Epidemiological evidence for Mycobacterium avium subspecies paratuberculosis as a cause of Crohn's disease.

Mycobacterium avium subspecies paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants including cattle, sheep, goats, and farmed deer. Recently, this bacterium has received an increasingly wide interest because of a rapidly growing body of scientific evidence which suggests that human infection with this microorganism may be causing some, and possibly all, cases of Crohn's disease. Recent studies have shown that a high percentage of people with Crohn's disease are infected with M. avium subsp. paratuberculosis; whether the association of this bacterium and Crohn's disease is causal or coincidental is not known. Crohn's disease is a gastrointestinal disease in humans with similar histopathological findings to those observed in the paucibacillary form of Johne's disease in cattle. The search for risk factors in Crohn's disease has been frustrating. However, epidemiologists have gathered enough information that points to an association between M. avium subsp. paratuberculosis and Crohn's disease. This paper reviews epidemiological models of disease causation, the major philosophical doctrines about causation, the established epidemiological criteria for causation, and the currently known epidemiological evidence of M. avium subsp. paratuberculosis as a possible cause of Crohn's disease.

A longitudinal study of Salmonella shedding and antimicrobial resistance patterns in North Dakota feedlot cattle.

Salmonella is one of the most frequent causes of foodborne illness worldwide, and transmission involves foods of animal origin such as beef. The objective of this study was to monitor the prevalence of Salmonella fecal shedding in feedlot cattle during the finishing period and to assess the antimicrobial resistance patterns of the isolated salmonellae. On arrival at the feedlot, 1 (0.7%) of the 144 steers was shedding Salmonella in its feces. After 28 days on feed, shedding was detected in 8 (5.6%) of the 144 steers. At the third sampling, 19 (13%) of 143 steers were shedding, and the number of shedders continued to increase to 89 (62%) of 143 at the last sampling. Salmonella shedding was significantly influenced (P < 0.0001) by sampling time but not by herd of origin. All Salmonella isolates identified belonged to serotype Typhimurium serovar Copenhagen, a type commonly isolated from Salmonella infections in humans. Antimicrobial resistance testing of the isolates revealed five multidrug resistance patterns, two of which accounted for 104 (95.4%) of 109 of the isolates. All the isolates were susceptible to ceftiofur, and all were resistant to spectinomycin, sulfathiazole, tiamulin, florfenicol, ampicillin, penicillin, chlortetracycline, oxytetracycline, and clindamycin. Data from this study indicate that a high prevalence of antimicrobial-resistant Salmonella strains can sometimes be found in feedlot cattle in North Dakota. These data will contribute to risk assessment of Salmonella shedding by cattle in feedlots and highlight the need to continue preharvest monitoring for this organism.

Comparison of two sampling methods for Escherichia coli O157:H7 detection in feedlot cattle.

Two sampling methods (rectoanal swabs and rectal fecal grabs) were compared for their recovery of Escherichia coli O157:H7 from feedlot cattle. Samples were collected from 144 steers four times during the finishing period by swabbing the rectoanal mucosa with cotton swabs and immediately obtaining feces from the rectum of each individual steer. The number of steers with detectable E. coli O157:H7 increased from 2 of 144 (1.4%) cattle on arrival at the feedlot to 10 of 144 (6.9%) after 1 month, 76 of 143 (52.8%) after 7 months, and 30 of 143 (20.8%) at the last sampling time before slaughter. Wilcoxon signed-rank tests indicated that the two sampling methods gave different results for sampling times 3 and 4 (P < 0.05) but not for sampling time 2 (P = 0.16). Agreement between the two sampling methods was poor (kappa < 0.2) for three of the four sampling times and moderate (kappa = 0.6) for one sampling time, an indication that in this study rectoanal swabs usually were less sensitive than rectal fecal grabs for detection of E. coli O157:H7 in cattle. Overall, the herd of origin was not significantly associated with E. coli O157:H7 results, but the weight of the steers was. Further investigation is needed to determine the effects of potential confounding factors (e.g., size and type of swab, consistency of feces, site sampled, and swabbing technique) that might influence the sensitivity of swabs in recovering E. coli O157:H7 from the rectoanal mucosa of cattle.

