Antibacterial Effect of Surface Pretreatment Techniques against Streptococcus Mutans.

OBJECTIVE: The aim of this study was to evaluate the antibacterial surface pretreatment methods against Streptococcus mutans within the infected dentin surface using a tooth cavity model. MATERIAL AND METHODS: Seventy-two cavities were prepared on caries-free third molars (n = 8). After sterilization, teeth were inoculated with S. mutans for 48 h. One cavity of each tooth was used to evaluate the infection. Following inoculation, infected cavity surfaces were treated either with (1) Er:YAG Laser (1W; 5x5s, Smart 2940D Plus, Deka Laser), (2) Ozone (80s; HealOzone, Kavo), (3) ErYAG-Ozone combination, (4) Er:YAG-Ozone-CHX combination, (5) Chlorhexidine (CHX), (6) Clearfil Protect Bond (PB), (7) potassium-titanyl-phosphate (KTP) Laser (1W; 60 s, SMARTLITE D, Deka Laser), (8) KTP-Ozone combination, and (9) KTP-Ozone-CHX. Standardized amounts of dentin chips were obtained from the cavity walls, and the number of bacteria recovered was counted. Kruskal-Wallis test was used for statistical analyzes. RESULTS: Both sole antibacterial materials, CHX or Protect Bond application, exhibited the most effective antibacterial activity with 125 and 156 CFU is an acronym of "colony forming unit" usullay mentioned by acronym. (CFU/ml), respectively, among the groups evaluated (P < 0.05). Er:YAG laser irradiation and its combinations with other antibacterial surface pretreatment applications also inhibited the bacterial growth with, respectively, 1444, 406, and 294 CFU/ml bacterial recovery being more efficient than KTP laser irradiation and ozone combinations. CONCLUSIONS: As an alternative device with photodynamic effects, Er:YAG and KTP laser irradiations and their further combinations during the cavity pretreatment procedure with chlorhexidine and ozone treatments exerted antibacterial effect against S. mutans, whereas chlorhexidine and antibacterial dentin bonding application solely have the highest antibacterial effects.

The effect of calcium phosphate-containing desensitizing agent on the microtensile bond strength of multimode adhesive agent.

OBJECTIVE: The aim of this study was to investigate the effect of calcium phosphate containing desensitizing pretreatments on the microtensile bond strength (MTBS) and microleakage of the multimode adhesive agent to dentin. MATERIALS AND METHODS: In this study, twelve noncarious, freshly extracted human third molar teeth for MTBS and 20 premolar teeth for the microleakage test were used. The teeth were restored using Clearfil Universal Bond + Clearfil APX and Teeth mate Desensitizer (TMD). For MTBS test, Group 1: Self-etch, Group 2: Etch and rinse (G1 and 2, nondesensitizer treatment served as a control), Group 3: TMD/self-etch, Group 4: Acid-etch/TMD/etch and rinse. For microleakage test, Class V adhesive cavities (3 mm × 2 mm × 2 mm) were prepared and restored as mentioned before. The restored teeth were subjected to thermal cycling. The MTBS test was performed in all procedures. The MTBS data were submitted to a one-way ANOVA and post hoc Tukey test (P P Results: Control groups exhibited a higher mean MTBS value than TMD groups, and there were statistical differences between the groups. TMD groups also demonstrated significantly less microleakage than control groups (P Conclusions: This study proves that the application of TMD with a multimode adhesive bonding system produced significantly lower MTBS and microleakage.

Canine Nervous System Lymphoma Subtypes Display Characteristic Neuroanatomical Patterns.

