Reduced formation of granulomata in gamma(delta) T cell knockout BALB/c mice inoculated with Mycobacterium avium subsp. paratuberculosis

The role of gamma(delta) T cells in the bovine immune response to Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) infection is poorly understood. Accordingly, using BALB/c mice that are innately susceptible to M. paratuberculosis, we compared wild-type and gamma(delta) T cell knockout BALB/c mice to study the protective roles of gamma(delta) T cells in M. paratuberculosis infection. Ten-week-old mice were inoculated intraperitoneally with either a low dose (4 x 10(6) colony-forming units [CFU]/mouse) or a high dose (4 x 10(9) CFU/mouse) of M. paratuberculosis strain ATCC 19698. Histopathologic and morphometric examinations showed reductions in the number and area of granulomatous lesions in the liver of the knockout mice at 18 weeks after inoculation with either the low or the high dose of the mycobacteria. Furthermore, at 18 weeks after inoculation, the bacterial load in the spleens of the knockout mice inoculated with the high dose was significantly lower than that of wild-type mice. No differences were found in bacterial load between the knockout and the wild-type mice in the low-dose groups. In contrast, in the livers of wild-type mice inoculated with either the low or high mycobacterial dose, increased areas of epithelioid granulomata were observed and the granulomata became disseminated widely during the experimental period. These findings in model mice suggest that gamma(delta) T cells, rather than restricting mycobacterial growth, may play a crucial role in development of epithelioid granulomata similar to those seen consistently in bovine paratuberculosis. The results of this study may have relevance to our understanding of the pathogenesis of paratuberculosis in ruminants, in which a prominent number of gamma(delta) T cells exist in the lymphoid system.     

HPLC does not differentiate Mycobacterium paratuberculosis from Mycobacterium avium

HPLC, which is gaining its place as identification tool in mycobacteriology laboratories, has been proposed to distinguish Mycobacterium paratuberculosis from Mycobacterium avium. We had reported no significant difference between M. avium and M. paratuberculosis reference strain ATCC 19698. Because of the advantages offered by such a method, we enlarged our observations to include more isolates of M. paratuberculosis. Within the double cluster of peaks obtained by both M. avium and M. paratuberculosis, we could not find a consistent difference typical of M. paratuberculosis. Therefore, the present study confirmed that M. avium and M. paratuberculosis could not be distinguished by HPLC, raising doubts of a straightforward use of HPLC to identify M. paratuberculosis.     

Mycobacterium paratuberculosis monoassociated nude mice as a paratuberculosis model

In this study, a paratuberculosis (Johne's disease) model was developed by intragastrically dosing gnotobiotic athymic nude mice with Mycobacterium paratuberculosis. The mice infrequently shed bacilli from their intestinal tracts during the first 4 months after inoculation. Following this time, increasing numbers of M. paratuberculosis (greater than 4.0 log10 bacilli per fecal pellet by 40 weeks) were recovered from the feces of the 12 mice that remained in the isolator. A similar pattern of recovery of M. paratuberculosis was obtained from the ileum, cecum, colon, and liver. Histopathologic lesions and acid-fast bacilli were rare during the first 4 months of infection and then, with time, increased in prevalence and severity. Mice maintained for 7 months or longer exhibited severe granulomatous inflammation and large numbers of acid-fast bacilli in the gastrointestinal tract and liver (up to 10(8) log10 colony forming units per gram wet weight). Five mice maintained for 7 months or more developed clinical signs consistent with those seen in paratuberculosis (weight loss, chronic diarrhea); three of these mice eventually died or became moribund and were euthanatized. M. paratuberculosis monoassociated mice released increased levels of tumor necrosis factor activity into their sera, as compared to uninfected control mice, when they were injected with bacterial lipopolysaccharide. The clinical signs, fecal shedding of M. paratuberculosis, granuloma formation, and progressive bacillary multiplication observed with these mice are consistent with naturally occurring M. paratuberculosis infection of ruminants (Johne's disease). This model will be useful for future studies of immunoregulation and antimicrobial therapy of paratuberculosis.     

