Experimental interspecies transmission studies of the transmissible spongiform encephalopathies to cattle: comparison to bovine spongiform encephalopathy in cattle.

Prion diseases or transmissible spongiform encephalopathies (TSEs) of animals include scrapie of sheep and goats; transmissible mink encephalopathy (TME); chronic wasting disease (CWD) of deer, elk and moose; and bovine spongiform encephalopathy (BSE) of cattle. The emergence of BSE and its spread to human beings in the form of variant Creutzfeldt-Jakob disease (vCJD) resulted in interest in susceptibility of cattle to CWD, TME and scrapie. Experimental cross-species transmission of TSE agents provides valuable information for potential host ranges of known TSEs. Some interspecies transmission studies have been conducted by inoculating disease-causing prions intracerebrally (IC) rather than orally; the latter is generally effective in intraspecies transmission studies and is considered a natural route by which animals acquire TSEs. The "species barrier" concept for TSEs resulted from unsuccessful interspecies oral transmission attempts. Oral inoculation of prions mimics the natural disease pathogenesis route whereas IC inoculation is rather artificial; however, it is very efficient since it requires smaller dosage of inoculum, and typically results in higher attack rates and reduces incubation time compared to oral transmission. A species resistant to a TSE by IC inoculation would have negligible potential for successful oral transmission. To date, results indicate that cattle are susceptible to IC inoculation of scrapie, TME, and CWD but it is only when inoculated with TME do they develop spongiform lesions or clinical disease similar to BSE. Importantly, cattle are resistant to oral transmission of scrapie or CWD; susceptibility of cattle to oral transmission of TME is not yet determined.

Minimum intravenous infectious dose of ovine progressive pneumonia virus (OPPV).

The minimum intravenous infectious dose for ovine progressive pneumonia virus (OPPV) WLC1 was determined using twenty-four 6month-old lambs. Twelve groups of two 6month-old lambs were inoculated intravenously (i.v.) with tissue culture fluid containing ovine progressive pneumonia virus (OPPV) WLC1 titers ranging from 10(7.6) TCID(50)/lamb down to 10(-3.4) TCID(50)/lamb and were monitored for seroconversion using the OPPV agar gel immunodiffusion assay (AGID). Fifteen of the 16 lambs given equal or greater than 10(0.6) TCID(50) seroconverted, and virus could be isolated from peripheral blood leukocytes in 13 out of the 15 of these lambs. None of the eight lambs receiving less than 10(0.6) TCID(50) seroconverted during the 12months. The results of this study indicated that 10(0.6) or 4 TCID(50)/lamb given i.v. was capable of establishing infection.

Age-related lesions in laboratory-confined raccoons (Procyon lotor) inoculated with the agent of chronic wasting disease of mule deer.

This communication documents age-associated pathologic changes and final observations on experimental transmission of chronic wasting disease (CWD) by the intracerebral route to raccoons (Procyon lotor). Four kits were inoculated intracerebrally with a brain suspension from mule deer with CWD. Two uninoculated kits served as controls. One CWD-inoculated raccoon was humanely killed at 38 months after inoculation, and 1 control animal died at 68 months after inoculation. Both animals had lesions that were unrelated to transmissible spongiform encephalopathy. Six years after inoculation, none of the 3 remaining CWD-inoculated raccoons had shown clinical signs of neurologic disorder, and the experiment was terminated. Spongiform encephalopathy was not observed by light microscopy, and the presence of abnormal prion protein (PrP(d)) was not detected by either immunohistochemistry or Western blot techniques. Age-related lesions observed in these raccoons included islet-cell pancreatic amyloidosis (5/6), cystic endometrial hyperplasia (3/4), cerebrovascular mineralization (5/6), neuroaxonal degeneration (3/6), transitional-cell adenoma of the urinary bladder (1/6), and myocardial inclusions (4/6). The latter 2 pathologic conditions were not previously reported in raccoons.

Transmission of chronic wasting disease of mule deer to Suffolk sheep following intracerebral inoculation.

