The postmortem diagnosis of the infection in its early stages has presented additional challenges, as mac-

Transcription

1 Vet Pathol 36: (1999) Bacterial Isolation, Immunological Response, and Histopathological Lesions during the Early Subclinical Phase of Experimental Infection of Goat Kids with Mycobacterium avium subsp. paratuberculosis Ó. G. SIGUR-D ARDÓTTIR, C. MCL. PRESS, F. SAXEGAARD, A Ø. EVENSEN Departments of Pathology (ÓGS, ØE) and Bacteriology (FS), National Veterinary Institute, Oslo, Norway; and Department of Morphology, Genetics and Aquatic Biology, Norwegian College of Veterinary Medicine, Oslo, Norway (CMP) Abstract. The diagnosis of Mycobacterium avium subsp. paratuberculosis infection is difficult, especially in the early stages of disease. This is due to the long incubation period, the variable lag phase associated with bacterial proliferation, and the multifocal distribution of slowly developing lesions. There are few previous studies of the early stages of experimental paratuberculosis in goats. In the present study, the ability of conventional diagnostic methods to detect M. a. paratuberculosis infection during the early stages of infection was assessed. Eight goat kids were experimentally infected with M. a. paratuberculosis and subjected to a series of immunological and bacteriological tests before being euthanatized at various times postinfection. At postmortem examination, the ages of the kids ranged from 1½ to 12 months. Of the eight goats infected, three had histopathological evidence of paratuberculosis. Two of these goats were positive with bacteriology, but only one was also positive with all immunological tests. One animal had a positive immunological response, but infection could not be demonstrated by bacteriologic or histopathologic examination. Histopathologic lesions were found in the jejunum, in the ileum, and in one mesenteric lymph node, but only the mesenteric lymph nodes and one retropharyngeal lymph node gave positive results following bacteriologic culture. The disparity between the localization of histopathologic lesions and bacteriologic results emphasizes the need for exhaustive sampling to confirm a diagnosis during the early phase of an infection. It also highlights the need for a better understanding of the biology of M. a. paratuberculosis and its interaction with the immune system of the host. Key words: Goats; granulomatous inflammation; Mycobacterium paratuberculosis; paratuberculosis. Paratuberculosis is a worldwide chronic infectious disease of primarily domestic ruminants. It is caused by Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis). Animals are infected at an early age, but disease usually does not become apparent until they are 2 years of age or older. 1,4,5 Most infected animals are able to eliminate the infection, but if an infection becomes established, there is initially a subclinical stage, followed by the clinical stage. 1,4,5,8,19 Paratuberculosis has not been as extensively studied in goats as in cattle and sheep, but the disease appears to be similar in these species. Studies have reported that focal infiltrates of neutrophilic leukocytes are present in the submucosa of the intestine a few days after experimental infection, and macrophages and multinucleated giant cells appear soon after. With time, the multifocal submucosal lesions expand, coalesce, and become visible grossly. Lesions are found in association with the lymphoid follicles of the Peyer s patches and are most likely to be encountered in the ileum. Similar inflammatory changes occur in the draining lymph nodes. Very few acid-fast bacilli are seen in early stages, but their number increases with the expansion of lesions. 4,5,13,21 In vivo diagnosis of M. a. paratuberculosis infection is difficult in the preclinical stages of the disease. Diagnostic tests include the bacteriologic examination of feces and immunological tests measuring humoral and cell-mediated immune responses. These diagnostic tests have low to moderate sensitivity and variable specificity. Diagnostic testing is more useful on a herd basis than in testing individual animals, and a combination of two or more tests is recommended. 3,4,13,19 The complement fixation test has been successfully employed in mapping the disease in goat herds in Norway. 8 The definitive diagnosis of paratuberculosis is based on the cultivation of M. a. paratuberculosis and/ or identification of specific gene segments, since histopathology cannot differentiate the mycobacterial infections. 4,6 The postmortem diagnosis of the infection in its early stages has presented additional challenges, as mac- 542

