DNA fingerprinting of Australian isolates of Mycobacterium avium subsp paratuberculosis using IS900 RFLP

OBJECTIVES: To evaluate additional restriction enzymes for IS900 RFLP of Mycobacterium avium subsp paratuberculosis and examine the genetic diversity among Australian isolates for epidemiological studies of Johne's disease. DESIGN AND PROCEDURE: Seventy-one isolates of M paratuberculosis from cattle, sheep, goat, alpaca and rhinoceros in six Australian States and the Northern Territory, reference strains and reference DNA from previously characterised strains were tested for genetic variation. Bst EII, Pvu II and Pst I restriction enzymes were used, and four others (Bam HI, Alu I, Xho I and Dra I) were assessed for their ability to detect polymorphisms. Multiple isolates from some animals were tested. RESULTS: Bam HI, was the most effective enzyme for identifying polymorphisms (12 types), followed by Bst EII (11 types). Both Pvu II and Pst I were relatively ineffectual. Fifteen different types were identified, 12 in clinical isolates. Most isolates were cattle (C) strains and fell into the C1 (n = 28) and C3 (n = 32) groupings. All isolates from alpaca were type C1, and bovine isolates were commonly C1 (n = 15) or C3 (n = 28). All of the sheep were infected with sheep (S) strains; no S strains were identified in cattle. Two of six isolates from one animal had single band differences. CONCLUSION: The epidemiological features of M paratuberculosis in Australia are similar to those reported in New Zealand, where cattle and sheep are commonly infected with different strains. However, because of the lack of polymorphism identified within the major groups, it is unlikely that DNA fingerprinting will have a significant role in epidemiological studies of Johne's disease, unless an unusual strain in being studied.     

Distribution of IS900 restriction fragment length polymorphism types among animal Mycobacterium avium subsp. paratuberculosis isolates from Argentina and Europe

Sixty-one Mycobacterium avium subsp. paratuberculosis isolates from cattle and deer from the Buenos Aires province, an important livestock region in Argentina, were typed by restriction fragment length polymorphisms (RFLP) analysis based on IS900. Four different RFLP patterns (designated ‘A’, ‘B’, ‘C’ and ‘E’) were identified in BstEII digests of genomic DNA. The most frequently observed type, pattern ‘A’, was found in 46 isolates (75%). The second, pattern ‘E’, included 8 isolates (13%), while the third, pattern ‘B’, included 6 isolates (10%). Pattern ‘C’ was found for only one isolate. All of the deer isolates were classified as pattern ‘A’, while cattle isolates represented all four RFLP patterns. Twenty-one isolates representing the four different BstEII-RFLP patterns were digested with PstI. Twenty isolates showed identical PstI-RFLP pattern. BstEII-RFLP patterns from Argentine cattle and deer were compared with patterns found in cattle, goat, deer, rabbit, and human isolates from Europe. The most common pattern in Argentina, pattern ‘A’, was identical to a less frequently occurring pattern R9 (C17) from Europe. The other Argentine patterns ‘B’, ‘C’ and ‘E’, were not found in the Europe. These results indicate that the distribution of M. avium subsp. paratuberculosis genotypes in the Buenos Aires province of Argentina is different from that found in Europe.     

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.     

IS900 Restriction Fragment Length Polymorphism (RFLP) Analysis of Mycobacterium avium subsp. paratuberculosis Isolates from Goats and Cattle in Norway

In Norway, paratuberculosis has been frequently diagnosed in goats, while cattle have been almost free of the infection. This difference in prevalence between goats and cattle has led to speculations about the existence of a Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) isolate that is non-pathogenic for cattle. There is little information available on genotypic variation of M. a. paratuberculosis isolated from animals in Norway. In the present study, genotypic information on 51 isolates from goats and four isolates from cattle in Norway was obtained by use of IS900 restriction fragment length polymorphism (RFLP) analysis. All isolates from cattle and 84% of the isolates from goats had the same RFLP pattern (B-C1). Five RFLP patterns not previously detected were found. No genotypic variation that could explain a difference in host origin was found between the isolates from cattle and the majority of the Norwegian goat isolates. This lack of difference indicates that the most common M. a. paratuberculosis isolates in Norway may infect both cattle and goats.     

