Correlating levels of type III secretion and secreted proteins with fecal shedding of Escherichia coli O157:H7 in cattle.

The locus of enterocyte effacement (LEE) of Escherichia coli O157:H7 (O157) encodes a type III secretion system (T3SS) for secreting LEE-encoded and non-LEE-encoded virulence proteins that promote the adherence of O157 to intestinal epithelial cells and the persistence of this food-borne human pathogen in bovine intestines. In this study, we compared hha sepB and hha mutants of O157 for LEE transcription, T3SS activity, adherence to HEp-2 cells, persistence in bovine intestines, and the ability to induce changes in the expression of proinflammatory cytokines. LEE transcription was upregulated in the hha sepB and hha mutant strains compared to that in the wild-type strain, but the secretion of virulence proteins in the hha sepB mutant was severely compromised. This reduced secretion resulted in reduced adherence of the hha sepB mutant to Hep-2 cells, correlating with a significantly shorter duration and lower magnitude of fecal shedding in feces of weaned (n = 4 per group) calves inoculated with this mutant strain. The levels of LEE transcription, T3SS activity, and adherence to HEp-2 cells were much lower in the wild-type strain than in the hha mutant, but no significant differences were observed in the duration or the magnitude of fecal shedding in calves inoculated with these strains. Examination of the rectoanal junction (RAJ) tissues from three groups of calves showed no adherent O157 bacteria and similar proinflammatory cytokine gene expression, irrespective of the inoculated strain, with the exception that interleukin-1ß was upregulated in calves inoculated with the hha sepB mutant. These results indicate that the T3SS is essential for intestinal colonization and prolonged shedding, but increased secretion of virulence proteins did not enhance the duration and magnitude of fecal shedding of O157 in cattle or have any significant impact on the cytokine gene expression in RAJ tissue compared with that in small intestinal tissue from the same calves.

Characterization of a US sheep scrapie isolate with short incubation time.

Scrapie is a naturally occurring fatal neurodegenerative disease of sheep and goats. Susceptibility to the disease is partly dependent upon the genetic makeup of the host. In a previous study it was shown that sheep intracerebrally inoculated with US scrapie inoculum (No. 13-7) developed terminal disease within an average of 19 months. We have since produced an inoculum, No. x124 from pooled brains of US-origin sheep scrapie, that results in incubations nearly threefold shorter. The present study documents clinicopathologic findings and the distribution of abnormal prion proteins (PrP(Sc)) by immunohistochemical (IHC) and Western blot (WB) techniques, in tissues of sheep inoculated with No. x124. All inoculated sheep developed clinical disease and were euthanatized within an average of 7.7 months postinoculation (MPI). Sheep that had valine/valine or alamine/valine at codon 136 of prion protein (PRNP) gene developed the disease faster and were euthanatized at an average of 4.3 and 5.6 MPI, respectively. Also, the inoculum was able to induce disease in a short time (7 MPI) in a sheep that was relatively resistant (QR at codon 171) to scrapie. This indicates that inoculum No. x124 appears to induce scrapie in shorter time than inoculum No. 13-7, especially in sheep homozygous or heterozygous for valine at codon 136.

Serial passage of sheep scrapie inoculum in Suffolk sheep.

Scrapie is a naturally occurring fatal neurodegenerative disease of sheep and goats. Susceptibility to the disease is partly dependent upon the genetic makeup of the host. In a recent study, it was shown that sheep intracerebrally inoculated with a US scrapie agent (No. 13-7) developed scrapie and survived for an average of 19 months post inoculation. In the present study, when this scrapie inoculum was further passaged for 3 successive generations, the survival time was reduced by approximately 8 months in scrapie-susceptible (QQ on prion protein gene [PRNP] at codon 171) Suffolk sheep. It is concluded that inoculum No. 13-7 appears to have been stabilized in susceptible (171 QQ) Suffolk sheep and may be considered a specific isolate of sheep scrapie agent in the USA and therefore that it can be used to evaluate other isolates of sheep scrapie in this country.

Experimental transmission of chronic wasting disease (CWD) of elk (Cervus elaphus nelsoni), white-tailed deer (Odocoileus virginianus), and mule deer (Odocoileus hemionus hemionus) to white-tailed deer by intracerebral route.

