Antemortem Tests Research Articles

Cholesterol Metabolism in Brain and Skin Fibroblasts from Sarda Breed Sheep With Scrapie-resistant and Scrapie-susceptible Genotypes

Scrapie is a fatal spongiform encephalopathy of sheep, a transmissible form of prion disease caused by neuronal accumulation of the aberrantly conformed prion protein (PrPsc). Currently, no ante-mortem diagnostic tests are available to detect this untreatable disease in the pre-clinical stage, thus making difficult to control its spread. Recent evidence suggests that the production of PrPsc can be modulated by the levels of membrane cholesterol in neuronal cells. Since cholesterol levels in cell membranes are dependent on cholesterol homeostasis in the whole organism, we studied cholesterol metabolism in brain tissues, plasma and skin fibroblasts of Sarda breed sheep with scrapie-resistant (ARR/ARR) and scrapie-susceptible (ARQ/ARQ) prion protein genotypes, both not infected (ARQ/ARQ-) and infected (ARQ/ARQ+) with scrapie. We found that, the levels of cytoplasmic cholesterol esters (CE) in brains and skin fibroblasts from sheep with the ARQ/ARQ genotype were consistently higher than those from sheep with the ARR/ARR genotype. Conversely, the levels of free cholesterol (FC) were lower in ARQ/ARQ, as compared to ARR/ARR sheep, thus resulting in a sharp reduction of the FC/CE ratio. Moreover, both uninfected and infected ARQ/ARQ sheep showed abnormally low levels of high density lipoprotein-cholesterol (HDL-C) in their plasma, as compared to ARR/ARR sheep. These data other than adding new strength to the notion that altered levels of intracellular cholesterol may indicate the presence of a lipid metabolic state that predisposes to infection with, and accumulation of, PrPsc in the brain, discriminate for the first time between two distinct but related cellular pools of cholesterol, namely membrane FC on one hand and cytoplasmic CE on the other.

EVALUATION OF CONTROL AND TREATMENT OF GONGYLONEMA SPP. INFECTIONS IN CALLITRICHIDS

Numerous cases of Gongylonema spp. infection with associated pathological lesions and clinical signs were identified in a collection of Goeldi's monkeys (Callimico goeldii) (GMs) at a zoological park during a 3-yr period. An increase in the incidence of clinical signs in the GMs and other callitrichid species prompted an investigation to determine the prevalence of infection within the collection and evaluate treatment protocols. Twenty-one callitrichids [nine GMs, four golden lion tamarins (Leontopithecus rosalia), six cotton-top tamarins (Saguinus oedipus), and two golden-headed lion tamarins (Leontopithecus chrysomelas)] were included in this study. Many of the animals had been positively diagnosed on past examinations. Repeated cytological evaluations of scrapings taken from the mucosa of the tongue were performed to diagnose infection. The animals were randomly divided into two groups and treated with either ivermectin (290 microg/kg p.o., q7 days for four doses) or mebendazole (70 mg/kg p.o. q24 hr for three doses). Follow-up scrapings were performed on all animals at days 35, 64, and 156. Numerous animals displayed clinical signs (facial pruritus, inflammation, and ptyalism) before and throughout the investigation; however, Gongylonema spp. infections were only confirmed by tongue scrapings in two animals. Fecal floatation by using a sodium nitrate solution for recovery of spirurid eggs also was performed, but it yielded no positive results. The low number of confirmed cases precluded comparative evaluation of the efficacy of the anthelmintic treatment protocols. However, both regimes seemed subjectively similar in decreasing clinical signs and were safe with no adverse effects. Diagnosis of Gongylonema spp. infection is challenging, even when severe clinical signs are present. This investigation further demonstrates the lack of a reliable ante-mortem test for the parasite and underscores the importance of treatment based on clinical signs. Until a more sensitive test is available, further comparison studies on treatment regimes will be difficult and likely unrewarding.

