A longitudinal study to evaluate the diagnostic potential of a direct faecal quantitative PCR test for Johne's disease in sheep

A longitudinal study was carried out to evaluate the diagnostic potential of the previously developed direct faecal real-time quantitative PCR (QPCR) assay (Kawaji et al., 2007) for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) infected sheep. Of the 58 sheep, 38 were orally inoculated with MAP, while 20 controls were maintained separately from the infected group throughout the trial. All animals were tested by QPCR, faecal culture and serum ELISA pre-inoculation and at 4, 8 and 13 months post-inoculation, and were necropsied at 13 months post-inoculation. Eighteen out of 38 inoculated sheep were detected by QPCR to be shedding MAP in faeces at 4 months post-inoculation, while only one sheep was positive in faecal culture at this time point. At 8 months post-inoculation, MAP DNA was detected in faeces of all inoculated sheep by QPCR, while MAP organisms were isolated from only 34% of the inoculated animals by faecal culture. The QPCR results for faecal samples that were collected at necropsy demonstrated that faecal QPCR was more sensitive than culture of intestinal tissues for MAP. The QPCR assay was confirmed to be a sensitive and specific ante-mortem diagnostic test for MAP in sheep, circumventing faecal culture which is a less sensitive and highly time consuming test. Quantification of MAP DNA in faeces by QPCR may provide immediate information to estimate the stage of the infection as well as the risk of transmission from infected animals.     

Exploring the use of molecular epidemiology to track bovine tuberculosis in Nigeria: an overview from 2002 to 2004

This study focused on the development of a reliable and cost-efficient DNA isolation procedure for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in faeces by previously developed IS900 and F57 quantitative real time PCR (qPCR) and their comparison with culture. The recovery of MAP DNA from the spiking experiments ranged from 29.1 to 102.4% of the input amount of MAP with median 37.9%. The limit of detection was determined to be 1.03 × 10(4) for F57 qPCR and 6.87 × 10(2)MAP cells per gram of faeces for IS900 qPCR, respectively. The developed technique for DNA isolation was coupled with IS900 qPCR and compared to traditional MAP culture using a cohort of 1906 faecal samples examined from 12 dairy cattle farms in our laboratory. From those 1906 original faecal samples, 875 were positive by IS900 qPCR and 169 by culture. None of the culture positive samples was negative by IS900 qPCR. This data facilitated development of a predictive model capable of estimating the probability of being culture positive by estimating the absolute number of MAP per gram of faeces as determined IS900 qPCR without performing the culture.     

Detection of Mycobacterium avium subsp. paratuberculosis by buoyant density centrifugation, sequence capture PCR and dot blot hybridisation

Detection of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) by polymerase chain reaction (PCR) is often hampered by the lack of efficient methods for sample treatment. We report a protocol for analysis of faecal samples based on buoyant density centrifugation in Percoll and IS900 sequence capture PCR combined with a dot blot assay for detection of low-grade infection of M. paratuberculosis. Serial dilutions of M. paratuberculosis genomic DNA and M. paratuberculosis bacteria were used to assess the sensitivity of the method. The final evaluation was performed with spiked faecal samples, which also were analysed by culture. The presence of PCR inhibitory substances in processed faecal samples was evaluated by including a PCR internal control. By using buoyant density centrifugation, sequence capture PCR, and dot blot hybridisation, we achieved a sensitivity of 10(3)CFU (colony forming units)/g of faeces. The detection limit by culture was assessed to 10(2)CFU/g of faeces. We conclude that the described protocol is a fast and sensitive alternative to bacterial culture of faecal samples.     

