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IS900 restriction fragment length polymorphism (RFLP) analysis of Mycobacterium avium subsp. paratuberculosis isolates from goats and cattle in Norway.

In Norway, paratuberculosis has been frequently diagnosed in goats, while cattle have been almost free of the infection. This difference in prevalence between goats and cattle has led to speculations about the existence of a Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) isolate that is non-pathogenic for cattle. There is little information available on genotypic variation of M. a. paratuberculosis isolated from animals in Norway. In the present study, genotypic information on 51 isolates from goats and four isolates from cattle in Norway was obtained by use of IS900 restriction fragment length polymorphism (RFLP) analysis. All isolates from cattle and 84% of the isolates from goats had the same RFLP pattern (B-C1). Five RFLP patterns not previously detected were found. No genotypic variation that could explain a difference in host origin was found between the isolates from cattle and the majority of the Norwegian goat isolates. This lack of difference indicates that the most common M. a. paratuberculosis isolates in Norway may infect both cattle and goats.

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Use of an enzyme-linked immunosorbent assay for serodiagnosis of clinical paratuberculosis in goats. Study by western blotting of false-positive reactions.

An enzyme-linked immunosorbent assay (ELISA) was performed for diagnosis of paratuberculosis in goats, using as antigen a protoplasmic extract (PPA-3). The test was developed on the basis of the results obtained with two serum reference pools, positive and negative respectively. To avoid day-to-day variations, dilutions of the positive serum pool were included in each plate to obtain an arbitrary system, transforming absorbance into immunoglobulin (Ig) G anti-Mycobacterium paratuberculosis units. The ELISA was used on sera of two reference groups of animals. One group consisted of 35 goats suspected of being infected with paratuberculosis, which was confirmed by histological findings and isolation of M. paratuberculosis. The negative group consisted of 61 healthy goats from a farm free of paratuberculosis. The test showed a sensitivity of 100% and a specificity of 91.8%. Absorption of sera with a Mycobacterium phlei suspension did not modify either the sensitivity or the specificity of the test. Sera from the negative group were analysed by Western blotting, and four of them recognized two fractions with a molecular weight of 17.3 and 28.1 kDa.

Absorption↗

Factors associated with ELISA scores for paratuberculosis in an Angus-Brahman multibreed herd of beef cattle.

Cow and calf genetic and environmental factors were evaluated for their association with ELISA scores for paratuberculosis in a multibreed population of beef cattle. The ELISA scores are a measure of the presence or absence of antibodies against Mycobacterium avium subsp. paratuberculosis in bovine serum. The linear mixed-model analysis used 352 ELISA scores from 238 cows: 51 Angus (A); 34 Brahman (B); 41 (3/4 A 1/4 B); 45 (1/2 A 1/2 B); 34 (1/4 A 3/4 B); and 33 Brangus (5/8 A 3/8 B). Cows were assumed to be unrelated. Year affected (P < 0.001) ELISA scores, but age of cow did not, which was expected to be significant because of the chronic progressive nature of this disease. Important regressions on fixed effects associated with cows were 1) a positive estimate of cow B breed effect (0.59 +/- 0.24; P < 0.017), indicating an upward trend of ELISA scores toward 100% B cows; 2) a negative estimate for weight change from before calving (late November) to the date of the blood sample in May (-0.0062 +/- 0.0019 score/kg; P < 0.002), indicating that poorer maintenance of cow weights was associated with higher ELISA scores; and 3) a positive estimate for days in lactation of cow on the date of the blood sample (0.0086 +/- 0.0034 score/d; P < 0.021), indicating the production of larger amounts of antibodies against Mycobacterium avium subsp. paratuberculosis as lactation progressed. Relevant regressions on fixed effects associated with calves were 1) calf birth weight (-0.022 +/- 0.010 score/kg; P < 0.035), and 2) calf gain from birth to the date of the cow blood sample (-0.0092 +/- 0.0027 score/kg; P < 0.001). These estimates indicate that cows that produced lighter calves at birth and/or calves with slower preweaning growth tended to have greater ELISA scores. Although the sensitivity (percentage of infected animals detected) of ELISA was only 50%, these results suggest that subclinical paratuberculosis may be negatively affecting cows and their offspring. Factors identified as associated with ELISA scores could help producers with culling decisions related to paratuberculosis control and eradication in beef cattle.