APPLICATION OF ASSAYS FOR THE DIAGNOSIS OF TOXOPLASMOSIS IN A COLONY OF WOOLLY MONKEYS (LAGOTHRIX LAGOTRICHA)

Toxoplasma gondii infection is a common cause of morbidity and mortality in New World primate species. Clinical abnormalities associated with toxoplasmosis can be nonspecific, making it difficult to make a definitive antemortem diagnosis and initiate appropriate treatment. Toxoplasmosis in New World primates can have a rapid clinical course, which may lessen the diagnostic utility of antemortem tests. However, while there are a variety of T. gondii serum antibody tests and T. gondii polymerase chain reaction (PCR) assays available that are not species specific, these assays have not been comparatively applied to New World primate cases. Woolly monkeys (Lagothrix lagotricha), a species of New World primate, are highly susceptible to fatal toxoplasmosis. Archived serum samples from 15 living and deceased woolly monkeys housed at the Louisville Zoological Garden (Louisville, Kentucky) were tested for T. gondii antibodies by a commercially available latex agglutination kit, a commercially available indirect hemagglutination kit, and the modified agglutination test. In addition, aliquots of the sera were assayed for T. gondii DNA using a PCR assay. Both woolly monkeys that died of disseminated toxoplasmosis were positive in all four assays, indicating that each could be used to aid in the diagnosis of toxoplasmosis in this species. We suspect that these assays have applications to other species of New World primates.

Assessment of clinical criteria to diagnose scrapie in Italy

A reliable ante-mortem test for the detection of scrapie in all genotypes has not yet been developed and clinical diagnosis remains a useful tool for surveillance purposes. This paper describes the results of a three-year study in which clinical signs consistent with scrapie were recorded according to standardized criteria in 245 sheep from 21 outbreaks in Italy in order to identify helpful criteria for the diagnosis of the disease. Thirty-seven sheep were scrapie-positive at post-mortem rapid testing, 23 showed weight loss, 20 had proprioceptive deficits, 17 demonstrated ataxia and nibble reflex, and some sheep had a combination of signs. Six scrapie-positive sheep were asymptomatic. The clinical protocol was easy to handle and appears to be a useful tool for improving passive surveillance. The data suggested that positive clinical history, nibble, and nibble combined with proprioceptive positioning deficit have a quite high negative predictive value. The protocol will be proposed as a tool for field inspection in passive surveillance in Italy.

An Overview of Prion Biology and the Role of Blood Filtration in Reducing the Risk of Transfusion-Transmitted Variant Creutzfeldt-Jakob Disease

Prions are infectious proteins believed to be responsible for a variety of progressive and fatal neurodegenerative diseases, collectively referred to as transmissible spongiform encephalopathies (TSE). By 1996, it was recognized that ingestion of beef from cattle afflicted with a TSE known as bovine spongiform encephalopathy, could result in a devastating human TSE known as variant Creutzfeldt-Jakob disease (vCJD). Two recent reports of probable transfusion-transmitted vCJD have raised concerns about the safety of the blood supply. The relatively long asymptomatic latency of vCJD, as well as the lack of sensitive and specific antemortem tests, increase the risk that asymptomatic, infected individuals may become blood donors. To this point, donor deferral has been a strategy used to reduce this risk. Nevertheless, this strategy may be unreliable and, furthermore, may threaten blood availability. Leukoreduction has also been helpful in reducing cell-associated infectious prion, which has been reported to reduce up to 42% of the infectivity in blood. Proprietary prion affinity surface modifications have been developed and applied to filters, which exploit an understanding of the unique chemical characteristics of prion surfaces. These have been successfully adapted to existing high-efficiency blood filter matrices for the reduction of prions present in blood components for transfusion.

Gene expression alterations in brains of mice infected with three strains of scrapie

BackgroundTransmissible spongiform encephalopathies (TSEs) or prion diseases are fatal neurodegenerative disorders which occur in humans and various animal species. Examples include Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in deer and elk, and scrapie in sheep, and experimental mice. To gain insights into TSE pathogenesis, we made and used cDNA microarrays to identify disease-associated alterations in gene expression. Brain gene expression in scrapie-infected mice was compared to mock-infected mice at pre-symptomatic and symptomatic time points. Three strains of mouse scrapie that show striking differences in neuropathology were studied: ME7, 22L, and Chandler/RML.ResultsIn symptomatic mice, over 400 significant gene expression alterations were identified. In contrast, only 22 genes showed significant alteration in the pre-symptomatic animals. We also identified genes that showed significant differences in alterations in gene expression between strains. Genes identified in this study encode proteins that are involved in many cellular processes including protein folding, endosome/lysosome function, immunity, synapse function, metal ion binding, calcium regulation and cytoskeletal function.ConclusionThese studies shed light on the complex molecular events that occur during prion disease, and identify genes whose further study may yield new insights into strain specific neuropathogenesis and ante-mortem tests for TSEs.