Transmission parameters of Mycobacterium avium subspecies paratuberculosis infections in a dairy herd going through a control program

A Johne's disease control program, including stringent management practices and a test-and-cull program (whole-herd fecal-samples taken twice a year), was implemented on a medium-sized Pennsylvania dairy farm that was suffering losses from clinical Johne's disease. The data that emerged from the control program, combined with birthdates, culling dates, lactation information and pedigrees, yielded an extensive longitudinal dataset. The dataset was processed through SAS 9.1 for statistical analysis; herd-level disease dynamics and dam-to-daughter transmission parameters were calculated. After the implementation of the program in 1984, prevalence dropped dramatically from 60% to less than 20% in 1989. After an apparent prevalence peak (25%) in 1991 due to improved test sensitivity, prevalence maintained a plateau of 10% from 1996 to 2000. After the implementation of the program, 9.5% of the offspring from test-negative dams and 26.8% of the offspring from known-infected dams became infected with Mycobacterium avium subspecies paratuberculosis (Map) (χ² = 14.7; p = 0.0001). Calves born shortly following the calving of an infected dam and calves growing up with a future high shedder were more likely to be infected compared to calves without this risk profile. It was concluded that, after the implementation of the control program, the most important causes of infections of susceptible calves were their own dams or infected animals which had calved recently.     

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.     

Evaluation of a high-throughput nucleic acid extraction method for the detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples by PCR

Johne’s disease (paratuberculosis) is an economically important disease of cattle worldwide. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious gram-positive bacterium. PCR is increasingly used in diagnostic laboratories for the detection of MAP in fecal samples given the rapid test turnaround time and sensitivity and specificity comparable to fecal culture. However, efficient extraction of DNA for sensitive detection of MAP by PCR is affected by the complex lipid-rich cell wall of MAP and the presence of PCR inhibitors in feces. We evaluated a high-throughput nucleic acid extraction method (MagMAX core nucleic acid purification kit with mechanical lysis module) in conjunction with an hspX gene PCR for the detection of MAP from bovine fecal samples, which resulted in correct identification of all negative (13 of 13) and positive (35 of 35) proficiency test samples obtained from the National Veterinary Services Laboratories. In addition, all 6 negative and 50 of 51 positive diagnostic specimens tested were categorized correctly.     

牛副结核分枝杆菌LAMP快速检测方法的建立

为建立牛副结核分枝杆菌(MAP)的快速检测方法,本研究采用环介导等温扩增(LAMP)技术,以MAP的IS900基因序列为靶基因设计特异性引物,建立了MAP特异性的LAMP快速检测方法。经反应条件优化,检测结果显示,建立的LAMP检测方法具有良好的特异性,最低见测量为0.53 pg;与其他病原菌无任何交叉反应。临床样品检测与行业标准的符合率为100%。该LAMP检测方法可以用于MAP的常规检疫。     

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.     

Efficacy of spheroplastic and cell-wall competent vaccines for Mycobacterium avium subsp. paratuberculosis in experimentally-challenged baby goats

