Compared to serology, the benefits of pooled fecal culture for flock diagnosis of ovine paratuberculosis are increased diagnostic sensitivity and a large reduction in cost. Pooling of samples is possible due to the massive numbers of M. avium
present in the feces of sheep with multibacillary disease. The average rate of excretion of M. avium
in such sheep was 1.09 × 108
organisms per g of feces (26
). As the analytical sensitivity of similar culture methods has been estimated to be 100 CFU per g of feces (3
), the pooling rate could be much greater than that used in this study without a loss of detection of multibacillary cases, and it was not surprising that all 20 multibacillary cases were detected when feces were diluted 1 in 50. However, paucibacillary cases may comprise a significant proportion of the cases of paratuberculosis in a flock, and fecal culture for such cases was susceptible to the dilution effect caused by pooling of samples. In an earlier study of undiluted feces with the same culture methods, a lower isolation rate (48.4%) was reported for paucibacillary cases (24
), confirming that the proportion of paucibacillary cases in a flock is important in determining the sensitivity of pooled fecal culture. Another factor limiting the pooling rate is the efficacy of mixing of pooled samples. It is essential to ensure that pooled samples are completely mixed, and this goal can be difficult to achieve. In this study, mixing was achieved most easily and completely using a high-speed blending apparatus.
Pooled fecal culture is a sensitive method for flock diagnosis of ovine paratuberculosis. In experiment 2, about 93% of 27 infected flocks were detected by pooled fecal culture, a remarkable outcome given that flock infection status was derived from lengthy historical information, often based on the results of sequential field and laboratory investigations. In many instances, a positive culture result was obtained from a single pool of 50 fecal pellets. Overall, pooled fecal culture appeared to be considerably more sensitive than serologic examination for detection of flock infection. In experiment 2, more than three times as many flocks were detected by pooled fecal culture than by serologic examination of the same sheep, while in experiment 3, with a much larger sample size, about 1.5 times as many farms were detected by pooled fecal culture than by serologic examination.
The absolute value for the diagnostic sensitivity of pooled fecal culture is not known but lies somewhere between the sensitivities of detection of individual multibacillary cases (83 to 100%) and paucibacillary cases (27 to 73%) when feces are pooled at a rate of 1 in 50. Thus, the probability of detection of flock infection is very high if multibacillary cases are present in a pool. In a separate study, excretion of M. avium
by sheep with multibacillary disease was shown to be continuous, so that one would expect a single fecal sample from such sheep to be satisfactory (26
). The proportion of multibacillary cases would be expected to vary from flock to flock and would be affected by factors such as time since introduction of infection to the flock, contamination levels in the pasture, and age of sheep but may reach 70% of clinical cases (4
The data obtained in experiments 1 and 4 permit analysis of the sample sizes required to detect infection using pooled fecal culture. Using conservative values of sensitivity of 95 and 40% for multibacillary and paucibacillary cases, respectively, and a 20:80 distribution of multibacillary and paucibacillary cases in a flock, the average sensitivity value is (0.95 × 0.2) + (0.4 × 0.8) = 0.51. Using an estimate of 50% for the average sensitivity of pooled fecal culture at the individual animal level, a sampling rate of 300 animals (six pools of 50) would provide a level of assurance exceeding 95% confidence of detecting a prevalence of infection of ≥2%. In experiment 4, the sample sizes required to detect flock infection were evaluated by statistical modelling of randomly sampled pools for 14 known infected flocks. Culturing of four pools was needed to be 95% certain of detecting infection in 13 flocks, and culturing of eight pools was needed for the other flock, which had a low prevalence of infection.
Despite pooled fecal culture having higher sensitivity than serologic examination, the results of this study suggest that it may be difficult to diagnose infection with pooled fecal culture in flocks with a low prevalence of sheep with ovine paratuberculosis. Several low-prevalence flocks were included in experiment 2, and pooled fecal culture failed to detect infection in one. Infection was confirmed in this flock only by intensive investigation conducted during another research trial. Three of 20 sheep in this flock were selected based on weak reactivity in an ELISA for anti-M. avium subsp. paratuberculosis antibodies, and 1 was found culture positive (intestinal samples) in the absence of histologic lesions of paratuberculosis. Despite these observations, in experiment 3, more than 30 flocks were found to be infected by pooled fecal culture, despite an apparent lack of clinical and serologic evidence of M. avium subsp. paratuberculosis infection. There were objective reasons to believe that these flocks were infected, and it is likely that many had either a very low prevalence of infection or sheep only in the early stages of the disease. Several have since been confirmed infected. Conversely, several flocks that were known to be infected based on serologic and histopathologic test results were not detected by pooled fecal culture (1 of 14 infected farms in experiment 2 and 5 of 63 infected farms in experiment 3). Thus, no single test at a single point in time can be used to rule out the presence of M. avium subsp. paratuberculosis infection in a flock of sheep. A high level of assurance that a flock is not infected with M. avium subsp. paratuberculosis can be gained only after successive negative tests.
