Correlation between geographic distance and genetic similarity in an international collection of bovine faecal Escherichia coli O157:H7 isolates.

Evidence from epidemiological and molecular studies of bovine Escherichia coli O157:H7 suggests that strains are frequently transmitted across wide geographic distances. To test this hypothesis, we compared the geographic and genetic distance of a set of international bovine Escherichia coli O157:H7 isolates using the Mantel correlation. For a measure of genetic relatedness, pulsed-field gel electrophoresis of six different restriction enzyme digests was used to generate an average Dice similarity coefficient for each isolate pair. Geographic distance was calculated using latitude and longitude data for isolate source locations. The Mantel correlation between genetic similarity and the logarithm of geographic distance in kilometers was -0.21 (P<0.001). The low magnitude of the Mantel correlation indicates that transmission over long distances is common. The occurrence of isolates from different continents on the same cluster of the dendrogram also supports the idea that Escherichia coli O157:H7 strains can be transferred with considerable frequency over global distances.

Rehabilitation after amputation.

The principles of amputee rehabilitation, from preamputation to reintegration into the work force and community, are reviewed. The authors discuss exercise techniques, training programs, and environmental modifications that have been found to be helpful in the rehabilitation of the amputee. The exercise programs presented here are divided into four main components: flexibility, muscle strength, cardiovascular training, and balance and gait. The programs include interventions by the physical, occupational, and recreational therapist under the supervision and guidance of a physician.

Development and validation of sensitive assays to quantitate gene expression after p53 gene therapy and paclitaxel chemotherapy using in vivo dosing in tumor xenograft models.

SCH58500 (ACN53) is a replication-deficient, type 5 adenovirus (Ad) expressing human wild-type p53 tumor suppressor. It is currently undergoing clinical trials as a cancer therapeutic. Many SCH58500 clinical trials incorporate an arm comparing traditional chemotherapy against chemotherapy combined with SCH58500. Paclitaxel was chosen for combination therapy in the preclinical study reported here due to its extensive use as a first-line therapy in ovarian cancer, its synergy with SCH58500 in preclinical cancer models, and its activation of p53-independent apoptosis, which might result in a "lowered threshold" for tumor cell death. SCID mice bearing human tumor xenografts were dosed with intratumoral vehicle, control Ad vector, or SCH58500, with or without paclitaxel. Real-time quantitative reverse transcriptase polymerase chain reaction assays were developed and validated to quantitate expression of p53, the p53 downstream effector gene p21, and the apoptosis-related genes, bax, bcl-2, and survivin. Protein expression was confirmed using immunohistochemical assays for p53 and p21. Only tumors injected with SCH58500 had detectable levels of exogenous p53 DNA and mRNA. After SCH58500 treatment, 3-11-fold elevations of p21 expression were observed in tumor xenografts containing nonfunctional p53 (MDA-MB-468, MDA-MB-231, MIAPaCa2, DU-145, and SK-OV-3), but no change in p21 mRNA in wild-type p53 PA-1 tumors. Immunohistochemical assays confirmed induction of p21 protein in MDAMB-468 and SK-OV-3 cells, but not in PA-1 cells. Ad vector alone or paclitaxel alone had no effect on p21 mRNA levels in most tumors. However, paclitaxel suppressed p21 expression induced by SCH58500 4-fold in DU-145 and SK-OV-3 tumors. Paclitaxel also affected expression of the housekeeping gene gapdh. There was no consistent pattern to the changes in bax, bcl-2, or survivin after SCH58500 treatment with or without paclitaxel between tumor types, although there were consistent responses within individual tumor lines. The mRNA ratios for bax/bcl-2 and bax/survivin were also not informative across tumor types. Of the genes examined, only p21 gave a predictable response 24 hours after p53 gene therapy and therefore, p21 expression may be useful for confirming SCH58500 activity in human tumor biopsies.

Evaluation of dietary and environmental risk factors for hyperthyroidism in cats.