Investigation of antigen-specific T-cell responses and subcutaneous granuloma development during experimental sensitization of calves with Mycobacterium avium subsp paratuberculosis

OBJECTIVE: To characterize the early cellular immune response to Mycobacterium avium subsp paratuberculosis (MAP) infection and evaluate the development of granulomatous inflammation at the SC injection site in experimentally inoculated calves. ANIMALS: Forty-eight 4-week-old calves. PROCEDURE: Calves received an SC injection of MAP strain 19698 (n = 25), sterile saline (0.9% NaCl) solution (20), or a commercial paratuberculosis vaccine (3); the inoculation site tissue and associated draining lymph node were excised at postinoculation day (PID) 0 (n = 36), 7 (14), 14 (6), 21 (8), and 60 (32). Sections of inoculation site tissues were evaluated immunohistochemically for T-cell subsets; lymph node mononuclear cells (LNMCs) were assessed for T-cell surface markers and for intracellular interferon-gamma via flow cytometry. RESULTS: At MAP inoculation sites, calves developed mild, focal granulomatous inflammation by PID 7; by PID 60, areas of inflammation contained macrophages with numerous lymphocytes. Compared with control calves, there was increased antigen-specific LNMC proliferation in MAP- and vaccine-inoculated calves at PID 60, although proliferation among lymphocyte subsets was not significantly different between MAP-inoculated and control calves; in vaccine-inoculated calves, CD4+ T-cells predominated. In MAP-inoculated and control calves, antigen-specific interferon-gamma production by LNMCs did not differ significantly; vaccine-inoculated calves had marked interferon-gamma expression by CD4+ T-cells. CONCLUSIONS AND CLINICAL RELEVANCE: In calves, SC administration of MAP resulted in granulomatous inflammation at inoculation sites and an antigen-specific T-cell proliferative response. Results suggest that this experimental system can be used to reproducibly generate antigen-specific T-cells during MAP infection for functional analysis.     

Temporal Mycobacterium paratuberculosis infection in T-cell receptor (TCR)-alpha and TCR-delta-deficient mice

Mycobacterium paratuberculosis is the causative agent of paratuberculosis (Johne's disease), a chronic inflammation of the terminal portion of the ileum in ruminants. The predominance of cell-mediated immunity in early stages of the disease suggests that T lymphocytes are essential to protect the host from infection with M. paratuberculosis. In this study, we investigated the role of alphabeta and gammadelta T cells in resistance to M. paratuberculosis infection using a T-cell receptor (TCR) knockout mouse model. Weanling TCR-alpha-deficient, TCR-delta-deficient, and C57BL/6 control mice (5-6 weeks of age) were acclimated for 2 weeks and then inoculated intraperitoneally with 10(8)CFU/ml of M. paratuberculosis (either strain 19698 or strain Ben). Groups of mice within each treatment group were euthanized at 1, 3 and 6 months post-inoculation. Sections of spleen, liver, ileum and mesenteric lymph node were prepared for bacterial culture and histologic examination. At all time points of infection and regardless of bacterial strain, TCR-alpha-deficient mice had higher levels of M. paratuberculosis colonization in their tissues compared to TCR-delta-deficient mice or C57BL/6 control mice. Lesions were located predominately in the liver and the ileum, depending upon period of infection, and lesion scores were higher for TCR-alpha-deficient mice compared to the other treatment groups. These results suggest that alphabeta T cells play a major role in resistance to infection with M. paratuberculosis and that gammadelta T cells may play a lesser role and potentially confound protective immune responses.     