To compare clinical and pathologic findings of chronic wasting disease (CWD) in a natural host, 3 groups (n = 5) of white-tailed deer (WTD) fawns were intracerebrally inoculated with a CWD prion of WTD, mule deer, or elk origin. Three other uninoculated fawns served as controls. Approximately 10 months postinoculation (MPI), 1 deer from each of the 3 inoculated groups was necropsied and their tissues were examined for lesions of spongiform encephalopathy (SE) and for the presence of abnormal prion protein (PrP(d)) by immunohistochemistry (IHC) and Western blot (WB). The remaining deer were allowed to live until they developed clinical signs of the disease which began approximately 18 MPI. By 26 MPI, all deer were euthanatized on humane grounds. Obvious differences in clinical signs or the incubation periods were not observed between the 3 groups of deer given CWD. In 1 of 3 nonclinical deer euthanatized at 10 MPI, minimal microscopic lesions of SE were seen in the central nervous system (CNS) tissues, and PrP(d) was observed by IHC in tissues of all 3 deer. In the clinical deer, CNS lesions of SE and PrP(d) accumulations were more severe and extensive. It is concluded that the 3 sources of CWD prion did not induce significant differences in time to clinical disease or qualitative differences in signs or lesions in WTD. However, this observation does not imply that these CWD agents would necessarily behave similarly in other recipient species.

Preliminary observations on the experimental transmission of chronic wasting disease (CWD) from elk and white-tailed deer to fallow deer.

To determine the transmissibility of chronic wasting disease (CWD) to fallow deer (Dama dama) and to provide information about clinical course, lesions and suitability of currently used diagnostic procedures for detection of CWD in this species, 13 fawns were inoculated intracerebrally with CWD brain suspension from elk (n=6) or white-tailed deer (n=7). Three other fawns were kept as uninfected controls. Three CWD-inoculated deer were killed 7.6 months post-inoculation (mpi). None had abnormal prion protein (PrPd) in their tissues. One sick deer died at 24 mpi and one deer without clinical signs was killed at 26 mpi. Both animals had a small focal accumulation of PrPd in the midbrain. Between 29 and 37 mpi, three other deer became sick and were killed. All had shown gradual decrease in appetite and some loss of body weight. Microscopical lesions of spongiform encephalopathy were not observed, but PrPd was detected in tissues of the central nervous system (CNS) by immunohistochemistry, western blot and by two commercially available rapid diagnostic tests. This study demonstrates that intracerebrally inoculated fallow deer amplified CWD PrPd from white-tailed deer and elk in the absence of lesions of spongiform encephalopathy. Four years after CWD inoculation, the remaining five inoculated and two control deer are alive and apparently healthy.

Experimental transmission of US scrapie agent by nasal, peritoneal, and conjunctival routes to genetically susceptible sheep.

Scrapie is a naturally occurring fatal neurodegenerative disease of sheep and goats. This study documents incubation periods, pathologic findings, and distribution of abnormal prion proteins (PrP(Sc)) by immunohistochemistry in tissues of genetically susceptible sheep inoculated with US sheep scrapie agent. Four-month-old Suffolk lambs (QQ at codon 171) were inoculated by 1 of 3 different routes (nasal, peritoneal, and conjunctival) with an inoculum (No. 13-7) consisting of a pool of scrapie-affected sheep brains. Except for 3 sheep, all inoculated animals were euthanized when advanced clinical signs of scrapie were observed between 19 and 46 months postinoculation (MPI). Spongiform lesions in the brains and labeling of PrP(Sc) in central nervous system and lymphoid tissues were present in these sheep. One intranasally inoculated sheep euthanized at 12 MPI had presence of PrP(Sc) that was confined to the pharyngeal tonsil. These results indicate that the upper respiratory tract, specifically the pharyngeal tonsil, may serve as a portal of entry for prion protein in scrapie-infected environments.

Polymorphisms of the prion gene promoter region that influence classical bovine spongiform encephalopathy susceptibility are not applicable to other transmissible spongiform encephalopathies in cattle.