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Destruction of Mycobacterium paratuberculosis, Salmonella spp., and Mycoplasma spp. in raw milk by a commercial on-farm high-temperature, short-time pasteurizer.

The 2002 NAHM's Dairy Survey indicated that 87.2% of dairy farms in the United States feed waste milk to their neonatal calves. Although cost-effective, this practice can lead to increased calf morbidity and mortality due to ingestion of pathogenic agents. In an effort to reduce the risk of infection, dairy producers are implementing on-farm pasteurization of the waste milk as a control procedure before feeding the milk to calves. In the present study, the efficacy of a commercial high-temperature, short-time (HTST) on-farm pasteurizer unit to destroy Mycobacterium paratuberculosis, Salmonella enterica spp., and Mycoplasma spp. in raw milk was evaluated. Replicate experiments were run for 3 isolates of M. paratuberculosis, 3 serovars of Salmonella (derby, dublin, typhimurium); and 4 species of Mycoplasma (bovis, californicum, canadense, serogroup 7) at 2 different levels of experimental inoculation. In addition, HTST pasteurization experiments were performed on colostrum experimentally inoculated with M. paratuberculosis. After culture of the pasteurized milk samples, no viable M. paratuberculosis, Salmonella, or Mycoplasma were recovered, regardless of species, strain, or isolate. Pasteurization of colostrum was also effective in the destruction of M. paratuberculosis but resulted in an average 25% reduction in colostral immunoglobulin. These results suggest that HTST pasteurization is effective in generating a safer product to feed to young calves.

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Thermal inactivation of Mycobacterium paratuberculosis in milk.

Thermal inactivation of Mycobacterium paratuberculosis, a suspected human pathogen, was determined in ultrahigh-temperature whole milk. Three strains of M. paratuberculosis were examined for survival at temperatures from 55 to 75 degrees C using a submerged glass capillary tube method. Clumped and declumped suspensions of the cultures were used to determine the rate of heat inactivation and survival at pasteurization temperatures. Methods for declumping M. paratuberculosis included the use of glass beads, vortexing, and passing the cells through a 26-gauge needle. The latter procedure was found to be superior over other methods and did not affect the viability of cells. Capillary tubes filled with milk containing 4 x 10(6) to 3 x 10(7) CFU/ml were heated at temperatures ranging from 55 to 75 degrees C. At 55 degrees C, minimal thermal inactivation was observed for clumped and declumped cells. At 58 degrees C, thermal inactivation ranging from 0.3 to 0.7 log reduction was observed for both clumped and declumped suspensions. D values at 60 degrees C ranged from 8.6 to 11 min and 8.2 to 14.1 min for clumped and declumped cells, respectively. At 63 degrees C, the D values ranged from 2.7 to 2.9 and 1.6 to 2.5 min for clumped and declumped cells, respectively. Survival of M. paratuberculosis at initial levels ranging from 44 to 10(5) CFU/ml at pasteurization treatment (63 degrees C for 30 min and 72 degrees C for 15 s) was also determined. No survivors were observed after incubating plates for up to 4 months on Middlebrook 7H11 agar and up to 2 months on Herrold's egg yolk medium. The sensitivity of the plating method was 1 CFU/250 microliters. These results demonstrate that low levels of M. paratuberculosis, as might be found in raw milk, will not survive pasteurization treatments.

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Efficacy of pasteurization conditions for the inactivation of Mycobacterium avium subsp. paratuberculosis in milk.