2 Vet Pathol 36:6, 1999 Subclinical Paratuberculosis in Goat Kids 543 roscopic lesions are usually absent and the multifocal distribution of small granulomas makes the histopathologic identification of lesions difficult and exhaustive. Bacteriologic methods have also proven to have low sensitivity. 4,7,19 Most studies of experimental M. a. paratuberculosis infection have not focused on the early stages of the disease. 10,13,17,21 However, in studies where the time of infection was known, M. a. paratuberculosis has been detected in tissues as early as days postinfection (PI). 11,12 The aim of the present study was to establish an experimental infection with M. a. paratuberculosis in goat kids and to compare the localization and nature of early histopathologic lesions with the results of bacteriologic examination and other diagnostic tests. Materials and Methods Animals and experiment design Twelve goat kids of the Norwegian breed were used in this study. The goats came from a herd with no history of paratuberculosis, but where the animals were regularly vaccinated against the infection. The goats were taken from their dams immediately after birth and fed milk from suckling bottles the first weeks of their lives. This was done to avoid a possible infection with caprine arthritis encephalitis virus. The goats were infected with M. a. paratuberculosis between 7 and 26 days of age. A bacterial suspension of M. a. paratuberculosis was added to their milk. Eight goats were infected and four goats were used as control animals. Each experimentally infected goat received 10 mg dry weight of bacteria for 10 consecutive days, a total of 100 mg bacteria per animal. The kids were fed cow colostrum the first week of life and then soured cow milk until 6 weeks of age. Hay was available to the kids from the third week of life, and from the fourth week, they received concentrates. When the experimental infection commenced, the goats were divided into two groups and kept in separated rooms with no contact between the two groups. The experimentally infected goats were euthanatized 3, 5, 9, 11, 17, 24, 37, and 49 weeks PI, at the ages of 1½, 2,2½, 3, 4, 6, 9, and 12 months, respectively (subsequently referred to as goat Nos. 1 8). The control animals were euthanatized when 1½, 2½, 6 and 12 months old, respectively (subsequently referred to as goat Nos. 9 12). Bacterial strains and culture methods A pool of secondary and tertiary cultures of five strains of M. a. paratuberculosis was used in this study. Bacteriologic procedure and bacterial identification was as described by Saxegaard. 17 The strains were isolated from the intestines and mesenteric lymph nodes of clinically affected goats in natural outbreaks of paratuberculosis. The pathologic material from these goats was decontaminated with 5% oxalic acid and inoculated on Dubos serum-agar medium with and without antibiotics, 18 and containing 2% mycobactin P extracted from Mycobacterium phlei (National Veterinary Institute, Oslo, Norway). The bacterial cultures were scraped from the culture media, finely ground in a mortar, and 100 mg dry weight was suspended in 1 ml of 0.1 M phosphatebuffered saline (PBS de Boer) solution (ph 7.2). The suspension was diluted further in PBS de Boer, divided into 10 portions, and stored at 4 C until inoculation was performed. A PCR technique was used to confirm the identities of the bacterial strains used. In brief, cultured mycobacteria were disrupted using a Mini-Beadbeater cell disrupter (Biospec Products, Bartlesville, OK) and zirconium beads. A 2- l sample was inoculated into 48 l of a prepared reaction mixture containing 67 mm Tris-HCl (ph 8.8), 