Chicken embryo lethality assay for determining the virulence of avian Escherichia coli isolates.

Multiple isolates of Escherichia coli from clinical cases of colibacillosis and E. coli from the intestinal tracts of normal broilers at slaughter were assayed by the embryo lethality test to determine their virulence. The assay was repeated five times in order to establish reproducibility and determine the statistical parameters of the test. This study showed that the inoculation of approximately 100 colony-forming units in the allantoic cavity of 12-day-old embryos discriminated between virulent and avirulent E. coli isolates. Gross lesions included cranial and skin hemorrhages in addition to encephalomalacia in embryos inoculated with virulent isolates. Abnormalities were observed by microscopic examination of the heart, brain, and liver in embryos inoculated with virulent isolates. Analysis of data indicated that the length of the test should be 4 days. In the virulent group, day 2 postinoculation had the most significant death patterns. Sample size calculations indicated that 11 embryos are sufficient for the assay. On the basis of death rates, isolates considered to be avirulent had an embryo death rate of <10%, moderately or secondary pathogens had a 10%-29% death rate, and virulent isolates had a death rate of >29%. An important aspect of this assay is the accessibility of good-quality fertile embryonated eggs.

Normal hematologic and serum biochemical reference intervals for juvenile wild turkeys.

Blood samples taken from 48 4-mo-old wild turkeys (Meleagris gallopova silvestris) were used to establish reference intervals for hematology and serum chemistry values. The study was conducted during September and October 1996. Packed cell volume, total and differential white cell counts, total protein, albumin, glucose, calcium, uric acid, triglyceride concentrations, as well as aspartate transaminase (AST) and lactate dehydrogenase (LDH) activities were assayed. Reference intervals from wild turkeys are similar to those reported for domestic turkeys.

In vitro effect of ethylenediaminetetraacetic acid-Tris on the efficacy of hatchery disinfectants.

Solutions of ethylenediaminetetraacetic acid (EDTA)-Tris showed synergistic or additive effects on gram-negative bacteria when combined with hatchery disinfectants consisting of phenol and detergent (Magnaphen-100), quaternary ammonium compound (QAC) and glutaraldehyde (Synergize), QAC (BioSentry 904), and hydrogen peroxide. The gram-positive bacteria reacted less favorably, with reaction mixtures showing all three levels of potentiation (synergistie, additive, and antagonistic). Combinations of EDTA-Tris and a commercial glutaraldehyde solution (Glutracide), when mixed with the test organisms, showed mostly antagonistic effects. Solutions of EDTA-Tris decreased the concentration of hatchery disinfectants required for bacterial killing by 75% in those situations in which synergistic potentiation occurred. EDTA-Tris is nontoxic to 12-day-old embryos. Serial passage of the test organisms in solutions of EDTA-Tris did not result in the development of resistant forms.

Inhibition of Salmonella typhimurium in the chicken intestinal tract by a transformed avirulent avian Escherichia coli.

An avirulent, wild-type avian Escherichia coli (E. coli Av) was electrotransformed with a plasmid coding for the production of microcin 24 (pGOB18) and was designated E. coli AvGOB18. The transformant inhibited the growth of seven serotypes of Salmonella commonly associated with colonization and contamination of poultry products and seven strains of E. coli O157:H7 in the in vitro colicin/microcin assay. The transformant did not inhibit the replication of multiple isolates of Listeria monocytogenes or Campylobacter jejuni in similar assays. The transformant is nonconjugative, indicating that the plasmid would not be transmitted to other intestinal microflora in the environment. The transformant also survived in sterile tap and deionized water incubated at 25 C and 37 C in the laboratory for 30 days and was recovered from drinkers and birds in in vivo floor pen studies. In in vivo studies, E. coli AvGOB18 did not colonize the intestinal tract of broiler chicks when given as a single or multiple dose and did not reduce the Salmonella load in the broilers. But Salmonella typhimurium was reduced significantly in the intestinal tracts of broiler chickens when E. coli AvGOB18 was administered continually in the water supply.

Mutation rate of avian intestinal coliform bacteria when pressured with fluoroquinolones.