Mycobacterium avium subsp. paratuberculosis, the causative agent of a chronic enteritis in ruminants (Johne's disease), has been linked to Crohn's disease in humans. This microorganism is shed by infected animals primarily in the feces but is also shed in the milk at much lower levels. Therefore, dairy products from infected animals may be one mode of transmission of this animal pathogen. This study was designed to evaluate the effectiveness of the holder and high-temperature short-time pasteurization standards on the destruction of M. paratuberculosis. One hundred eighty experiments were conducted in this study using a slug-flow pasteurizer unit and a laboratory scale pasteurizer unit. Ultrahigh-temperature milk was inoculated at two concentrations, 10(8) and 10(5) CFU/ml, with three different field strains of M. paratuberculosis. Five different time-temperature combinations were evaluated: 62.7 degrees C for 30 min, 65.5 degrees C for 16 s, 71.7 degrees C for 15 s, 71.7 degrees C for 20 s, and 74.4 degrees C for 15 s. Three replicates of each experiment were run for the pasteurizer unit, time-temperature combination, and strain of M. paratuberculosis. Treatment of milk regardless of bacterial strain or pasteurizer unit resulted in an average 5.0- and 7.7-log kill for the low and high concentrations of inoculum, respectively. Milk treated for cheese production (65.5 degrees C for 16 s) resulted in a much lower and more variable kill. Results from this study indicate that the current U.S. minimum standards for batch and high-temperature short-time pasteurization of grade A milk significantly reduced the survivability of M. paratuberculosis, but some bacteria survived subpasteurization heat treatment of milk used for cheese manufacture.

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Mycobacterium avium subspecies paratuberculosis triggers intestinal pathophysiologic changes in beige/scid mice.

We investigated whether infection of beige/scid mice with Mycobacterium avium subspecies paratuberculosis can induce intestinal pathophysiologic changes. Six-week-old beige/scid mice were inoculated intraperitoneally with M. paratuberculosis, then were killed 32 weeks after inoculation when the small intestine was evaluated for physiologic and morphologic abnormalities. All infected mice developed clinical disease. The lamina propria of the intestine from infected mice was mildly infiltrated with mononuclear cells containing acid-fast bacteria, and had significantly increased villus width. In vitro physiologic studies in Ussing chambers indicated that M. paratuberculosis infection caused significant abnormalities in intestinal transport parameters. Baseline short circuit current and potential difference were abnormally high in tissues from infected, compared with control mice, indicative of increased ion secretion. Baseline conductance was significantly decreased in infected mice, suggesting that intestinal tissue from infected mice was less permeable to ions. The change in short circuit current following transmural electrical and glucose stimulation was significantly reduced in intestines from infected mice, suggesting that inflamed intestine had neural and/or epithelial cell damage. We conclude that infection of beige/scid mice with M. paratuberculosis triggers significant intestinal pathophysiologic changes consistent with chronic inflammation. These functional abnormalities may contribute to the pathogenesis of the wasting syndrome seen in bovids with paratuberculosis. This animal model provides evidence that T cell-independent mechanisms are sufficient to cause mucosal pathophysiologic changes and inflammation in response to a specific pathogen, and may be of relevance to inflammatory bowel disease in humans.

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Economic decision analysis model of a paratuberculosis test and cull program.

A spreadsheet program was written to perform decision tree analysis for control of paratuberculosis (Johne's disease), when testing all adults in a herd and culling all animals with positive test results. The program incorporated diagnostic test sensitivity, specificity, and test cost with the cost or value of each of the 4 possible outcomes; true-positive, true-negative, false-positive, and false-negative test results. The program was designed to repeat the analysis for the independent variable pretest paratuberculosis prevalence (0 to 100%). Model output was graphed as profit or loss in dollars vs pretest prevalence. The threshold was defined as the pretest prevalence at which benefit-cost equaled zero. Reed-Frost disease modeling techniques were used to predict the number of Mycobacterium paratuberculosis-infected replacement heifers resulting from infected cows during a control program. Sensitivity analysis was performed on variables of the decision tree model; test sensitivity, specificity, test cost, and factors affecting the cost of paratuberculosis to a commercial dairy. A test and cull program was profitable when paratuberculosis caused greater than or equal to 6% decrease in milk production if the pretest prevalence was greater than 6%, test sensitivity was 50%, test specificity was 98%, and the testing cost was $4/cow. Test specificities greater than 98% did not markedly affect the threshold for tests with a 50% sensitivity and costing $4/cow. Test sensitivity had minimal effect on the threshold. Using a diagnostic test with a 50% sensitivity and a 98% specificity as an example, test cost was shown to affect the threshold prevalence at which the test and cull program became profitable.(ABSTRACT TRUNCATED AT 250 WORDS)

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An epidemiological study of paratuberculosis in dairy cattle in Ontario: study design and prevalence estimates.

