INTRODUCTION

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Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease or paratuberculosis, a granulomatous enteropathy of economic importance in ruminants throughout the world. Johne's disease is a chronic infection; in the end stages of the disease, large numbers of M. avium subsp. paratuberculosis organisms may be shed in feces, thereby contaminating the pasture and providing opportunity for transmission of infection to other hosts. Control of Johne's disease is achieved by identification and culling of infected hosts and sometimes entire herds or flocks. Diagnosis is based on serology and pathology, with culture of M. avium subsp. paratuberculosis from affected tissues being the definitive test. Culture from feces is also used to confirm infection in live animals.

For more than 2 decades a dramatic difference has been noted in most countries in the ease of culture of M. avium subsp. paratuberculosis from cattle and other ruminants compared to sheep. In Australia, culture from sheep has been largely unsuccessful. Similarly, in the United Kingdom, New Zealand, United States of America, Morocco, South Africa, and Iceland, isolation rates from sheep have been very low, or very lengthy incubations have been required to demonstrate scanty growth (Table 1). The existence of different strains of M. avium subsp. paratuberculosis in sheep and cattle might explain the above observations (20); it is already known that cattle and sheep tend to be infected with different genotypic variants of M. avium subsp. paratuberculosis (8). Complicating this interpretation, researchers in India, Iran, Spain, and China have reported reasonable success in culturing M. avium subsp. paratuberculosis from sheep (Table 1). This suggests that the reported differences in the culturability of strains of M. avium subsp. paratuberculosis from sheep might also be due to methodological differences between laboratories. Disinfection protocols and type of media are both known to influence in vitro survival and growth of M. avium subsp. paratuberculosis (35) and other mycobacteria (29). In one Indian study, hexadecylpyridinium chloride disinfection and culture on Herrold's egg yolk medium resulted in a 3.7% isolation rate (33), while in another Indian study, benzalkonium chloride disinfection and glycerol serum agar resulted in a 57% isolation rate (22). In Spain, M. avium subsp. paratuberculosis from sheep produced minute colonies that required a stereo microscope for visualization on Lowenstein-Jensen medium after up to 40 weeks of incubation (1, 20), whereas growth on Middlebrook 7H11 medium was read easily with the naked eye within 16 weeks (1).

Culture of M. avium subsp. paratuberculosis offers many advantages over other tests for confirmation of Johne's disease. It is a definitive test, it is cheaper than pathology-based confirmation, and it can be used to advantage in large-scale investigations. Recently we found that it was possible to culture M. avium subsp. paratuberculosis from tissues and feces of sheep after disinfection in hexadecylpyridinium chloride by using modified Middlebrook 7H9 radiometric medium (38). The aims of the present study were to evaluate this cultural method on a greater number of samples, to determine the sensitivity of culture in relation to the present "gold standard" test (histopathology), and to evaluate a range of solid media for the culture of strains of M. avium subsp. paratuberculosis that infect sheep in Australia.

	MATERIALS AND METHODS

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Clinical samples. Feces, mesenteric lymph nodes, and intestinal tissues were collected from sheep known or suspected to have Johne's disease. The status of all animals was evaluated by histological examination of terminal ileum and three other sites of ileum 2 m apart, cecum (one site), proximal colon (one site), and caudal mesenteric lymph node (one site). Feces were collected from the rectum of animals on the farm or during postmortem examination. Feces and tissues for culture were stored at 4°C overnight and then, if required, at 20 or 80°C pending examinations.

Acid-fast stained smears and histopathology. Smears were prepared from feces, scrapings of intestinal mucosa, and cultures; dried in an oven at 65°C: and stained by a Ziehl-Neelsen technique (13). Tissues were fixed in 10% buffered neutral formalin at the time of necropsy, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin and by a Ziehl-Neelsen method (23). Sheep with granulomatous enteritis were defined as having multibacillary Johne's disease if there were numerous acid-fast bacilli (AFB) in at least some high-power fields and as having paucibacillary Johne's disease when there were few or no AFB in any high-power field.