2 mm MgCl 2, 100 M each of the four deoxyribonucleotide triphosphates, 0.2 M each of the oligonucleotide primers and 0.5 U of Taq DNA polymerase (Perkin Elmer). The reaction mixes were overlaid with 50 l of liquid paraffin and subjected to an initial denaturation step of 5 minutes followed by 35 cycles of amplification under the following conditions: denaturation at 94 C for 1 minute, annealing at 55 C for 1 minute, and extension at 72 C for 1 minute in a thermal cycler. The primers for IS 900 were P11: 5-CGTCGTTTATTACCA- TGCAG-3 and P36: 5-GGCCGTCGCTTAGGCTTCGA- 3; 15 which were derived from positions of the IS 900 sequence. 9 The amplified products were detected by agarose gel electrophoresis, stained with ethidium bromide, and visualized by ultraviolet transillumination. IS 900 was identified by the appearance of a 278-bp band of DNA. 9,15 The five strains were found to be IS 900 positive. Antemortem examination Diagnostic tests. All animals were examined for immunologic evidence of paratuberculosis infection by using a delayed-type hypersensitivity skin test, a serum complement fixation test (CFT), and an ELISA test. The animals were tested every month, from 1 to 11 months PI. The control animals were also tested. Delayed-type hypersensitivity test. A delayed-type hypersensitivity (DTH) test was performed by using an intradermal injection of 0.1 ml of Norwegian standardized avian tuberculin supplied by the National Veterinary Institute, Oslo, Norway. The test was performed according to the manufacturer s instructions. The injection was performed once a month, alternately in the left flank and the right flank. Preinjection skin fold thickness, measured with slide callipers, varied from 2.8 to 5.4 mm. The reaction was read after 48 and 72 hours. An increase of 3.5 mm or more was considered a positive reaction. Serological tests. Jugular blood samples were collected, allowed to clot, and then centrifuged. The serum was harvested and stored at 70 C until tested. A CFT was performed on the serum as described by Saxegaard 17 and tested in dilutions of 1 : 5, 1 : 10, and 1: 20. An indirect ELISA test was performed according to the manufacturer s instructions, including the recommended performance controls (Herd Check Mpt Ab, IDEXX Laboratories, Inc., Westbrook, ME). Bacteriology. Blood and fecal samples from the experimentally infected animals were cultured at monthly intervals. Fecal samples of two to three pellets of manure were collected on each of the first 3 days after the infection, and blood samples were collected on the first day after the infection. Middelbrook 7H9 broth (DIFCO Laboratories, PO Box

3 544 SigurUardóttir, Press, Saxegaard, and Evensen Vet Pathol 36:6, , Detroit, MI ) with 2% mycobactin J (Allied Monitor, Inc., PO Box 71, Fayette, MO 65248) was added to the blood samples, and 2 weeks later the samples were sown onto the Dubos serum-agar medium. The culture procedure previously described was applied to the blood and fecal samples, except that mycobactin P was replaced by mycobactin J. Postmortem examination The goats were euthanatized with intravenous pentobarbital. Samples of the internal organs were collected for bacteriology, and other samples were fixed in 10% neutral buffered formalin for histopathologic studies. Bacteriology. Samples from the distal jejunum and/or ileum, colon, tonsil, and liver and from the parotid, medial retropharyngeal, mandibular, ileocolic, jejunal, colic, hepatic, and pancreoduodenal lymph nodes were collected for bacteriology. Bacteriologic examination of the organ material was performed as previously described, except that mycobactin P was replaced by mycobactin J. Histopathology. For histopathology, the same organs were collected as for bacteriology. In addition, samples were taken from the rumen, abomasum, duodenum, cecum, rectum, thymus, spleen, heart, lung, kidney, pancreas, and adrenal glands and from the renal, mediastinal, and superficial inguinal lymph nodes. Tissues collected from the intestine were as follows: proximal and distal duodenum and the proximal, mid-, and distal jejunum, four sites from the proximal to the distal ileum, two sites