An observational study involving 304 dairy herds and three abattoirs was conducted between 1986 and 1989 to investigate the epidemiology of paratuberculosis in dairy cattle in Ontario. The objectives of this paper were to describe the method of data collection for the study, to present descriptive production statistics and to present estimates of the prevalence of paratuberculosis among dairy cattle in Ontario. Mycobacterium paratuberculosis was isolated from the distal ileum and/or the ileocecal lymph node of 5.5% of 400 cull cows. Based on a lipoarabinomannan antigen enzyme-linked immunosorbent assay (LAM-ELISA) on sera, the predicted true prevalence of paratuberculosis among 14,923 dairy cattle from 304 herds, was 6.1%. Nineteen percent of 2,943 fecal cultures were uninterpretable because of overgrowth with contaminating bacteria or fungi. It was concluded that the true prevalence of paratuberculosis among dairy cattle in Ontario was no greater, and may be less than the true prevalence among dairy cattle from various regions of the United States. However, at a practical level, for the purposes of trade policy, the present study suggests that the functional prevalence is very similar in the two countries.

Abattoirs↗

Crossed immunoelectrophoretic analysis of Mycobacterium paratuberculosis.

Antigenic analysis of M. paratuberculosis revealed extensive cross-reactivity with M. avium; however, the number of cross-reactive antigens found was dependent on the strain of M. avium tested. One antigen was shown to be the common antigen while another appeared to be iron-regulated in its production. A commercial polyclonal antibody to M. paratuberculosis produced a CIE precipitin pattern comparable to that of the antibody produced for the present study. An antigen designated no. 6 was consistently precipitated by sera from cattle infected with M. paratuberculosis. This antigen exhibited complete cross-reaction with M. avium and partial cross-reaction with M. phlei. Among three commercially available complement fixation (CF) antigen that could be precipitated by M. paratuberculosis antibodies. A commercial antigen for use in an agar gel immunodiffusion test for Johne's disease diagnosis produced 12 precipitins with the M. paratuberculosis antibody, one of which was identical with antigen 6.

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Isolation of Mycobacterium paratuberculosis from washed bovine ova after in vitro exposure.

To establish whether Mycobacterium paratuberculosis could be cultured from Dulbecco phosphate-buffered saline solution (DPBSS) and to test 3 sampling methods, DPBSS supplemented with 2% fetal bovine serum was inoculated with M paratuberculosis at concentrations of 10(4), 10(3), 10(2), 10(1), and 10(0) colony-forming units/ml. The inoculated media was sampled after mixing, after centrifugation, and after centrifugation and decontamination with 0.75% hexadecylpyridinium chloride. The samples were inoculated onto 3 slants of Herrolds egg yolk medium supplemented with sodium pyruvate and mycobactin J and 1 slant without mycobactin J. Mycobacterium paratuberculosis was isolated following all 3 sampling methods for all concentrations. Treatment with hexadecylpyridinium chloride decreased the number of colonies isolated. To test the efficacy of a 10-step wash procedure for removing M paratuberculosis from bovine ova, washed zona pellucida intact bovine ova were incubated in DPBSS supplemented with 2% fetal bovine serum containing concentrations of 10(4), 10(3), 10(2), 10(1), and 10(0) colony-forming units of M paratuberculosis/ml for 12 hours at 22 C. Ten zona pellucida intact ova were removed from each concentration and washed by passing through 10 changes of DPBSS supplemented with 15% fetal bovine serum. The media from each wash step was inoculated onto slants of Herrolds egg yolk medium. The ova were included with the tenth wash step. Mycobacterium paratuberculosis was isolated from 1 of 10 tenth-wash steps at the 10(4) concentration and 5 of 10 tenth-wash steps at 10(3).

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Evaluation of the agar gel immunodiffusion test for diagnosis of subclinical paratuberculosis in cattle.