Epidemiological survey of swine cysticercosis using ante-mortem and post-mortem examination tests in the southern highlands of Tanzania

Pig keeping is known to be popular in the regions of the southern highlands zone (Mbeya, Iringa and Ruvuma) of Tanzania where more than 60% of pigs under the small-scale production system are raised. However, no epidemiological surveys on porcine cysticercosis have been conducted in the zone in spite of unofficial reports indicating the disease to be a widespread problem. To estimate prevalence rates and risk factors for porcine cysticercosis in Chunya and Iringa Rural Districts and Ruvuma Region (Songea and Mbinga Districts), 722, 808 and 302 live pigs, respectively, were examined by lingually and the prevalence of swine cysticercosis was found to be 7.6%, 8.4% and 16.9% for Chunya and Iringa Rural Districts, and Ruvuma Region, respectively. Structured observations and questionnaire interviews were used to analyse pig rearing practices and household use of latrines in Chunya and Iringa Rural Districts only. The analysis of effect of pig management practices, lack of a latrine, eating undercooked pork, home slaughter and no inspection of pork and lack of knowledge of T. solium on their association to tongue positivity in pigs was done by means of a contingency table. Odds ratios (OR) with corresponding 95% confidence intervals (95% CI) and p-values were calculated. Structured questionnaire interviews identified factors associated with the disease prevalence in both Chunya and Iringa Rural Districts were free-ranging of pigs, home slaughtering of pigs and pork not being inspected. While in Chunya and Iringa Rural Districts lack of latrine and barbecuing were found a risk factor, respectively. To control the disease in the study areas of the southern highlands there is a need for significant improvements regarding the use of latrines, confinement of pigs, pork inspection and thorough cooking of pork.

Abattoir surveillance: The U.S. experience

Abattoir, or slaughter, surveillance has been an important component of bovine tuberculosis control and eradication programs in the U.S., and has adapted to changes in the livestock market from farm to table, and the threat of bovine tuberculosis from a wildlife reservoir. The purpose of this overview was to describe the current goals of U.S. bovine tuberculosis slaughter surveillance, describe the elements of slaughter surveillance in the U.S., describe enhancements to the slaughter surveillance system, and discuss future challenges for the U.S. bovine tuberculosis surveillance program. Government regulations and the scientific literature were examined to provide information for this paper. The control and eradication of bovine tuberculosis in livestock falls to the United States Department of Agriculture and two agencies within the Department: the Food Safety and Inspection Service (FSIS) and the Animal and Plant Health Inspection Service (APHIS). FSIS conducts routine slaughter surveillance for disease or conditions that render carcasses unsuitable for human consumption, while APHIS is involved in antemortem bovine tuberculosis testing, and necropsy and investigation of bovine tuberculosis cases identified through slaughter surveillance or antemortem testing. Results from the previous 5 years of surveillance are presented. Enhancements have been added to the current surveillance system to improve its performance. An incentive program has been used to increase the numbers of tissues submitted for laboratory examination, the state of Michigan is implementing electronic animal identification under a pilot program, and expansions to the current system are being developed to accommodate new livestock industries. The success of these programs and challenges for the future are discussed.

Tonsillar Biopsy Test for Chronic Wasting Disease: Two Sampling Approaches in Mule Deer and White-tailed Deer

Preclinical antemortem testing of deer (Odocoileus spp.) for chronic wasting disease (CWD) can be important for determining prevalence rates and removing infected individuals from wild populations. Because samples with high numbers of tonsillar follicles are likely to provide earlier detection of CWD than samples with fewer follicles, the method of obtaining follicular samples may be critical when investigating disease prevalence. Between January 2003 and January 2005, white-tailed deer (O. virginianus) in southeast and southwest Minnesota and white-tailed and mule deer (O. hemionus) in Wind Cave National Park, South Dakota, were sampled using dorso-lateral and ventral-medial approaches for collecting tonsillar follicles. We obtained significantly more follicles using a dorso-lateral (median number of follicles = 19) rather than a ventral-medial (median number of follicles = 5.5) approach. No differences were observed in collection of tonsillar follicles that were related to sex, age class, or species of deer. We recommend the dorso-lateral approach for assessing CWD prevalence in deer populations.