Two regulatory region polymorphisms in the prion gene of cattle have been reported to have an association with resistance to classical bovine spongiform encephalopathy (BSE). However, it is not known if this association also applies to other transmissible spongiform encephalopathies (TSE) in cattle. In this report, we compare the relationship between these 2 polymorphisms and resistance in cattle affected with naturally occurring atypical BSE as well as in cattle experimentally inoculated with either scrapie, chronic wasting disease, or transmissible mink encephalopathy. Our analysis revealed no association between genotype and resistance to atypical BSE or experimentally inoculated TSE. This indicates the promoter polymorphism correlation is specific to classical BSE and that atypical BSE and experimentally inoculated TSE are bypassing the site of influence of the polymorphisms. This genetic discrepancy demonstrates that atypical BSE progresses differently in the host relative to classical BSE. These results are consistent with the notion that atypical BSE originates spontaneously in cattle.

Susceptibility of cattle to first-passage intracerebral inoculation with chronic wasting disease agent from white-tailed deer.

Fourteen, 3-month-old calves were intracerebrally inoculated with the agent of chronic wasting disease (CWD) from white-tailed deer (CWDwtd) to compare the clinical signs and neuropathologic findings with those of certain other transmissible spongiform encephalopathies (TSE, prion diseases) that have been shown to be experimentally transmissible to cattle (sheep scrapie, CWD of mule deer [CWDmd], bovine spongiform encephalopathy [BSE], and transmissible mink encephalopathy). Two uninoculated calves served as controls. Within 26 months postinoculation (MPI), 12 inoculated calves had lost considerable weight and eventually became recumbent. Of the 12 inoculated calves, 11 (92%) developed clinical signs. Although spongiform encephalopathy (SE) was not observed, abnormal prion protein (PrPd) was detected by immunohistochemistry (IHC) and Western blot (WB) in central nervous system tissues. The absence of SE with presence of PrPd has also been observed when other TSE agents (scrapie and CWDmd) were similarly inoculated into cattle. The IHC and WB findings suggest that the diagnostic techniques currently used to confirm BSE would detect CWDwtd in cattle, should it occur naturally. Also, the absence of SE and a distinctive IHC pattern of CWDwtd and CWDmd in cattle suggests that it should be possible to distinguish these conditions from other TSEs that have been experimentally transmitted to cattle.

Abnormal prion protein in ectopic lymphoid tissue in a kidney of an asymptomatic white-tailed deer experimentally inoculated with the agent of chronic wasting disease.

Chronic wasting disease (CWD), a transmissible spongiform encephalopathy (TSE) of deer and elk, is one of a group of fatal, neurologic diseases that affect several mammalian species, including human beings. Infection by the causative agent induces accumulations of an abnormal form of prion protein (PrPres) in nervous and lymphoid tissues. This report documents the presence of PrPres within ectopic lymphoid follicles in a kidney of a white-tailed deer that had been experimentally inoculated by the intracerebral route with CWD 10 months previously. The deer was nonclinical, but spongiform lesions characteristic of TSE were detected in tissues of the central nervous system (CNS) and PrPres was seen in CNS and in lymphoid tissues by immunohistochemistry. The demonstration of PrPres in lymphoid tissue in the kidney of this deer corroborates a recently published finding of PrPres in lymphoid follicles of organs other than CNS and lymphoid tissues in laboratory animals with TSE (scrapie).

First and second cattle passage of transmissible mink encephalopathy by intracerebral inoculation.

To compare clinicopathologic findings of transmissible mink encephalopathy (TME) with other transmissible spongiform encephalopathies (TSE, prion diseases) that have been shown to be experimentally transmissible to cattle (sheep scrapie and chronic wasting disease [CWD]), two groups of calves (n = 4 each) were intracerebrally inoculated with TME agents from two different sources (mink with TME and a steer with TME). Two uninoculated calves served as controls. Within 15.3 months postinoculation, all animals from both inoculated groups developed clinical signs of central nervous system (CNS) abnormality; their CNS tissues had microscopic spongiform encephalopathy (SE); and abnormal prion protein (PrP(res)) as detected in their CNS tissues by immunohistochemistry (IHC) and Western blot (WB) techniques. These findings demonstrate that intracerebrally inoculated cattle not only amplify TME PrP(res) but also develop clinical CNS signs and extensive lesions of SE. The latter has not been shown with other TSE agents (scrapie and CWD) similarly inoculated into cattle. The findings also suggest that the diagnostic techniques currently used for confirmation of bovine spongiform encephalopathy (BSE) would detect TME in cattle should it occur naturally. However, it would be a diagnostic challenge to differentiate TME in cattle from BSE by clinical signs, neuropathology, or the presence of PrP(res) by IHC and WB.