Tissue and fecal samples. (i) Group A. Sheep were selected based on clinical, serological, and/or pathological findings. Individual samples of ileum, or occasionally jejunum, from a group (n = 36) representing 34 different farms from New South Wales, Australia, were stored at 80°C for up to 14 months, thawed at 37°C, and then prepared for culture as described below. Twenty-two of these sheep had histological evidence of Johne's disease. Individual fecal samples from a different group (n = 29), representing nine different farms from the central tablelands district of New South Wales, were stored at 80°C for up to 11 months (n = 8) or for 4 days (n = 20) or at 20°C for 3 months (n = 1), thawed at 37°C, and then prepared for culture as described below. Twenty-six of these sheep had histological evidence of Johne's disease.

(ii) Group B. Sheep (n = 30) were selected based on knowledge of their histological classification. Samples of feces and ileum from each of 10 sheep with multibacillary Johne's disease, representing five different farms from New South Wales, and feces and tissues from each of 20 sheep with paucibacillary Johne's disease, representing the same five farms and another four farms, were cultured. These samples, which had been stored at 80°C for 1 to 4 months, were thawed at 37°C and then prepared for culture as described below.

(iii) Group C. Sheep (n = 31) from a farm with a high prevalence of Johne's disease were selected because of low body condition score. The farm was located in the southern tablelands district of New South Wales. Feces were collected and stored at 80°C for several weeks. There were 11 sheep with no intestinal lesions, 6 with paucibacillary Johne's disease, and 14 with multibacillary Johne's disease.

(iv) Group D. Sheep (n = 40) were selected from two farms based on their reaction in a serological test for Johne's disease. The two farms, located in the central tablelands district of New South Wales, were suspected to have sheep with Johne's disease. Twenty sheep were chosen from each farm on the basis of their having the highest apparent levels of anti-M. avium subsp. paratuberculosis antibodies as determined by an absorbed enzyme-linked immunosorbent assay. Terminal ileum and feces were collected from each sheep and stored at 80°C for up to 2 weeks before culture. Thirteen sheep, all from one farm, had histological evidence of Johne's disease; 11 had multibacillary disease while 2 had paucibacillary disease.

Preparation of samples and culture methods. The double-incubation method of Whitlock and Rosenberger (36) was used to prepare feces. Briefly, 2 to 5 g of feces was placed in a 15-ml polypropylene tube containing a swab stick. The stick was used to break up the feces in 10 to 12 ml of sterile normal saline. After mixing, the tube was allowed to stand for 30 min at room temperature. A 5-ml aliquot of the surface fluid was transferred to a 35-ml polystyrene tube containing 25 ml of 0.9% hexadecylpyridinium chloride (Sigma Chemical Co., St. Louis, Mo.) in half-strength brain heart infusion broth (Oxoid, Basingstoke, England) and allowed to stand at 37°C for 24 h. The tube was centrifuged at 900 × g for 30 min. The pellet was collected; resuspended in 1 ml of sterile water with vancomycin (100 µg/ml), nalidixic acid (100 µg/ml), and amphotericin B (50 µg/ml) (all Sigma reagents); and incubated for 48 to 72 h at 37°C.

Evaluation of need for additives in BACTEC 12B medium. The standard radiometric medium was based on those described by Collins et al. (10) and Cousins et al. (12) and consisted of enriched Middlebrook 7H9 medium (BACTEC 12B; Becton Dickinson, Sparks, Md.) with 200 µl of PANTA PLUS (Becton Dickinson), 1 ml of egg yolk, 5 µg of mycobactin J (Allied Monitor Inc., Fayette, Mo.) and 0.7 ml of water (Table 2). The requirement for egg, Mycobactin J, and PANTA PLUS in this medium was determined by omitting some or all of these additives; samples were from group A. Two tissue samples and two fecal samples containing high numbers of AFB were used in the first experiment. In a second experiment three fecal samples and three tissue samples containing high numbers of AFB, three fecal samples and three tissue samples containing moderate numbers of AFB, and four fecal samples and four tissue samples containing low numbers of AFB were cultured in BACTEC 12B medium either with all three additives or without any additive.

