PrP genotype in Sarda breed sheep and its relevance to scrapie

Summary.  Several PrP gene polymorphisms modulate sheep scrapie susceptibility. Recently, an increase of scrapie outbreaks has been reported in Italy. A vaccine containing sheep brain homogenate was used in most of the outbreaks. We investigated PrP gene polymorphisms in scrapie-affected and clinically healthy Sarda breed sheep from a flock exposed to the aforementioned vaccine, and in affected Sarda sheep from unexposed flocks. All affected animals were (Gln/Gln)₁₇₁ homozygous. Moreover, we observed no variation for Ala₁₃₆ and a new polymorphism (Lys to Asn) at codon 176. Our findings confirm the correlation between scrapie and (Gln/Gln)₁₇₁ in breeds with no variation for Ala₁₃₆. Accepted June 18, 2001 Recevied March 19, 2001     

PrP gene polymorphism in sheep breeds in Slovakia and susceptibility to scrapie

We analyzed the prion protein (PrP) genotype based on the codons 136, 154 and 171 and assigned to five risk groups (R1-R5) in healthy and scrapie-affected sheep in Slovakia. In healthy (asymptomatic) population, 119 Merino, 106 Improved Valachian, 117 Tsigai, and 48 Suffolk breeds were tested. Among the asymptomatic sheep, the low-risk genotypes R1 and R2 were most abundant in Suffolk (94%) and Merino (84%) breeds, followed by Tsigai (58%) and Improved Valachian (40%) breeds. The medium-risk group R3 was most frequent in Improved Valachian (31%) breed, followed by Tsigai (21%), Merino (10%), and Suffolk (6%) breeds. The occurrence of high-risk groups R4 and R5 was none in Suffolk breed, followed by Merino (6%), Tsigai (21%), and Improved Valachian (30%) breeds. Since 2003, altogether 48 cases of scrapie have been confirmed in Tsigai (38), Merino (4), Improved Valachian (2), Improved Valachian x Tsigai (3), and Suffolk (1) breeds. Among sheep with scrapie, Merino breed belonged to the medium-risk group R3. The majority of scrapie-affected Tsigai sheep were classified into high-risk R5 (50%) and medium-risk R3 (42%) groups. We showed an association of scrapie with medium- and high-risk groups of PrP genotype in Slovakia. In particular, the glutamine at position 171 appears to be of major importance for the susceptibility to scrapie.     

Effects of agent strain and host genotype on PrP accumulation in the brain of sheep naturally and experimentally affected with scrapie

Different cellular and neuroanatomical types of disease-specific prion protein (PrP(d)) accumulation in the brain were identified in sheep of different breeds and PrP genotypes exposed to experimental or natural scrapie infection. Immunohistochemical examination of the brains of 43 sheep with clinical signs compatible with scrapie revealed 12 different PrP(d)types, which were subjectively quantified in eight different brain regions. The PrP(d)types were grouped into four PrP(d)patterns, the relative magnitude of which provided the PrP(d)profile of each sheep examined. The analysis of the differences in magnitude and relative proportion of each of these PrP(d)types and patterns indicated (1) an effect of the scrapie strain on the PrP(d)profile, and (2) a possible effect of the host genotype on the magnitude of PrP(d)accumulation in the brain, apparently related to the incubation period. Furthermore, intraneuronal deposition of PrP(d)was the type most closely associated with the development of clinical disease. We conclude that different scrapie strains can be distinguished by PrP immunohistochemical examination of brains of affected animals.     

Transmission of classical scrapie to wild-type mice: the influence of the ovine PrP sequence on lesion profiles

Susceptibility of sheep to classical scrapie is determined by polymorphisms in the coding region of the prion protein gene (PRNP), mainly at codons 136, 154 and 171. It has recently been shown that lesion profiles from classical field scrapie isolates that transmitted to RIII mice can be classified into different groups. There was also strong, but not absolute, association between the different groups and codon 136. Here, we examine the hypothesis that additional polymorphisms in the open reading frame sequence of the ovine PRNP may account for the different groups of lesion profiles observed following transmission to mice.     