To determine the transmissibility of chronic wasting disease (CWD) to sheep, 8 Suffolk lambs of various prion protein genotypes (4 ARQ/ARR, 3 ARQ/ARQ, 1 ARQ/VRQ at codons 136, 154, and 171, respectively) were inoculated intracerebrally with brain suspension from mule deer with CWD (CWDmd). Two other lambs were kept as noninoculated controls. Within 36 months postinoculation (MPI), 2 inoculated animals became sick and were euthanized. Only 1 sheep (euthanized at 35 MPI) showed clinical signs that were consistent with those described for scrapie. Microscopic lesions of spongiform encephalopathy (SE) were only seen in this sheep, and its tissues were determined to be positive for the abnormal prion protein (PrP(res)) by immunohistochemistry and Western blot. Three other inoculated sheep were euthanized (36 to 60 MPI) because of conditions unrelated to TSE. The 3 remaining inoculated sheep and the 2 control sheep did not have clinical signs of disease at the termination of the study (72 MPI) and were euthanized. Of the 3 remaining inoculated sheep, 1 was found to have SE, and its tissues were positive for PrP(res). The sheep with clinical prion disease (euthanized at 35 MPI) was of the heterozygous genotype (ARQ/VRQ), and the sheep with subclinical disease (euthanized at 72 MPH) was of the homozygous ARQ/ARQ genotype. These findings demonstrate that transmission of the CWDmd agent to sheep via the intracerebral route is possible. Interestingly, the host genotype may play a notable part in successful transmission and incubation period of CWDmd.

Determination of sheep prion gene polymorphisms from paraffin-embedded tissue.

Amino acid polymorphisms of the prion protein (PrP) greatly influence the susceptibility of sheep to scrapie. Selective breeding to increase the prevalence of PrP gene alleles associated with scrapie resistance is a flock management practice that is important for scrapie control programs. Determination of sheep PrP alleles typically has required extraction of DNA from host tissues that are freshly derived or stored frozen. We describe application of a DNA extraction procedure for formalin-fixed, paraffin-embedded tissues (PET) for the purpose of PCR amplification and nucleotide sequencing of relevant codons (136-171) of the sheep PrP gene. Tissues derived from 96 sheep were studied. The DNA sequence identity was confirmed in 87 of 94 matched samples of PET and frozen tissue specimens. DNA from brainstem PET of 2 sheep, from which fresh tissue was not available, was amplified and sequenced after formalin fixation for 7-70 days. This method will allow retrospective analysis of PrP genetics of sheep subsequent to postmortem diagnosis of scrapie when nonfixed tissue is unavailable for DNA extraction; however, it is not recommended that submission of fixed tissue supplant collection of fresh tissues for the purpose of determining PrP gene polymorphisms.

Experimental transmission of chronic wasting disease agent from mule deer to cattle by the intracerebral route.

This communication reports final observations on experimental transmission of chronic wasting disease (CWD) from mule deer to cattle by the intracerebral route. Thirteen calves were inoculated intracerebrally with brain suspension from mule deer naturally affected with CWD. Three other calves were kept as uninoculated controls. The experiment was terminated 6 years after inoculation. During that time, abnormal prion protein (PrP(res)) was demonstrated in the central nervous system (CNS) of 5 cattle by both immunohistochemistry and Western blot. However, microscopic lesions suggestive of spongiform encephalopathy (SE) in the brains of these PrP(res)-positive animals were subtle in 3 cases and absent in 2 cases. Analysis of the gene encoding bovine PRNP revealed homozygosity for alleles encoding 6 octapeptide repeats, serine (S) at codon 46, and S at codon 146 in all samples. Findings of this study show that although PrP(res) amplification occurred after direct inoculation into the brain, none of the affected animals had classic histopathologic lesions of SE. Furthermore, only 38% of the inoculated cattle demonstrated amplification of PrP(res). Although intracerebral inoculation is an unnatural route of exposure, this experiment shows that CWD transmission in cattle could have long incubation periods (up to 5 years). This finding suggests that oral exposure of cattle to CWD agent, a more natural potential route of exposure, would require not only a much larger dose of inoculum but also may not result in amplification of PrP(res) within CNS tissues during the normal lifespan of cattle.

Experimental transmission of sheep scrapie by intracerebral and oral routes to genetically susceptible Suffolk sheep in the United States.

Scrapie is a naturally occurring fatal neurodegenerative disease of sheep and goats. Susceptibility to the disease is partly dependent on the genetic makeup of the host. This study documents clinicopathological findings and the distribution of abnormal prion proteins (PrPres) by immunohistochemical and Western blot techniques, in tissues of genetically susceptible sheep inoculated with US sheep scrapie agents. Four-month-old Suffolk lambs (QQ or HQ at codon 171) were inoculated (5 intracerebrally and 19 orally) with an inoculum (#13-7) consisting of a pool of scrapie-affected sheep brains. Intracerebrally inoculated animals were euthanized when advanced clinical signs of scrapie were observed. Orally inoculated animals were euthanized at predetermined time points (4, 9, 12, 15, and 21 months postinoculation [PI]) and thereafter when the animals had terminal signs of disease. All intracerebrally inoculated animals exhibited clinical signs of scrapie and were euthanized between 13 and 24 months PI. Spongiform lesions in the brains and PrPres deposits in central nervous system and lymphoid tissues were present in these sheep. In orally inoculated sheep, clinical signs of scrapie were seen between 27 and 43 months PI in 5/9 animals. The earliest detectable PrPres was observed in brainstem and lymphoid tissues of a clinically normal, orally inoculated sheep at 15 months PI. Three of the 4 clinically normal sheep were positive at 15, 20, and 49 months PI by PrPres immunohistochemistry.