Serologic Diagnosis of Sarcocystosis in Psittacine Birds: 16 Cases

Abstract In psittacine birds, the diagnosis of sarcocystosis is usually made on postmortem examination, and antemortem testing has been limited to muscle biopsy. We studied the applications of a serologic assay for antibody and plasma protein electrophoresis for changes in protein fractions in the antemortem diagnosis of sarcocystosis in psittacine birds. Several confirmed cases of sarcocystosis in psittacine birds were examined. To detect antibody, an indirect immunofluorescent assay was employed using Sarcocystis falcatula merozoites. Plasma samples from birds that demonstrated chronic neurologic signs had reactivity to the S falcatula antigen, and increases were found in the beta and gamma fractions by plasma protein electrophoresis. Conversely, in birds with the classic peracute onset of dyspnea and subsequent death, test results by either methodology were negative. These findings support the use of these 2 diagnostic tools as aids in the antemortem diagnosis of the nonperacute form of sarcocystosis i...

Diagnostic criteria for degenerative suspensory ligament desmitis in Peruvian Paso horses

Degenerative suspensory ligament desmitis (DSLD) has arisen in recent years as a health issue of concern to breeders and owners of Peruvian Paso horses. Lack of understanding of this disease and a “gold-standard” antemortem test has led to widespread confusion and uncertainty in the diagnosis of the disease in these horses. Using observations and measurements from a group of horses with histologically confirmed DSLD and from an age-matched group of horses without evidence of DSLD, estimates of the sensitivity and specificity of various measurements were calculated. The results of these analyses suggested four steps for the diagnosis of DSLD when screening large numbers of horses and as part of pre-purchase examinations. These four steps included: palpation of the suspensory ligaments, observance of baseline lameness, fetlock flexion tests, and sonographic examinations of the suspensory ligament at mid-cannon and both branches. These four steps accurately provide the information needed to make a diagnosis of degenerative suspensory ligament desmitis in Peruvian Pasos.

Intrauterine and transmammary transmission of Mycobacterium avium subsp paratuberculosis in sheep.

To investigate intrauterine infection of foetuses with Mycobacterium avium subsp paratuberculosis and the presence of infection in mammary secretions of sheep. A study of 142 late-pregnant ewes and their foetuses from two heavily infected flocks. Infection of ewes was determined at necropsy by histopathology and culture of tissues and mammary secretions. Antemortem tests (clinical assessment, faecal culture and serology) were also applied. Foetuses from 59 infected ewes and 47 apparently uninfected ewes were examined by culture and histopathology. Five of five ewes with clinical ovine Johne's disease had infected foetuses. Only one of 54 subclinically affected ewes, and none of 47 uninfected ewes had an infected foetus. M a paratuberculosis was cultured from mammary secretions or mammary glands of only two of 76 ewes, both of which were clinical cases and had infected foetuses. Although intrauterine or transmammary transmission of Mycobacterium avium subsp paratuberculosis may occur frequently in clinically affected sheep, these are less common in subclinically infected ewes. Therefore these modes of transmission are unlikely to compromise existing control programs for ovine Johne's disease on most farms, especially if programs include the immediate culling of clinically affected sheep.

Antemortem Identification of Bovine Spongiform Encephalopathy from Serum Using Infrared Spectroscopy

Since 1986, more than 180 000 clinical cases of bovine spongiform encephalopathy (BSE) have been observed in the U.K. alone. Most of these cases were confirmed by postmortem examination of brain tissue. However, BSE-related risk assessment and risk management would greatly benefit from antemortem testing on living animals. A serum-based test could allow for screening of the cattle population; thus, even a BSE eradication program would be conceivable. Here we report on a novel method for antemortem BSE testing, which combines infrared spectroscopy of serum samples with multivariate pattern recognition analysis. A classification algorithm was trained using infrared spectra of bovine sera from more than 800 animals (including BSE-positive, healthy controls and animals suffering from classical viral or bacterial infections). In two validation studies, sensitivities of 85 and 84% and specificities of 86 and 91% were achieved, respectively. The combination of classification algorithms increased the sensitivity and specificity of BSE detection to 96 and 92%, respectively.