The purpose of this study was to determine the rate at which resistance developed in avian coliform bacteria when exposed to nalidixic acid, sarafloxacin, or enrofloxacin. In in vitro studies, the rates of mutation of avian isolates of Escherichia coli and Salmonella were determined following nalidixic acid, sarafloxacin, or enrofloxacin pressure. The rates of mutation were similar for nalidixic acid and sarafloxacin, whereas a lower rate of mutation was seen after enrofloxacin pressure. In in vivo studies, the quinolones were administered in the drinking water to broiler chickens at a concentration of 40 ppm for five consecutive days. Samples of feces were inoculated onto appropriate media and the frequency of resistance was determined. The frequency rates of resistance to nalidixic acid and sarafloxacin were similar. Enrofloxacin-medicated birds did not develop enrofloxacin-resistant coliform bacteria. The in vitro and in vivo data appear to correlate.

Colonization of the chicken trachea by an avirulent avian Escherichia coli transformed with plasmid pHK11.

Recombinant plasmid pHK11 was transformed into an avirulent, wild-type avian Escherichia coli (E. coli Av) in order to study the plasmid's effect on colonization of the chicken trachea. The transformant (E. coli Av + pHK11) produced colicin V (ColV), had type F1 fimbriae, and was motile. The E. coli Av recipient possessed type F1 fimbriae but was nonmotile; it did not produce ColV. Four-day-old chicks were inoculated in the trachea with 100 microliters of an overnight culture (approximately 10(8) colony-forming units) of E. coli Av, E. coli Av + pHK11, or sterile brain-heart infusion (BHI) broth. A group of uninoculated chicks was also included. Samples of the trachea were taken on days 4 and 10 postinoculation and compared histologically and bacteriologically. Birds inoculated with E. coli Av + pHK11 had enhanced tracheal colonization and showed increased histologic changes as compared with those inoculated with E. coli Av or BHI broth or uninoculated controls. These results indicate that production of ColV and motility enhance the colonization of the trachea and may be involved in the cause of pathologic lesions.

Analysis of plasmids cloned from a virulent avian Escherichia coli and transformed into Escherichia coli DH5 alpha.

Three of four plasmids from a virulent wild-type avian Escherichia coli were cloned or transformed into an avirulent laboratory recipient E. coli DH5 alpha and tested for the ability to confer a virulence phenotype. The three plasmids transformed into E. coli DH5 alpha were 5, 6, and 56 kb. A fourth plasmid of 64 kb was not successfully transformed. Parameters used to measure virulence included presence of type 1 pili and a smooth lipopolysaccharide (LPS) layer, motility, production of Colicin V, resistance to host complement, and embryo lethality. The 5-kb plasmid encoded for ampicillin resistance, whereas the 6-kb plasmid encoded for tetracycline resistance. The 56-kb plasmid encoded for streptomycin, sulfisoxazole, and tetracycline resistance. Twelve-day embryos inoculated with 467 colony-forming units of E. coli DH5 alpha containing the 56-kb plasmid had increased death rates (45%) in the embryo lethality assay and a decreased weight of surviving embryos with cranial hemorrhages as compared with embryos inoculated with similar amounts of E. coli DH5 alpha (0%) and phosphate-buffered saline (0%). Embryos inoculated with the wild-type virulent E. coli had 90% deaths. The 56-kb plasmid also had homology with a probe for Colicin V production (cvaC). No differences in LPS layer, complement resistance, motility, Colicin V activity, type 1 pili, cell-free supernatant proteins, or outer membrane proteins were observed in the transformants when compared with nontransformed E. coli DH5 alpha.

Analysis and characterization of Mycoplasma gallisepticum isolates from Pennsylvania.

Because Mycoplasma gallisepticum F strain vaccine can be pathogenic in chickens and is pathogenic in turkeys, we monitored the spread of MG F strain into unvaccinated flocks by screening field and experimental isolates. Thirteen MG isolates obtained from various sources in Pennsylvania were screened using several techniques capable of differentiating between MG strains. DNA restriction enzyme analysis (REA), Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein profiles, non-isotopic DNA probes, and a monoclonal antibody specific for F strain were used to characterize each of the 13 isolates. Three of the 13 isolates were identical to F strain; two of these were obtained from challenge studies, and one was a field isolate from a multiple-age commercial egg farm where the F strain vaccine had been used in the past. The remaining 10 isolates were different from MG F strain but were quite similar if not identical to each other according to REA; SDS-PAGE protein profiles show similarities between the 10 isolates. The results suggested that F strain vaccine is not a major cause of field outbreaks of MG in Pennsylvania.