Transmission of sheep scrapie to elk (Cervus elaphus nelsoni) by intracerebral inoculation: final outcome of the experiment.

This is a final report of an experimental transmission of sheep scrapie agent by intracerebral inoculation to Rocky Mountain elk (Cervus elaphus nelsoni). It documents results obtained in experimental (n = 6) and control (n = 2) elk. During the first 2 years postinoculation (PI), 3 animals died or were euthanized because of infection or injuries other than spongiform encephalopathy (SE). In years 3 and 4 PI, 3 other inoculated elk died after brief terminal neurological episodes. Necropsy of these animals revealed moderate weight loss but no other gross lesions. Microscopically, characteristic lesions of SE were seen throughout the brain and spinal cord, and the tissue was positive for proteinase K-resistant prion protein (PrPres) by immunohistochemistry (IHC) and by Western blot. Scrapie-associated fibrils (SAF) were observed by negative-stain electron microscopy in the brain of elk with neurologic signs. PrPres and SAF were not detected in the 3 inoculated elk necropsied during the first 2 years or in the 2 control animals. Retrospective analysis of the gene-encoding cervid PrP revealed a polymorphism at codon 132. The elk with SE were either homozygous (MM) or heterozygous (LM). These findings confirm that intracerebral inoculation of sheep scrapie agent results in SE with accumulations of PrPres in the central nervous system of elk. Based on morphologic and IHC findings, the experimentally induced SE cannot be distinguished from chronic wasting disease of elk with currently available diagnostic techniques.

Antimicrobial anionic peptide binds in vivo to Mannheimia (Pasteurella) haemolytica attached to ovine alveolar epithelium.

Endogenous antimicrobial peptide activity in vivo has rarely been demonstrated. To assess this, Mannheimia haemolytica (log(10) 10.20 cfu) was deposited into the lungs of adult sheep, which were killed at 0, 5, 10 and 20 min for necropsy. At 0 min, M. haemolytica appeared normal and monoclonal antibody to antimicrobial anionic peptide (AP) and Protein A-colloidal gold identified AP already bound to the bacterial surface. At 5-20 min, many organisms were distorted with flocculated intracellular constituents characteristic of AP cellular damage indicating that AP can bind to and presumably help inactivate organisms in vivo.

Neuroaxonal dystrophy in raccoons (Procyon lotor) from Iowa.

During a 12-month period (1998-1999), microscopic evidence of neuroaxonal dystrophy (NAD) in medullae oblongata of raccoons (Procyon lotor) was observed in 17/39 (47% prevalence in adults) from Iowa, USA. Three of the animals were kits (<3 months), 26 were between 1 and 2 years, and 10 were over 7 years. Lesions were not seen in the medullae of the 3 kits. In young adults, the lesions were mild and were seen in 7 animals. More severe lesions were present in the 10 older raccoons. Grossly, the brains were unremarkable. Microscopically, NAD was confined to the dorsal caudal medulla, where certain nuclei (predominantly gracilis and cuneate) were bilaterally affected. Severely affected animals had vacuolar degeneration of neurons or neuronal loss and extensive areas of spongiosis. Tests for the presence of PrP(res) in the brain were negative. Spongiotic areas often contained axonal spheroids. Degenerate neurons and axons occasionally contained amphophilic periodic acid-Schiff-positive granular material. There was a paucity of inflammatory cells in the affected areas. Since lesions were not present in kits, were either absent or mild in young adults, and were severe in older raccoons, the findings may be related to advancing age. Neuroaxonal dystrophy has not been previously reported in raccoons. Retrospective examination of raccoon brains from the eastern and northwestern areas of the country revealed very low prevalence of NAD. Because of the apparently high prevalence of this condition at this geographic location, factors other than age (genetic, nutritional, and/or environmental) may influence this degenerative process in the brains of raccoons in Iowa.

