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Recently some mycobactin-dependent mycobacteria were isolated from patients with Crohn's disease. These mycobacteria should be very similar to M. paratuberculosis which is closely related to M. avium. The author has investigated the relationship between M. avium, M. paratuberculosis and the mycobacteria associated with Crohn's disease.
Ten male Holstein-Friesian calves naturally infected by Mycobacterium paratuberculosis were experimentally re-infected orally at an average of 17 days. Monthly measurements were conduced of the following activities, in the period between post infection days 160 and 400: total protein (TPR), albumin (ALB), cholesterol (CHOL), triglycerides (TRIG), Zn and Cu concentrations as well as sorbitol dehydrogenase, lactate dehydrogenase (LDH), alpha-hydroxybutyrate dehydrogenase (alpha-HBDH), gamma-glutamyltransferase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), alkaline phosphatase and fructose-1,6-diphosphate aldolase (ALD). TPR, ALB, TRIG, and CHOL were reduced by day 400, in conjunction with disorders of digestion and absorption. Increased activities of CK, ALD, LDH, alpha-HBDH, AST and ALT primarily indicated damage to skeletal muscle and/or liver. Serum CK and ALD activities as well as TRIG and TPR concentrations may serve as aids to specific diagnosis of paratuberculosis, particularly in the advanced stage of the disease.
Total DNA was extracted from M. paratuberculosis (ATCC 19698) and from M. avium complex (ATCC 25291) cultivated on RVB-10 enriched liquid media. Restriction endonuclease analysis was conducted of Total DNA using 34 enzymes and DNA digestion profiles were compared. Fifteen enzymes revealed important differences between the two species. Two pairs of enzymes (EcoRII, BstNI) and (MboI, Sau3AI) provide evidence for the presence of dcmI and dam methylation in DNA of M. avium complex and M. paratuberculosis. The differences in DNA fragments of these two species could be of potential value in differentiating these clinically significant mycobacteria.
Cattle and swine slurry and a mixture of equal parts of both, was mixed with a culture of M. paratuberculosis, 0.1 mg per ml (1 mg = 33 X 10(6) viable units) and stored under anaerobic conditions at 5 degrees and 15 degrees C. At 5 degrees C the survival time for M. paratuberculosis was 252 days in all 3 kinds of slurry, and at 15 degrees C it was 98 days in cattle slurry, 182 days in swine slurry, and 168 days in mixed slurry.
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.
During 1983 and 1984, ileocecal lymph node specimens were obtained from clinically normal cattle at 76 US Department of Agriculture-inspected cull cattle slaughterhouses in 32 states and Puerto Rico. Mycobacterium paratuberculosis was isolated from the lymph nodes of 119 of 7,540 cattle. The prevalence of bovine paratuberculosis was 1.6% overall, with 2.9% in dairy culls and 0.8% in beef culls. The prevalence for females and males did not appear to differ significantly.
The prevalence of bovine paratuberculosis in New England was determined to be 18% based on a random survey of abattoir cattle. Six tissues from each of 100 animals were examined by histologic and bacteriologic methods. The cecal lymph node and the ileocecal valve yielded positive cultures most frequently, but isolations were also made from liver, tonsil, colon, and ileum. On the basis of the prevalence data, the economic significance of paratuberculosis to the New England dairy industry was estimated in excess of +15.4 million annually. Field studies identified 25 infected herds in New England. One of the infected herds from Connecticut was sold at public auction, an act which resulted in the dissemination of 95 potentially infected cattle to 28 farms in 8 different states.
The lymphocyte-transformation (LT) test was evaluated for its potential application as a field test for bovine paratuberculosis. Using a whole blood technique, samples from 3 consecutive collection periods were subjected to 3 mycobacterial antigens and to phytohemagglutinin. The results obtained from LT were compared with conventional serologic and cultural methods. A positive LT response to johnin purified-protein derivative (PPD) or avian PPD (or both) was noted in 40% to 60% of the animals tested. The complement-fixation test yielded 4% to 6.7% positive results, the immunodiffusion test between 1.2% and 1.4%, and the direct fecal culture between 2.4% and 6%. The mean of the stimulation indices of all positively responding animals was highest with johnin PPD. Specific stimulation to mammalian PPD occurred between 2.4% and 6% of the animals. The efficacy of the LT test for determining the incidence of infection with Mycobacterium paratuberculosis is discussed.
Paratuberculosis was diagnosed in a herd of 62 fallow deer (Dama dama) and 38 sika deer (Cervus nipon) maintained in an animal park in Ohio. A comparison was made of delayed-type hypersensitivity responses, results of in vitro lymphocyte immunostimulation tests, and isolation of Mycobacterium paratuberculosis on fecal culture in 19 deer.
