Herd-level economic losses associated with Johne's disease on US dairy operations

Johne's disease (‘paratuberculosis') is a chronic, infectious, wasting disease that affects dairy cattle. Estimation of its impact on herd productivity and corresponding economic loss on US dairy operations was part of the USDA National Animal Health Monitoring System's (NAHMS) 1996 national dairy study. Johne's-positive herds experience an economic loss of almost US$ 100 per cow when compared to Johne's-negative herds due to reduced milk production and increased cow-replacement costs. For Johne's-positive herds that reported at least 10% of their cull cows as having clinical signs consistent with Johne's disease, economic losses were over US$ 200 per cow. These high-prevalence herds experienced reduced milk production of over 700 kg per cow, culled more cows but had lower cull-cow revenues, and had greater cow mortality than Johne's-negative herds. Averaged across all herds, Johne's disease costs the US dairy industry, in reduced productivity, US$ 22 to US$ 27 per cow or US$ 200 to US$ 250 million annually.     

Ovine Johne's disease in Australia--the first 20 years

OBJECTIVE: To review the history of ovine Johne's disease in Australia. PROCEDURE: Relevant publications and reports were identified and reviewed to document the spread of ovine Johne's disease (OJD) from 1980 until the end of 2000, as well as the response of industry and government to the spread of this disease. RESULTS: OJD was first diagnosed in the central tablelands region of New South Wales in 1980. Since then it has spread, either from the initial focus or through separate introductions so that by December 2000 a total of 823 infected flocks had been identified. Cases have been confirmed in New South Wales, Victoria, the Australian Capital Territory, on Flinders Island in Tasmania, on Kangaroo Island in South Australia and in Western Australia. In early 1999, agreement was reached to fund and implement a 6-year, $40 million National OJD Control and Evaluation Program (NOJDP). This program is jointly funded by the sheep industries (national and state), and Commonwealth and State governments, and is managed by Animal Health Australia. CONCLUSION: A national program is now in place to support the control of OJD and research to determine the feasibility and cost-effectiveness of eradication. The development of new diagnostic techniques, such as abattoir surveillance and pooled faecal culture, provide opportunities to refine surveillance strategies and to define better the distribution and prevalence of this disease, as required by the national program. Effective control measures, combined with quality surveillance data, will enable informed decision making for the future national management of OJD.     

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.     

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.     

A comparison of methods to estimate the prevalence of ovine Johne's infection from pooled faecal samples

OBJECTIVE: To compare estimates of ovine Johne's infection prevalence produced by several alternate methods based on pooled faecal culture (PFC) results with prevalence estimates based on individual faecal culture (IFC). PROCEDURE: Seven methods for estimating prevalence of infection based on PFC results were incorporated in a computer program, including methods for imperfect test sensitivity and specificity, for variable pool size and a Bayesian method that incorporates prior knowledge about test performance and prevalence. These methods were then used to analyse PFC data at one observation 30 months post-vaccination in a field trial of a killed vaccine for the control of OJD, undertaken on three farms in New South Wales. RESULTS: Prevalence estimates, for three methods that assume a perfect test, were close to the IFC estimate, whereas for three other methods that assume an imperfect test, the estimated prevalence was generally higher than the IFC estimate. In comparison, the Bayesian approach produced more variable estimates that were substantially higher than the IFC estimate when an inappropriately high prior estimate of prevalence was used. CONCLUSION: Despite the limitations of each method, two methods provided accurate and reasonable estimates of the prevalence assessed by IFC in all instances, and are appropriate for the analysis of data from this vaccine trial. One of these methods also has the advantage of allowing for variable pool size. However, further research is needed to develop a method that will simultaneously account for variation in pool size and in test sensitivity and specificity.     