A Mycobacterium avium subspecies paratuberculosis (MAP) vaccine that reduced the incidence of clinical disease or reduced fecal shedding of MAP would aid control of Johne's disease (JD). The objective of the present study was to evaluate the efficacy of four MAP vaccine combinations, including cell-wall competent (CWC) alum adjuvant, CWC-QS21 adjuvant, cell-wall deficient (CWD) alum adjuvant and CWD-QS21 adjuvant vaccines. Eighty baby goats were vaccinated at 1 and 4 weeks of age with one of these vaccines or a sham control vaccine consisting of alum adjuvant. Kids were challenged orally with approximately 6.0 × 10⁹ organisms in four divided doses of 1.5 × 10⁹ organisms using a goat isolate of MAP. Vaccinated challenged and challenged control groups had 10 and 6 kids per group, respectively. Half of the kids within each group were necropsied at either 6 or 9 months post-challenge. Gross and microscopic lesions and relative number of acid-fast bacilli were evaluated and scored at necropsy. Results indicated all challenged kids had some lesions compatible with JD suggesting none of the vaccines prevented infection. Three vaccines (CWC-alum, CWC-QS21 and CWD-QS21) reduced lesion scores by 46–51% at 9 months. CWD-alum vaccine resulted in a more severe (+33.5%) lesion score than sham-vaccinated challenged control. Lesion scores were greater at 9 months than at 6 months post-challenge in the sham-vaccinated challenged group and CWD-alum vaccinated group, while lesion scores were generally stable with remaining vaccines. Mean fecal CFU/g were significantly different across time from challenge to 9-month necropsy (p = 0.043) and the CWC-QS21 vaccine group had a marked reduction in fecal CFU/g at all time points post-challenge. A reduction in MAP CFU/g was also detected in necropsy tissues from kids given the CWC-alum, CWC-QS21 and CWD-QS21 vaccines, and increased CFU/g were detected in tissues from kids given the CWD-alum vaccine. Immunological tests evaluated included, humoral response evaluation by AGID, ELISA and Western blot, and cell mediated response by comparative PPD skin testing (M. avium, Old Johnin, M. bovis and Lot 2 Johnin PPD's), and production of MAP induced γ-interferon. Vaccination also resulted in false-positive PPD skin test reactions for M. avium PPD, Old Johnin PPD and γ-interferon tests. When a 2-mm cutoff above normal skin thickness was used to define positive skin test reactions, false-positive reactions for M. bovis were detected in only 2 of 32 kids given a vaccine with QS21 adjuvant.     

Description of the Infection Status in a Norwegian Cattle Herd Naturally Infected by Mycobacterium avium subsp. paratuberculosis

The Norwegian surveillance and control programme for paratuberculosis revealed 8 seroreactors in a single dairy cattle herd that had no clinical signs of Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) infection. Paratuberculosis had been a clinical problem in goats several years previously in this herd. All 45 cattle were culled and a thorough investigation of the infection status was conducted by the use of interferon-γ (IFN-γ) immunoassay, measurement of antibodies, and pathological and bacteriological examination.In the IFN-γ immunoassay, 9 animals gave positive results, and 13 were weakly positive, while 19 animals were negative. In the serological test,10 animals showed positive reactions, and 5 were doubtful, while 30 animals gave negative reactions. There appeared to be a weak trend toward younger animals having raised IFN-γ and older animals having raised serological tests. Histopathological lesions compatible with paratuberculosis were diagnosed in 4 animals aged between 4 and 9 years. Three of these animals had positive serological reaction and one animal gave also positive results in the IFN-γ immunoassay. Infection was confirmed by isolation of M. a. paratuberculosis from 2 of these 4 animals. One single bacterial isolate examined by restriction fragment length polymorphism (RFLP) had the same profile, B-C1, as a strain that had been isolated from a goat at the same farm several years previously.Despite many animals being positive in one or both of the immunological tests, indicative of a heavily infected herd, none of the animals showed clinical signs and only one cow was shown to be shedding bacteria. A cross-reaction with other mycobacteria might have caused some of the immunoreactions in these animals. It is also possible that the Norwegian red cattle breed is resistant to clinical infection with M. a. paratuberculosis.     

Mycobacterium avium subsp. paratuberculosis infection in wildlife on three deer farms with a history of Johne's disease

Abstract

AIM: To determine the prevalence of Mycobacterium avium subsp. paratuberculosis (Map) infection in wildlife, in pastoral landscapes with a recent history of clinical Johne's disease in livestock.

METHODS: A total of 449 wild mammals and birds from three farms in the South Island of New Zealand with recent histories of clinical Johne's disease in their deer herds were trapped and examined for gross pathological changes in the gastrointestinal tract. Additionally, individual mesenteric lymph nodes from 380 mammals, and segments of gastrointestinal tract from 32 birds were excised, homogenised and cultured for viable Map bacilli. The prevalence of Map infection was then calculated for the various species. Faecal samples from those mammals which had culture-positive tissues were further cultured for the presence of Map.