Lewis rats are not susceptible to oral infection with Mycobacterium avium subsp. paratuberculosis

Pathogenesis studies of Mycobacterium avium subsp. paratuberculosis infection in ruminants are hampered by the long incubation time of the disease. A laboratory animal model with a shorter incubation time would facilitate research in this field. Although small rodents are usually considered to be resistant to M.a. paratuberculosis infection, several susceptible murine strains have been found. To our knowledge, there are no detailed reports with regard to susceptibility in rats. The Lewis rat is a valuable model for inflammatory bowel disease studies as well as autoimmune diseases involving mycobacteria as inducing agents. In this study Lewis rats were used to investigate their potential as a small laboratory animal model for paratuberculosis.In total 28 female Lewis rats were orally inoculated with M.a. paratuberculosis. The rats were first inoculated at 3 weeks of age, and 12 more inoculations followed in increasing intervals during the 3 months to follow. Eight control rats received a sham inoculation. Over 9 months, two rats from each group were sacrificed at regular intervals and immunological and histopathological examinations were performed on the gastrointestinal tract, the liver and the spleen.None of the rats developed lesions which were indicative of mycobacterial infection as determined by histology with HE and Ziehl-Neelsen staining. The bacteria could not be recultured from samples taken from the gut, the liver or the spleen. The immunological tests however, showed that bacteria had entered via the intestinal tract. From this study it appears that Lewis rats are resistant to oral inoculation with M. a. paratuberculosis, and not suitable as a model to study the immunopathogenesis of paratuberculosis as it occurs in ruminants.     

Intravenous johnin and tuberculin tests in cattle vaccinated with Mycobacterium paratuberculosis cells and subsequently inoculated with Mycobacterium bovis.

Calves at 30 days of age were vaccinated with a killed whole-cell Mycobacterium paratuberculosis vaccine. Four months later, these calves were inoculated with Mycobacterium bovis. The intravenous tuberculin and johnin tests were applied both before and after inoculation. The results of the hematologic investigation had extremes at both high and low values and were too unsuitable for statistical analysis. The intravenous tuberculin test is considered unsuitable for diagnosis of bovine tuberculosis in cattle vaccinated against paratuberculosis.     

Development of a Johne's disease infection model in laboratory rabbits following oral administration of Mycobacterium avium subspecies paratuberculosis

To assess the rabbit as a model for the study of Johne's disease pathogenesis, a breeding group of adult and juvenile New Zealand white rabbits were orally challenged with three doses of the Mycobacterium avium subspecies paratuberculosis wildtype bovine strain, CLIJ623, on three occasions. Faecal culture, post-mortem tissue bacteriological culture and histopathology were used to monitor the disease progression in the rabbits for more than 2 years. Of 4 adult and 16 juvenile orally dosed rabbits M. paratuberculosis organisms were recovered bacteriologically from two and three animals, respectively, using the BACTECtrade mark radiometric culture system. Tissue sites from which the bacteria were recovered included the mesenteric lymph nodes, ileocaecal valve, vermiform appendix, caecum, proximal colon and jejunum. Body weight loss, reduced abdominal fat and mild lesions were observed at necropsy in four infected rabbits. Diarrhoea and persistent faecal shedding of bacteria were not observed. Faecal culture did not yield any cultivable mycobacterial organisms on solid media.     

Analysis by ELISA and Western blotting of antibody reactivities in cattle infected with Mycobacterium paratuberculosis after absorption of serum with M phlei

Preabsorption of cattle serum with Mycobacterium phlei was of value in eliminating falsely positive reactions in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against M paratuberculosis. Specific antibody titres from 16 animals naturally infected with M paratuberculosis were unaffected by absorption. Analysis by Western blotting indicated that a different set of antigens of M paratuberculosis were recognised by serum from falsely positive reactors compared with that from animals with established infection. After experimental infection the time required for seroconversion in the ELISA in nine calves lay between 10 and 28 months, although one animal had not seroconverted after 30 months when the experiment ended. All animals shed M paratuberculosis in their faeces before seroconversion.     

Skin testing, fecal culture, and lymphocyte immunostimulation in cattle inoculated with Mycobacterium paratuberculosis.