Experimental second passage of chronic wasting disease (CWD(mule deer)) agent to cattle.

To compare clinicopathological findings in first and second passage chronic wasting disease (CWD(mule deer)) in cattle, six calves were inoculated intracerebrally with brain tissue derived from a first-passage CWD-affected calf in an earlier experiment. Two uninoculated calves served as controls. The inoculated animals began to lose both appetite and weight 10-12 months later, and five subsequently developed clinical signs of central nervous system (CNS) abnormality. By 16.5 months, all cattle had been subjected to euthanasia because of poor prognosis. None of the animals showed microscopical lesions of spongiform encephalopathy (SE) but PrP(res) was detected in their CNS tissues by immunohistochemistry (IHC) and rapid Western blot (WB) techniques. Thus, intracerebrally inoculated cattle not only amplified CWD PrP(res) from mule deer but also developed clinical CNS signs in the absence of SE lesions. This situation has also been shown to occur in cattle inoculated with the scrapie agent. The study confirmed that the diagnostic techniques currently used for diagnosis of bovine spongiform encephalopathy (BSE) in the US would detect CWD in cattle, should it occur naturally. Furthermore, it raised the possibility of distinguishing CWD from BSE in cattle, due to the absence of neuropathological lesions and to a distinctive multifocal distribution of PrP(res), as demonstrated by IHC which, in this study, appeared to be more sensitive than the WB technique.

Second passage of sheep scrapie and transmissible mink encephalopathy (TME) agents in raccoons (Procyon lotor).

To determine the transmissibility and pathogenicity of sheep scrapie and transmissible mink encephalopathy (TME) agents derived from raccoons (first passage), raccoon kits were inoculated intracerebrally with either TME (one source) or scrapie (two sources-each in separate groups of raccoons). Two uninoculated raccoon kits served as controls. All animals in the TME-inoculated group developed clinical signs of neurologic dysfunction and were euthanatized between postinoculation month (PIM) 6 and 8. Raccoons in the two scrapie-inoculated groups manifested similar clinical signs of disease, but such signs were observed much later and the animals were euthanized between PIM 12 and 18. Necropsy revealed no gross lesions in any of the raccoons. Spongiform encephalopathy was observed by use of light microscopy, and the presence of protease-resistant prion protein (PrPres) was detected by use of immunohistochemical (IHC) and Western blot analytic techniques. Results of IHC analysis indicated a distinct pattern of anatomic distribution of PrPres in the TME- and scrapie-inoculated raccoons. These findings confirm that TME and sheep scrapie are experimentally transmissible to raccoons and that the incubation periods and IHC distribution for both agents are distinct. Therefore, it may be possible to use raccoons for differentiating unknown transmissible spongiform encephalopathy (TSE) agents. Further studies, with regard to the incubation period and the pattern of PrPres deposition by use of IHC analysis in bovine spongiform encephalopathy and for other isolates of scrapie, chronic wasting disease, and TME in raccoons are needed before the model can be further characterized for differentiation of TSE agents.

Evaluation of a diet free of animal protein in germfree swine.