Primary and secondary nervous system involvement occurs in 4% and 5%-12%, respectively, of all canine non-Hodgkin lymphomas. The recent new classification of canine malignant lymphomas, based on the human World Health Organization classification, has been endorsed with international acceptance. This histological and immunocytochemical classification provides a unique opportunity to study the histologic anatomic distribution patterns in the central and peripheral nervous system of these defined lymphoma subtypes. In this study, we studied a cohort of 37 dogs with lymphoma, which at necropsy had either primary (n = 1, 2.7%) or secondary (n = 36; 97.3%) neural involvement. These T- (n = 16; 43.2%) or B-cell (n = 21; 56.8%) lymphomas were further classified into 12 lymphoma subtypes, with predominant subtypes including peripheral T-cell lymphoma (PTCL) or diffuse large B-cell lymphoma (DLBCL), respectively. This systematic study identified 6 different anatomically based histologically defined patterns of lymphoma infiltration in the nervous system of dogs. Different and distinct combinations of anatomical patterns correlated with specific lymphoma subtypes. Lymphoma infiltration within the meningeal, perivascular, and periventricular compartments were characteristic of DLBCL, whereas peripheral nerve involvement was a frequent feature of PTCL. Similarly cell counts above 64 cells/µL in cerebrospinal samples correlated best with marked meningeal and periventricular lymphoma infiltration histologically. Prospective studies are needed in order to confirm the hypothesis that these combinations of histological neuroanatomic patterns reflect targeting of receptors specific for the lymphoma subtypes at these various sites.

Characterization of an Inherited Neurologic Syndrome in Toyger Cats with Forebrain Commissural Malformations, Ventriculomegaly and Interhemispheric Cysts.

BACKGROUND: In children, frequent congenital malformations with concomitant agenesis of the corpus callosum are diagnosed by neuroimaging in association with other cerebral malformations, including interhemispheric cysts and ventriculomegaly. Similar studies providing full characterization of brain defects by in vivo magnetic resonance imaging (MRI), and correlations with the pertinent anatomic pathologic examinations are absent in veterinary medicine. HYPOTHESIS/OBJECTIVES: Congenital brain defects underlie the neurologic signs observed in Toyger cats selectively bred for a short ear phenotype. ANIMALS: Using proper pedigree analysis and genetic evaluations, 20 related Oriental-derived crossbred Toyger cats were evaluated. Seven clinically healthy (carrier) cats and 13 clinically affected cats that had neurologic signs, short ear phenotype and concomitant complex brain anomalies were studied. METHODS: Complete physical and neurologic examinations and MRI were performed in all clinically healthy and affected cats. Postmortem and histopathologic examinations were performed in 8 affected cats and 5 healthy cats. RESULTS: Neurologic and MRI investigations confirmed 13 clinically affected cats with structural brain abnormalities. Ventriculomegaly with frequent concomitant supratentorial interhemispheric, communicating ventricular type-1b cysts and multiple midline and callosal malformations were detected in all cats displaying neurologic signs. Genetic analysis confirmed autosomal recessive mode of inheritance with no chromosomal abnormalities. CONCLUSIONS AND CLINICAL IMPORTANCE: Neuroanatomic dissections and histopathology were helpful for evaluation of abnormalities in midline brain structures, and for the full characterization of cysts. However, MRI was more sensitive for detection of small cysts. In this feline model, MRI diagnosis had extremely good correlation with pathologic abnormalities noted in the subset of animals that were examined by both modalities.

Unusual Necrotizing Encephalitis in Raccoons and Skunks Concurrently Infected With Canine Distemper Virus and Sarcocystis sp.

Canine distemper virus commonly infects free-ranging, terrestrial mesopredators throughout the United States. Due to the immunosuppressive effects of the virus, concurrent opportunistic infections are also common. Among these, secondary systemic protozoal infections have been described in a number of species. We report an unusual presentation of necrotizing encephalitis associated withSarcocystissp in four raccoons and one skunk concurrently infected with canine distemper virus. Lesions were characterized by variably sized necrotizing cavitations composed of abundant mineral admixed with inflammatory cells and protozoa.Sarcocystissp was confirmed via immunohistochemistry using a monoclonal antibody toSarcocystis neurona The pathologic changes are similar to lesions in human AIDS patients infected withToxoplasma gondii.