Fourteen calves at 21 days of age were experimentally inoculated with 100 mg (wet weight) of Mycobacterium paratuberculosis. Three calves were inoculated orally, 4 intravenously, and 7 subcutaneously. Lymphocyte immunostimulation, fecal culture, and intradermal tuberculin skin testing were done between 112 to 150 days following exposure. Lymphocyte immunostimulation test results, conducted at 112 days after inoculation, showed all animals positive to Mycobacterium avium purified protein derivative. Fecal culture results, taken at 120 days after inoculation, showed that 2 of 3 animals inoculated intravenously were positive, whereas only 2 of 7 inoculated subcutaneously were positive (8 of 14 total were positive). Intradermal skin testing results at 150 days with M avium purified protein derivative showed 13 of the 14 calves were positive. Calves were examined at necropsy 153 days after inoculation, and M paratuberculosis was isolated from tissues of each of the 14 calves.     

Divergent cytokine responses of macrophages to Mycobacterium avium subsp. paratuberculosis strains of Types II and III in a standardized in vitro model

Based on epidemiological and clinical observations, different strains of Mycobacterium avium subsp. paratuberculosis (MAP) are suspected to significantly differ in their virulence for ruminants. In the pathogenesis of paratuberculosis, macrophages represent the principal target cell for MAP. In order to judge the ability of different MAP-genotypes to modulate macrophage responses, the cytokine responses of the monocyte cell line THP-1 were studied after challenge with three different MAP strains under standardized conditions. The bovine field isolate J1961 (major Type II) and the ovine field isolate JIII-86 (Type III) were compared with the laboratory adapted reference strain ATCC 19698 (Type II). Strains were shown by three different typing methods (IS900-RFLP-, MIRU-VNTR-, and SSR-analysis) to substantially differ in several genotypic features. Macrophage function was assessed by quantifying mRNA of the cytokines TNF-α, IL-1?, and IL-10 by quantitative RT-PCR. Secreted TNF-α protein was measured by a cytotoxicity test, IL-1? and IL-10 using ELISA tests. The three MAP strains of various genotypes differ in their effect on human macrophages depending on challenge dose and infection time. These differences concerned both the mRNA level and secreted protein amounts of proinflammatory cytokines TNF-α, IL-1β and anti-inflammatory cytokine IL-10. Type III strain produced less IL-10 and IL-1β mRNA and protein but more TNF-α protein at 2h than the Type II strains. In summary, our results support the hypothesis that strain characteristics might have relevance for the host response towards MAP and, consequently, for the pathogenesis of paratuberculosis.     

IFN-gamma diagnostic tests in the context of bovine mycobacterial infections in Belgium

In countries where cattle tuberculosis caused by Mycobacterium bovis (Mbov) and paratuberculosis caused by Mycobacterium avium subsp. paratuberculosis (Mptb) are present, testing strategies for the Mbov eradication have to discriminate between these two infections. Present indirect tests are based on the analysis of the specific cellular immune response (DTH, IFN-gamma) against crude mycobacterial antigens (avian and bovine PPD). In this study, we compared the evolution of the IFN-gamma responses of animals experimentally infected with Mbov, Mptb, or inoculated with Mycobacterium phlei. Mbov inoculation induced a strong IFN-gamma response that allows rapid classification of the status of the animals following interpretation criteria set up by us. Experimental inoculation with M. phlei induced sensitisation to mycobacterial antigens as detected by the IFN-gamma test but these reactions were of short duration, therefore, repeated testing allows us to define these animals as aspecific reactors. IFN-gamma response induced after oral inoculation of calves with Mptb was of low intensity and ratio of responses measured against avian versus bovine PPD did not allow a clear diagnostic at least for the six first month of infection.     

Experimental disease in young chickens induced by a Mycobacterium paratuberculosis isolate from a patient with Crohn's disease.

The susceptibility of young chickens to infection with an isolate of Mycobacterium paratuberculosis which had been recovered from diseased tissues of a patient with Crohn's disease was determined. Two-week-old Leghorn-Cochin chicks were inoculated with strain Linda. Six birds received 10(7) organisms orally, six intraperitoneally, and five intracardially. Six uninoculated birds served as contact controls. Birds from each group were necropsied at two-week intervals. Focal granulomatous lesions occurred in two of six chickens which were inoculated orally, in six of six inoculated intraperitoneally, in three of five inoculated intracardially, but in none of six controls. Of the 11 birds with lesions, acid-fast bacilli were demonstrated in five. Immunoperoxidase reactivity paralleled the presence of acid-fast bacilli.     