OBJECTIVE: To identify dietary and environmental risk factors for hyperthyroidism in cats. DESIGN: Case-control study. ANIMALS: 100 cats with hyperthyroidism and 163 control cats. PROCEDURE: Medical records were examined, and owners completed a mailed questionnaire. Data collected included information regarding demographic variables, environmental exposures, and diet, including preferred flavors of canned cat food. RESULTS: Case cats were significantly less likely to have been born recently than control cats. Housing; exposure to fertilizers, herbicides, or plant pesticides; regular use of flea products; and presence of a smoker in the home were not significantly associated with an increased risk of disease, but cats that preferred fish or liver and giblets flavors of canned cat food had an increased risk. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that cats that prefer to eat certain flavors of canned cat food may have a significantly increased risk of hyperthyroidism.

Design, characterization, and structure of a biologically active single-chain mutant of human IFN-gamma.

A mutant form of human interferon-gamma (IFN-gamma SC1) that binds one IFN-gamma receptor alpha chain (IFN-gamma R alpha) has been designed and characterized. IFN-gamma SC1 was derived by linking the two peptide chains of the IFN-gamma dimer by a seven-residue linker and changing His111 in the first chain to an aspartic acid residue. Isothermal titration calorimetry shows that IFN-gamma SC1 forms a 1:1 complex with its high-affinity receptor (IFN-gamma R alpha) with an affinity of 27(+/- 9) nM. The crystal structure of IFN-gamma SC1 has been determined at 2.9 A resolution from crystals grown in 1.4 M citrate solutions at pH 7.6. Comparison of the wild-type receptor-binding domain and the Asp111-containing domain of IFN-gamma SC1 show that they are structurally equivalent but have very different electrostatic surface potentials. As a result, surface charge rather than structural changes is likely responsible for the inability of the His111-->Asp domain of to bind IFN-gamma R alpha. The AB loops of IFN-gamma SC1 adopt conformations similar to the ordered loops of IFN-gamma observed in the crystal structure of the IFN-gamma/IFN-gamma R alpha complex. Thus, IFN-gamma R alpha binding does not result in a large conformational change in the AB loop as previously suggested. The structure also reveals the final six C-terminal amino acid residues of IFN-gamma SC1 (residues 253-258) that have not been observed in any other reported IFN-gamma structures. Despite binding to only one IFN-gamma R alpha, IFN-gamma SC1 is biologically active in cell proliferation, MHC class I induction, and anti-viral assays. This suggests that one domain of IFN-gamma is sufficient to recruit IFN-gamma R alpha and IFN-gamma R beta into a complex competent for eliciting biological activity. The current data are consistent with the main role of the IFN-gamma dimer being to decrease the dissociation constant of IFN-gamma for its cellular receptors.

Design and analysis of an engineered human interleukin-10 monomer.

A monomeric form of human interleukin 10 (IL-10M1) has been engineered for detailed structure-function studies on IL-10 and its receptor complexes. Wild type IL-10 (wtIL-10) is a domain swapped dimer whose structural integrity depends on the intertwining of two peptide chains. wtIL-10 was converted to a monomeric isomer by inserting 6 amino acids into the loop connecting the swapped secondary structural elements. Characterization of IL-10M1 by mass spectroscopy, size exclusion chromatography, cross-linking, and circular dichroism shows that IL-10M1 is a stable alpha-helical monomer at physiological pH whose three-dimensional structure closely resembles one domain of wtIL-10. As previously reported, incubation of wtIL-10 with a soluble form of the IL-10Ralpha (sIL-10Ralpha) generates a complex that consists of 2 wtIL-10 molecules and 4 sIL-10Ralphas. In contrast, IL-10M1 forms a 1:1 complex with the sIL-10Ralpha. Characterization of the interaction using isothermal titration calorimetry confirmed the 1:1 stoichiometry and yielded a dissociation constant of 30 nm with an apparent binding enthalpy of -12.2 kcal/mol. Despite forming a 1:1 complex, IL-10M1 is biologically active in cellular proliferation assays. These results indicate that the 1:1 interaction between IL-10M1 and IL-10Ralpha is sufficient for recruiting the signal transducing receptor chain (IL-10Rbeta) into the signaling complex and eliciting IL-10 cellular responses.

Respiratory diseases of rabbits.

Respiratory diseases are second only to gastroenteric diseases in importance in rabbits. Pasteurellosis is the primary respiratory disease affecting domestic rabbits, but other bacteria (e.g., Bordetella broniseptica and Staphylococcus spp) are significant opportunistic pathogens. The primary manifestations are upper respiratory disease (e.g., rhinitis, sinusitis, conjunctivitis, and dacryocystitis). Various antimicrobials are effective for treatment.