Cross species transmission of ovine Johne's disease from sheep to cattle: an estimate of prevalence in exposed susceptible cattle

OBJECTIVE: To determine the prevalence of infection of cattle with the sheep strain of Mycobacterium avium subsp paratuberculosis at least two years after exposure at < 6 months old. DESIGN: Prospective survey One thousand seven hundred and seventy-four cattle from 12 properties (Farms A to L) were sampled by ELISA and faecal culture to detect evidence of infection with M a paratuberculosis. All properties had a known history of Johne's disease (JD) in sheep, and sampled cattle were likely to be susceptible to JD at the time they were first exposed, being at an age of 6 months or less. In addition, opportunistic investigations were undertaken of ELISA reactor cattle discovered during testing for the Australian Johne's Disease Market Assurance Program for Cattle (Farms M and N). RESULTS: All animals in the survey gave negative results on serology while one animal from a herd of 349 gave a positive faecal culture result. Follow-up faecal culture, post-mortem and histopathology on the latter animal were negative, suggesting that it was a passive faecal shedder or carrier. Two occurrences of OJD transmission to cattle were detected during the opportunistic investigations. CONCLUSION: These observations confirm existing beliefs about the risk of transmission of OJD to cattle, that the risk of transmission is low. However transmission occurs sporadically. An estimated upper limit of prevalence of S strain M a paratuberculosis infection in susceptible exposed cattle in the OJD high prevalence area of New South Wales is 0.8%, assuming a common prevalence within herds.     

Evaluation of serum and milk ELISAs for paratuberculosis in Danish dairy cattle

A milk and a serum ELISA for detection of antibodies against Mycobacterium avium ssp. paratuberculosis (MAP) were evaluated against the complement-fixation test (CFT) and culture of faecal samples from 580 cows collected between August 1996 and December 1996. Milk and serum were obtained concurrently from six dairy herds infected with MAP and from two dairy herds without history of infection with MAP.

A cut-off value of 7 OD% was used in the ELISAs. At this cut-off value, all six culture-positive herds were positive in the serum ELISA but one was negative in the milk ELISA. All six culture-positive herds were positive in the CFT. In the two culture-negative herds, the serum and the milk ELISA deemed all serum samples negative at this cut-off value, whereas four serum samples from one of these herds were positive in the CFT. The highest cut-off value enabling the milk ELISA to record all six culture-positive herds as positive was 4 OD%. The highest cut-off value enabling the serum ELISA to record all six culture-positive herds as positive was 17 OD%. Individual-sample relative sensitivities of the ELISAs ranged from 49 to 64% and relative specificities were 80–96% at the cut-off values of 4, 7 and 17 OD%.     

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.     

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.     

Economic evaluation of Johne's disease control programs implemented on six Michigan dairy farms

Johne's disease (JD) is an incurable, chronic infectious disease prevalent in dairy herds throughout the US and the world. The substantial economic losses caused by JD have been well documented. However, information on the costs of controlling the disease is limited, yet necessary, if producers are to make sound decisions regarding JD management. The purpose of this paper is to describe a method for evaluating the cost-effectiveness of management changes to control JD on infected dairy farms. A 5-year longitudinal study of six dairy herds infected with JD was performed. Each herd implemented a JD control program upon study enrollment. Prevalence of JD within each herd was monitored with annual testing of all adult cows using fecal culture and/or serum ELISA. Individual cow production and culling information was collected to estimate the annual economic losses caused by JD. An economic questionnaire was developed and administered to each herd annually to estimate costs directly attributable to the JD control program. Based on the costs of the control program, and using the losses to estimate the potential benefits of the control program, the net present value (NPV) of the control program was calculated for each herd during the study and projected into the future for a total of 20 years. The NPV was calculated for four different scenarios: (1) assuming a linear decline in losses beyond the observed period of the study with JD eradication by year 20 of the control program; (2) assuming losses and JD prevalence remain constant at the rate equal to that of the last observed year while continuing the control program; (3) assuming linear increase in losses at rate equal to that in scenario 1 with no control program; and (4) assuming losses remain constant at same level as the beginning of the study with no control plan implemented. The NPV varied greatly across the herds. For scenario 1, only three herds had a positive NPV; and only two herds had a positive NPV under scenario two. In the absence of a control program, the NPV's were always negative. The costs of the JD control programs implemented on these herds averaged $30/cow/year with a median of $24/cow/year. The annual losses due to JD averaged $79/cow/year with a median of $66/cow/year. Investing in a JD control program can be cost-effective.     