Experimental conjunctival infection of lambs with a strain of Chlamydia psittaci isolated from the eyes of a sheep naturally affected with keratoconjunctivitis

Five ram-lambs were inoculated into the left conjunctival sac with the 15R isolate of Chlamydia psittaci, recovered from a sheep with keratoconjunctivitis. A sixth ram-lamb was kept in contact with them. The five lambs developed varying degrees of acute conjunctivitis and 14 days later C psittaci could be recovered from the inoculated eyes, from which Branhamella ovis was also isolated. The eyes were examined regularly for four months; C psittaci could not be re-isolated but the eyes developed varying degrees of follicular conjunctivitis. After four months the sheep were treated with corticosteroids in an attempt to reactivate a latent chlamydial infection but no chlamydiae could be isolated. Five months after the start of the experiment the six lambs were inoculated with 15R into the left conjunctival sacs. Acute conjunctivitis developed which was not as severe as after the first inoculation, but C psittaci could only be recovered from the left eyes of three sheep three days after inoculation. The eyes remained chronically affected by follicular conjunctivitis. Six months after the start of the experiment the left eyes were again inoculated with 15R; on this occasion acute conjunctivitis did not develop and chlamydiae could not be isolated. Chronic follicular conjunctivitis persisted until the experiment was terminated three months later.     

Evaluation of diagnostic tests for Johne's disease in young cattle

OBJECTIVE: To investigate the development of immune responses in calves experimentally and naturally infected with Mycobacterium paratuberculosis and to evaluate the potential for diagnostic tests to detect infected calves. DESIGN: Sequential testing of four treatment groups of calves over a 2 year period. PROCEDURE: Twenty-nine calves were allocated to four groups. Group D calves were orally dosed with M paratuberculosis, group N calves naturally exposed to M paratuberculosis, group V calves vaccinated for M paratuberculosis, and group C were control calves (not infected or vaccinated). Blood and faecal specimens were collected from each calf at monthly intervals to 18 months of age and then every 2 months until they were slaughtered between the ages of 21 and 29 months. Specimens were tested using absorbed EIA, IFN-gamma EIA and faecal culture. The infection status of the calves was confirmed by extensive histopathological examination and tissue culture. RESULTS: M paratuberculosis infection was confirmed in 10 calves, comprising six of eight orally dosed calves, three of five naturally exposed calves and one of nine vaccinated calves. The six artificially infected calves and one naturally infected calf were detected shedding M paratuberculosis in their faeces. Results with positive absorbed EIA were obtained from one artificially infected calf, one naturally infected calf and three vaccinated calves. All calves including controls had positive results on at least one occasion using the IFN-gamma EIA. In addition, seven calves had positive bovine tuberculosis results using the IFN-gamma EIA, even though bovine tuberculosis has been eradicated from Australia. CONCLUSION: Detection of M paratuberculosis infection in young cattle continues to be difficult using current tests.     

Pathogenicity of Mycobacterium fortuitum and Mycobacterium smegmatis to goldfish, Carassius auratus

Despite the ubiquitous presence of atypical mycobacteria in the environment and the potential risk of infection in humans and animals, the pathogenesis of diseases caused by infection with atypical mycobacteria has been poorly characterized. In this study, goldfish, Carassius auratus were infected either with the rapidly growing fish pathogen, Mycobacterium fortuitum or with another rapidly growing mycobacteria, Mycobacterium smegmatis. Bacterial persistence and pathological host response to mycobacterial infection in the goldfish are described. Mycobacteria were recovered from a high percentage of inoculated fish that developed a characteristic chronic granulomatous response similar to that associated with natural mycobacterial infection. Both M. fortuitum and M. smegmatis were pathogenic to fish. Fish infected with M. smegmatis ATCC 19420 showed the highest level of giant cell recruitment compared to fish inoculated with M. smegmatis mc(2)155 and M. fortuitum. Of the three strains of mycobacteria examined, M. smegmatis ATCC 19420 was the most virulent strain to goldfish followed by M. fortuitum and M. smegmatis mc(2)155, respectively.     