Diagnosis and PrP genotype target of scrapie in clinically healthy sheep of Massese breed in the framework of a scrapie eradication programme

Summary. The application of a selective culling programme in two scrapie affected flocks of Massese breed sheep is described. The genetic susceptibility of this breed and the sensitivity of different diagnostic methods in the pre-clinical diagnosis of scrapie were also investigated. Overall, 2,068 clinically healthy sheep underwent PrP genotyping, providing the basis for selective culling. The prevalence of scrapie infection was investigated in susceptible sheep by two independent diagnostic methods. All the sheep older than 18 months (n = 620) were tested by Prionics^® Check Western rapid test on the obex, with a prevalence of infection of 3.9%. Furthermore, 385 sheep underwent immunohistochemistry (IHC) on retropharyngeal lymph node (RPLN), with a prevalence of infection of 5.2%. Overall, 32 sheep were diagnosed with pre-clinical scrapie. Of these, 31 were positive by Western blot on the spleen, 29 by IHC on the RPLN and tonsil, 28 by IHC on the obex, 24 by rapid test, and only 18 by IHC on the third eyelid. All the scrapie positive sheep were of the ARQ/ARQ, ARQ/AHQ or ARQ/VRQ genotypes. No significant differences in scrapie prevalence were observed among these genotypes. The estimated risk of the three targeted alleles was also similar, suggesting that in this breed the VRQ allele was not at higher risk for scrapie, compared to the ARQ and AHQ alleles.     

Onset of accumulation of PrPres in murine ME7 scrapie in relation to pathological and PrP immunohistochemical changes.

In a murine scrapie model, three different methods (immunohistochemistry, Western blotting and histoblotting) for determining disease-specific PrP accumulation were compared. The incubation period of ME7 scrapie in the F1 cross of C57 BL and VM/Dk mice is about 230 days. Mice show hippocampal neuronal loss from 160-180 days post-inoculation (dpi), CA1 neuron dendritic spine atrophy at 126 dpi, and axon terminal degeneration and synaptic loss from 84-98 dpi. Infectivity titres of at least 100 are present from 40 dpi. PrP was detected immunohistochemically at 60 dpi in the hippocampus and in the thalamus. Thus, PrP accumulation in the hippocampus precedes even the earliest neurodegenerative changes. Low amounts of PrP immunolabelling were found between 60 dpi and 126 dpi, after which the intensity increased markedly. The histoblot method detected PrPres in one of four mice at 100 dpi. Western blotting of whole brains first identified the PrPres at 80 dpi. Thus, in our hands, the most sensitive method for detecting disease-specific accumulations of PrP was immunohistochemical examination. However, immunohistochemical methods are unable to distinguish the normal and abnormal isoforms of PrP. It is therefore possible that the initial accumulation of PrP takes place as PrPsen and that the translation of PrPsen to PrPres does not take place until the later stages of the disease process. The accumulation of disease-specific PrP lags behind the development of infectivity titres. The relative rates of increase of infectivity titre and PrP accumulation are different, suggesting that these parameters may be measures of different biological events.     

Detection of PrP(Sc) in rectal biopsy and necropsy samples from sheep with experimental scrapie

Scrapie diagnosis is based on the demonstration of disease-associated prion protein (PrP(Sc)) in brain or, in the live animal, in readily accessible peripheral lymphoid tissue. Lymphatic tissues present at the rectoanal line were readily obtained from sheep without the need for anaesthesia. The presence of PrP(Sc) in such tissue was investigated in sheep infected orally with scrapie-infected brain material. The methods used consisted of immunohistochemistry and histoblotting on biopsy and post-mortem material. PrP(Sc) was detected in animals with PrP genotypes associated with high susceptibility to scrapie from 10 months after infection, i.e., from about the time of appearance of early clinical signs. In the rectal mucosa, PrP(Sc) was found in lymphoid follicles and in cells scattered in the lamina propria, often near and sometimes in the crypt epithelium. By Western blotting, PrP(Sc) was detected in rectal biopsy samples of sheep with the PrP genotype VRQ/VRQ, after electrophoresis of material equivalent to 8 mg of tissue. This study indicated that rectal biopsy samples should prove useful for the diagnosis of scrapie in sheep.     