Prion diseases

The term “transmissible spongiform encephalopathy” (TSE) refers to a group of rare and fatal neurodegenerative disorders that affect animals and humans. In contrast to all other neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease, TSE can be acquired by infection. It is believed that all TSEs share a common mechanism that involves the conversion of a normal cellular protein, prion (PrP c), to a rogue infectious isoform, scrapie prion (PrP Sc). It is the propagation and accumulation of the PrP Sc in the central nervous system that causes TSE. Hence, prion disease has been used synonymously with TSE. Since the discovery of bovine spongiform encephalopathy (BSE), the variant Creutzfeldt-Jakob disease (vCJD) has taken center stage among all human TSEs. It is believed that vCJD is acquired from cattle affected with BSE and that vCJD represents the first incidence of TSE transmission from animals to humans. Currently, the only in vitro diagnostic test for human and animal TSE is a complex postmortem test. It is unlikely that the rate of human prion disease will increase dramatically in the future. Nevertheless, a rapid and sensitive antemortem test is needed to monitor the development and potential spread of prion diseases in animals and humans.

A model of avian mycobacteriosis: clinical and histopathologic findings in Japanese quail (Coturnix coturnix japonica) intravenously inoculated with Mycobacterium avium.

Mycobacterial infections are an important cause of morbidity and mortality in birds and a considerable diagnostic challenge until the disease is advanced. In order to develop more clinically useful antemortem tests, a biological model was created that replicated naturally occurring disease. Japanese quail (Coturnix coturnix japonica; n = 8) were inoculated intravenously with Mycobacterium avium. Two additional birds served as uninoculated controls. Mean survival time of the inoculated birds was 68 +/- 13 days postinoculation (PI). Seven of the eight inoculated birds died naturally. Clinical and postmortem abnormalities in inoculated birds were characteristic of naturally occurring mycobacteriosis. Abnormal clinical findings included decreased activity, feather erection, and sudden death. Mean body weight and packed cell volume declined and mean total white blood cells (primarily heterophils, bands, and monocytes) increased from 28 days PI onward. Similar to birds that are naturally infected with mycobacteriosis, the inoculated birds were thin and had severe hepatosplenomegaly on postmortem examination. All eight birds had lesions in the liver, spleen, intestine, lung, gonads, and serosa. Less commonly affected tissues included bone marrow, thymus, gizzard, heart, pancreas, and brain. Lesions were invariably severe in the liver and spleen. These gross postmortem findings were consistent with natural infections of avian mycobacteriosis. Mycobacterium avium was isolated from the liver, spleen, and intestine of all inoculated birds. Both control birds remained disease free and culture negative. This inoculation protocol is a reliable and practical means of inducing avian mycobacteriosis for further study.

Testing for Mycobacterium avium subsp. paratuberculosis infection in asymptomatic free-ranging tule elk from an infected herd.

Forty-five adult tule elk (Cervus elaphus nannodes) in good physical condition were translocated from a population located at Point Reyes National Seashore, Marin County (California, USA), to a holding pen 6 mo prior to release in an unfenced region of the park. Because infection with Mycobacterium avium subsp. paratuberculosis (Mptb) had been reported in the source population, the translocated elk underwent extensive ante-mortem testing using three Johne's disease assays: enzyme linked immunosorbent assay (ELISA); agar gel immunodiffusion assay (AGID), and fecal culture. Isolation of Mptb was made from fecal samples in six of 45 elk (13%). All AGID results were negative while ELISA results for 18 elk (40%) were considered elevated. Elevated ELISA results or Mptb isolation from fecal samples were obtained for 22 of 45 elk (49%); these elk were euthanized and necropsied. Mycobacterium avium subsp. paratuberculosis was isolated from tissue in 10 of 22 euthanized elk (45%); of these 10 cases of confirmed infection, eight had elevated ELISA results (80%) and four were fecal culture positive (40%). One of 10 cases had histopathologic lesions consistent with Mptb infection. Mycobacterium avium subsp. paratuberculosis was also isolated from tissue from one of eight fetuses sampled. The number of tule elk found to be infected was unexpected, both because of the continued overall health of the source herd and the normal clinical status of all study animals.