Quantitative molecular viral loads in 7 horses with naturally occurring equine herpesvirus-1 infection.

REASONS FOR PERFORMING STUDY: Data associating quantitative viral load with severity, clinical signs and survival in equine herpesvirus-1 myeloencephalopathy (EHM) have not been reported. OBJECTIVES: To report the clinical signs, treatment, and temporal progression of viral loads in 7 horses with naturally occurring EHM and to examine the association of these factors with survival. STUDY DESIGN: Retrospective case series. METHODS: The population included 7 horses with EHM presented to the University of California, Davis William R. Pritchard Veterinary Medical Teaching Hospital from May to September 2011. Horses were graded using a neurological grading scale. Daily quantitative PCR was performed on nasal secretions and whole blood. Treatment, survival, outcome and histopathology were reported. RESULTS: At presentation, one horse was neurological grade 5/5, 3 were grade 4/5 and 3 were grade 3/5. All were treated with anti-inflammatory drugs, valacyclovir and management in a sling if necessary. All were infected with equine herpesvirus-1 of DNA polymerase D752 genotype. Peak viral load in nasal secretions and blood of 5 survivors ranged from 6.9 × 10(3) to 2.81 × 10(5) (median 5.11 × 10(4) ) and from 143 to 4340 gB gene copies/million eukaryotic cells (median 3146), respectively. The 2 nonsurvivors presented with grade 3/5 neurological signs and progressed to encephalopathy. Peak viral load was higher in nonsurvivors, with levels in nasal secretions of 1.9 × 10(9) and 2.2 × 10(9) and in blood of 2.05 × 10(4) and 1.02 × 10(5) gB gene copies/million eukaryotic cells. Case fatality was 2/7. CONCLUSIONS: Nonsurvivors had viral loads 1000-fold higher in nasal secretions and 10-fold higher in blood than survivors. There was no relationship between severity of clinical signs at presentation and survival. Thus, encephalopathy and high viral load were negatively associated with survival in this population. Further research should be performed to determine whether high viral loads are associated with encephalopathy and poor prognosis. The Summary is available in Chinese - see Supporting information.

Effect of QMix irrigant on the microhardness of root canal dentine.

BACKGROUND: The aim of this study was to evaluate the effects of QMix, EDTA + CHX, EDTA + NaOCl and maleic acid on the microhardness of root canal dentine. METHODS: Forty recently extracted human maxillary canine teeth were longitudinally sectioned into 80 segments and then embedded in an autopolymerizing acrylic resin. The microhardness of the dentine in the specimen was measured with a Vickers diamond indenter at the coronal, middle and apical thirds of the roots. Finally, the specimens were divided randomly into four groups: 17% EDTA + 2.5% NaOCl; 17% EDTA + 2% CHX; QMix; and 7% maleic acid. Post-treatment microhardness values were obtained and the decrease in microhardness was calculated as a percentage. Microhardness values were statistically analysed using the Kruskal-Wallis and Mann-Whitney U tests. RESULTS: Maleic acid significantly decreased microhardness in all regions, compared to the other groups. In the coronal and middle regions, there was no significant difference among the other groups. In the apical region, there was no significant difference between QMix and 17% EDTA + 2% CHX but these groups presented significant dentine microhardness reduction compared to the 17% EDTA + 2.5% NaOCl group. CONCLUSIONS: While maleic acid showed the greatest reduction in dentine microhardness, it was found that QMix, 17% EDTA + 2% CHX and 17% EDTA + 2.5% NaOCl cause the same reduction in the microhardness of root canal dentine in the coronal and middle regions.

Bilaterally symmetric focal cortical dysplasia in a golden retriever dog.