is an obligate pathogen and parasite of animals that is identified by slow growth, mycobactin dependency (22
), and the presence of IS900
). The specificity of pooled fecal culture is assumed to be equivalent to the specificity of culture in confirming the taxonomic characteristics of M. avium
, that is, 100%. Although we have no evidence that any of the following occurred, factors that could affect apparent specificity include misidentification of individual sheep or flocks, sample cross contamination in the field, sample mislabeling in the field, sample mislabeling and transcription errors in the laboratory, sample cross contamination in the laboratory at any stage of the testing protocol, false-positive reactions in PCR, and errors in reporting from the laboratory. These potential problems can be prevented by quality control protocols, care, and common sense.
There are several explanations for the presence of M. avium
in fecal samples from individual animals or groups of animals that lacked other clinicopathologic evidence of infection. First, fecal shedding may occur in the absence of an antibody response. Excretion in feces is known to occur months before seroconversion in sheep (2
). Second, fecal shedding may occur in the absence of detectable histologic lesions regardless of serologic status. Culturing of feces or intestinal tissues from individual sheep has been shown to be a more sensitive indicator of the presence of M. avium
than histopathologic examination for infected flocks (24
). One obvious reason for this finding is the focal nature of lesions in some sheep and the relatively small amounts of tissue that can be examined histologically. Third, in infected flocks, there may be passive excretion of ingested M. avium
. This excretion may continue for up to 1 week after ingestion (8
). Another factor that could affect the apparent specificity of culture is the existence of strains of M. avium
of low virulence, but there is no evidence for such strains, and variations in virulence are not considered in the description of the taxon. The validity of the results obtained during experiment 3 is supported by the matching of positive pooled fecal culture results with districts of New South Wales that are known to contain flocks infected with M. avium
and the absence of positive results from most other districts.
Results for nine farms (11 pools) in experiment 3 were classified inconclusive by pooled fecal culture. This classification was necessitated by technical limitations in the conduct of REA, namely, insufficient DNA to conduct the test in agarose gels with ethidium bromide staining. REA is necessary to increase the level of confidence that a positive result by PCR is in fact due to the DNA from M. avium
). Alternate methods would enable the conduct of REA even when only small amounts of DNA are available. One of the simplest would be to evaluate REAs in acrylamide gels with silver staining. It is important to note that when REA was performed on the IS900
PCR product in this study, results consistent with M. avium
were obtained in every instance.
The collection and examination of fecal pellets from large groups of sheep for the purpose of diagnosing paratuberculosis have not been undertaken before, and it was therefore considered important to assess the practicality of the technique. Most respondents to the survey noted that collection of fecal samples was practical unless sheep had been kept in yards too long and had already defecated. Transport of fecal samples from farm to laboratory is an important issue. Efforts are required to ensure that the interval between sampling and receipt at the laboratory is as short as possible, preferably overnight, and samples should not be left in transit over a weekend in order to minimize problems with growth of irrelevant microorganisms (R. J. Whittington, unpublished data). The practicality of laboratory aspects of pooled fecal culture is similar to that of individual sample culture, but there is an additional requirement for facilities and equipment to carry out homogenization of the pooled pellets, a rate-limiting step. Facilities are therefore required to store fecal samples pending homogenization, and at present it is suggested that storage be done at −80°C to minimize the loss of viability of M. avium
In considering the requirements for sampling from a flock, the necessity for randomization should not be overlooked unless whole-flock examination is undertaken. Biased samples did not improve the rate of detection in this study (Table ), in contrast to earlier reports on the benefits for serologic diagnosis of using samples biased toward sheep with poor body condition (12
). In the present study, sheep that were positive by the pooled fecal culture test generally had no clinical signs of paratuberculosis and may have been in uniform body condition within farms; when biased selection was used, it may have been based merely on age. In other words, the infected sheep were likely to have been in relatively early stages of the disease and/or the prevalence of infection was low. Under such conditions, the biasing of collection in favor of sheep with poor body condition would not be effective.
The costs of pooled fecal culture are lower than those of serologic testing. Collection costs (in Australian dollars) are approximately $0.50 per sheep for feces compared to $1.00 per sheep for blood, excluding the costs of containers, needles, and syringes. Serologic testing is undertaken for approximately $6 per head, compared to $2 per head for pooled fecal culture at a pooling rate of 50. Thus, the costs of flock diagnosis using pooled fecal culture are approximately 30% of those using serologic testing, assuming that equal numbers of sheep are tested. The apparent higher sensitivity of pooled fecal culture also means that fewer sheep need to be sampled to provide equivalent confidence of detecting infected flocks, further reducing costs. Considering that there are over 30,000 flocks of sheep in New South Wales, Australia, and that 30% of these might require testing, the economic benefits of pooled fecal culture are considerable.