and 45 days (N [number of samples], 46; mean, 22.7; median, 19.5) using the CB-18 and microscopic screening method, and between 21 and 63 days (N, 47; mean, 31; median, 28) using HEYM (P < 0.001). Time to detection also varied with MAP concentration (P < 0.001). Higher concentrations of MAP were detected earlier than lower concentrations and this finding was independent of the method used (P = 0.479). The two methods had similar detection limits but the modified culture method reduced the time to detect MAP in raw milk for the majority of concentrations.

Comparison of Four Different Culture Media for Isolation and Growth of Type II and Type I/III Mycobacterium avium subsp. paratuberculosis Strains Isolated from Cattle and Goats

Culture is considered the definitive technique for Johne's disease diagnosis, and it is essential for later applications of certain molecular typing techniques. In this study, we have tested four solid media (Herrold's egg yolk medium [HEYM] with sodium pyruvate and mycobactin [HEYMm-SP], HEYM with mycobactin and without sodium pyruvate [HEYMm], Middlebrook 7H11 with mycobactin [Mm], and Löwenstein-Jensen with mycobactin [LJm]) for isolation of Mycobacterium avium subsp. paratuberculosis strains in 319 tissue samples from cattle herds and goat flocks. We have shown that each of the two main groups of M. avium subsp. paratuberculosis (type II and type I/III) has different requirements for growth in the culture media studied. The recommended solid media for isolation of type I/III strains are LJm and Mm, since the combination of both media allowed the recovery of all these strains. The most widespread culture medium, HEYM, is not suitable for the isolation of this group of M. avium subsp. paratuberculosis strains. Regarding the type II strains, HEYMm-SP was the medium where more strains were isolated, but the other three media are also needed in order to recover all type II strains. The incubation period is also related to the strain type. In conclusion, because the type of strain cannot be known in advance of culture, coupled with the fact that cattle and goats can be infected with both groups of strains, we recommend the use of the four solid media and the prolongation of the incubation period to more than 6 months to detect paratuberculous herds/flocks and to determine the true prevalence of the infection.

Application of a semi-dependent latent model in the Bayesian estimation of the sensitivity and specificity of two faecal culture methods for diagnosis of paratuberculosis in sub-clinically infected Greek dairy sheep and goats

In this study, we compared the frequency of isolation of Mycobacterium avium subsp. paratuberculosis (MAP) from faecal samples grown on Herrold's egg-yolk medium (HEYM) or on Lowenstein–Jensen (LJ) medium and estimated the sensitivity (Se) and specificity (Sp) of the methods separately in sub-clinically infected Greek dairy sheep and goats, using latent-class models and Bayesian estimation procedures. Faecal and blood samples were collected from 400 animals ≥1 year old in April–May 2002. The HEYM supported growth of MAP better than the LJ method and their agreement was very poor (weighted kappa = 0.062 (95% CI: −0.098, 0.222)). There was no evidence of dependence between the Ses whereas the Sps were positively correlated. Thus, a semi-dependent model that assumed independence of Ses and accounted for the dependence of Sps was adopted. Under this model, the parallel interpretation of the results of the two methods gave median estimates and 95% credible intervals (CrIs) for Se par, Sp par of 15% (CrIs: 3, 45%), 96% (92, 98%) in sheep and 16% (6, 36%) and 97% (94, 99%) in goats.

The association of sub-clinical paratuberculosis with the fertility of Greek dairy ewes and goats varies with parity

Our cross-sectional study investigated the association of sub-clinical Mycobacterium avium subsp. paratuberculosis (MAP) infection with failing to produce a live offspring the season of lambing/kidding (November 2001 to January 2002) before testing (in April–May 2002), in four dairy-sheep and/or goat flocks in Greece (369 animals ≥1.5-year-old). From each selected animal 10 ml of blood and 10 g of feces from the rectum were obtained. The harvested sera were tested for antibodies to MAP with a commercial ELISA test kit; the feces were cultured on Herrold's egg-yolk medium supplemented with mycobactin J and antibiotics. An animal was considered sub-clinically infected when found either seropositive or culture positive. The true prevalence of sub-clinically infected animals, adjusted for the sensitivity and specificity of the parallel testing, was 14% (0.1–28%) and 35.9% (9.2–62.7%) in sheep and goats, respectively. The association of fertility of sheep and goats with sub-clinical paratuberculosis was investigated in random-effects logistic models. Sub-clinically infected animals (compared to uninfected) had OR for live offspring the previous year of 5.4 for parity <4, OR = 0.05 for parity >6, and a non-significant OR for the middle parity category.