The use of laser scanning cytometry to assess depth of penetration of adenovirus p53 gene therapy in human xenograft biopsies.

SCH58500 is an agent for gene therapy of cancer, consisting of a replication-deficient type 5 adenovirus (Ad5) expressing the human p53 tumor suppressor gene (Ad5/p53). An important question about the use of Ad5/p53 gene therapy is how to achieve the therapeutically effective delivery of an Ad5/p53 vector to the tumor. We wanted to determine the effective depth of penetration of an Ad5/p53 vector by dosing the vector in an experimental human xenograft/SCID model. To assess depth of penetration, we developed a novel methodology for scanning tissue sections by laser scanning cytometry (LSC). SCID mice were given intraperitoneal injections of either p53(null) SK-OV-3 human ovarian tumor cells or p53(mut) DU-145 human prostate tumor cells to establish xenograft solid tumors. Mice were then dosed once or twice at 24-hour intervals by intraperitoneal injection with SCH58500 (Ad5/p53), an adenovirus construct expressing beta-galactosidase (Ad5/beta-gal), or a buffer control. Additional groups of mice received a single intraperitoneal dose of 10 mg/kg paclitaxel either alone or coadministered with Ad5/p53. Twenty-four hours after each last dose, the human solid tumor xenograft and relevant mouse tissue were removed from each mouse for the analysis of Ad5/p53 penetration. Immunohistochemistry (IHC) for beta-galactosidase protein revealed a depth of penetration of between 1 and 10 cells from the tumor surface. In some mice, hepatocytes in the periportal regions of liver lobules were also positive, indicating systemic absorption of adenovirus from the peritoneal cavity. IHC staining for p53 and p21 proteins in SK-OV-3 solid tumor xenografts revealed similar Ad/p53 penetration. LSC was used to map and quantitate apoptosis in both tumor and liver tissue biopsies, with over 450,000 nuclei from liver tissue and 150,000 nuclei from tumor tissue being evaluated. LSC analysis demonstrated a high level of apoptosis in the tumors that had been removed from Ad5/p53-dosed mice (12.7-19.7%). This level of apoptosis was significantly higher (P < 0.05) than was observed for liver tissues taken from Ad5/p53-dosed mice (2.7-8.0%) or tumor tissues taken from either Ad5/beta-gal-dosed mice (3.0-6.4%) or buffer control-dosed mice (3.0-5.3%). Scan bit maps from the extensive LSC analyses confirmed that apoptosis was present to about the same depth (1-10 cells) as had been identified by IHC for beta-galactosidase, p53, and p21 proteins. Paclitaxel coadministered with Ad5/p53 had no effect on Ad5 penetration into solid tumors in vivo as measured by IHC for p53 or p21 protein. However, the combination therapy did cause an elevation in the number of tumor cells undergoing apoptosis.

Changes in antimicrobial resistance among Salmonella enterica Serovar typhimurium isolates from humans and cattle in the Northwestern United States, 1982-1997.

We compared antimicrobial resistance patterns of Salmonella enterica serovar Typhimurium (ST) of isolates from humans (n = 715) and cattle (n = 378) in the Pacific Northwest from 1982 through 1997. The major changes in antimicrobial resistance can be attributed to the widespread clonal dissemination of multidrug-resistant definitive phage type 104 ST.

Natural transmission of bovine leukemia virus in dairy and beef cattle.

Many potential routes of bovine leukemia virus (BLV) transmission are reviewed in this article. Vertical transmission, in utero, or through colostrum and milk, accounts for a relatively small proportion of infections. Iatrogenic horizontal transmission, through procedures permitting the transfer of blood between cattle, has been shown to be a major route of transmission in most settings. Contact transmission stems from a mixture of natural sources of blood, exudates, and tissues that enter the body through mucosal surfaces or broken skin. Careful analysis of management procedures and environmental conditions present in individual dairy and beef herds affords the greatest opportunity to develop effective BLV prevention programs.

Food animal and poultry retroviruses and human health.