A 10-year-old golden retriever dog was referred with a 24-h history of generalized seizures. Magnetic resonance imaging of the brain found no abnormalities on 3 mm transverse sections and the dog was subsequently humanely destroyed. Microscopically there was bilaterally symmetrical focal disorganization of cortical grey matter within the tips of the right and left suprasylvian gyri of the temporal cortex. The focal abnormal cortical lamination was characterized by loss of pyramidal neurons with abnormal, irregular, angular, remaining neurons occasionally forming clusters, surrounded by fibrillary astrogliosis and microgliosis and vascular proliferation. These histological findings are consistent with focal cortical dysplasia, a cerebral cortical malformation that causes seizures in people, but not reported previously in the dog.

Necrotizing meningoencephalitis in atypical dog breeds: a case series and literature review.

BACKGROUND: Canine necrotizing meningoencephalitis (NME) is a fatal, noninfectious inflammatory disease of unknown etiology. NME has been reported only in a small number of dog breeds, which has led to the presumption that it is a breed-restricted disorder. HYPOTHESIS/OBJECTIVES: Our objective was to describe histopathologically confirmed NME in dog breeds in which the condition has not been reported previously and to provide preliminary evidence that NME affects a wider spectrum of dog breeds than previously reported. ANIMALS: Four dogs with NME. METHODS: Archives from 3 institutions and from 1 author's (BS) collection were reviewed to identify histopathologically confirmed cases of NME in breeds in which the disease has not been reported previously. Age, sex, breed, survival from onset of clinical signs, and histopathologic findings were evaluated. RESULTS: Necrotizing meningoencephalitis was identified in 4 small dog breeds (Papillon, Shih Tzu, Coton de Tulear, and Brussels Griffon). Median age at clinical evaluation was 2.5 years. Histopathologic abnormalities included 2 or more of the following: lymphoplasmacytic or histiocytic meningoencephalitis or encephalitis, moderate-to-severe cerebrocortical necrosis, variable involvement of other anatomic locations within the brain (cerebellum, brainstem), and absence of detectable infectious agents. CONCLUSIONS AND CLINICAL IMPORTANCE: Until now, NME has only been described in 5 small dog breeds. We document an additional 4 small breeds previously not shown to develop NME. Our cases further illustrate that NME is not a breed-restricted disorder and should be considered in the differential diagnosis for dogs with signalment and clinical signs consistent with inflammatory brain disease.

Pathogenesis of scrapie in ARQ/ARQ sheep after subcutaneous infection: effect of lymphadenectomy and immune cell subset changes in relation to prion protein accumulation.

It is well established that the infectious agent of scrapie can replicate in the lymphoreticular system (LRS). However, the effects of removal of LRS target tissues on the pathogenesis of the infection and the accumulation of disease-associated prion protein (PrP(d)) in LRS tissues on specific immune cell subsets are poorly understood aspects. To address these questions 16 ARQ/ARQ sheep were subcutaneously inoculated in the drainage area of the prefemoral lymph node with brain homogenate derived from Suffolk sheep naturally infected with scrapie. Fourteen sheep were then subjected to either early (14-17 days post-inoculation [dpi]) or late (175-201 dpi) lymphadenectomy and culled at preclinical or clinical stages of infection. Neither late nor even early lymphadenectomy prevented infection or had any effect on the accumulation of PrP(d) in the LRS or CNS suggesting a rapid organic dissemination of the infectious agent after inoculation. Lymph nodes from eight scrapie inoculated sheep selected on the basis of the amount of PrP(d) in their LRS tissues (negative, low or high) were examined for six different immune cell markers. The PrP(d) negative lymph nodes from two sheep with no evidence of scrapie infection showed lower numbers of cluster of determination (CD) 21 positive cells than PrP(d) positive nodes, irrespective of their location (hind leg or head). However, quantitative differences in the expression of this marker were not detected when comparing lymph nodes with low and high levels of PrP(d) accumulation, suggesting that proliferation of CD21 positive cells is related to scrapie infection, but not directly linked to the magnitude of PrP(d) accumulation. An additional observation of the study was that sheep that were methionin-threonine at codon 112 of the prion protein gene showed lower attack rates than methionine homozygotes (67% and 100%, respectively) and also generally lower levels of PrP(d) accumulation in the LRS and brain and increased survival times, suggesting an influence of such polymorphism in the susceptibility to scrapie.