RESULTS: Gross pathological changes were identified in the gastrointestinal tract of four brushtail possums, one cat, six ferrets, 12 hares, six hedgehogs, three rabbits, one stoat, and one paradise shelduck. Infection with Map in the gastrointestinal tract was confirmed in only three of these cases, one each of brushtail possums, hares and hedgehogs. In contrast, Map infection in the absence of gross pathological changes was frequently recorded in enteric tract tissues of mammals and birds. Among mammals, Map infection was recorded in 18/73 (25%) brushtail possums, 4/23 (17%) cats, 15/42 (36%) hedgehogs and 29/113 (26%) rabbits. Among birds, intestinal tract tissue Map infection was recorded in 3/17 (18%) paradise shelducks. Among 64 of the 74 mammals which had Map culture-positive tissues, 38% (n=5) of hedgehogs and 11% (n=3) of rabbits also had culture-positive faecal samples.

CONCLUSIONS: This study is the first to identify that Map infection can be prevalent in wildlife in New Zealand. There was a high prevalence of Map infection among both scavenging and grazing wild animals. Both mammals and birds are capable of harbouring viable Map organisms in their gastrointestinal tract; further, viable Map was excreted into the environment via faeces by hedgehogs and rabbits.

CLINICAL RELEVANCE: Previous studies overseas have postulated a role of wildlife as reservoirs of Map infection and possible vectors of Johne's disease to livestock. Here, brushtail possums, hedgehogs and rabbits and in particular were identified as potential wildlife hosts for Map infection in NewZealand. This suggests that several wildlife species could contribute to the persistence of Map infection within a wildlife/livestock complex, and potentially, perhaps more importantly, to the spread of infection between farms.     

Inter-laboratory comparison of radiometric culture for Mycobacterium avium subsp. paratuberculosis using raw milk from known infected herds and individual dairy cattle in Victoria

Objective To compare the results of radiometric culture conducted in three Australian laboratories for Mycobacterium avium subsp. paratuberculosis (Mptb) using bulk vat and individual animal milk samples. Procedure Milk samples were collected from 15 cows exhibiting clinical signs of Johne's disease, and subsequently confirmed as infected with Mptb, and from the bulk milk vats on 91 farms running herds known to be infected with Mptb. Each milk sample was divided into three equivalent samples and one of each of the replicates was forwarded to the three participating laboratories. The identity and nature of the samples was protected from the study collaborators. The laboratories processed the samples and undertook radiometric culture for Mptb using their standard method. Results of testing were provided to the principal investigator for collation and analysis. Results In total, 2 (2.2%) of 91 vat-milk samples and 8 (53.3%) of 15 individual cows' milk samples returned positive radiometric milk culture results. Only one sample, from a clinical case of Johne's disease, was identified as positive by more than one laboratory. There were differences in the absolute frequency with which Mptb was identified in the milk samples by the collaborating laboratories. Conclusions Mptb was cultured from a very small percentage of Australian raw bulk milk samples sourced from known infected herds. By contrast, Mptb was successfully cultured from half of the milk samples collected from clinically affected cows. There was no statistical difference between laboratories in the proportion of vat samples or individual animal milk samples in which Mptb was detected.     

Further analysis of VNTR and MIRU in the genome of Mycobacterium avium complex, and application to molecular epidemiology of isolates from South America

All members of Mycobacterium avium complex are serious pathogens for humans and animals. The aim of this study was to look for and analyze VNTR-MIRU loci in the genome of M. avium complex and their preliminary application to test these isolates. In the present study, we identified 22 novel VNTR-MIRU by using Tandem Repeat software: five with a structure similar to MIRU and 17 without MIRU structure; these latter were designated as VNTR. Most VNTR were located within predicted coding regions. Most MIRU were intercistronic with their extremities overlapping the termination and initiation codons of their flanking genes. Some of these VNTR-MIRU exhibited polymorphism among M. avium complex isolates due to insertion or deletion of whole repeats and/or of nucleotide sequence degeneration. We determined the variability of six VNTR-MIRU loci in 21 M. avium subsp. hominissuis and 26 M. avium subsp. paratuberculosis. The analysis identified 15 different alleles with the combination of six VNTR-MIRU in the 21 M. avium subsp. hominissuis with 16 different IS1245 RFLP and four different profiles with PCR-restriction analysis of hsp65 (PRA). However, neither the six VNTR-MIRU loci nor the PRA were able to distinguish M. avium subsp. paratuberculosis isolates with five different IS900 RFLP profiles. In conclusion, some of the VNTR-MIRU loci identified were useful to differentiate M. avium subsp. hominissuis but not M. avium subsp. paratuberculosis isolates here included. However, we observed polymorphism in VNTR-MIRU loci between M. avium subsp. hominissuis and M. avium subsp. paratuberculosis genomes, which could be important in the understanding of the obvious differences in the pathogenic effects of these mycobacteria.     