Antimicrobial effect of combinations of EDTA-Tris and amikacin or neomycin on the microorganisms associated with otitis externa in dogs.

Combinations of EDTA-Tris and two aminoglycoside antibiotics (amikacin and neomycin) were tested for synergistic activities against the microorganisms associated with otitis externa in dogs and for the solutions' stability over time. Synergistic activity was observed when EDTA-Tris plus amikacin and EDTA-Tris plus neomycin were tested against Staphylococcus intermedius, Proteus mirabilis, Pseudomonas aeruginosa, and Escherichia coli, but not against Candida albicans. Stability studies over a 3-month period indicated that the test solutions were stable at room temperature and that their antimicrobial activity was maintained.

Phenotypic expression of recombinant plasmids pKT107 and pHK11 in an avirulent avian Escherichia coli.

An avirulent wild-type avian Escherichia coli strain (Av) was electrotransformed with plasmids coding for complement resistance (pKT107) and Colicin V (ColV) production (pHK11) in order to study the effects of complement resistance and ColV production on virulence. Transformants were also compared with the wild type for embryo lethality, uptake by macrophages, motility, growth rate, plasmid content, and hemolysis. Growth rates and complement resistance patterns of strain Av and transformant Av+pHK11 were similar, but Av+pHK11 caused a significantly greater number of deaths in embryos and acquired motility. Transformant Av+pKT107 had a lower rate of phagocytosis, a slower growth rate, and a greater sensitivity to complement, and it changed from being non-hemolytic to expressing alpha-hemolytic action. The 35-kb plasmid present in the wild type was not present in the transformants. Although some of the results demonstrate the difficulties encountered in using wild-type organisms as recipients in virulence studies, the results with Av+pHK11 indicate that ColV production plus the acquisition of motility contributes to the virulence of avian E. coli.

Effect of normal intestinal flora of chickens on colonization by virulent colicin V-producing, avirulent, and mutant colicin V-producing avian Escherichia coli.

Colonization of the intestinal tracts of newly hatched chicks with Escherichia coli was attempted by swabbing test organisms onto the air-shell of 19-day-old embryos. Test organisms consisted of two virulent E. coli isolates, one avirulent isolate, and one laboratory-derived mutant of the avirulent isolate carrying a recombinant plasmid coding for Colicin V production. Chicks were cultured weekly for 3 weeks for total E. coli and for the test organisms using selective media. Control chicks were sampled on weeks 1 and 5, and the normal E. coli intestinal microflora were examined for the production of colicins. The two virulent E. coli isolates maintained colonization of the chicks for the 3-week test period, with titers decreasing from 10' to 10'- colony-forming units (CFU)/g of intestine. The avirulent isolate and laboratory mutant did not consistently colonize the intestinal tracts. The majority of intestinal samples taken from the control chicks at 1 and 5 weeks had colicin-producing E. coli that were inhibitory to the test organisms.

Association of K-1 capsule, smooth lipopolysaccharides, traT gene, and Colicin V production with complement resistance and virulence of avian Escherichia coli.

A group of complement-resistant, virulent avian Escherichia coli isolates were compared with a group of complement-sensitive, avirulent avian isolates for the presence of K-1 capsule, smooth lipopolysaccharides (LPS), the traT gene, and Colicin V (ColV) production. These parameters were selected because of their reported association with complement resistance and virulence in E. coli. Lethality in chicken embryos has also been shown to be correlated with virulence of avian E. coli for chickens. The complement-resistant, virulent E. coli isolates did not possess a K-1 capsule. Production of ColV and the presence of smooth LPS were significantly correlated with embryo lethality. There was no correlation between the presence of traT and embryo lethality. These results suggest that complement resistance and virulence in avian E. coli are associated with ColV production and smooth LPS but not with K-1 antigen or traT.