Pooled faecal culture for the detection of Mycobacterium avium subsp paratuberculosis in goats

OBJECTIVE: To evaluate pooled faecal culture for herd diagnosis of caprine Johne's disease and relate these findings to faecal shedding rates of Mycobacterium avium subsp paratuberculosis (Map). DESIGN: Radiometric broth culture was applied to several pooling dilutions, and shedding rates were estimated from a regression equation based on bacterial growth rates and known processing losses during radiometric culture. PROCEDURE: Sixteen faecal samples from goats naturally infected with sheep (n = 3) or cattle (n = 13) strains of Map, were diluted in normal goat faeces from 1 in 5 to 1 in 50. Cultures were confirmed by IS900 polymerase chain reaction and restriction endonuclease analysis, and mycobactin dependency. The numbers of viable Map in the culture inocula were determined by endpoint titration (most probable number) of nine samples and related to a cumulative growth index. RESULTS: A pooling dilution of 1 in 25 with an incubation period of 10 weeks detected 13 of 16 culture positive goats, all shedding > or = 2 x 10(4) Map per gram of faeces. Two samples containing very low numbers of Map (< 2 x 10(3)/g) were only culture positive from undiluted faeces. Thirteen of 16 goats were considered to be shedding low to moderate concentrations of Map (< 2 x 10(5)/g faeces). CONCLUSIONS: These data support a pooling dilution of 1 in 25 for application of pooled faecal culture as a diagnostic tool in caprine Johne's disease control. A test based on this dilution would reduce laboratory costs of whole herd testing in goats by approximately 40% relative to serology and 75 to 90% relative to individual faecal culture.     

Radiometric pooled faecal culture for the detection of Mycobacterium avium subsp paratuberculosis in low-shedder cattle

OBJECTIVE: To identify the optimum pooling rate for pooled faecal culture (PFC) as a diagnostic tool in bovine Johne's disease control, for detection of cattle shedding low concentrations of Mycobacterium avium subsp paratuberculosis (Map). METHOD: Thirteen target animals were selected by delayed growth of Map from initial individual radiometric faecal cultures (first growth index at 5 weeks or later). A procedure based on radiometric culture and IS900 polymerase chain reaction and restriction endonuclease analysis confirmation was then used for PFC. RESULTS: Eight samples (stored for up to 17 months at -80 degrees C) yielded Map on subsequent culture, either from undiluted faeces or those mixed with normal cattle faeces at dilution rates from 1 in 5 to 1 in 50. From a regression equation, culture-positive animals were considered to be shedding relatively low levels of Map (< 6 x 10(4)/g of faeces). Pooling dilutions of more than 1 in 5 reduced PFC sensitivity. A minimum incubation period of 10 weeks at a dilution of 1 in 5 is recommended to detect such infected cattle. This pooling rate in radiometric culture is probably capable of detecting cattle shedding < or = 5 x 10(3) Map organisms/g of faeces, representing an estimated inoculum per culture vial of fewer than 20 viable organisms. CONCLUSION: Map was detected in more than 50% of the stored faecal samples from cattle shedding low concentrations of the organism. A pooling rate of 5 samples per pool is required to reliably detect infected low-shedder cattle using PFC based on radiometric culture.     

Detection of Mycobacterium avium subsp. paratuberculosis in faeces using different procedures of pre-treatment for real-time PCR in comparison to culture

One of the most relevant aspects in the diagnosis of paratuberculosis (Johne’s disease) in cattle is the availability of a method for the rapid and sensitive detection of Mycobacterium avium subsp. paratuberculosis (MAP) in order to facilitate the prompt removal of pathogen-shedding animals from a herd. To meet this requirement, methods for pre-treatment of bovine faecal samples and subsequent extraction of DNA for detection of MAP by real-time PCR were compared with MAP culture results. A total of 116 bovine faecal samples that showed weak (64.7%), moderate (18.1%) or strong (17.2%) growth of MAP on solid HEY medium were investigated.For PCR, supernatants, sediments or bacterial pellets were obtained from faecal samples by pre-treatment before extraction of MAP DNA based on silica membranes or magnetic particles. Samples then were tested by MAP IS900 and ISMav2 real-time PCR with an analytical sensitivity of 6 and 28 genome equivalents (GE) per mL, respectively.The best results were obtained by including a microfiltration step in the sample pre-treatment in combination with silica membrane-based mini-columns or magnetic particles for DNA extraction. This approach enhanced the detection rate of MAP in IS900 real-time PCR from 58.6% to 84.5% using silica membrane mini-columns and from 61.2% to 64.7% using magnetic particles.     