Evaluation of other media. BACTEC 12B medium consists of Middlebrook 7H9 broth with the addition of bovine serum albumin (0.5%, wt/vol), catalase (192 U per 4 ml), casein hydrolysate (0.1% wt/vol), and radiolabelled palmitic acid (Table 2). Solid media with similar composition were prepared with Middlebrook 7H10 and 7H11 agars (Difco) as the basal media (Table 2). A range of other media were selected either because they have been reported to support the growth of M. avium subsp. paratuberculosis from sheep or were selected empirically from media that have been used for culture of a diverse range of mycobacterial species.

Preparation of BACTEC samples and colonies for PCR. The preparation of BACTEC samples and colonies was conducted as previously described (38). Briefly, the rubber stopper-lid of the radiometric culture vial was disinfected with 70% ethanol, the contents were mixed by inverting the tube, and 200 µl of medium was removed and transferred to a microcentrifuge tube. Absolute ethanol (500 µl) was added, and the tube was left to stand for 2 min and then was vortexed for 5 s and centrifuged at 8 × g for 10 min at 22°C. The supernatant was transferred to a clean tube and then centrifuged at 18,000 × g for 5 min. The resulting bacterial pellet was washed twice in 200 µl of sterile phosphate-buffered saline, resuspended in 50 µl of sterile distilled water, and lysed at 100°C for 20 min. A 5-µl aliquot of the lysate was added to each PCR mixture. The lysate was then stored at 20°C. If PCR results were negative, lysates (45 µl) were thawed and DNA was purified from the entire lysate by binding to silica in a column using 6 M guanidine thiocyanate according to the manufacturer's instructions (Wizard PCR Preps DNA purification system; Promega Corporation, Madison, Wis.) and 5 µl of purified DNA solution was added to a second PCR mixture. For solid media, a crude suspension of DNA was obtained by suspending a colony in distilled water, washing the cells three times in water, suspending the cells in 100 µl of water, and boiling the washed cells for 20 min.

IS900 PCR. A reaction volume of 50 µl containing 5 µl of the DNA sample; 250 ng of each of the M. avium subsp. paratuberculosis IS900 primers, P90 (5'GAA GGG TGT TCG GGG CCG TCG CTT AGG) and P91 (5'GGC GTT GAG GTC GAT CGC CCA CGT GAC) (27); 200 µM concentrations of each of the nucleotides dATP, dTTP, dGTP, and dCTP; 66.8 mM Tris-HCl; 16.6 mM (NH₄)₂SO₄; 2.5 mM MgCl₂; 1.65 mg of bovine serum albumin per ml; 10 mM -mercaptoethanol; and 2 U of Taq polymerase, in buffer (10 mM Tris-HCl, 0.1 mM EDTA [pH 8.8]) was used. Amplification was undertaken in 200-µl tubes in a 96-place thermal cycler (Corbett Research, Sydney, Australia) under the following conditions: one cycle of denaturation at 94°C for 2 min followed by 37 cycles of denaturation at 94°C for 30 s, annealing at 62°C for 15 s, and extension at 72°C for 1 min. Products of approximately 400 bp were predicted and evaluated by electrophoresis at 94 V for 45 min in 2% agarose gels stained with ethidium bromide. The specificity of the reaction for IS900 was confirmed during optimization experiments by Southern hybridization with an internal probe and then routinely by restriction endonuclease analysis of the PCR product with AlwI.

	RESULTS

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Evaluation of modified BACTEC 12B radiometric medium. Samples of tissues and feces (group A) from sheep from numerous farms were evaluated to ensure that culturability was not restricted to unrepresentative strains of M. avium subsp. paratuberculosis. A GI of >999 was recorded from 27 of 36 (75%) tissue cultures after 2 to 6 weeks of incubation and from 22 of 29 (76%) fecal cultures after 3 to 6 weeks of incubation (Table 3). Culture from feces of sheep with paucibacillary disease appeared to be less efficient than that from sheep with multibacillary disease, and histopathology appeared not to detect all infected individuals from affected farms (Table 3). These findings were confirmed in experiments with samples from groups B, C, and D, which comprised sheep selected for culture based on histological, clinical, and serological criteria, respectively.