Concurrent bacteriologic culture of feces and agar gel immunodiffusion (AGID) testing was performed on all cows and bred heifers over 14 months old in 10 dairy herds during a 32-month period to determine the effectiveness of the AGID test for the detection of subclinical paratuberculosis. Herds were sampled 5 times and, when possible, culled animals were tested again at slaughter. During 5 herd-wide samplings, Mycobacterium paratuberculosis was isolated from 139 fecal specimens obtained from 109 cattle. Results of the AGID test were simultaneously positive 40 of 139 times (28.8%). Thirty-six of the 109 cattle (33.0%) determined to be infected had a positive AGID test result at some point during the 5 herd-wide samplings. When results of tests performed at time of slaughter were included, 117 cattle were identified as infected by culture methods; 55 of these (47.0%) were AGID test-positive at some point during the study. The upper limit of the maximal false-positive rate for the AGID test was 2.1%. On the basis of colony counts from cultures, subclinically infected cows shedding higher numbers of M paratuberculosis in their feces were more likely to have positive AGID test results (P less than 0.0001). In known infected cattle, neither the culture nor AGID test results were consistently positive on repeated testing. Of 48 official calfhood paratuberculosis vaccinates tested as adults, 3 had positive AGID test results and in 1 of these, M paratuberculosis was also isolated from the feces, indicating that the rate of false-positive AGID test results in calfhood vaccinates is low.

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Evaluation of the serological response of sheep in one flock to Mycobacterium paratuberculosis by crossed immunoelectrophoresis.

Sera from 74 sheep culled from one flock on the basis of performance and response to immunological tests for paratuberculosis or maedi visna were used to evaluate the serological response to a sonicated antigen of Mycobacterium paratuberculosis by crossed immunoelectrophoresis. A total of seven precipitating components was demonstrated. Four components (A,C,V,W) were detected in low frequency only with sera from animals with paratuberculosis while two components (X,Y) were detected in high frequency with sera from animals with or without paratuberculosis. One component (D) was observed in high frequency with sera from animals with paratuberculosis. The magnitude of the serological response to the D component as measured by crossed immunoelectrophoresis correlated well with bacterial load and generally agreed with the quantitative assessment by agar gel immunodiffusion. A development time for crossed immunoelectrophoresis of 24-72 hours after electrophoresis was required to achieve correlation with agar gel immunodiffusion.

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Identification and control of paratuberculosis in a large goat herd.

Mycobacterium paratuberculosis infection was detected in 2 goats in 1974 and in 5 goats in 1975; 5 of which were from a single herd. The magnitude of the subsequent epizootic in the goat herd was not recognized until 1977, when results of bacteriologic culture of fecal and tissue specimens, antibody determinations (agar-gel immuno-diffusion test), and histopathologic studies became available. By 1984, paratuberculosis had been diagnosed in 124 goats. Nearly all the goats were being used in antiserum production and had been given Freund complete adjuvant and human antigens. From 1974 to 1986, herd size varied from 100 to 300. The yearly incidence of paratuberculosis decreased from 13.2% (27 of 204 goats) in 1977 to 0% in 108 goats in 1985. The prevalence was higher in does. In goats that arrived on the farm in 1975 and before, 49 of 121 (40.5%) does developed paratuberculosis vs 41 of 120 (34.2%) wethers. In goats arriving on the farm in 1976 and after, 25 of 274 (8.5%) does and 9 of 216 (4.1%) wethers developed paratuberculosis. The average incubation period was approximately 4 years from arrival on the farm in every year except 1978, regardless of whether the goat was born on the farm or was purchased elsewhere.(ABSTRACT TRUNCATED AT 250 WORDS)

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Paratuberculosis in cattle and free-living exotic deer.

Paratuberculosis was studied among dairy cows and exotic deer that shared grazing areas at Point Reyes National Seashore, California. Of the 10 dairy herds tested, 5 (50%) were infected with Mycobacterium paratuberculosis (based on results of fecal culture). Mycobacterium paratuberculosis was cultured from 9 (8.7%) of the 103 bovine fecal samples and from 4 (3.9%) of the 103 bovine rectal mucosa scapings tested. Of 89 fecal samples from 52 axis deer (Axis axis) and 37 fallow deer (Dama dama), 5 (9.6%) and 3 (8.1%), respectively, contained M paratuberculosis. Culture of intestinal necropsy samples from the same deer indicated that 3 (5.8%) of the axis deer and 2 (5.4%) of the fallow deer were infected with M paratuberculosis. The cows were tested for serum antibodies by the complement-fixation test and by radioimmunoassay. Of 95 sera tested by complement fixation, 15 (15.8%) were positive, as were 15 (14.7%) of 102 sera tested by radioimmunoassay. Culture results and serologic test results were compared on a herd basis.