Rapid PCR detection of Salmonella in horse faecal samples

A rapid polymerase chain reaction (PCR) assay was developed for detecting Salmonella in faeces of horses and assessed on samples from horses admitted to a veterinary hospital. Direct detection was achieved by amplification of part of ompC after extraction of DNA from faeces using a spin column method to reduce the amount of inhibitory substances in samples. An internal positive control was included to detect false negative results. While the sensitivity of the PCR assay was less than culture when assessed on faeces inoculated with Salmonella, its sensitivity on faecal samples obtained from horses was much greater than culture. Salmonella DNA was detected in 40% of faecal samples using the PCR assay while Salmonella were cultured from only 2% of the samples. The PCR assay has potential for use in either routine diagnosis or for detection of the carrier status in animals.     

Dam Mycobacterium avium subspecies paratuberculosis (MAP) infection status does not predetermine calves for future shedding when raised in a contaminated environment: a cohort study

Uptake of Mycobacterium avium subsp. paratuberculosis (MAP) by calves in the first days of life from colostrum, milk and faeces is regarded an important moment of transmission. The objective of this study was to quantify the association between the MAP status of dams as determined by the presence of MAP DNA and antibody in colostrum and that of DNA in faeces and the environment with subsequent MAP shedding of their daughters. A cohort of 117 dam-daughter pairs giving birth/being born on eight commercial dairy farms with endemic paratuberculosis was followed where colostrum, faecal and environmental samples (dust) were analysed for the presence of MAP using an IS900 real-time PCR. Antibodies in colostrum were measured by ELISA. Analysis of dust samples showed that on all farms environmental MAP exposure occurred continuously. In significantly more colostrum samples (48%) MAP DNA was detected compared to faecal samples (37%). MAP specific antibodies were present in 34% of the colostrum samples. In total MAP DNA was present in faecal samples of 41% of the daughters at least once during the sampling period. The association between faecal shedding in the offspring and the dam MAP status defined by MAP PCR on colostrum, MAP PCR on faeces or ELISA on colostrum was determined by an exact cox regression analysis for discrete data. The model indicated that the hazard for faecal shedding in daughters born to MAP positive dams was not significantly different compared to daughters born to MAP negative dams. When born to a dam with DNA positive faeces the HR was 1.05 (CI 0.6; 1.8) and with DNA positive colostrum the HR was 1.17 (CI 0.6; 2.3). When dam status was defined by a combination of both PCR outcomes (faeces and colostrum) and the ELISA outcome the HR was 1.26 (CI 0.9; 1.9). Therefore, this study indicates that neither the presence of MAP DNA in colostrum, MAP DNA in faeces nor the presence of MAP antibodies in colostrum of the dam significantly influences the hazard of MAP shedding in their subsequent daughters up to the age of two years when raised in a contaminated environment.     

Comparison of faecal culture and IS900 PCR assay for the detection of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> 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.     

Evaluation of radiometric faecal culture and direct PCR on pooled faeces for detection of Mycobacterium avium subsp. paratuberculosis in cattle