from the cecum, the proximal, mid-, and distal colon, and the proximal and distal rectum. The formalin-fixed material was processed by routine paraffin embedding, and 2 3-m-thick sections were cut, mounted, and stained with haematoxylin and eosin (HE) and the Ziehl-Neelsen (ZN) method for acid-fast bacilli. Immunohistochemistry. Formalin-fixed paraffin-embedded sections of jejunum, ileum, and one lymph node with histopathologic lesions were cut at 2 3 m and laid on gelatincoated slides for immunohistochemistry. Sections were deparaffinized at 56 C, washed in xylene, and rehydrated through a series of ethanol solutions (100%, 96%, 70%) at room temperature. All of the subsequent incubations were performed at room temperature, and all washings were for 5 minutes in Tris-buffered saline (ph 7.4). The procedure was as previously described. 20 In brief, blocking was performed with 5% bovine serum albumin (BSA) (Sigma Chemical Co., PO Box 14508, St. Louis, MO 63178) for 20 minutes. The sections were then incubated for 1 hour with rabbit anti M. paratuberculosis serum (DAKO A/S, Glostrup, Denmark), diluted at 1 : 4,800, and then washed. Biotinylated goat anti-rabbit immunoglobulin (DAKO A/S, Glostrup, Denmark), diluted at 1 : 300, was then applied as the secondary antibody for 30 minutes. After washing, sections were incubated for 30 minutes with Streptavidin alkaline phosphatase (Amersham International plc, Amerham Place, Little Chalfont Buckinghamshire, England HP7 9NA), diluted at 1 : 1,000, and then washed. Sections were then incubated with a filtered solution of Fast red (Sigma Chemical Co., PO Box 14508, St. Louis, MO 63178) for 20 minutes, washed and counterstained with haematoxylin for 2 minutes, and mounted in an aqueous mounting medium. As some granulomatous lesions were small, tissue sections were not available for immunohistochemistry from all identified lesions. Immunohistochemistry was performed on 12 of 16 identified histopathologic lesions ascribed to M. a. paratuberculosis infection. Performance controls included application of a nonimmune serum (normal rabbit serum, 0.5 mg IgG/ml) at the same dilution as the immune serum. Tissue sections from a known positive animal were used as controls and incubated with both immune and nonimmune serum. Estimation of the end point dilution value (the highest dilution giving a positive reaction that was discernible from background) was performed for antibody solutions using 60-minute incubation at 37 C. 20 Antemortem examination Results Clinical symptoms. None of the goats showed clinical symptoms of paratuberculosis. One goat developed diarrhea at a few days of age. The condition resolved itself several days before the animal was experimentally infected. Goat No. 3 had transient, slightly softer stools 6 days after the bacterial inoculation was started. Goat No. 8 developed generalized seborrhea at the age of 8 months. The seborrhea was most noticeable around the eyes and on the pinnae. The animal received no treatment, and the condition gradually improved but never completely disappeared. No other clinical abnormality was noted in the goats. Diagnostic tests Delayed-type hypersensitivity test. Two goats were positive by the DTH test (Table 1). Goat No. 7 had positive reactions 6 and 8 months PI, and goat No. 8 had a positive reaction 3 months PI. The other infected animals (Nos. 3, 4, 5, and 6) and the three tested control animals (Nos. 10, 11, 12) were negative at all times. Two of the infected animals (Nos. 1 and 2) and one control (No. 9) were not tested. Serological tests. Two experimentally infected goats were serologically positive (Table 1). With the CFT, goat No. 7 was positive 5 and 6 months PI, and goat No. 8 was positive 10 and 11 months PI. With the ELISA test, only goat No. 7 was positive 8 months PI. Serum for the ELISA test was not available from goat No. 12 at 11 months of age or from goat No. 8 at 10 and 11 months PI. All goats were tested with ELISA 1 day before infection, and the results were negative for all animals. Bacteriology. All blood and fecal samples were negative following bacteriologic examination for M. a. paratuberculosis.