BACKGROUND: Two experiments were conducted in which germfree pigs or pigs monoassociated with Lactobacillus paracasei subspecies paracasei were fed either a traditional milk-based diet (Esbilac) or an experimental diet free of animal protein (DFAP). METHODS: Throughout the 16-day study, animals' clinical condition, total weight gain, feed conversion, and bacterial contamination were monitored. At the conclusion of the study the animals were killed, necropsied and tissues sampled for L. paracasei isolation. RESULTS: General pig disposition remained consistent between treatment groups and trials, except for two animals that developed mild diarrhoea during trial 1. All pigs remained viable during the study irrespective the diet fed or probiotic inoculation. Germfree pigs fed the Esbilac diet gained on average a total of 1034 +/- 63.0 g, and had a feed conversion ratio of 0.17 +/- 0.01 g of gain per 1 ml of diet. Germfree pigs fed the experimental diet gained on average a total of 599 +/- 151 g, and had a feed conversion ratio of 0.10 +/- 0.02 g of gain per 1 ml of diet. Monoassociated pigs fed the Esbilac diet gained on average a total of 862 +/- 70.3 g, and had a feed conversion ratio 0.14 +/- 0.01 g of gain per 1 ml of diet. Monoassociated pigs fed the experimental diet gained on average a total of 563 +/- 96.8 g, and had a feed conversion ratio of 0.09 +/- 0.02 g of gain per 1 ml of diet. Lactobacillus paracasei established extensively in pigs fed either the Esbilac or experimental diets. Lactobacillus paracasei had no effect (P >0.05) on piglet growth and did not display any interactions based on the diet fed. Measured growth parameters were statistically different (P <0.05) based on the diet fed and variance seen between trials. CONCLUSION: In conclusion, a DFAP has been developed and has been shown to be capable of sustaining life in piglets up to 16 days of age.

Congeners of SMAP29 kill ovine pathogens and induce ultrastructural damage in bacterial cells.

SMAP29, an ovine cathelicidin, was systematically altered to create a family of 23 related peptides for MIC and minimum bactericidal concentration determinations. SMAP28, SMAP29, and a derivative of SMAP29 called ovispirin were all antimicrobial. However, many congeners of SMAP29 and ovispirin were not as active as the parent molecules. With immunoelectron microscopy, SMAP29 was seen on membranes and within the cytoplasm of Pseudomonas aeruginosa PAO1.

Genetic variation in response of turkeys to experimental infection with Bordetella avium.

One hundred ninety-six male and female turkeys representing two genetic lines were experimentally infected with Bordetella avium. The lines of turkeys included a randombred control line (RBC2) and a subline (F) of RBC2 selected for increased 16-wk body weight. No difference was found between lines RBC2 and F in the number of days to onset of clinical signs, and no mortality due to B. avium infection was observed in either line. Interestingly, however, a significant depression (12%) occurred in body weight of F line poults infected with B. avium, but no significant depression occurred in body weight of RBC2 poults.

Absence of protection against challenge with aspergillus fumigatus by adoptive transfer of splenocytes from convalescent turkeys.

Only limited protective immunity against aspergillosis after experimental immunization of turkeys has been previously demonstrated. No studies evaluating the efficacy of transfer of immunity in preventing aspergillosis in birds have been reported. This study consisted of two trials assessing the level of protection against Aspergillus fumigatus challenge afforded by transfer of splenocytes from convalescent turkeys. Three treatment groups of 12-to-14-wk-old Beltsville small white (BSW) turkeys comprising the splenocyte donors were prepared by one of the following: 1) intra-air sac (IA) challenge with A. fumigatus conidia 5 wk prior to transfer; 2) IA challenge and then intravenous (i.v.) injection of killed conidia 1 wk prior to transfer; or 3) sham inoculations. Splenocytes from each group were pooled, enriched for mononuclear leukocytes by density gradient centrifugation, and diluted in cell culture medium (CM). Cell viability was assessed by dye exclusion. Each splenocyte preparation was administered intravenously to one of three recipient groups consisting of 10 BSW turkeys each. A control group (n = 10) was given cell-free CM. Recipients were challenged with viable A. fumigatus conidia 16 hr after splenocyte transfer by unilateral IA (trial 1) or i.v. (trial 2) inoculation. Lesion scores postchallenge revealed no differences between turkeys given splenocytes from convalescent vs. naive (control) turkeys. IA exposure produced ipsilateral lesions in air sacs and lung, whereas i.v. exposure produced severe miliary hepatitis. Donor cell function was confirmed by mitogen blastogenesis; however, cells were nonresponsive to A. fumigatus antigens, regardless of previous exposure status.