Activation of thymocyte deoxyribonucleic acid degradation by endogenous glucocorticoids.

Immature lymphocytes in the thymus gland are killed by treatment with exogenous glucocorticoids. This steroid-mediated lymphocytolysis is preceded by numerous alterations in lymphocyte metabolism, including a DNA-degrading process in which the genome is cleaved at internucleosomal intervals. To date, this process has only been characterized by treating lymphocytes in vitro with glucocorticoids or by exogenous treatment of whole animals with adrenal steroids. To determine whether thymocyte DNA degradation could be activated by endogenous glucocorticoids, 4-wk-old chicks were treated with porcine adrenocorticotropic hormone (ACTH). This procedure elevated serum corticosterone levels approximately 80-fold within 2 h of hormone treatment. Following ACTH administration, thymocyte DNA was isolated and analyzed by agarose gel electrophoresis. The ACTH activated a DNA-degrading process that generated internucleosomal fragments of DNA identical in size to those observed following exogenous treatment with synthetic or naturally occurring glucocorticoids. Furthermore, this response could be inhibited by the glucocorticoid antagonist RU486 (17 beta-hydroxy-11 beta, 4-dimethylaminophenyl-17 alpha-propynl-estra-4,9,diene-3-one), indicating that adrenal steroids activate this process via the glucocorticoid receptor. These results demonstrate that lymphocyte DNA degradation does not result solely from exogenous glucocorticoid treatment; moreover, endogenous glucocorticoids can mediate this process and may thereby play an important role in thymic gland function.

Glucocorticoid-mediated activation of DNA degradation in avian lymphocytes.

Little information is known about the molecular mechanism of programmed cell death in the avian species. In the current study we have analyzed this process in chickens using a glucocorticoid-lymphocyte model system. Three-week-old male broiler chicks were treated in vivo with the synthetic glucocorticoid, dexamethasone. Following this treatment genomic DNA was isolated from thymocytes and analyzed by agarose gel electrophoresis. Dexamethasone activated a DNA degrading process in which the genome was specifically cleaved at internucleosomal intervals. This steroid-induced response occurred prior to thymocyte cell death and was time and glucocorticoid dose dependent, as well as tissue and steroid specific. Only the glucocorticoid class of steroid hormones could elicit this response and DNA degradation was only detectable in lymphoid tissues that contained immature lymphocytes. Internucleosomal DNA degradation could also be elicited via administration of adrenocorticotrophic hormone, a treatment that elevates endogenous glucocorticoids. Based on these data, glucocorticoid-activated DNA degradation of the avian thymocyte genome appears to be a steroid receptor-mediated process which involves the activation of an endogenous nuclease that cleaves the genome at internucleosomal sites. Degradation of the thymocyte genome occurs prior to cell death and may represent an initial event in a cascade of hormone-mediated processes that culminate in a type of cellular suicide referred to as programmed cell death.

Glucocorticoid activation of deoxyribonucleic acid degradation in bursal lymphocytes.

Treatment of animals with exogenous adrenal steroids or elevation of endogenous glucocorticoids results in a profound involution of lymphoid tissue. In rodent species, this lymphoinvolution is accompanied by lymphocyte cell death and extensive degradation of the genome prior to lymphocytolysis. In the present study, this process was investigated in the bursa of Fabricius of domestic fowl. Four-wk-old chicks were treated with a single injection of dexamethasone, and bursal regression and cell viability were monitored over a 72-h period. Following hormone treatment, DNA was extracted from bursal lymphocytes and analyzed by agarose gel electrophoresis. Dexamethasone treatment resulted in a rapid regression of bursal tissue that could be detected as soon as 6 h posttreatment, but lymphocyte viability was not altered until 24 h afterward. The DNA isolated from bursal lymphocytes of glucocorticoid-treated birds appeared to be degraded at internucleosomal sites and generated a "ladder" of discrete DNA fragments when analyzed by agarose gel electrophoresis. This form of hormone-induced cell death, referred to as programmed cell death, may play a key role in glucocorticoid-mediated immunosuppression.