Minimal involvement of the circumventricular organs in the pathogenesis of spontaneously arising and experimentally induced classical bovine spongiform encephalopathy.

In sheep infected experimentally with the bovine spongiform encephalopathy (BSE) agent, amplification of infectivity in peripheral organs during early preclinical stages is thought to contribute to high titres of the agent being detected in blood, with subsequent haematogenous neuroinvasion through the circumventricular organs (CVOs). In contrast, little disease-associated prion protein (PrP(d)) or infectivity is detected in the peripheral tissues of cattle during the preclinical and clinical stages of BSE. The aim of this study was to investigate immunohistochemically the role of haematogenous neuroinvasion in cattle with spontaneously arising and experimentally induced BSE. There was almost complete absence of PrP(d) in the peripheral organs of BSE infected cattle. Additionally, there was minimal involvement of the CVOs during preclinical disease and there was progressive caudorostral accumulation of PrP(d) in the brain. These findings do not support haematogenous neuroinvasion in the bovine disease.

Increased immunohistochemical labelling for prion protein occurs in diverse neurological disorders of sheep: relevance for normal cellular PrP function.

The classical prion diseases (e.g. scrapie of sheep and goats and bovine spongiform encephalopathy of cattle) are characterized by the accumulation of abnormal forms of the prion protein (PrP), usually recognized by their relative resistance to proteolysis compared with the physiological cellular forms of PrP. However, novel prion diseases have been detected in sheep, cattle and man, in which the abnormal PrP has less resistance to proteolysis than identified previously. These more subtle differences between abnormal and normal forms of PrP can be problematic in routine diagnostic tests and raise questions in respect of the range of PrP disorders. Abnormal accumulations of PrP in atypical and classical prion diseases can be recognized by immunohistochemistry. To determine whether altered PrP expression or trafficking might occur in nosological entities not previously connected with prion disease, the brains of sheep affected with diverse neurological conditions were examined for evidence of altered PrP labelling. Such altered immunolabelling was detected in association with either basic lesions or specific diseases. Some reactive glial cells and degenerate neurons found in several different recognized disorders and non-specific inflammatory processes were associated with abnormal PrP labelling, which was absent from brains of healthy, age-matched sheep. The results agree with previous indications that normal PrP function may be linked with the oxidative stress response, but the data also suggest that PrP functions are more extensive than simple protective responses against stress insults.

Neuropathological changes correlate temporally but not spatially with selected neuromodulatory responses in natural scrapie.

AIM: Neuropathological changes classically associated with sheep scrapie do not always correlate with clinical disease. We aimed to determine if selected neuromodulatory responses were altered during the course of the infection as it has been described in Creutzfeldt-Jakob disease and experimental bovine spongiform encephalopathy. METHODS: Hemi-brains from healthy sheep and natural scrapie cases at two stages of infection were examined for biochemical alterations related to the expression of type I metabotropic glutamatergic receptors (mGluR(1) ) and type I adenosine receptors I (A(1) R), and of selected downstream intermediate signalling targets. Immunohistochemistry for different scrapie-related neuropathological changes was performed in the contralateral hemi-brains. RESULTS: PrP(d) deposition, spongiform change, astrocytosis and parvalbumin expression were significantly altered in brains from clinically affected sheep compared with preclinical cases and negative controls; the latter also showed significantly higher immunoreactivity for synaptophysin than clinical cases. Between clinically affected and healthy sheep, no differences were found in the protein levels of mGluR(1) , while phospholipase Cß1 expression in terminally ill sheep was increased in some brain areas but decreased in others. Adenyl cyclase 1 and A(1) R levels were significantly lower in various brain areas of affected sheep. No abnormal biochemical expression levels of these markers were found in preclinically infected sheep. CONCLUSIONS: These findings point towards an involvement of mGluR(1) and A(1) R downstream pathways in natural scrapie. While classical prion disease lesions and neuromodulatory responses converge in some affected regions, they do not do so in others suggesting that there are independent regulatory factors for distinct degenerative and neuroprotective responses.