Relationships between the presence of Johne's disease and farm and management factors in dairy cattle in England

The data collected by a postal questionnaire sent to 3772 randomly selected dairy farmers in England and the border regions in Wales were used to estimate the relationships between the presence of clinical Johne's disease and farm and management factors associated with that disease. Two binary outcomes (case reported in 1993, case reported in 1994) and 27 predictor variables were considered. Only two variables were consistently and significantly associated with clinical disease in multivariable analysis. Farms on which Channel Island breeds were predominant were associated with an increased risk of reporting disease (odds ratios (ORs) ranged from 10.9 to 12.9). The presence of farmed deer on the farm also increased the risk of reporting disease (ORs ranged from 15.2 to 209.3). There were other significant but inconsistent associations involving the source of replacements, age of first-offering hay, type of concentrate feed to calves, and calving in individual pens when the cows were at grass. Since Johne's disease is predominantly subclinical, these contributing factors may play important roles in switching subclinical infection to overt disease.     

Current value of historical and ongoing surveillance for disease freedom: surveillance for bovine Johne's disease in Western Australia

‘Confidence’ in freedom from disease is generally derived from multiple sources of varied surveillance information, and typically this surveillance evidence has been accumulated over time. In the state of Western Australia (WA) the main surveillance evidence supporting Free Zone status in the national bovine Johne's disease (BJD) program comprises periodic surveys and the ongoing clinical diagnostic system. This paper illustrates a simple approach to current valuation of historical surveillance information, based on the calculated sensitivity of the surveillance processes, the time elapsed since the data were accumulated, and the probability of new introduction of disease into the population during that elapsed time. Surveillance system components (SSCs) contributing to the overall sensitivity of the surveillance system were the clinical diagnostic system and periodic targeted surveys. Sensitivity of each component was estimated using a stochastic scenario tree model of the surveillance process as implemented. Probability of introduction of BJD into WA during each time period was estimated retrospectively from a stochastic import risk analysis model applied to actual cattle importation data. The probability that the WA cattle population was free from infection (at design prevalences of 0.2% of herds and 2% of animals within an infected herd) was estimated following each of 11 years, giving a median probability that the State was free of BJD (at these design prevalences) at the end of 2005 of 0.89. The meaning of this result is discussed.     

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.     

Ovine Johne's disease: the risk associated with sheep imported into Western Australia

Objective To assess the risk of Johne's disease not being detected in sheep imported from New South Wales into Western Australia.
Design A stochastic simulation model.
Procedure The process of importing sheep was broken down into steps and numbers or probabilities assigned to each. Controls on the movement of sheep included surveillance tests in source flocks and serological tests on sheep in consignments before and after transportation to Western Australia. The model calculated the risk of occurrence of Johne's disease in Western Australia and the success of the agar gel immunodiffusion test in identifying consignments with infected sheep.
Results Negative surveillance tests in source flocks reduced the risk to about one twentieth of that when no surveillance tests were required. On average, Johne's disease was predicted to be introduced once in every 3 to 7 years when no testing of either the source flock or the sheep in consignments was required. When negative surveillance tests only were required the interval increased to once in every 63 to 111 years and, with the additional requirement that all sheep in each consignment must have a negative test before and after transport, the interval further increased to once in every 125 to 333 years. When only sheep in consignments were tested, the interval was calculated to be 8 to 14 years.
Conclusion A requirement that imports be derived from flocks which had negative surveillance tests to Johne's disease would provide significantly greater protection for the sheep industry in Western Australia.     

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.     

Benefits of obtaining test-negative Level 4 classification for beef producers in the Voluntary Bovine Johne's Disease Control Program

The US Voluntary Bovine Johne's Disease Control Program (VBJDCP) stipulates the national standards for Johne's disease (JD) control, and herds classified as test-negative at Level 4 of the VBJDCP have the greatest likelihood of being non-infected. A questionnaire survey of owners of VBJDCP test-negative Level 4 beef herds was conducted to describe perceived benefits of attaining Level 4 status. Thirty-nine of the 40 producers returned completed or partially completed surveys. Sixty-four percent (23/36) of herds contained 50 or less test eligible cattle. Twenty-seven percent (10/37) of producers reported increased marketing opportunities as a goal for enrollment in the VBJDCP. Classification at test-negative Level 4 status in the VBJDCP led to increased marketing opportunities for more than one-third (13/35) of the producers. Twenty-five percent (9/36) of the producers reported significant and 39% (14/36) marginal benefits (financial and non-financial) as a result of participation in the VBJDCP. The median (range) reported annual benefit was $0 ($0, $10,000), whilst the median (range) annual cost of implementing and sustaining the VBJDCP on ranches was $200 ($0, $5000). It is suggested that greater publicity about the VBJDCP in the beef cattle industry will increase its chances of success by increasing awareness amongst producers concerned about herd health/disease monitoring, and through improved marketing opportunities.     