Dilution rates for pooled faecal culture (PFC) and direct IS900 polymerase chain reaction (D-PCR) tests were evaluated on faecal samples from infected cows mixed with uninfected faeces in dilutions from 1 in 5 to 1 in 50. PFC was performed by radiometric culture, with confirmation by IS900 PCR and restriction endonuclease analysis (PCR/REA) on growth, and by mycobactin dependency testing on solid medium. Using 37 culture positive faecal samples from 12 subclinical cows, 83.8% and 94.6% of samples were confirmed positive in the PFC assay at dilutions of 1 in 50 and 1 in 30, respectively. Lower dilutions (1 in 5 to 1 in 20) provided only marginally better sensitivity, and confirmation of PFC growth by PCR/REA was significantly more sensitive than mycobactin dependency. D-PCR had significantly lower sensitivity than PFC confirmed by PCR/REA, with pools of 1 in 50, 30, 10 and 5 yielding positive results in 64.9%, 70.3%, 78.4% and 83.8% of samples, respectively. Cattle considered to be shedding 1.5 x 10(6) viable M. avium subsp. paratuberculosis (Map)/g faeces (on the basis of estimated losses in processing and growth rates in radiometric broth) were positive at dilutions up to 1 in 50 in the PFC and D-PCR. Those shedding 5 x 10(5) viable Map/g were positive in the PFC at dilutions up to 1 in 40, but required a 1 in 10 dilution or less for D-PCR. The results suggest that for cattle shedding relatively high concentrations of Map in faeces (>2 x 10(5) viable Map/g), maximal dilutions of 1 in 30 for PFC and 1 in 10 for D-PCR would be applicable.     

Detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples: comparison of three polymerase chain reaction-based diagnostic tests with a conventional culture method.

Three commercially available assays, designed to specifically detect the presence of Mycobacterium avium subsp. paratuberculosis (MAP) in fecal samples by IS900-PCR, were compared with a conventional culture method. Fecal samples from 100 dairy cows were tested. Fifty-four (67.5%) of 80 culture-positive samples were positive for an assay that detects MAP DNA by dot spot hybridization of polymerase chain reaction products (kit A), 48 (60%) were positive by an assay using ethidium bromide staining for agar gel visualization of amplification products (kit B), and 49 (61.3%) were positive by an assay in which amplified products are detected by a colorimetric detection system (kit C). Relative sensitivity of all tests increased in proportion to the presence of MAP in fecal samples. Specificity was 100% based on results from 20 culture-negative samples from an MAP-free herd.     

Temporal patterns and quantification of excretion of Mycobacterium avium subsp paratuberculosis in sheep with Johne's disease

OBJECTIVES: To determine the frequency of excretion of Mycobacterium avium subsp paratuberculosis in Merino sheep with Johne's disease and to quantify excretion in a group of Merino sheep. DESIGN: A pen and laboratory experiment. PROCEDURE: Seven sheep selected from an affected flock on the basis of acid-fast bacilli in the sheep's faeces were housed and total daily faecal output was collected, weighed and subjected to culture for M avium subsp paratuberculosis. An end-point titration method was used to enumerate viable M avium subsp paratuberculosis in a 15 day pooled sample from five sheep that had acid-fast bacilli in their faeces while housed. RESULTS: Four sheep with subclinical multibacillary Johne's disease excreted M avium subsp paratuberculosis each day for 11 days of cultural observation. A further three sheep were intermittent excreters but lacked other evidence of infection with M avium subsp paratuberculosis. The average number of viable bacteria excreted was 1.09 x 10(8) per gram of faeces while total daily excretion was 8.36 x 10(10) viable M avium subsp paratuberculosis per sheep. Examination of faecal smears stained with Ziehl Neelsen was an unreliable means of assessing daily excretion in individual animals except in those with severe lesions. CONCLUSION: Excretion of M avium subsp paratuberculosis in Merino sheep with multibacillary Johne's disease occurred daily, proving that environmental contamination can be continuous on farms with endemic ovine Johne's disease. Faecal culture is a useful method for detecting infection as it does not appear to be affected by the timing of collection of a sample from sheep with multibacillary disease however, to maximise the sensitivity of disease surveillance using faecal culture, sampling rates should be adjusted to take account of the proportions of multibacillary and paucibacillary cases.     