4 Vet Pathol 36:6, 1999 Subclinical Paratuberculosis in Goat Kids 545 Table 1. Results of diagnostic tests and pathological findings for goats infected with Mycobacterium avium subsp. paratuberculosis.* Goat No. No. Infected No. Control Age at Infection (days) Age at Euthanasia Months 1½ 2 2½ ½ 2½ 6 12 Weeks PI Serology CFT ELISA DTH Test Bacteriology Histopathologic Histochemical Staining Lesions (paratuberculosis) ZN IHC *Cf complement fixation test; not done; DTH test delayed-type hypersensitivity test; ZN Ziehl-Neelsen staining; IHC immunohistochemical staining; PI postinfection. Postmortem examination Macroscopic lesions. The body conditions of all animals were good at postmortem examination. The only macroscopic lesion suggestive of M. a. paratuberculosis infection was seen in the midjejunum of goat No. 7. The lesion consisted of a few, often coalescing, pale nodules approximately 1.0 cm in diameter. The nodules were located on the anti-mesenteric border of the intestine and were visible from both the serosal and the mucosal surfaces. The lymphatic vessels on the serosal surface of the jejunum were distended. Incidental findings included a thread-like (hair/ plant) material embedded in the ileal mucosa of goat Nos. 2 and 8 and the jejunal mucosa of goat No. 5. Microscopic lesions. Lesions compatible with M. a. paratuberculosis infection were found in three experimentally infected animals (Table 1). The lesions were located in the jejunum (goat Nos. 5, 6, and 7), the ileum (goat Nos. 5 and 6), and the jejunal lymph node (goat No. 6) and consisted predominantly of small, nodular, nonencapsulated, inflammatory cell infiltrates. The lesions were located in the submucosa, between or involving the capsule of Peyer s patch lymphatic nodules (Figs. 1, 2). A few lesions in all three animals were large enough to replace one or several Peyer s patch nodules and often extended into the overlying lamina propria. In some sections of jejunum from two animals (Nos. 5 and 7), there were very small clusters of inflammatory cells or single multinucleated giant cells (MGCs) scattered in the lamina propria and the submucosa. The inflammatory cells included large macrophages and, in most lesions, also MGCs of both the Langhans and foreign-body types. In some lesions, large macrophages with ample cytoplasm and often indistinct cell boundaries were present. In the jejunal lymph node of goat No. 6, there were three small granulomatous foci. These lesions were located in the perinodal connective tissue, in the outer cortex, and in the diffuse lymphatic tissue of the deep cortex, respectively. ZN staining demonstrated acid-fast bacilli in all or some of the granulomatous lesions in all three animals (Nos. 5, 6, and 7), but seldom in large numbers. The immunohistochemical staining for M. a. paratuberculosis detected bacterial antigen in all or most of the lesions in two animals (Nos. 5 and 7). This method did not detect bacterial antigen in some lesions for which ZN staining gave positive results. Other histopathologic lesions seen in the intestine of the goat kids were foreign-body reactions. Three animals (Nos. 5, 9, and 10) had granulomas or granuloma-like lesions in the intestine that were not ascribed to paratuberculosis infection. In two of three lesions, inflammation was centered around a particulate foreign material that was birefringent in polarized light (Fig. 3). Lesions were located in the submucosa of the proximal and distal ileum, respectively. There was an infiltration of large macrophages and a few granulocytes. One lesion also had MGCs of both the Langhans and the foreign-body types. The third lesion, found in the submucosa of the midileum of goat No. 10, consisted of a nodular infiltration of large macrophages and neutrophilic granulocytes. The center of the lesion was missing, and at the periphery there was fibrosis. It was concluded that the lesion represented a foreign-body