Lesions attributed to vaccination of sheep with Gudair for the control of ovine paratuberculosis: post farm economic impacts at slaughter

OBJECTIVE: To determine i) the prevalence of lesions at the site of vaccination with Gudair, and ii) the incidence of discounting of slaughtered sheep carcasses due to the presence of these lesions. DESIGN: A survey of the prevalence of injection lesions and actual discounts applied to 20 consignments of vaccinates slaughtered in abattoirs in New South Wales. PROCEDURE: Consignments of sheep previously vaccinated against ovine Johne's disease (OJD) were assessed on the slaughter chain for the prevalence of vaccination site lesions and any costs associated with carcass trimming were estimated. In addition a telephone survey was conducted to determine the experiences and risk attitudes of 8 abattoirs in south east New South Wales likely to have previously processed vaccinates. RESULTS: The prevalence of lesions observed was 18% for adult (mutton) and 65% for lamb carcasses. The value of the trim removed was insignificant, the labour cost of its removal was nil and no carcass was downgraded to a lower value grade. CONCLUSION: Under the market conditions existing at the time the study was conducted, in sheep vaccinated at the recommended site high on the neck, it is unlikely that OJD vaccination site lesions will be a significant cost to producers or the processing industry and will represent only a very small proportion of the total cost of OJD control by vaccination.     

Sample handling substantially affects Johne's ELISA

Detection methods for Mycobacterium avium subsp. paratuberculosis (MAP) are imperfect, yet crucial for diagnosis of Johne's disease. Our purpose was to test for significant and biologically relevant changes in Johne's ELISA results associated with how field-collected blood samples were transported to the laboratory, prepared and stored prior to testing, while removing potential confounding by test kit and laboratory variables. Blood samples were collected from 21 cows that previously had MAP ELISA scores ranging from negative to highly positive. Samples for immediate laboratory processing were subjected to different transportation temperatures (on ice, 26 °C) and preparation methods (serum separated, hemolyzed and serum separated, clotted whole blood), but were tested using the same ELISA kit in the same laboratory. Samples for laboratory processing after one week of storage were subjected to different storage temperatures (4 °C, −20 °C) and preparation methods (serum separated, hemolyzed and serum separated, clotted whole blood), and again were tested using the same ELISA kit in the same laboratory. Finally, samples were evaluated by time to processing (one day, one week) and storage temperature (4 °C, −20 °C). Data were checked for normality and analyzed with repeated measures ANOVAs. Significantly (P = 0.027) higher MAP ELISA scores were recorded for whole blood and hemolyzed samples transported at 26 °C than serum separated samples. Sample storage for one week at −20 °C resulted in significantly (P < 0.001) lower MAP ELISA scores, regardless of handling method, compared to samples stored at 4 °C for one week. Method of sample preparation, as well as transportation temperature and medium-term storage temperature, affects MAP ELISA results. Such discrepancies will inevitably result in improper classification of MAP-infected cattle, impeding both biosecurity measures on uninfected farms and MAP control programs.     

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.     

The contribution of molecular biology to Mycobacteriumavium subspecies Paratuberculosis research

Molecular biology has contributed to our knowledge and understanding of the structure of Mycobacteriumavium subspecies paratuberculosis and has been particularly useful in determining those components that elicit immune responses in the host or discriminate M. avium paratuberculosis from other closely related environmental mycobacteria. As such, it has made a significant impact in the field of diagnosis, and has been instrumental in the development of specific and sensitive diagnostic tests. The next decade will see exciting new developments in paratuberculosis research as a consequence of substantial advances made in the construction of gene transfer systems in mycobacteria. These will provide opportunities for applying new strategies to determine the genetic basis for pathogenesis and the mechanisms of drug resistance and will offer new prospects for the rational design of efficient vaccines.