Detection of Mycobacterium avium subsp. paratuberculosis in skeletal muscle and blood of ewes from a sheep farm in New Zealand

Abstract

AIM: To determine whether viable Mycobacterium avium subsp. paratuberculosis (Map) is present in skeletal muscle and blood in ewes with and without Johne's disease confirmed histologically.

METHODS: A total of 51 mixed-aged ewes in poor body condition from a farm with a history of clinical Johne's disease were culled and examined at necropsy. BACTEC radiometric culture was performed on samples of skeletal muscle from the biceps femoris, mononuclear cells in peripheral blood (hereafter referred to as blood), and ileum. Histological sections and Ziehl-Neelsen (ZN)-stained impression smears of terminal ileum and mesenteric lymph nodes were examined. Ewes were defined as having confirmed Johne's disease if there was histopathological evidence typical of the disease within the ileum and adjacent lymph nodes.

RESULTS: Eighteen of 21 (86%) ewes with confirmed clinical Johne's disease were culture-positive for Map from sites peripheral to the alimentary tract, comprising 15 from skeletal muscle and 13 from blood. Five of 30 (17%) ewes that did not have Johne's disease were culture-positive, with four from skeletal muscle and one from blood. The likelihood that ewes with confirmed Johne's disease had systemic Map infection compared with ewes without was determined as OR=30 (95% CI=6.3–142.0; p<0.001).

CONCLUSION: The prevalence of Map infection of skeletal muscle and blood in ewes with confirmed Johne's disease was 71% and 62% respectively, and in unaffected ewes was 13% for muscle and 3% for blood.

CLINICAL RELEVANCE: Skeletal muscle and blood are potential sources of exposure of humans to Map, and the risk appears higher from sheep with Johne's disease.     

Detection of Mycobacterium paratuberculosis in formalin-fixed paraffin-embedded tissues by the polymerase chain reaction.

The polymerase chain reaction (PCR) utilizing primers specific for the IS900 sequence of Mycobacterium paratuberculosis was applied to tissue sections of formalin-fixed, paraffin-embedded ileum from cattle with Johne's disease and the results compared to those obtained with acid-fast (Ziehl-Neelsen) and immunohistochemical staining. The PCR was positive in 19/21 tissues (90%) while Ziehl-Neelsen staining was positive in 18/21 (86%) and immunohistochemical staining in 21/21 (100%). The Ziehl-Neelsen and immunohistochemical stains are not, however, specific for M. paratuberculosis. The PCR for detection of M. paratuberculosis using the IS900 sequence is a specific and relatively sensitive method for confirmation of Johne's disease and its application to formalin-fixed, paraffin-embedded tissues may be useful for confirmation of dubious cases, for retrospective studies and for epidemiological analyses.     

Survival of Mycobacterium avium subsp paratuberculosis in amitraz cattle dip fluid

OBJECTIVE: To determine the survival time of Mycobacterium avium subsp paratuberculosis in amitraz-based cattle dip fluid derived from an active dip site in northern New South Wales. PROCEDURE: Following inoculation of triplicate 5 L containers with faeces (0.5 g/L) from a clinical case of bovine paratuberculosis, samples collected up to 8 weeks after inoculation were examined by conventional and radiometric culture. M a paratuberculosis colonies were enumerated on solid media. RESULTS AND CONCLUSIONS: M a paratuberculosis survived in amitraz cattle dip fluid for up to 2 weeks, but not 3 weeks. Where 1% of solids in dip fluid is derived from a clinical case of paratuberculosis, dip fluid may contain viable M a paratuberculosis for at least 2 weeks. These findings have implications for the management of cattle dip sites.     