Wildlife (Boselaphus tragocamelus)-small ruminant (goat and sheep) interface in the transmission of 'Bison type' genotype of Mycobacterium avium subspecies paratuberculosis in India

Information on Mycobacterium avium subspecies paratuberculosis (MAP) genotypes infecting different animal species in India is limited. Presence of MAP was investigated in free ranging antelopes (locally known as Nilgai/blue bulls/Boselaphus tragocamelus) using direct microscopy, culture, IS900 PCR and IS1311 PCR-REA. IS900 elements of MAP from Nilgai and previously isolated from goats were sequenced and compared to establish inter-species transmission between free ranging Nilgai and closed farm herds and flocks of goats and sheep sharing common grazing and water resources. Fecal samples were collected from two geographical regions (Mathura and Kanpur Dehat districts) separated by 300km, in North India. Of the 42 fecal samples cultured, MAP colonies were recovered from 23.8% samples (Nilgai). Of the 10 positive fecal samples, two were in 'Super shedder' (>1000cfu/g) category and rest were moderate (<10-100cfu/g) shedders. None of the Nilgai from Kanpur Dehat was positive in culture. The 229bp fragment targeting specific IS900 sequence was amplified from template DNA isolated from all the positive MAP cultures of Nilgai. Using IS1311 PCR-REA, MAP colonies were genotyped as 'Bison type'. Goatherds and a sheep flock located at Central Institute for Research on Goats (CIRG), shared 303.52ha of land (Mathura district of Uttar Pradesh) with Nilgai and were endemic for MAP infection. MAP strains isolated from goats and sheep have been genotyped as 'Bison type'. Nucleotide sequence of the insertion elements (900) from MAP 'Bison type' strain (S5) of goat origin and MAP (B42) from Nilgai showed difference of 2 (1%) base pairs at the 11th and 12th position (Genbank accession number EU130943). Study is first report on sharing (inter-species transmission) of a new 'Bison type' genotype of MAP between free ranging wildlife (Nilgai population) and domestic animals (farm goatherds and sheep flocks) in India.     

IS900-PCR-based detection and characterization of Mycobacterium avium subsp. paratuberculosis from buffy coat of cattle and sheep

Johne's disease is one of the main causes of economic losses in ruminants and a major health hazard in the developing and developed world. Up till now, many microbiological, serological and molecular methods have been tried for the detection of Mycobacterium avium subsp. paratuberculosis (MAP). In this study, we attempt a PCR-based detection of IS900, distinct insertion sequences of MAP from the buffy coat of cattle (n=262) and sheep (n=78), and direct genotyping by single strand conformational polymorphism (SSCP). A total of 30 (11.45%) cattle and one sheep (1.28%) were positive for MAP-IS900. This IS900-based PCR detection proved highly specific, particularly when tested on other non-MAP strains. SSCP analysis grouped the MAP-IS900 into four distinct clusters based on different band patterns. Nucleotide sequence variability between MAPs detected from sheep (GenBank accession ) and cattle (GenBank accession -) was noticed in the study. Although, in recent years IS900-PCR-based detection of MAP from WBCs is being used in human, its use in animals is still limited. Our work not only supports its use in animals but also suggests further IS900-SSCP-based MAP-genotyping, coupled with DNA sequencing, as a promising tool for rapid and effective Johne's disease surveillance.     

Comparison of BACTEC 460 and MGIT 960 systems for the culture of Mycobacterium avium subsp. paratuberculosis S strain and observations on the effect of inclusion of ampicillin in culture media to reduce contamination

To compare the utility and diagnostic accuracy of BACTEC and MGIT culture systems, a total of 41 pooled faecal samples, each containing faeces from one sheep infected with the S strain of Mycobacterium paratuberculosis and four uninfected sheep was cultured. The MGIT culture system did not support the growth of the S strain of M. paratuberculosis from faeces within the time frame of the experiments, although a laboratory adapted S strain grew slowly in MGIT provided that sufficient bacteria were inoculated. In contrast, C strain grew rapidly in MGIT. The sensitivity of culture was calculated relative to the infection status of the animals, none of which had clinical signs of ovine Johne's disease. The overall sensitivity of pooled faecal culture in the BACTEC culture system was 21.9% (95% confidence limits, 10.5-37.6), a figure dependant on the proportion of multibacillary cases. The sensitivities of the BACTEC culture system for pools containing animals with multibacillary and paucibacillary lesions were 100.0% (95% confidence limits, 47.2-100.0) and 17.8% (95% confidence limits 6.06-36.8), respectively. The contamination rate of BACTEC cultures was 9.7% compared to 14.3% for MGIT. The effect of 100 microg/ml ampicillin on the S strain of the M. paratuberculosis was examined and in both BACTEC and MGIT media it delayed growth by about 1 week. The composition of MGIT medium, particularly presence of vancomycin hydrochloride, slowed the growth of the S strain. The low content of egg yolk was considered to be another possible factor. The radiometric BACTEC culture system remains the best alternative for the culture of S strain and is recommended in circumstances where the genotype (s) of the strains present in a region/farm is either unknown or S strain.     