Requirement for mycobactin J, egg yolk, and PANTA PLUS in BACTEC 12B medium. In the first experiment, for which all three additives were included, all tissue and fecal cultures produced GIs of 999 within 4 to 5 weeks, and the GIs remained high in later weeks. Similar results were obtained without PANTA PLUS. In the absence of mycobactin J, egg yolk, or any additive, neither fecal culture developed a GI while both tissue cultures developed a low GI, which peaked at week 1 to 2 and then declined. In the second experiment, a GI of 999 occurred in 8 of the 10 fecal cultures by week 6 if all additives were present, but growth did not occur in any fecal culture in the absence of the additives. A GI of 999 was present in all 10 tissue cultures by week 7 in the presence of the three additives, while no growth was recorded for 7 of these cultures in the absence of the additives. The other three cultures had very low GIs (12 to 74) at week 3, which then declined. Samples with poor growth included those that contained high or medium numbers of AFB. In all cases GI was associated with positive PCR results. It was concluded that both egg yolk and mycobactin J were essential for isolation of M. avium subsp. paratuberculosis from feces and dramatically improved the growth from tissues.

Evaluation of solid media. In the first experiment modified BACTEC 12B medium was converted to a solid medium slope by the addition of agar. Two weeks after inoculation a GI of 999 was recorded in all 10 vials that had been inoculated with medium from primary BACTEC 12B cultures, and after 4 weeks all 10 slopes were covered with numerous small (diameter, <1 mm), white, circular, raised, convex, shiny colonies. Colony diameters were 1 mm at 6 weeks and did not change in appearance thereafter. A colony from each vial was confirmed to be M. avium subsp. paratuberculosis by IS900 PCR. This experiment confirmed the potential for growth on solid medium and revealed colony morphology, but the high cost of BACTEC medium would preclude its use in this manner.

	DISCUSSION

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The culture of M. avium subsp. paratuberculosis from sheep generally has been an unrewarding task (Table 1), despite the fact that the organism can be cultured readily from other ruminants with Johne's disease. The reasons for the difference in culturability are probably related to the existence of different strains of M. avium subsp. paratuberculosis in the different ruminant hosts, a feature of the biology of the organism which has been confirmed by genomic analysis (4, 8). In Australia, where M. avium subsp. paratuberculosis has been frequently isolated from cattle but almost never cultured from sheep, distinct strains of M. avium subsp. paratuberculosis are present in cattle and sheep populations (11, 37). However, the work reported here indicates that culture of M. avium subsp. paratuberculosis from sheep is possible if appropriate media are used. Three closely related basal media, namely, Middlebrook 7H9, 7H10, and 7H11, were found to support the growth of ovine strains of M. avium subsp. paratuberculosis when supplemented appropriately. Middlebrook 7H11 agar has been used with success for the culture of M. avium subsp. paratuberculosis from sheep in Spain, but egg yolk and mycobactin J were not required (1).

Culture from intestinal tissues was more sensitive than culture from feces, because feces from some sheep with paucibacillary Johne's disease were culture negative. For some sheep that lacked histological evidence of Johne's disease tissues but not feces were also culture positive. Where matched radiometric cultures of feces and tissues from the same sheep were undertaken, no cultures were positive from feces and not tissues. The success of culture from feces is clearly related to the likelihood that sufficient numbers of M. avium subsp. paratuberculosis are being shed from the intestinal lesions. A similar trend was noted in a Spanish study where the success of culture from tissues was directly related to the number of AFB observed in lesions (30).

Egg yolk and mycobactin J were essential additives for isolation of ovine strains of M. avium subsp. paratuberculosis in Middlebrook 7H9 broth (as modified BACTEC 12B radiometric medium). The specific components of egg yolk that stimulate growth are not yet known. PANTA PLUS does not appear to affect the growth of M. avium subsp. paratuberculosis from sheep but might play a useful role in reducing contamination of the cultures.

Colonies of M. avium subsp. paratuberculosis were reliably obtained for the first time in Australia with solid media based on Middlebrook 7H10 and 7H11 agars, after growth on the surface of solidified BACTEC 12B radiometric medium was first demonstrated. All three media were of very similar composition, although the modified BACTEC 12B radiometric medium had lower concentrations of most ingredients, including egg yolk and mycobactin J, than did the two agar media (Table 2). Further experiments to determine optimal concentrations of these ingredients may be worthwhile.