In summary, studies reported to date have largely failed to demonstrate human infection with animal and poultry retroviruses or an association between human diseases and these viruses. A number of studies, most of them serologic, have attempted to demonstrate human infection with these viruses. The lack of antibodies in apparently exposed groups of persons suggests an absence of infection. However, another possible explanation is that humans may be immunologically unresponsive to infection with these viruses. Although attempts to infect normal human cells in vitro with many of these viruses have not been reported, BLV and BIV appear to grow poorly or not at all. On the other hand, ALSV subgroup D infect and transform human cells in vitro. However, the production of infectious virus in vitro has been low or nonexistent. This may explain the absence of antibodies in human populations. Furthermore, many of the methods used to detect infection, either directly or indirectly, have either low sensitivity or problems with specificity. Several epidemiologic studies have tried to show a relationship between human and animal leukemia or lymphoma. In many of these studies the actual exposure to retroviruses is unknown and exposure to animals may merely represent exposure to other risk factors that are more important but were either not considered or are undefined; alternatively, a common exposure may be responsible for malignancy in humans and animals with no interspecies relationship. Based on the reported studies, these viruses appear unlikely to be responsible for any significant occurrence of human disease, particularly lymphoid malignancies. Although a definitive statement of no risk to human health is probably unwarranted, the evidence to date indicates that the risk is low and perhaps nonexistent. Thus, no specific public health recommendations regarding retrovirus-infected animals or poultry are warranted at this time.

Detection of Antibodies to Pasteurella multocida by capture enzyme immunoassay using a monoclonal antibody against P37 antigen.

As infection with Pasteurella multocida is common in rabbits, an enzyme immunoassay (EIA) was developed for its detection. A murine immunoglobulin G monoclonal antibody was used to capture a 37-kDa polypeptide of P. multocida serotype A:12 in an EIA to detect antibodies to P. multocida. The 37-kDa antigen was selected since it was previously shown to be a major immunogen during P. multocida infection in rabbits. The sensitivity of the P37 EIA, determined with sera from 56 rabbits infected with P. multocida, was 98%. Specificity, evaluated with sera from 62 rabbits from colonies free of P. multocida, was 92%. Titration curves of sera from rabbits immunized with P. multocida serotype A:3 or A:12 coincided, indicating that the P37 EIA was equally efficient in detecting antibodies to the two major serotypes of the organism. Comparison of the P37 EIA with the current serodiagnostic test, a bacterial lysate EIA, revealed relatively good correlation (r = 0.68). However, specificity was greatly improved, as 34% of uninfected rabbits were falsely positive by the lysate EIA whereas only 3% of uninfected rabbits were falsely positive by the P37 EIA. The coefficient of variation for same-day tests was 10%, and that for interday tests was 15%, indicating good reproducibility. The greater sensitivity and specificity of the P37 EIA should significantly enhance diagnostic capability to identify rabbits infected with P. multocida.

The effect of combined rotavirus and Escherichia coli infections in rabbits.

In rabbits, experimentally induced rotavirus infection results in soft feces only; thus it is unlikely that it is the sole cause of the severe, often fatal diarrhea of weanling rabbits with which it is associated. To determine whether rotavirus acts synergistically with another pathogen, New Zealand White rabbits (10 to 38 weeks old) were inoculated with rotavirus (L:ALA:84) and/or Escherichia coli 015:H-(RDEC-1) via orogastric tube. A single dose of high-titer (10(6) fluorescent focus-forming units) rotavirus was used, whereas E. coli was administered in various doses (10(2) to 10(9) CFU) to determine the titer of E. coli that induced only mild diarrhea but, when combined with rotavirus, resulted in diarrheal disease. Doses of E. coli > 10(6) CFU resulted in infection in almost all rabbits 10 to 16 weeks old, as detected by fecal shedding, regardless of whether rotavirus was inoculated simultaneously. However, inoculation of > 10(6) CFU of E. coli, in conjunction with rotavirus, resulted in increased morbidity and mortality due to diarrheal disease compared with E. coli alone. Inoculation of rabbits 28 to 38 weeks old with similar doses of rotavirus and E. coli caused infection but failed to induce diarrhea, indicating that older rabbits were more resistant to the pathogenic effects of these two agents. A synergistic effect between rotavirus and E. coli occurred, causing more severe diarrheal disease in weanling rabbits than that resulting from either pathogen alone.

Descriptive features of Dientamoeba fragilis infections.