Detection of Mycobacterium avium subsp paratuberculosis in formalin-fixed paraffin-embedded intestinal tissue by IS900 polymerase chain reaction

OBJECTIVES: To evaluate and compare methods for DNA extraction from formalin-fixed, paraffin-embedded tissues and methods for detection of Mycobacterium avium subsp paratuberculosis by IS900 PCR for confirmation of Johne's disease in ruminants. DESIGN: A laboratory study. PROCEDURE: Three methods of DNA extraction of differing complexity and two PCR protocols using different pairs of IS900 primers were compared. Sensitivity and specificity were assessed using samples from ruminants with and without histological evidence of Johne's disease. RESULTS: The simplest method of DNA extraction, which involved two cycles of boiling and freezing followed by centrifugation, gave more consistent results than two methods that required solvent extraction of paraffin, proteinase digestion and DNA purification. The sensitivity of detection of M avium subsp paratuberculosis in paraffin blocks stored for 1 to 6 years from 34 cases of Johne's disease in sheep, cattle and goats was 88% for a 229 bp IS900 PCR assay and 71% for a 413 bp assay, using the detection of acid-fast bacilli by Ziehl Neelsen staining of histological sections from the same blocks as the gold standard test. PCR results correlated with the abundance of acid-fast organisms in the tissues. No false positive reactions were detected. CONCLUSION: PCR for identification of M avium subsp paratuberculosis in formalin-fixed, paraffin-embedded intestinal tissues from ruminants is a rapid and useful method. A simple method of sample preparation is effective. Amplification of short fragments of IS900 is more effective than amplification of longer fragments.     

A survey for Mycobacterium avium subspecies paratuberculosis in the Royal Zoological Society of Antwerp

Paratuberculosis is a chronic intestinal disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (Map). Very little is known about the status of paratuberculosis in European zoos. In this study, the presence of Map in the animal collection of the Royal Zoological Society of Antwerp (RZSA) was investigated. Faecal and post mortem samples from 48 ruminants were used to set up cultures. DNA from faeces, tissue and positive cultures were tested by IS900 polymerase chain reaction (PCR). Additionally, 448 serum samples were tested with an ELISA kit. All culture samples were negative whereas PCR gave three positives on biopsy samples and one positive on faecal samples. With the ELISA, 21 sera could be classified as positive. There is evidence that Map is present in the RZSA but no high level faecal shedders could be detected. Further investigations are required in other European Zoos in order to complete the picture of Map infections.     

Sensitivity and specificity of pooled faecal culture and serology as flock-screening tests for detection of ovine paratuberculosis in Australia

The flock-level sensitivity of pooled faecal culture and serological testing using AGID for the detection of ovine Johne’s disease-infected flocks were estimated using non-gold-standard methods. The two tests were compared in an extensive field trial in 296 flocks in New South Wales during 1998. In each flock, a sample of sheep was selected and tested for ovine Johne’s disease using both the AGID and pooled faecal culture. The flock-specificity of pooled faecal culture also was estimated from results of surveillance and market-assurance testing in New South Wales.