Characterization of strains of ovine transmissible spongiform encephalopathy with a short PrPd profiling method.

Scrapie is the transmissible spongiform encephalopathy (TSE) that naturally affects sheep and goats; these species are also susceptible to experimental infection with the bovine spongiform encephalopathy (BSE) agent. Discrimination between different strains of sheep scrapie and ovine BSE has been achieved by descriptive and quantitative profiling of deposits of the disease-associated prion protein (PrPd) in different areas of the brain, but this process is time-consuming and difficult to standardize between laboratories. The present paper describes an alternative PrPd profiling method that is less demanding and addresses these difficulties. It is based on the scoring of similar 14 PrPd types in 11 precisely defined areas of the telencephalon. When applied to 48 archived cases of experimental sheep BSE, SSBP/1, CH1641 and natural scrapie, it gave comparable results to the original profiling method, previously conducted on the same brains, and allowed differentiation between the different infectious sources. This new 'short PrPd profiling' method has the advantages of being less time-consuming and easier to standardize, so that it can be readily adopted by different laboratories to provide comparable results.

Pathological phenotype of sheep scrapie after blood transfusion.

Blood transfusion practices have resulted in iatrogenic cases of variant Creutzfeldt-Jakob disease (vCJD) and it is known that sheep blood is also infectious in the pre-clinical stages of natural scrapie and experimentally induced bovine spongiform encephalopathy (BSE). Further investigations have also shown that the pathological phenotype of sheep BSE and human vCJD is maintained after blood transfusion. The present study describes the pathological phenotype, in terms of accumulation of the disease-associated prion protein in brain and lymphoreticular tissues, in sheep receiving blood from donors infected with natural scrapie. The immunohistochemical examinations undertaken showed a degree of phenotypic variability within and between scrapie donors and recipients, which might be attributable to the presence of more than one scrapie strain amongst the donor sheep or to a host adaptation process, or to the interaction of both, rather than to the influence of the route of infection.

Prevalence and factors affecting the formation of second molar distal caries in a Turkish population.

The aim of this study was to evaluate the prevalence of second molar distal caries in a Turkish population and to determine the factors that affect it. Clinical records and panoramic radiographs of partially erupted mandibular third molars were reviewed in this retrospective study. The analysis outcome measures were the patients' age, second molar distal caries, third molar angulation and second and third molar contact point localization. Prevalence of second molar distal caries in the population was 20%. This prevalence was 47% when the third molar had an angulation of 31-70 degrees (majority of mesioangular third molars) and 43% at 70-90 degrees (all horizontal third molars). The contact point on the second molar amelocemental junction and increasing age had significant effects on caries formation. The results revealed that second molar distal caries justifies prophylactic third molar removal and partially erupted third molars that have an angulation of 30-90 degrees with a contact point on the amelocemental junction should be removed to prevent second molar distal caries.

Neuroinvasion in sheep transmissible spongiform encephalopathies: the role of the haematogenous route.

BACKGROUND: It is generally believed that after oral exposure to transmissible spongiform encephalopathy (TSE) agents, neuroinvasion occurs via the enteric nervous system (ENS) and the autonomic nervous system. As a result, the dorsal motor nucleus of the vagus nerve is the initial point of disease-associated prion protein (PrP(d)) accumulation in the brain. HYPOTHESIS AND AIM: If direct ENS invasion following oral infection results in an early and specific brain targeting for PrP(d) accumulation, such topographical distribution could be different when other routes of infection were used, highlighting distinct routes for neuroinvasion. METHODS: An immunohistochemical study has been conducted on the brain of 67 preclinically infected sheep exposed to natural scrapie or to experimental TSE infection by various routes. RESULTS: Initial PrP(d) accumulation consistently occurred in the dorsal motor nucleus of the vagus nerve followed by the hypothalamus, regardless of the breed of sheep, PrP genotype, TSE source and, notably, route of infection; these factors did not appear to affect the topographical progression of PrP(d) deposition in the brain either. Moreover, the early and consistent appearance of PrP(d) aggregates in the circumventricular organs, where the blood-brain barrier is absent, suggests that these organs can provide a portal for entry of prions when infectivity is present in blood. CONCLUSIONS: The haematogenous route, therefore, can represent a parallel or alternative pathway of neuroinvasion to ascending infection via the ENS/autonomic nervous system.