Sensitivity and specificity of two serological tests for the detection of ovine paratuberculosis

OBJECTIVE: To determine the sensitivity and specificity of an absorbed ELISA and an AGID test for the detection of clinical and subclinical paratuberculosis in sheep. DESIGN: By testing a panel of sera from 1257 Australian Merino and crossbred sheep greater than 1 year of age, of which 1137 sheep were not infected with Mycobacterium avium subsp paratuberculosis and 120 sheep had paratuberculosis. PROCEDURE: Sera were collected from 457 sheep in Victoria and 800 sheep in Western Australia. Presence of M a paratuberculosis infection in Victorian sheep was determined by histological examination of intestinal tissues, whereas sheep from Western Australia were presumed to be free of Johne's disease. The ability of an absorbed ELISA to discriminate between infected and uninfected sheep was described by test sensitivity and specificity, the distribution of ELISA OD, and the area under a receiver operating characteristic curve. RESULTS: The absorbed ELISA had a specificity of 98.2 to 99.5% (CI) and a sensitivity of 35 to 54% (CI). In sheep from infected flocks in Victoria, the AGID test had a specificity of 99 to 100% (CI) and a sensitivity of 38 to 56% (CI). The sensitivity of serological tests was higher in sheep with a body condition representative of the lower quintile of their flock of origin. CONCLUSION: The AGID test and absorbed ELISA are useful tests for the detection of ovine paratuberculosis. Although the tests had a similar accuracy, they detected different subpopulations of infected sheep with only moderate overlap. The AGID test had a higher specificity than the absorbed ELISA.     

Molecular epidemiology of Mycobacterium avium subspecies paratuberculosis: IS900 PCR identification and IS1311 polymorphism analysis from ruminants in the Punjab region of India

Johne's disease is chronic granulomatous infectious enteritis of animals caused by Mycobacterium avium subspecies paratuberculosis. A total of 153 animals from 19 dairy farms, 2 gaushalas (unproductive-animal rehabilitation centers), 2 goat and 2 sheep farms from different districts of the Punjab region were selected on the basis of clinical signs of disease. All samples from cattle (n = 86), buffalo (n = 34), goat (n = 25) and sheep (n = 26) were subjected to Ziehl-Neelsen staining and DNA extraction by a freeze and thaw method. Ziehl-Neelsen staining detected 71% samples positive for acid-fast bacilli whereas IS900 PCR detected 55% positive for Map DNA. IS1311 PCR-REA analysis of IS900 positive samples revealed ‘Bison’ type as the most prevalent (82%) genotype of Map, infecting all domestic ruminants. ‘Cattle’ type was present in a minority of cases (15%) from cattle, buffaloes and goats. This is the first report of ‘Cattle’ type Map from buffalo and goat species in India.     

Simulation modeling to evaluate the persistence of Mycobacterium avium subsp. paratuberculosis (MAP) on commercial dairy farms in the United States

We developed a series of deterministic mathematical models of Mycobacterium avium subsp. paratuberculosis (MAP) transmission on commercial US dairies. Our models build upon and modify models and assumptions in previous work to better reflect the pathobiology of the disease. Parameter values were obtained from literature for animal turnover in US dairy herds and rates of transition between disease states.

The models developed were used to test three hypotheses. (1) Infectious transmission following intervention is relatively insensitive to the presence of high-shedding animals. (2) Vertical and pseudo-vertical transmission increases prevalence of disease but is insufficient to explain persistence following intervention. (3) Transiently shedding young animals might aid persistence.

Our simulations indicated that multiple levels of contagiousness among infected adult animals in combination with vertical transmission and MAP shedding in infected young animals explained the maintenance of low-prevalence infections in herds. High relative contagiousness of high-shedding adult animals resulted in these animals serving as the predominant contributor to transmission. This caused elimination of infection in herds using the test-and-cull intervention tested in these simulations. Addition of vertical transmission caused persistence of infection in a moderately complicated model. In the most complex model that allowed age-based contacts, calf-to-calf transmission was required for persistence.