The overall flock-sensitivity of pooled faecal culture was 92% (95% CI: 82.4 and 97.4%) compared to 61% (50.5 and 70.9%) for serology (assuming that both tests were 100% specific). In low-prevalence flocks (estimated prevalence <2%), the flock-sensitivities of pooled faecal culture and serology were 82% (57 and 96%) and 33% (19 and 49%), respectively, compared to 96% (85 and 99.5%) and 85% (72 and 93%), respectively, in higher-prevalence flocks (estimated prevalence ≥2%). A Bayesian approach incorporating prior knowledge on flock-specificity of pooled culture produced similar estimates and probability intervals. These estimates assume conditional independence of the two tests, and therefore might have over-estimated the true flock-sensitivities of the tests if the flock-sensitivities of pooled faecal culture and serology were correlated.

The estimated minimum flock-specificity of pooled culture when used for surveillance and assurance testing was 99.1% (96.9 and 99.9%). Surveillance and assurance programs in Australia are designed to provide a flock-sensitivity of 95% for an assumed prevalence of 2%. Pooled faecal culture is performing at close to this level—whereas the flock-sensitivity of serology appears to be lower than expected, particularly in lower prevalence flocks.     

Risk of exposure to Coxiella burnetii from ruminant livestock exhibited at Iowa agricultural fairs

Coxiella burnetii is a zoonotic pathogen typically associated with clinical and asymptomatic infection in ruminant livestock. A re‐emerging pathogen of significant public health importance, C. burnetii has caused recent epidemics in the United States and Europe, and public livestock exhibitions are increasingly scrutinized as a potential source of C. burnetii exposure. Although C. burnetii prevalence data among North American domestic ruminants are extremely limited, contemporary studies suggest that this pathogen is both geographically widespread and highly prevalent on a herd basis, especially in dairy cattle and goat populations. We utilized a real‐time PCR assay to detect C. burnetii faecal shedding by clinically normal, non‐periparturient beef cattle, meat goats and sheep exhibited at Iowa agricultural fairs. Individual faecal samples were collected from beef cattle, meat goats and sheep exhibited at twelve Iowa county fairs during the summer of 2009. The sample pool was blocked by species and fair, and ten samples from each block were randomly selected for the diagnostic assay; this test pool is considered sufficient to identify with 95% confidence a shedding animal in a population prevalence of 2.85% (cattle and sheep) and 6.25% (goats). Detection of C. burnetii DNA was determined through use of a real‐time PCR assay validated for use in bovine, ovine and caprine faeces; threshold of detection is one DNA copy per PCR (sensitivity 95.8%, specificity 100%). All tested samples were negative for C. burnetii DNA. We conclude that non‐dairy, non‐periparturient ruminants exhibited at Iowa fairs are unlikely to shed C. burnetii in their faeces and that this population should not be considered to be a significant exposure risk to other livestock or fair attendees.     

Earthworms (Oligochaeta,Lumbricidae) and mycobacteria

The objective of the study was to define the role of earthworms in the survival of mycobacteria in animal populations. In 13 sampling sites mycobacteria were detected in 53 (5.5%) samples of faeces and parenchymatous tissues from animals, in 25 (7.3%) environmental and in nine (8.2%) earthworm samples. In cattle and goat farms affected by Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) of IS900 restriction fragment length polymorphism (RFLP) type B-C1 was isolated from 37 (4.6%) faecal samples, three (1.4%) environmental and one (3.1%) earthworm sample. Investigations of aviaries affected by avian tuberculosis detected M. avium of genotype IS901+ and IS1245+ in six (7.9%) bird's faecal and in four (4.4%) environmental samples. M. avium (genotype IS901- and IS1245+) was detected in four (4.4%) and M. abscessus in one (1.1%) environmental sample. M. avium of genotype IS901- and IS1245+ and M. gastri were isolated from three (6.4%) earthworm samples. In pig farm with mycobacteriosis M. avium of genotype IS901- and IS1245+ was detected in five (20.0%) faecal samples from pigs and in four (12.9%) environmental samples. M. scrofulaceum was isolated in one (4.6%) sample of Lumbricus rubellus. In laboratory experiments identical RFLP types of M. paratuberculosis were isolated from bodies and faeces of earthworms 1-2 days after the last contact with the faeces contaminated with the same RFLP type of M. paratuberculosis. The results suggest that earthworms may become vectors of mycobacteria.