Bovine viral diarrhea virus isolated from fetal calf serum enhances pathogenicity of attenuated transmissible gastroenteritis virus in neonatal pigs.

A bovine viral diarrhea virus (BVDV-C) was isolated from swine tissue culture cells used to attenuate the transmissible gastroenteritis virus (TGEV) after 68 passes. Piglets given a pure culture of BVDV-C developed clinical signs similar to those of a mild TGEV infection and recovered by 10 days postexposure. Villous blunting and fusion was observed in the small intestine, and a lymphocyte depletion was observed in Peyer's patches in the ileum. Piglets given a combination of BVDV-C and attenuated TGEV developed clinical signs similar to those of a virulent TGEV infection and were euthanized. The combined infection induced a generalized lymphocyte depletion throughout the lymphatic system and villous atrophy in the intestinal tract. Piglets exposed to a another type I strain of BVDV (NY-1) either alone or in combination with the attenuated TGEV had mild clinical signs similar to those of a TGEV infection. Moderate villous atrophy in the ileum and a lymphocyte depletion in the mesenteric lymph node were observed in these piglets postmortem. The data indicate a potential problem for diagnostic laboratories in relation to a diagnosis of virulent TGEV infections and in the field for young piglets exposed to a BVDV-contaminated TGEV vaccine.

Bacterin-induced protection of turkeys against fowl cholera following infection with Bordetella avium.

Groups of Beltsville small white turkeys, passively immunized and not passively immunized against Bordetella avium, were challenged with live B. avium at 2 days of age. Birds not passively immunized developed severe bordetellosis with early onset, whereas passively immunized birds developed mild bordetellosis with late onset. Following convalescence, birds with and without exposure to B. avium were vaccinated against fowl cholera with a water-in-oil bacterin. The birds were given a homologous challenge with serotype A: 3 Pasteurella multocida. Although no difference in protection against fowl cholera was seen between vaccinated birds that were previously infected with B. avium and those that were not, survivability was better in birds given two doses rather than 1 dose of bacterin.

Early pulmonary lesions in turkeys produced by nonviable Aspergillus fumigatus and/or Pasteurella multocida lipopolysaccharide.

This study assessed the potential of lipopolysaccharide (LPS) purified from Pasteurella multocida to cause pulmonary pathology or exacerbate lesions produced by gamma-irradiated nonviable Aspergillus fumigatus conidia when administered via the intra-air sac route in turkeys. LPS provoked suppurative airsacculitis, pleuritis, and pneumonia. Nonviable conidia produced airsacculitis and transient pneumonitis but did not elicit multinucleate giant cells, which are a feature of the inflammatory process in A. fumigatus infection. LPS in combination with A. fumigatus conidia resulted in accelerated pulmonary inflammation and apparently delayed clearance of conidia from pulmonary tissues. This study presents a model of aseptic airsacculitis and pneumonia with clinical relevance.

Susceptibility of convalescent turkeys to pulmonary aspergillosis.

Pulmonary lesions resulting from Aspergillus fumigatus inoculation were assessed in convalescent turkeys and compared with those in previously noninoculated (control) turkeys. In addition, lesions observed in small Beltsville white (SBW) turkeys were compared with those in broad-breasted white (BBW) turkeys challenged with the same inoculum. Turkeys were challenged by unilateral posterior thoracic air sac (PTAS) inoculation, rechallenged via the contralateral air sac after 5 wk, and then necropsied 1 wk later. Pulmonary lesions induced by the initial challenge had resolved in 6 of 10 SBW and 9 of 10 BBW turkeys. However, convalescence did not protect against pulmonary aspergillosis subsequent to rechallenge; 10 of 10 SBW and 9 of 10 BBW developed granulomatous pulmonary lesions on the side of reexposure. A greater proportion of control SBW turkeys developed pneumonia and airsacculitis following challenge as compared with the BBW breed. Lesions were limited to the lower respiratory tract in all turkeys and were confined to the ipsilateral lung and PTAS in the singly inoculated control turkeys. This study demonstrates that convalescence from pulmonary aspergillosis does not confer protection against rechallenge but may, instead, decrease resistance to subsequent infection.