5 546 SigurUardóttir, Press, Saxegaard, and Evensen Vet Pathol 36:6, 1999 Fig. 1. Midjejunum; goat No. 6. Focal, nonencapsulated, granulomatous inflammation (black arrows) in the submucosa. HE. Bar 96 m. reaction, although no foreign material was present in the histologic sections taken. Both control and experimentally infected animals had very mild inflammatory cell infiltration in the triads of the liver, and most of the experimentally infected animals also had small inflammatory cell foci scattered in the liver parenchyma. The infiltrates consisted of a few lymphocytes, small macrophages, a few plasma cells and an occasional granulocyte. Acid-fast bacilli were not detected in ZN-stained sections of liver. Bacteriologic findings. Bacteriologic examination of organs collected at postmortem examination detected infection in two animals (Table 1). M. a. paratuberculosis was isolated from the ileocolic and colic lymph nodes of goat No. 6 and from the medial retropharyngeal, ileocolic, and jejunal lymph nodes of goat No. 7. Discussion The present study used large oral doses of M. a. paratuberculosis bacteria administered over an extended period to establish a paratuberculosis infection in goat kids. Three of eight animals showed histopathologic lesions and two of these were positive on bacteriological examination. Only the older goat kids, beyond 4 months of age (17 weeks or more PI), showed evidence of an established infection, and the younger animals, 3 months of age or less (11 weeks PI), were negative for all tests and examinations performed. Other investigators have reported lesions and isolated the

6 Vet Pathol 36:6, 1999 Subclinical Paratuberculosis in Goat Kids 547 Fig. 2. Midileum; goat No. 5. Nodular, granulomatous inflammation in the submucosa (black arrows). M multinucleated giant cells. HE. Bar 96 m. organism from tissues as early as days PI. 11,12 However, most experimental investigations of M. a. paratuberculosis infection have not examined animals until several months to over a year after infection. 10,13,21 The detection of paratuberculosis during the early subclinical stage of the infection has proven difficult because of the protracted and variable incubation period, involving a period of bacteriostasis followed by gradual bacterial replication. It is thus only after the initial lag period in bacterial replication that bacteria can be detected in cultures from tissues and an immunological response can be measured. 3 Microbiological examination of tissues has been claimed to provide the most sensitive method for the diagnosis of paratuberculosis. 6,8 However, with appropriate and extensive tissue sampling, histopathologic examination may detect a greater number of infected animals than microbiological examination. 7 Nevertheless, the culture of the organism is the definitive method for the diagnosis of paratuberculosis infection. 4,6 In the present study, histopathologic examination and histochemical staining produced evidence of infection in more animals than bacteriologic examination. However, it should be noted that the histological and immunohistochemical methods used in the present study are not able to differentiate between M. a. paratuberculosis and other mycobacteria. The limited design of the present experiment could account for this difference. The use of an extended period of administration of the bacteria and the examination of more animals

7 548 SigurUardóttir, Press, Saxegaard, and Evensen Vet Pathol 36:6, 1999 Fig. 3. Proximal ileum; goat No. 9. Granulomatous inflammation in the submucosa, surrounding a foreign body (long black arrow). Lymphoid nodules of Peyer s patch (p) are on either side. Acid-fast bacilli were not demonstrated. Short black arrows muscularis mucosa. ZN. Bar 48 m. at each time during the study could have increased the likelihood of detecting an infection at an earlier stage. The problems currently faced in the early postmortem diagnosis of paratuberculosis were illustrated by the oldest animal in the present study. This 12-monthold animal (No. 8) produced negative results on postmortem examination, although the immunologic tests indicated exposure to the bacteria. While more extensive histologic and bacteriologic sampling protocols may have revealed lesions or the presence of viable bacteria in this goat, other investigators have reported that a certain percentage of individuals are able to eliminate the infection. 