Efecto de la inmunización pasiva sobre la reacción de Shwartzman en el pulmón del conejo empleando lipopolisacárido de Pasteurella haemolytica / The effect of passive immunization on the Shwartzman reaction in the lung of rabbits induced by Pasteurella haemolytica lipopolysaccharide

La reacción local de Shwartzman (RS) puede inducirse en el pulmón del conejo, empleando lipopolisacárido (LPS) de Pasteurella haemolytica. Se ha considerado que las lesiones provocadas por este fenómeno reflejan, al menos en parte, aquellas que se reconocen en casos naturales de pasteurelosis neumónica (PN) en el ganado. En este estudio se examinó la influencia de la inmunidad pasiva en el desarrollo de las lesiones pulmonares provocadas por la RS. Se emplearon dos grupo de conejos, el primer grupo recibió, por vía subcutánea, 4 ml de suero hiperinmune de conejo, preparado contra la cepa de P. haemolytica 82-25. El título de este suero contra el LPS de la bacteria fue de 1:2560, determinado por la prueba de hoaglutinación pasiva. El segundo grupo no recibió suero hiperinmune, sino solución salina fisiológica (SSF). Veinticuatro horas después, los grupos fueron subdivididos. La RS se provocó inoculando por vía endotraqueal 50 µg del LPS de la misma cepa de P. haemolytica y, 24 h más tarde, administrando 100 µg del mismo compuesto por vía endovenosa; a estos inóculos se les denominó preparatorio y desencadenante, respectivamente. Este procedimiento se realizó tanto en animales inmunizados como no inmunizados. Además, se incluyeron animales que recibieron solamente el inóculo preparatorio o el desencadenante. Todos los animales fueron sacrificados a las 36 h posinoculación; es decir, 60 h después de la inmunización pasiva y de la administración de SSF. Se emplearon secciones del pulmón derecho para observar los cambios patológicos, mientras que el pulmón izquierdo se empleó para realizar lavados bronquioalveolares (LBA) y determinar el número total de células y su conteo diferencial. Las lesiones reconocidas en los pulmones de los animales con RS fueron similares en severidad a las que se registraron en los conejos que recibieron únicamente la inoculación endotraqueal del LPS. Los animales que recibieron la inmunización pasiva presentaron una desproporción en las lesiones discretas, pero en otros, éstas fueron mucho más severas que en los animales no inmunizados. Estadísticamente, se identificaron diferencias entre los animales inmunizados y los no inmunizados en lo que concierne a número total de células y conteos diferenciales, siendo los valores mayores para los conejos inmunizados

Inducción de la reacción de Shwartzman en el pulmón del conejo mediante el empleo de lipopolisacárido de Pasteurella haemolytica / Induction of the Shwartzman reaction in the rabbit lung employing Pasteurella haemolytica lipopolysacharide

Se indujo la reacción de Shwartzman (RS) en el pulmón de conejo empleando lipopolisacárido (LPS) de Pasteurella haemolytica con el fin de comparar las lesiones provocadas con las que se presentan naturalmente en la pasteurelosis neumónica (PN). Para inducirla, primero se administró una dosis de LPS de Pasteurella haemolytica (50 mg) por vía endotraqueal (inoculación preparatoria) seguida 24 h más tarde por otra dosis del mismo LPS (100 mg) por vía endovenosa (inoculación desencadenante) (Grupo 5). Doce horas después, los animales fueron sacrificados. El pulmón derecho se destinó a estudios de histopatología, mientras que el izquierdo se empleó para realizar lavados bronquioalveolares (LBA). Para comparar se incluyeron grupos que recibieron solamente el LPS de P. haemolytica por vía endotraqueal (Grupo 3) o endovenosa (Grupo 4) (la inoculación faltante correspondió a solució salina fisiológica [SSF]), así como otro grupo al que se le administró LPS de Escherichia coli tal y como se describió al principio para inducir la RS (Grupo 2), y otro más que recibió SSF de la misma manera (Grupo 1). Las lesiones más notorias se presentaron en los animales que recibieron LPS de P. haemolytica por vía endotraqueal y en los que se les provocó la RS con el mismo LPS. Sólo los polimorfonucleares (PMN), monocitos y linfocitos recuperados por LBA resultaron diferentes entre los grupos, particularmente en los animales que recibieron el LPS de P. Haemolytica por vía endotraqueal y a los que se les indujo la RS con el mismo LPS. Se concluye que el LPS de P. Haemolytica es capaz de provocar una reacción flogística en el pulmón tanto por vía endotraqueal como endovenosa, aunque la respuesta es mucho más intensa en la primera, además, la RS no provoca una respuesta inflamatoria más intensa que la sola inoculación endotraqueal del LPS. Finalmente, se puede afirmar también que el conejo es un buen modelo para evaluar la respuesta inflamatoria pulmonar provocada por el LPS de P. haemolytica