Penelope S. Gibbs

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

Mycobacterium avium subspecies paratuberculosis is the causative agent of Johnes 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 Crohns disease. Recent studies have shown that a high percentage of people with Crohns disease are infected with M. avium subsp. paratuberculosis; whether the association of this bacterium and Crohns disease is causal or coincidental is not known. Crohns disease is a gastrointestinal disease in humans with similar histopathological findings to those observed in the paucibacillary form of Johnes disease in cattle. The search for risk factors in Crohns disease has been frustrating. However, epidemiologists have gathered enough information that points to an association between M. avium subsp. paratuberculosis and Crohns 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 Crohns disease.

Location of Increased Serum Survival Gene and Selected Virulence Traits on a Conjugative R Plasmid in an Avian Escherichia coli Isolate

SUMMARY. Avian colibacillosis is a costly disease for the poultry industry. The mechanisms of virulence employed by the etiologic agent of this disease remain ill defined. However, accumulated evidence suggests that complement resistance and the presence of the increased serum survival gene (iss) in an avian Escherichia coli isolate may be indicative of its ability to cause disease. This association of iss with the E. coli implicated in avian disease may mean that iss and/or, perhaps, the genes associated with it are important contributors to avian E. coli virulence. For this reason, we have begun a search for isss location in the bacterial genome. Thus far, iss in an avian E. coli isolate has been localized to a conjugative R plasmid and estimated to be about 100 kilobase (kb) in size, encoding resistance to tetracycline and ampicillin. Hybridization studies have revealed that this plasmid contains sequences with homology to tsh, a gene associated with virulence of avian E. coli; intI1, a gene encoding the integrase of Class 1 integrons; and certain genes of the aerobactin- and CoIV-encoding operons. Sequences homologous to merA, a gene of the mercury resistance operon, were not identified on this R plasmid. This plasmid, when transferred into an avirulent, recipient strain by conjugation, enhanced the transconjugants resistance to complement but not its virulence, in spite of the plasmids possession of several putative virulence genes and traits. Such results may reflect the multifactorial nature of virulence, the degree of the recipients impairment for virulence, or an inability of the embryo assay used here to detect this plasmids contribution to virulence. Additionally, this plasmid contains genes encoding antimicrobial resistances, which may provide a selective advantage to virulent E. coli in the production environment. Further study will be needed to determine whether this plasmid is widespread among virulent E. coli and to ascertain the implications that this link between virulence and antimicrobial resistance genes may have for poultry management.

Comparative effect of direct-fed microbials on fecal shedding of Escherichia coli O157:H7 and Salmonella in naturally infected feedlot cattle.

The effect of direct-fed microbials (DFM) on fecal shedding of Escherichia coli O157:H7 and Salmonella in naturally infected feedlot cattle was evaluated in a clinical trial involving 138 feedlot steers. Following standard laboratory methods, fecal samples collected from steers were evaluated for change in the detectable levels of E. coli O157:H7 and Salmonella shed in feces after DFM treatment. Sampling of steers was carried out every 3 weeks for 84 days. A significant reduction (32%) in fecal shedding of E. coli O157:H7 (P < 0.001), but not Salmonella (P = 0.24), was observed among the treatment steers compared with the control group during finishing. The probability of recovery of E. coli O157:H7 from the feces of treated and control steers was 34.0 and 66.0%, respectively. Steers placed on DFM supplement were almost three times less likely to shed E. coli O157:H7 (odds ratio, 0.36; 95% confidence interval, 0.25 to 0.53; P < 0.001) in their feces as opposed to their control counterparts. The probability of recovery of Salmonella from the feces of the control (14.0%) and the treated (11.3%) steers was similar. However, the DFM significantly reduced probability of new infections with Salmonella among DFM-treated cattle compared with controls (nontreated ones). It appears that DFM as applied in our study are capable of significantly reducing fecal shedding of E. coli O157:H7 in naturally infected cattle but not Salmonella. The factors responsible for the observed difference in the effects of DFM on E. coli O157:H7 and Salmonella warrants further investigation.

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.