3 5 The multifocal nature and slow development of the initial lesions of paratuberculosis make the postmortem identification of convalescent and subclinically infected individuals uncertain at best. An important finding in the present study with implications for the postmortem diagnosis of early paratuberculosis was the disparity in localization of bacteriologic and histopathologic findings. Bacteria were not cultured from any tissue that showed histopathologic changes, even though granulomas in the tissues were shown with ZN and immunohistochemical staining to contain bacteria. These findings would indicate that these bacteria were present at a higher frequency than viable bacteria in the lymph nodes and argue that the detected bacteria in the intestine were not viable or were in a nonproliferative state. Similar findings for both natural and experimental infections have been reported, 7,8,13,16 and this disparity between bacteriologic and histologic detection of M. a. paratuberculosis has been attributed to low numbers of viable bacteria, inadequate culture conditions, and/or sampling protocols. 3,4,7,19 Nevertheless, an alternative explanation exists: that the presence of viable bacteria in the lymph nodes represents an escape from the local immune defenses in the gut. The inability to reconcile the localization of bacteriologic and histopathologic findings in the early subclinical phase of paratuberculosis highlights the need for a better understanding of the biology of M. a. paratuberculosis and its interaction with the host s immune system. The localization of early histopathologic lesions in the intestine may provide an insight into the initial uptake and distribution of M. a. paratuberculosis. An association between the lesions of paratuberculosis and Peyer s patches has been reported in numerous studies. 3,11,14,16 It has further been shown that in calves M. a. paratuberculosis bacteria are transported across the specialized epithelium overlying the Peyer s patches. 14 Most lesions in the present study were located in the

8 Vet Pathol 36:6, 1999 Subclinical Paratuberculosis in Goat Kids 549 submucosa between or involving the capsule of the lymphoid nodules of the Peyer s patches, and this localization is consistent with the trapping of bacteria in the resident and migratory phagocytic cell populations. However, in the present study, lesions in two animals were also located in the lamina propria, a finding that has been reported by others. 2,4,5,10 The small, focal nature of the present lesions argues against these lesions being an extension of larger inflammatory foci located in the submucosa or the dome, suggesting other possible routes of infection. 3,5 The ileum has been considered the primary site of infection and the most consistently and severely affected segment of the intestine. 4,11,14,21 However, in line with the observations of a number of investigators, 2,10,19,22 most lesions in the present study were found in the jejunum and not in the ileum. Differences between animal species, bacterial strains, routes of experimental infection, host ages and immune statuses, and experimental protocols have been suggested as possible explanations for these varying observations. Small foci of inflammatory cells were present in the portal triads of the livers of all goats in the present study. While liver lesions, usually without acid-fast bacilli, have been described in paratuberculosis, 1,2,4,10 the presence of mild, nongranulomatous, inflammatory changes in both control goats and experimentally infected goats possibly indicates the presence of an intercurrent disease. With the exception of a mild seborrhea of unknown etiology in one goat, clinical disease was not identified in the goats. Small inflammatory foci in the triads of the liver are a common postmortem finding, but the influence of such a finding on the immunologic and pathologic responses to mycobacterial infection is difficult to determine. An unexpected finding in the present study was the presence of foreign-body-type inflammatory lesions in the intestines of three goats, one experimentally infected animal and two control animals (Nos. 5, 9, and 10). These lesions resembled some of the paratuberculosis lesions identified in the present study, both in terms of inflammatory cell reaction and location in the intestinal wall (Figs. 2, 3). The presence of foreignbody-like granulomas should thus be considered in the evaluation of histopathologic changes associated with paratuberculosis. While advances in the immunologic and genetic tools available for the diagnosis of paratuberculosis hold hope for the future, the postmortem diagnosis of early subclinical paratuberculosis still remains within the realm of bacteriology and histopathology. The present study has reported the establishment of an experimental infection in goats that