Single and mixed infections of neonatal pigs with rotaviruses and enteroviruses: virological studies.

Three rotaviruses and three enteroviruses were isolated from pigs with diarrhea. The three enteroviruses and one of the rotaviruses were recovered from pigs infected with both viruses. Separation of rotaviruses and enteroviruses from tissues containing both viruses was effected by pancreatin treatment, terminal dilution, and inoculation onto different cell lines. The three rotaviruses were group A serotype 1, and the enteroviruses were serotypes 2, 3 and 7. Cell culture preparations of these six viruses were inoculated into colostrum-deprived neonatal pigs. All of the rotavirus and enterovirus isolates established intestinal and systemic infection and were shed in the feces after oral inoculation. Concurrent infection with both viruses resulted in only minor alteration of systemic distribution and did not alter fecal shedding of either virus.     

Experimental transmission of rabbit syphilis

Rabbit syphilis was experimentally transmitted from clinical cases to healthy rabbits. The purpose was to evaluate changes in RPR titers during the course of infection and to detect the pathogenic organism. Two of three littermate rabbits were inoculated topically. One rabbit became symptomatic, with typical clinical signs on its genitalia about 8 weeks after inoculation and a marked rise in RPR titers, another remained asymptomatic with a moderate rise in titers, and the control rabbit remained negative. These results supported the specific relationship between clinical signs and RPR titers. Histopathological examination of the skin lesion from the symptomatic rabbit revealed spirochetes.     

Experimental infection of rabbits with bovine herpesvirus-4: acute and persistent infection

A strain of bovine herpesvirus-4 (BHV-4) isolated from bovine cases of mammary pustular dermatitis was used for experimental infection of rabbits. The strain is serologically indistinguishable from the group prototype Movar 33/63 and from the American isolate DN599. Groups of rabbits were inoculated by various routes. Intravaginal and conjunctival inoculations resulted in vulvovaginitis and conjunctivitis, respectively, and in shedding of virus. The rabbits seroconverted for the virus, with high titers of antibodies (indirect fluorescent antibody test) that persisted throughout the experiment. Treatment with dexamethasone, beyond the acute infection, did not produce recrudescence of disease or shedding of the virus. Rabbits were killed at various times, from 3 to 6 months post-infection, and the virus was recovered from explant cultures of spleen and by cocultivation of spleen cells with bovine lung cells. These results demonstrate the usefulness of the rabbit as a model for studying the pathogenesis of BHV-4 infection in cattle.     

Characterization of Mycobacterium paratuberculosis antigenic proteins

The characterization of a purified antigen from Mycobacterium paratuberculosis, recently made commercially available for use in serodiagnosis by enzyme-linked immunosorbent assay (ELISA), of paratuberculosis in cattle was described. This assay had 89% specificity and 83% sensitivity for M paratuberculosis infection. The protein/polypeptide composition of the purified antigen was compared with that of a crude protoplasmic extract of strain 18 M paratuberculosis used in the agar-gel immunodiffusion test and ELISA and with that of sonicated strain 19698 M paratuberculosis organisms grown on Dorset-Henley synthetic liquid medium. The sonicated M paratuberculosis contained 27 major proteins/polypeptides; the crude protoplasmic extract, 18; and the purified antigen contained 14 proteins/polypeptides, using sodium dodecyl-sulfate polyacrylamide-gel electrophoresis analysis. The serologic reactivity of these proteins/polypeptides were defined, using the enzyme-linked immuno-electrotransfer blot technique. The sonicated M paratuberculosis contained 20 serologically reactive proteins/polypeptides (34,000 to 84,000 daltons); the crude protoplasmic extract contained 3 (37,000 to 45,000 daltons); and the purified extract contained a diffuse polypeptide band (34,000 to 38,000 daltons). Identification by enzyme-linked immuno-electrotransfer blot technique of M paratuberculosis antigens reactive in the ELISA will allow us to further study these antigens in the ELISA to improve sensitivity and specificity of the diagnostic test.