Occurrence of Mycobacterium avium subsp. paratuberculosis in Untreated Water in Northern Ireland

Mycobacterium avium subsp. paratuberculosis is the known cause of Johne's disease of both domestic and wild ruminants and has been implicated as a possible cause of Crohn's disease in humans. The organism is shed in the feces of infected animals and can survive for protracted periods in the environment and hence could be present in catchment areas receiving agricultural runoff. A limited survey was undertaken in Northern Ireland to test for M. avium subsp. paratuberculosis in untreated water entering nine water treatment works (WTWs) over a 1-year period. Three detection methods were employed, viz., immunomagnetic separation-PCR and culture on Herrold's egg yolk medium (HEYM) and BACTEC 12B medium, the latter both supplemented with mycobactins. Of the 192 untreated water samples tested, 15 (8%) tested M. avium subsp. paratuberculosis positive by one or more of the three detection methods. M. avium subsp. paratuberculosis was successfully isolated from eight untreated water samples, three by BACTEC culture and five by culture on HEYM. Although the highest incidence of M. avium subsp. paratuberculosis was found in spring, overall, there was no statistically significant difference between the seasons. No significant correlation was found between numbers of coliforms or fecal coliforms and the presence of M. avium subsp. paratuberculosis. In general, a higher incidence of M. avium subsp. paratuberculosis was found in untreated water entering those WTWs that had a high mean water pH value over the sampling period. This work indicates the need to determine the efficacy of water treatment processes to either kill or remove M. avium subsp. paratuberculosis from untreated water and the possible risks posed by contact with recreational water sources.

MAP Super-shedders

Traditionally, fecal cultures were either positive or negative for growth of Mycobacterium paratuberculosis (MAP). Nearly all culture-positive cattle were judged to be infected, shedding MAP, thus a threat to spread the disease to susceptible cattle and were culled from the herd as quickly as possible. Over time it was recognized that differences in MAP shedding existed among culture-positive cattle. Cattle were classified as low, moderate or high shedders based on the visible colonies of MAP on the surface of solid media, typically Herrold's egg yolk media (HEYM). Rarely did authors provide the estimated MAP colony forming units (cfu) per gram of manure. Additionally, culture methods were not standardized among diagnostic laboratories in the US, which made it difficult to compare results from one laboratory to another. Laboratories rarely used internal quality control fecal samples. As the infection and disease progressed, the intensity of fecal shedding of MAP increased, culminating in clinical disease which was associated with the greatest numbers of MAP present in manure. Since cattle with clinical signs lose weight and have decreased milk production causing them to be unprofitable, they are soon culled from the herd. If not culled from the herd, they would continue to lose weight and become emaciated with muscle weakness, leading to recumbency requiring euthanasia. The fewest colonies reported is one colony on one of four tubes ofHEYM, which translates into approximately six cfu/gram of feces, assuming the original sample was two grams; 5 ml was transferred from the fecal water tube to the second decontamination tube with HPC. That centrifugation technique was used and the pellet was re-suspended in 1 ml of antibiotic brew, with 200 ul of the re-suspended pellet inoculated onto each of four tubes of HEYM, all with mycobactin J. Low shedders typically had less than 10 colonies on the four tubes ofHEYM. Moderate shedders ranged from 11 to approximately 70 colonies of MAP, while high shedders had more than 70 colonies on any of the four tubes. Calculated on MAP cfu per gram of feces this would be up to 60 cfu for low shedders, 61 to 300 cfu per gram for moderate shedders and more than 300 cfu per gram for heavy shedders.&#x0D; Over the past five years most laboratories in the US report the number of visible MAP colonies on each tube of HEYM, but rarely enumerate above 50 to 70 colonies per tube. Accounting for sample preparation, 50 colonies on each of four tubes represents an estimated 1,050 colony forming units of MAP per gram of manure. The objective of this study was to determine the range of MAP cfu/gram of manure in cattle classified as heavy shedders.