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Agar gel immunodiffusion test for diagnosis of clinical paratuberculosis in cattle.

During a 19-month period, the agar gel immunodiffusion (AGID) test was used as an aid in differential diagnosis of paratuberculosis in 51 cattle with a history of chronic weight loss and/or chronic diarrhea. Thirty-three cattle were AGID test-positive for paratuberculosis. Twenty-eight cattle (87.5%) yielded Mycobacterium paratuberculosis from bacterial culture of feces. Four cattle were confirmed as having paratuberculosis on necropsy. One had a negative fecal culture but was lost to follow-up. Thus, 32 of the 33 AGID test-positive cattle (96.9%) were confirmed as paratuberculous by culture or necropsy. Of the 18 cattle that were AGID test-negative, 17 (94%) also were culture-negative after 12 to 20 weeks' incubation of the culture tubes. Direct fecal smears from 29 confirmed paratuberculous cattle were examined. Twenty-three (79.3%) had smears positive for M paratuberculosis. Fifteen culture-negative cattle were examined by direct smear; the results were negative for all.

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Comparative lymphocyte stimulation studies on whole blood from vaccinated and nonvaccinated cattle with paratuberculosis.

The lymphocyte stimulation test was evaluated in the diagnosis of bovine paratuberculosis, using whole blood samples from cattle. These samples were incubated with a purified protein derivative of Mycobacterium paratuberculosis. Samples from vaccinated and nonvaccinated cattle from paratuberculosis-free herds and from paratuberculous herds were used. Vaccinated and nonvaccinated groups from the paratuberculous herds each included fecal culture-positive cattle, fecal culture-negative cattle, and calves. All groups included some cattle with a stimulation index (SI) greater than or equal to 2, and all groups except 2 of the 4 vaccinated groups had some cattle with a SI less than or equal to 2. In paratuberculosis-free herds, 16% of the nonvaccinated cattle had a SI greater than or equal to 2, and 0.7% had a SI greater than or equal to 4. In paratuberculous herds, 42% of the nonvaccinated cattle that were negative on fecal culture had a SI greater than or equal to 2 and 24% had a SI greater than or equal to 4. Vaccinated cattle greater than 1 year old could not be distinguished from infected cattle by means of the lymphocyte stimulation test. If a SI of 2 or 4 had been used as a basis to cull individual animals, some paratuberculous cattle would have been identified incorrectly as noninfected, and some paratuberculosis-free cattle would have been identified incorrectly as infected.

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The role of restricted food intake in the pathogenesis of cachexia in severe combined immunodeficient beige mice infected with Mycobacterium paratuberculosis.

A paired feeding experiment was conducted to investigate if reduced food intake is a reason for the body weight loss previously observed in severe combined immunodeficient beige (SCID bg) mice infected with Mycobacterium paratuberculosis. Mice were paired on the basis of age, litter and sex. One of each pair was injected intraperitoneally with 10(5) viable M. paratuberculosis organisms. The remainder served as uninfected pairfed mates. Each uninfected mouse was restricted to the amount of food (per gram body weight) that its infected paired mate ate in the previous 24 hour period starting at four weeks postinfection until 12 weeks postinfection when the mice were necropsied. The mean body weights of the two groups were not significantly different (p < 0.05) at the start of the experiment (infected 27.6 +/- 2.1 g, pairfed 27.3 +/- 3.4 g) but the pairfed group weighed less after 12 weeks of restricted food intake. Mycobacterium paratuberculosis was isolated from the spleen, liver, gut and fecal pellets of the infected but not the uninfected mice. Acid-fast bacilli were seen histologically in the liver, spleen and intestines of the infected mice only. Analysis of carcass compositions indicated that both infected and pairfed mice lost dry matter. Despite the loss in dry matter, the infected mice appeared to have maintained their body weights due to an increased retention of body water (presumably due to edema of inflammation). These results suggest that infection of SCID bg mice with M. paratuberculosis causes a reduction in their food intake (presumably due to reduced appetite) which, in turn, contributes to a loss in dry matter. We suggest that this loss in dry matter is one of the initial events that eventually lead to cachexia, and that it precedes the body weight loss that inevitably occurs in SCID bg mice chronically affected with M. paratuberculosis.

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