The colonies on modified 7H10 agar developed to a maximum diameter after about 6 weeks, and their size was inhibited by crowding. The colonies were easily visualized, but they were made more obvious by including methylene blue in the medium. Detection of growth on solid medium was slower than that in modified BACTEC 12B medium and also appeared to be less sensitive. Of the animals with paucibacillary Johne's disease, 60% were identified by culture of feces in modified BACTEC 12B, compared to 50% by culture on modified 7H10 or 7H11 agar. Although this difference was not statistically significant, if proven in a larger study, it would tend to favor the use of the more expensive liquid radiometric medium. Liquid radiometric culture also appeared to be more sensitive than culture on solid medium for diagnosis of Johne's disease in cattle (38).

A range of solid media have been used to culture M. avium subsp. paratuberculosis from sheep in other countries, but none of these supported the growth of M. avium subsp. paratuberculosis from Australian sheep in this study. Although some of the media have been evaluated in Australia in the past, mostly with negative results, several isolates of M. avium subsp. paratuberculosis were obtained in New South Wales in the 1980s from Herrold's egg yolk medium cultures of sheep intestinal tissues. Some of these isolates have been shown to be bovine strains by restriction fragment length polymorphism analysis using IS900 probes (11, 37). This observation and the fact that M. avium subsp. paratuberculosis can be cultured from sheep on a range of solid media in several countries (Table 1) suggests that there are a number of variants of M. avium subsp. paratuberculosis in sheep populations and that these have quite different cultural requirements. It is also possible that some isolates obtained from sheep in some studies are not strains of M. avium subsp. paratuberculosis. However, it would be useful to compare a range of isolates from different countries under standard conditions, because the pattern of culturability on different media might be a useful phenotypic typing method for epidemiological purposes. For example, M. avium subsp. paratuberculosis has been isolated from sheep in Spain on Herrold's egg yolk medium, Lowenstein-Jensen medium, and Middlebrook 7H11 agar without egg yolk (1, 20, 30). Only the latter medium supports the growth of M. avium subsp. paratuberculosis from sheep in Australia, and then only when egg yolk is added. Furthermore, Spanish isolates of M. avium subsp. paratuberculosis from sheep do not require mycobactin J (1) whereas those from sheep in Australia display the conventional dependence on mycobactin J. This suggests fundamental differences between the strains of M. avium subsp. paratuberculosis present in sheep in Australia and Spain.

The development of a GI in radiometric medium inoculated with samples from sheep with Johne's disease does not guarantee the presence of M. avium subsp. paratuberculosis. There are now several options for confirmation of M. avium subsp. paratuberculosis in radiometric culture medium. Detection of IS900 by PCR on an aliquot taken from the primary culture is a rapid and effective test but is quite costly. Subculture from the primary radiometric culture to modified Middlebrook 7H10 agar with and without mycobactin J to check for the presence of colonies of appropriate morphology containing acid-fast organisms with mycobactin dependence is an alternative and cheaper method. However, a further incubation of at least 4 to 6 weeks is required, during which overgrowth by irrelevant microorganisms may be a problem. Environmental mycobacteria and other contaminants can be detected in radiometric medium by concurrent subculture to a general-purpose medium such as Lowenstein-Jensen agar. In Australia, the strain of M. avium subsp. paratuberculosis can be determined provisionally by concurrent subculture to Herrold's egg yolk medium, where no growth would be expected from typical ovine strains of M. avium subsp. paratuberculosis.

The development of a solid medium for culture of M. avium subsp. paratuberculosis from sheep in Australia has benefits other than those immediately applicable to diagnosis. It will now be possible to produce clonal isolates for antigen production for use in serological tests and vaccines and for in vivo inoculation experiments. It will also be possible to evaluate enumeration methods based on colony counts.

In conclusion, the results of this study confirm that culture of M. avium subsp. paratuberculosis from intestinal tissues and feces of sheep is a sensitive method of diagnosis of ovine Johne's disease. It is particularly notable that some sheep were detected by culture and not by histopathology of intestinal tissues, the current gold standard diagnostic test in Australia. These results support the preliminary data from an earlier study (38) and suggest that culture of M. avium subsp. paratuberculosis should be the test against which the diagnostic performance of other laboratory tests for ovine Johne's disease is assessed.