A total of 237 cases of Dientamoeba fragilis were identified by a state public health laboratory in 1985 and 1986. Dientamoeba fragilis was the only parasite found in about two-thirds of the cases. Compared to Giardia cases diagnosed in a similar time period, D. fragilis occurred more frequently in females and in children 5-9 years old; it was also more likely to be detected in spring and summer months. Giardia occurred more frequently in children 0-4 years old. Seventy-nine per cent of 70 interviewed D. fragilis cases reported symptoms associated with infection; nearly 80% had diarrhoea or loose stools. Interviewed cases reported more household and non-household exposure to children 5-9 years old than children of other ages. The difference in age and sex distribution of D. fragilis and Giardia cases may be related to the life cycle and mode of transmission of the two protozoans.

Characterization of recombinant extracellular domain of human interleukin-10 receptor.

The extracellular region of the human interleukin-10 (hIL-10) receptor was expressed using a myeloma cell line and was purified to homogeneity by ligand-affinity chromatography. SDS-polyacrylamide gel electrophoresis analysis indicated that the soluble receptor is glycosylated and has an apparent molecular mass of 35,000-45,000. Under native conditions, soluble hIL-10 receptor was determined by gel filtration to be a monomeric protein. Soluble hIL-10 receptor was able to inhibit the binding of 125I-hIL-10 to the full-length receptor and was able to antagonize the effect of human IL-10 in cell proliferation and cytokine synthesis inhibition. The apparent dissociation constant (Kd) of soluble hIL-10 receptor was determined to be 563 +/- 59 pM, approximately 2- to 10-fold higher than that found on intact cells (Tan, J. C., Indelicato, S. R., Narula, S. K., Zavodny, P. J., and Chou, C.-C. (1993) J. Biol. Chem. 268, 21053-21059; Liu, Y., Wei, S. H.-Y., Ho, A. S.-Y., de Waal Malefyt, R., and Moore, K. W. (1994) J. Immunol. 152, 1821-1829). When hIL-10 binds soluble hIL-10 receptor in solution, a single complex was detected by gel filtration, and the complex was found to consist of two hIL-10 dimers and four soluble receptor monomers, suggesting that hIL-10 may induce a novel mode of oligomerization of the receptor upon binding.

Cerebral larva migrans caused by Baylisascaris sp in pet rabbits.

Cerebral larva migrans was diagnosed histologically in 4 pet rabbits that developed progressive neurologic disease. Larvae of Baylisascaris sp were isolated from brain tissues in 2 rabbits. The clinical syndrome of progressive torticollis and ataxia manifested by these rabbits is commonly associated with otitis and labyrinthitis attributable to bacterial infection; however, the middle ears were normal on radiographic and postmortem examinations. The severe encephalopathy that developed in these rabbits was indicative that just a few Baylisascaris larvae may cause extensive brain injury. During the summer, all of the affected rabbits were maintained outdoors in suburban areas, where raccoons, the final host of B procyonis, are commonly observed. Raccoon feces containing B procyonis eggs constitute a health risk for rabbits, as well as for human beings.

Prevalence of coronavirus antibodies in rabbits.

Antibodies to coronavirus were detected by an indirect fluorescent antibody test in rabbit sera from six rabbitries. The prevalence ranged from 3 to 40% in different rabbitries and most seropositive rabbits were more than 4 months old. A rabbitry with high prevalence of antibodies and high incidence of diarrhea could serve as a source of virus and aid in studying the natural history of coronavirus infection in rabbits.

Use of survival analysis to compare cull rates between bovine leukemia virus seropositive and seronegative dairy cows.

Bovine leukemia virus (BLV) infection and culling of cows in a commercial dairy herd were evaluated to determine whether a relation existed between the 2 factors. Cattle from the study population, a Holstein dairy herd consisting of approximately 400 milking cows, were tested for antibodies to BLV, using the agar gel immunodiffusion test, semiannually for 2 years, annually for 2 years, and when cattle were culled. Complete records of BLV test results were available for 849 (79%) of the 1,078 cattle that had at least 1 test during the study period. Using the Cox hazard model, the cull hazard rates (culls/cow-months) were greater for BLV seropositive cows than for seronegative cows > 36 months old. Hence, among older dairy cows, BLV-infected cows were culled prematurely, compared with uninfected cows.