Oral inoculation of sheep with the agent of bovine spongiform encephalopathy (BSE). 1. Onset and distribution of disease-specific PrP accumulation in brain and viscera.

Sixty-three Romney sheep aged 6 months, consisting of three groups (PrP(ARQ/ARQ), PrP(ARQ/ARR), and PrP(ARR/ARR)genotypes) of 21 animals, were infected orally with brain tissue from BSE-infected cattle. Sub-groups of the 21 PrP(ARQ/ARQ) animals were killed, together with uninfected controls 4, 10, 16, 22 or 24-28 (after the development of full clinical disease) months post-inoculation (mpi). One sheep from each of the two groups of four killed at 4 or 10 mpi were shown by immunohistochemical examination to possess disease-specific PrP accumulations in single lymph nodes. At 16 mpi, such accumulations were detected in two of four infected sheep in some viscera and in the spinal cord and brain. At 22 mpi, three of five infected sheep had widespread disease-specific PrP accumulations in all tissues examined, but the remaining two animals gave positive results only in the central nervous system. Clinical disease appeared at 20-28 mpi. Three sheep killed with advanced clinical signs showed widespread PrP accumulation in brain, spinal cord and peripheral tissues. These results confirmed that PrP(ARQ/ARQ) Romney sheep are susceptible to experimental infection with the BSE agent. The different sites at which initial PrP accumulations were detected suggested that the point of entry of infection varied. Once established, however, infection appeared to spread rapidly throughout the lymphoreticular system. The results suggested that in some BSE-infected sheep neuroinvasion occurred in the absence of detectable PrP accumulations in the viscera or peripheral nervous system. In contrast to cattle with BSE, however, most sheep showed disease-specific PrP accumulations in the lymphoreticular system. In this respect, BSE-infected resembled scrapie-infected sheep; it is possible, however, that future research will reveal differences in respect of targeting of cell types within the lymphoreticular and peripheral nervous systems. The PrP(ARQ/ARR)and PrP(ARR/ARR)sheep were also killed in sub-groups at intervals after inoculation. Up to 24 mpi, however, none of these animals showed disease-specific PrP accumulations. Further results will be reported later.     

Search for healthy carriers of scrapie: an assessment of subclinical infection of sheep in an Icelandic scrapie flock by three diagnostic methods and correlation with PrP genotypes

Summary.  Subclinical infection in scrapie of sheep, characterized by a long incubation period, may be of importance for the spread of the disease. We screened brain samples from all 65 sheep in a scrapie-affected flock for subclinical infection and correlated with results of PrP genotyping, which is of relevance for the epidemiology and the question, whether by breeding for resistant genotypes one would be breeding for healthy carriers. The sensitivity of three methods was compared, i.e. histopathological examination for vacuoles (HP), immunohistochemical staining (IHC) and Western blotting (WB) for PrP^Sc. Five sheep showed definite clinical signs and histological scrapie lesions, and signs of infection were detected in 25 of 60 asymptomatic sheep, by HP and/or IHC and WB. The IHC was slightly more sensitive than HP and WB. Sheep with subclinical infection were, with one exception, either homo- or heterozygotes for 136-V, as were four of the five sheep with clinical scrapie. The incidence of the VRQ allelic variant in the flock was unusually high compared to the Icelandic sheep population probably contributing to the high prevalence of both clinical and subclinical infection in the flock. Neither sheep with definite scrapie nor detectable subclinical infection, were of the resistant AHQ genotype, indicating that Icelandic AHQ sheep are not healthy carriers of scrapie infection. Received September 7, 2001 Accepted December 13, 2001     

Occurrence and distribution of infection-specific PrP in tissues of clinical scrapie cases and cull sheep from scrapie-affected farms in Shetland