An indirect immunoperoxidase procedure for the diagnosis of paratuberculosis was described. Formalin-fixed ileocecal tissue containing large numbers of Mycobacterium paratuberculosis organisms was used as the source of antigen. Goat anti-bovine immunoglobulin G labeled with peroxidase was used as the conjugate in the test system. The method is relatively simple to do and may prove to be valuable as a routine screening test.
An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies to Mycobacterium paratuberculosis in the sera of cattle. This assay was designed to minimize the nonspecific ELISA reactions caused by immunoglobulin (Ig)M by measuring only IgG1 antibodies against a protoplasmic antigen from the organism. The ELISA detected IgG1 antibodies in the sera of 58% of cattle with positive fecal cultures for M paratuberculosis compared with detection of 45% of culture-positive animals with an immunodiffusion test. In addition to its sensitivity, the ELISA apparently is highly specific because only 4% of the sera from fecal culture-negative animals gave a false-positive result.
Paratuberculosis is enzootic in the Great Lakes region and northeast US, causing severe economic losses. Sheep, pigs and deer can serve as intermediate hosts. Diagnosis is difficult and there is no entirely satisfactory serodiagnostic test. While treatment is generally considered ineffective, vaccines have been used successfully for prevention. Measures that can be taken in herds to reduce losses include raising replacement animals separate from adults, slaughtering animals shedding M paratuberculosis in their feces, and decontaminating the premises.
Paratuberculosis in sheep usually is manifested as emaciation and decreased wool production. Diarrhea occurred in only 18% of affected animals. Significant hematologic changes included decreased RBC count, hemoglobin level and hematocrit. Necropsy revealed pallor, cachexia and serous fluid in body cavities. Staining of intestinal mucosal scrapings and mesenteric lymph node impression smears for acid-fast organisms revealed bright-red clumps of Mycobacterium paratuberculosis bacilli. Fecal examination identified 70% of affected animals, intradermal injection of johnin 60%, and avian tuberculin 39%.
A large goat herd naturally infected with Mycobacterium paratuberculosis has been monitored for new infections for approximately 21/2 years, using both agar gel immunodiffusion and bacterial culture of feces. On the basis of postmortem confirmation of infection status, both methods had equal diagnostic sensitivity and specificity. The agar gel immunodiffusion test offered distinct advantages in ease of performance and rapidity of test results. It was concluded that serious consideration should be given to the immunodiffusion test as a diagnostic tool in control programs for caprine paratuberculosis.
Mycobacterium paratuberculosis, the cause of Johne's disease, was isolated from the feces of a donor bull in an artificial insemination stud. During isolation and observation for 21 months, the organism was recovered from all of 26 fecal samples and from 8 of 31 semen samples. At necropsy, it was isolated from the intestine and adjacent lymph nodes, lung, spleen, seminal vesicles, and prostate gland but not from the testicles. We concluded that routine fecal cultures at bull studs will reveal infected bulls before they become genitally infected and shed M paratuberculosis in semen.
A dairy herd (102 cattle) which had been enrolled under a paratuberculosis control program for two years utilizing a complement fixation test (carbohydrate antigen) and intradermal skin test (johnin PPD) was subjected to two further herd tests and followed to slaughter to determine infection status by culture and histology. Mycobacterium paratuberculosis infection was demonstrated in 37 of the animals of which only five were considered reactors on the basis of the last two herd tests applied. Cultural and histopathological evaluation indicated the testing procedures had eliminated heavily infected animals. The limitations of these testing procedures under free stall housing conditions are discussed.
Fecal culture for Mycobacterium paratuberculosis and a complement fixing serological test using a carbohydrate antigen were compared for diagnostic efficiency in cattle naturally infected with M. paratuberculosis. Serological reactivity was associated with the persistent fecal shedding of large numbers of bacteria and in the absence of false positives in the population studied, was considered an efficient method for the identification of this segment of infected cattle. Minimally infected cattle, which inconsistently shed small numbers of bacteria, were generally serologically negative. One persistent fecal shedder demonstrated a fluctuating serological response to the carbohydrate antigen following abortion. The advantages and disadvantages of this serological diagnostic procedure in relationship to fecal culture are discussed.
Paratuberculosis is the most serious chronic bacterial disease of ruminants. Regarding the increasing incidence of the disease in the Czech Republic in connection with changing situation in agriculture and, among other factors, with import of animals we are presenting a summary of the latest data. We assume that high economic losses and expected participation of Mycobacterium paratuberculosis in Crohn's disease in men will evoke a broad interest in the disease among people from various branches of veterinary and human diagnosis.