Pathogenetical significance of porencephalic lesions associated with intracerebral inoculation of sheep with the bovine spongiform encephalopathy (BSE) agent.

UNLABELLED: Decreased rates of transmission of transmissible spongiform encephalopathies (TSEs) to sheep have been attributed to some polymorphisms of the prion protein (PrP) and to a 'species barrier' on interspecies experiments. In addition, the blood-brain barrier may be a further impediment to TSE neuroinvasion. The intracerebral (I/C) route is generally considered the most efficient for TSE transmission, as it may help to bypass those factors. Therefore, susceptibility of particular species to specific TSE agents is conducted by this route. AIMS: This study characterizes the traumatic brain lesions associated with the I/C injection of the bovine spongiform encephalopathy agent in sheep, assesses the relevance of such lesions in the outcome of clinical disease and provides insight into the mechanisms of PrP(d) conversion and amplification following I/C challenge. METHODS: A total of 27 hemibrains have been macroscopically and immunohistochemically examined to investigate the presence of lesions compatible with the needle track and the PrP(d) distribution, respectively. RESULTS: No residual inoculum was found and the extension and severity of the traumatic brain lesions were unrelated to the clinical outcome. Sheep with PrP(d) accumulation in the brain also showed conspicuous focal aggregates in the porencephalic lesions and in the circumventricular organs. In contrast, sheep without PrP(d) deposits in the brain were also negative in the traumatic lesions. CONCLUSION: Overall, these findings suggest that the efficiency of the I/C route is due to effective absorption and blood recirculation of infection, rather than to primary amplification at the site of injection.

Occurrence and cellular localization of PrPd in kidneys of scrapie-affected sheep in the absence of inflammation.

Following a preliminary description of disease-associated prion protein (PrPd) deposition in the kidneys of scrapie-affected sheep, detailed studies have been undertaken in order to evaluate the factors that could account for such PrPd accumulation and to determine the precise location of PrPd in the renal papillae. Immunohistochemical (IHC) examinations for PrPd were conducted in kidneys collected at post-mortem from 30 naturally and 37 experimentally infected sheep. In addition, PrPd detection by western blot analysis (WB) and ultrastructural examination was carried out in a selection of kidneys. PrPd-specific, multifocal IHC labelling with antibody R145 was achieved in the kidneys of 44% and 51% of the naturally and experimentally infected sheep, respectively. The specificity of these results was confirmed by further IHC and WB using several PrP antibodies raised to different amino acid sequences, and by examination of control tissues. PrPd was shown to accumulate in the interstitium of the renal papillae, in association with the cell membrane and lysosomes of fibroblast-like cells, or extracellularly, in close contact with collagen and basal membranes. These deposits were unrelated to inflammatory changes in the kidney as shown by routine histology and by IHC for different immune cell markers. PrPd accumulated in the kidney of sheep that showed widespread PrPd deposition in the lymphoreticular system and had long incubation periods; these findings argue for a haematogenous origin of renal PrPd, although the precise site and mechanism-glomerular filtration and reabsorption at Henle's loop, or extravasation from vasa recta capillaries, or both-by which PrPd leaves the blood to accumulate in the interstitium of renal papillae remain to be determined. Either of these pathogenetic mechanisms could lead to environmental contamination via urine.