The prion protein (PrP) genotypes of all cull sheep originating from four scrapie-affected farms in Shetland in 1998-1999 were determined and a representative sample of the different genotypes was selected for necropsy. Samples of brain and selected viscera were removed from 159 such sheep aged 2-11 years. These samples were examined immunohistochemically and by Western blotting for infection-specific forms of PrP. None of the sheep bearing the following genotypes showed any evidence of PrP accumulation in brain, intestine, selected lymph nodes or the cranial mesenteric ganglia: ARQ/ARQ (n = 41), ARQ/ARH (n = 12), ARH/ARH (n = 2), ARQ/ARR (n = 24), ARR/ARR (n= 2). In five of 71 sheep bearing a single VRQ allele, PrP accumulation was detected immunohistochemically in viscera or brain, or both. These results suggested that only a small proportion of susceptible sheep showed evidence of infection (accumulation of PrP) on the farms studied, and that even sheep of the most susceptible genotype (VRQ/VRQ) did not invariably develop disease in an infected environment. Furthermore, there was no evidence that, in sheep of semi-resistant or fully resistant genotypes, infection could be sequestered within the lymphoreticular system or peripheral nervous system and thereby provide a possible "carrier" source of infection. Rather, the data suggested that some sheep, possibly because they had been exposed to a relatively low infective dose, became infected and accumulated the infective agent over a protracted pre-clinical phase of the disease. Such sheep might be potentially infective for many years. In two VRQ/ARR genotype sheep, PrP was confined to the brain. Infection-specific PrP was also confined to the brain in two of 24 clinical cases of VRQ/ARQ scrapie. Thus, direct neuroinvasion, apparently without a prior phase of replication in the lymphoreticular system, occurred in a proportion of VRQ/ARQ sheep. Possibly it may occur in all sheep of the VRQ/ARR genotype. The factors responsible for direct neuroinvasion are not understood. However, it cannot be attributed to genotype alone.     

Early accumulation of pathological PrP in the enteric nervous system and gut-associated lymphoid tissue of hamsters orally infected with scrapie

Previous research revealed the existence of coupling mechanisms (e.g. iso-directionality) at the level of perception and action. The present experiment investigated how the strength of the perception-action coupling affected synchronization performance. Arm movements were to be synchronized with a moving light that traveled back and forth from the left to the right side of a runway. Four experimental conditions were administered representing the orthogonal combination of two viewing conditions (intermittent vs. continuous) and two synchronization modes (in-phase, i.e. arm moving in the same direction as the light vs. anti-phase, i.e. arm moving in the opposite direction). Performance outcome measures, movement kinematics, and relative phase were used to examine the data. The results revealed a better synchronization performance when the arm and light traveled in the same direction (iso-directionality) during the continuous viewing condition. Apparently, the strength of the perception–action coupling has a severe impact on the quality of the synchronization of an arm movement to an external event.     

Nematode parasites and scrapie: experiments in sheep and mice

To demonstrate the possible role of nematode parasites in the modification of host susceptibility to scrapie, experiments were conducted using sheep naturally exposed to scrapie, chosen by their genotype at the PrP gene, and infected with Teladorsagia circumcincta. Two 4-year duration experiments demonstrated that the nematode infection shortened the development of scrapie with a significant regression between the level of infection and age at first scrapie symptoms (P<0.006). Investigations by ELISA tests in different species of nematode parasites of the digestive tract collected from scrapie infected ewes did not reveal the presence of PrP^Sc. In scrapie-infected C57BL mice, infected or not with Heligmosoides polygyrus at various times, parasitized animals showed a slight but significantly longer survival period. Assays on transmission by the larvae hatching from eggs collected from scrapie-infected mice were unsuccessful. We concluded that nematodes modify host susceptibility to scrapie, but their role in the horizontal transmission of the disease was not demonstrated.     

Serum enzyme levels and influencing factors in three indigenous Ethiopian sheep breeds

Serum enzymes were studied in 377 apparently healthy sheep from three indigenous sheep breeds of Ethiopia. The effect of breed, age, sex and season on alanine aminotransferase (ALT)/glutamic pyruvic transaminase (GPT), aspartate aminotransferase (AST)/glutamic oxalacetic transaminase (GOT), alkaline phosphatase (ALP) and acid phosphatase (AcP) levels was assessed. The mean serum enzymes levels of the indigenous Menz, Tukur and Wello sheep breeds ranged from 17.2–17.7 IU l⁻¹, AST/GOT from 50.4–56.6 IU l⁻¹, ALP from 93.2–103.9 IU l−¹, and AcP from 2.47–2.56 IU l⁻¹, were within the normal range for sheep elsewhere. Season had significant influence on all serum enzymes except for the AcP in Menz breed. Sex had significant effect on AST/GOT for Menz and on ALP for all sheep breeds, with consistently higher values in males than in females. Age was significant only on ALP in the Menz and Tukur breeds. The serum enzyme levels of these indigenous sheep breeds can be used as normal reference values for Ethiopian sheep breeds adapted to similar agro-ecology and production system.     