may prove useful in the further investigation of the diagnosis of paratuberculosis during the crucial stage of the establishment of the infection. Acknowledgement We thank Sigrun Nilsen for her skillful technical assistance with the bacteriology. References 1 Barker IK, Van Dreumel AA, Palmer N: The alimentary system. In: Pathology of Domestic Animals, ed. Jubb KVF, Kennedy PC, and Palmer N, 4th ed., vol. 2, pp Academic Press, San Diego, CA, Buergelt CD, Hall C, McEntee K, Duncan JR: Pathological evaluation of paratuberculosis in naturally infected cattle. Vet Pathol 15: , Chiodini RJ: Immunology: resistance to paratuberculosis. Vet Clin North Am Food Anim Pract 12: , Chiodini RJ, Van Kruiningen HJ, Merkal RS: Ruminant paratuberculosis (Johne s disease): the current status and future prospects. Cornell Vet 74: , Clarke CJ: The pathology and pathogenesis of paratuberculosis in ruminants and other species. J Comp Pathol 116: , Collins DM, Gabric DM, de Lisle GW: Identification of two groups of Mycobacterium paratuberculosis strains by restriction endonuclease analysis and DNA hybridization. J Clin Microbiol 28: , Condron RJ, Schroen CJ, Black CA, Ridge SE, Hope AF: Histological confirmation of subclinical infection with M. paratuberculosis in cattle. In: Proceedings of the 4th International Colloquium on Paratuberculosis, ed. Chiodini RJ, Collins MT, and Bassey EOE, pp International Association for Paratuberculosis, Rehoboth, MA, Fodstad FH, Gunnarsson E: Postmortem examination in the diagnosis of Johne s disease in goats. Acta Vet Scand 20: , Green EP, Tizard ML, Moss MT, Thompson J, Winterbourne DJ, McFadden JJ, Hermon-Taylor J: Sequence and characteristics of IS900, an insertion element identified in a human Crohn s disease isolate of Mycobacterium paratuberculosis. Nucleic Acids Res 17: , Harding HP: Experimental infection with Mycobacterium Johnei. II. The histopathology of infection in experimental goats. J Comp Pathol 67:37 53, Juste RA, García Marín JF, Peris B, SáezdeOcáriz C, Badiola JJ: Experimental infection of vaccinated and non-vaccinated lambs with Mycobacterium paratuberculosis. J Comp Pathol 110: , Kluge JP, Merkal RS, Monlux WS, Larsen AB, Kopecky KE, Ramsey FK, Lehmann RP: Experimental paratuberculosis in sheep after oral, intratracheal, or intravenous inoculation: lesions and demonstration of etiologic agent. Am J Vet Res 29: , Levi ML: Experimental study of Johne s disease in goats. J Comp Pathol 58:38 63, Momotani E, Whipple DL, Thiermann AB, Cheville NF:

9 550 SigurUardóttir, Press, Saxegaard, and Evensen Vet Pathol 36:6, 1999 Role of M cells and macrophages in the entrance of Mycobacterium paratuberculosis into domes of ileal Peyer s patches in calves. Vet Pathol 25: , Moss MT, Green EP, Tizard ML, Malik ZP, Hermon- Taylor J: Specific detection of Mycobacterium paratuberculosis by DNA hybridisation with a fragment of the insertion element IS900. Gut 32: , Pérez V, García Marín JF, Badiola JJ: Description and classification of different types of lesion associated with natural paratuberculosis infection in sheep. J Comp Pathol 114: , Saxegaard F: Experimental infection of calves with an apparently specific goat-pathogenic strain of Mycobacterium paratuberculosis. J Comp Pathol 102: , Saxegaard F: Isolation of Mycobacterium paratuberculosis from intestinal mucosa and mesenteric lymph nodes of goats by use of selective Dubos medium. J Clin Microbiol 22: , Thomas GW: Paratuberculosis in a large goat herd. Vet Rec 113: , Thoresen OF, Falk K, Evensen Ø: Comparison of immunohistochemistry, acid-fast staining, and cultivation for detection of Mycobacterium paratuberculosis in goats. J Vet Diagn Invest 6: , Van Kruiningen HJ, Chiodini RJ, Thayer WR, Coutu JA, Merkal RS, Runnels PL: Experimental disease in infant goats induced by a Mycobacterium isolated from a patient with Crohn s disease. A preliminary report. Dige Dis Sci 31: , Whitlock RH, Rosenberger AE, Sweeney RW, Spencer PA: Distribution of M. paratuberculosis in tissues of cattle from herds infected with Johne s disease. In: Proceedings of the 5th International Colloquium on Paratuberculosis, ed. Chiodini RJ, Hines ME II, and Collins MT, pp International Association for Paratuberculosis, Rehoboth, MA, 1996 Request reprints from Ó. G. SigurUardóttir, Department of Pathology, National Veterinary Institute, PO Box 8156 Dep., 0033 Oslo (Norway). olof.sigurdardottir@vetinst.no.