Characterizing Avian Escherichia coli Isolates with Multiplex Polymerase Chain Reaction

Abstract SUMMARY. Colibacillosis caused by Escherichia coli infections account for significant morbidity and mortality in the poultry industry. Yet, despite the importance of colibacillosis, much about the virulence mechanisms employed by avian E. coli remains unknown. In recent years several genes have been linked to avian E. coli virulence, many of which reside on a large transmissible plasmid. In the present study, a multiplex polymerase chain reaction (PCR) protocol to detect the presence of four of these genes is described. Such a protocol may supplement current diagnostic schemes and provide a rapid means of characterizing the E. coli causing disease in poultry. The targets of this procedure included iss, the increased serum survival gene; tsh, the temperature sensitive hemagglutinin gene; cvi, the ColV immunity gene; and iucC, a gene of the aerobactin operon. Organisms, known for their possession or lack of these genes, were used as a source of the template DNA to develop the multiplex PCR protocol. Identity of the amplicons was confirmed by size, DNA:DNA hybridization with specific gene probes, and DNA sequencing. When the multiplex PCR protocol was used to characterize 10 E. coli isolates incriminated in avian colibacillosis and 10 from the feces of apparently healthy birds, nine of the isolates from apparently healthy birds contained no more than one gene, while the 10th contained all four. Also, eight of the isolates incriminated in colibacillosis contained three or more genes, while the remaining two contained two of the target genes. Interestingly, the isolates of sick birds containing only two of the targeted genes killed the least number of embryos, and the isolate of healthy birds that contained all the genes killed the most embryos among this group. These genes were not found among the non–E. coli isolates tested, demonstrating the procedures specificity for E. coli. Overall, these results suggest that this protocol might be useful in characterization and study of avian E. coli.

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.

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.

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.

Prediction of Chicken Embryo Lethality with the Avian Escherichia coli Traits Complement Resistance, Colicin V Production, and Presence of the Increased Serum Survival Gene Cluster (iss)

Abstract Differentiating between virulent and avirulent avian Escherichia coli isolates continues to be a problem for poultry diagnostic laboratories and the study of colibacillosis in poultry. The ability of a laboratory to conduct one simple test that correlates with virulence would simplify studies in these areas; however, previous studies have not enabled researchers to establish such a test. In this study, the occurrence of certain phenotypic and genotypic traits purported to contribute to avian E. coli virulence in 20 avian E. coli isolates was correlated with the results of embryo challenge studies. This analysis was undertaken in an effort to determine which trait(s) best identified each avian E. coli isolate as virulent or avirulent. Traits selected were complement resistance, production of colicin V (ColV), motility, type F1 pili expression, presence of the temperature-sensitive hemagglutinin gene (tsh), and presence of the increased serum survival genetic locus (iss). ColV production, complement resistance, and presence of the iss genetic element were the three traits most highly correlated with high embryo lethality. A logistic regression model was used to predict the embryo lethality results on the basis of the most frequent isolate characteristics. Results indicate that ColV, complement resistance, and iss are significant predictor variables for the percentage of embryo lethality resulting from challenge with a specific avian E. coli isolate. However, no single trait has the ability to predict virulent isolates 100% of the time. Such results suggest the possibility that the embryo lethality assay may prove to be the one test needed to determine if an avian E. coli isolate is virulent.

Comparison of Several Challenge Models for Studies in Avian Colibacillosis

In previous studies, the embryo lethality assay (ELA) discriminated between virulent and avirulent avian Escherichia coli isolates, and also proved to be highly correlated with mortality and morbidity results of the intravenous (IV) challenge model. In the current study, the same 20 avian E. coli isolates were used in subcutaneous (subQ) and intratracheal (IT) chicken challenge models in order to determine whether the results from the prior ELA challenges and/or the IV challenge model correlate with these models. The correlation observed between the two previous ELA trials and the combined mortality/morbidity percentages of the subQ challenge model were r = 0.792, P > 0.0001 for the first ELA trial and r = 0.738, P = 0.0002 for the second ELA trial. The IV challenge results were more highly correlated with the subQ challenge results (mortality/morbidity comparison, r = 0.894, P < 0.0001). The IV challenge mortality results were slightly correlated (r = 0.4810, P=0.0319) with the IT challenge results. Several of the isolates differed in their ability to produce mortality and/or morbidity with the different challenge models. The mortality/morbidity results of the IV and subQ challenges and the mortality results of the ELA were all positively correlated with the ability of an E. coli isolate to produce Colicin V (ColV) (r = 0.7131, P = 0.0004). The IT mortality results were slightly correlated with the production of ColV (r = 0.455, P = 0.049). The IT challenge results were only slightly correlated with resulting IV mortality and ColV production. Previous results indicate that the ELA correlates extremely well with the IV challenge model. The current study demonstrates that ELA also correlates well with the subQ challenge model. Overall, the conclusion of this study is that the ELA, IV, and subQ challenge models similarly demonstrate the ability to discriminate between virulent and avirulent avian E. coli isolates.