Natural variation in mite antigen density in house dust and relationship to residential factors

To investigate the year-to-year variation of mite antigen density (Der p I, Der f1) in dust from mattresses and the relevance of residential factors for antigen load, information derived from an epidemiologic study including two surveys carried out in the households of a cohort of elementary school children (n= 1291) was analysed. When considering residences with measurements taken in both years in question (n= 1050), rank-correlation indicated a predominance of stability for both antigens (Der p I: r_s= 0-82, p=0.0001; Der f I: r_s=0.72, P= 0.0001), Using multiple regression analyses, significant associations between antigen concentrations and a variety of residential factors were found. Use of a blanket of animal hair, use of a cover or underblanket, wet spots in the bedroom, higher relative humidity and a low storey level were significantly associated with increased concentrations of Der p I, whereas inverse relationships between this antigen and room temperature, number of persons per m² as well as use of underfloor heating were seen. Regarding Der f I, older mattresses, use of a cover or underblanket, higher weight of sampled dust, high educational level and higher ratio of inhabitants per m² were significantly associated with increased concentrations of the antigen. On the other hand, lower Der f I concentrations were found when interior sprung mattresses were used and when the mattress was ‘treated regularly’. In conclusion, two measurements, 1 year apart from each other, show that stability of mite antigen concentrations predominated. Our data suggest that allergic patients should be advised against living in lower storeys and damp homes and to use a newer or encased mattress and to give preference to a residence with underfloor heating.     

川崎病血脂异常及其与冠状动脉病变的关系

目的:研究川崎病患儿血脂水平及其与冠状动脉病变的关系。方法:检测川崎病患儿治疗前后外周血三酰甘油、胆固醇、低密度脂蛋白胆固醇和高密度脂蛋白,以健康儿童对照,并分析血脂水平变化与冠状动脉病变的关系。结果:川崎病患儿治疗前血脂异常,与健康儿童差异有统计学意义。治疗10~14d后未并发冠脉病变患儿血脂异常回复,而并发冠脉病变患儿血脂持续异常。结论:川崎病存在明显的脂质代谢紊乱,而且并发冠状动脉病变的川崎病血脂回复正常较为缓慢,其可能成为早期诊断川崎病并发冠状动脉病变的早期诊断指标。     

Expression of cyclooxygenase-2 and its relationship to p53 accumulation in colorectal cancers

PURPOSE: Cyclooxygenase (COX)-2 is an inducible isoform responsive to cytokines, mitogens, and growth factors, and is believed to be an important enzyme related to colorectal cancer (CRC). Existing evidence suggests that COX-2 expression is normally suppressed by wild-type p53 but not mutant p53, suggesting that loss of p53 function may result in the induction of COX-2 expression. The aim of this study was to determine the relationship between COX-2 expression and p53 levels in CRC. MATERIALS AND METHODS: Patients with sporadic colorectal adenocarcinoma (n=161) who underwent curative surgery in Chosun University Hospital were enrolled in this study. Expression of COX-2 and p53 proteins was examined by immunohistochemistry in paraffin-embedded cancer tissue blocks, and the relationship between COX-2 and/or p53 expression with clinicopathologic parameters was analyzed. RESULTS: Expression of COX- 2 was positive in 47.8% of colorectal cancers, and significantly associated with the depth of tumor invasion (p= 0.042). In contrast, p53 was positive in 50.3% of the cases, and was associated with both age (p=0.025) and the depth of tumor invasion (p=0.014). There was no correlation between COX-2 expression and p53 expression (p=0.118). CONCLUSION: These results suggest that COX-2 expression might play an important role in the progression of colorectal cancer. However, COX-2 expression was not associated with mutational p53. Further studies are needed to clarify the regulatory mechanisms governing COX-2 overexpression in colorectal cancers.