Motor impairment in normal aging, clinically possible Parkinson's disease, and clinically probable Parkinson's disease: longitudinal evaluation of a cohort of prospective brain donors

This study presents data on the antemortem evaluations of a cohort of individuals registered in a brain donation program. Clinical evaluation determined that many individuals were unaware they had clinical signs of Parkinson's disease (PD) (rest tremor, bradykinesia, rigidity). Quantitative motor testing (timed tapping test and Purdue pegboard test) revealed a graded reduction in performance in those clinically found to have clinically possible and clinically probable PD. Longitudinal examinations over 4 years revealed some individuals progressed from control to clinically possible PD and clinically possible PD to clinically probable PD. This study underscores the importance of longitudinal antemortem testing of prospective brain donors as well as the potential value of quantitative motor testing.

Targeted screening of high-risk cattle populations for BSE to augment mandatory reporting of clinical suspects

In Switzerland, the first case of bovine spongiform encephalopathy (BSE) was diagnosed in November 1990. Case numbers peaked in 1995, with a total of 352 BSE cases identified by 30 April 2000. Reporting of clinically suspect cattle is currently the most commonly used method world-wide to detect BSE cases. The effectiveness of mandatory reporting depends on a variety of factors; for other diseases passive surveillance underestimates the incidence of clinical cases. The efficiency of passive surveillance systems for BSE will remain unknown until screening tests able to identify clinically affected cattle have been applied in several countries. This paper provides the first detailed description of a targeted screening programme for BSE. Two populations of cows >24 months of age were included in the targeted screening: (i) cows found dead or culled on site where the carcass was submitted to rendering (fallen stock) and (ii) cows with health-related problems unfit for routine slaughter that were slaughtered under emergency procedures (emergency slaughter). Between 1992 and 1999, on average 81 clinical BSE suspects per year were reported to the veterinary authorities (passive surveillance), of which 43% were confirmed with BSE. A total of 30 clinical cases were captured by passive surveillance and an additional 20 BSE cases detected by targeted screening between May 1999 and April 2000. The odds of finding a BSE case was 49 times higher in the fallen stock and 58 times higher in emergency-slaughtered cattle when compared to passive surveillance. The targeted screening of fallen stock and emergency-slaughtered cattle considerably increased the number of detected cases in this 12-month period. Targeted-screening cases were on average 4 months younger than the clinical suspect cases. In conclusion, post-mortem testing of fallen stock and emergency-slaughtered cows >24 months for BSE is an important active surveillance element within a total surveillance system that principally is based on mandatory reporting of clinical suspect cases. Without ante-mortem screening tests to detect BSE-infected cattle during the incubation period, a combination of effectively functioning passive and active BSE surveillance strategies might be the only approach to assess the BSE situation reliably in a given country or region — and it is necessary to substantiate claims of freedom from the disease.

Prevalence of antibodies to NeosporaCaninumin dogs

An IFAT was used to determine the prevalence of Neospora-specific IgG antibodies in serum from Alabama horses. Serum samples ( n=536) were from asymptomatic horses routinely submitted for equine infectious anaemia virus infection testing. We also subjected a 13-year-old horse with CNS disease to necropsy examination for isolation and in vitro cultivation of protozoal organisms. In antemortem tests, this horse was positive for antibodies to Neospora sp. in the IFAT and western immunoblot. Results of the prevalence survey indicated that IgG antibodies to Neospora were present in 62 (11.5%) of the 536 serum samples. Endpoint titres for the positive samples were 1:50 (35/6.5%), 1:100 (19/3.5%), 1:200 (7/1.3%) and 1:1600 (1/0.2%). Tachyzoites were first seen in cultured bovine turbinate cells 32 days after inoculation with spinal cord homogenates from the horse with CNS disease. Tachyzoites reacted with known N. caninum-positive serum from horses, cows, dogs and mice, but did not react with murine anti- Toxoplasma gondii or equine anti- Sarcocystis neurona serum. Ultrastructural features of tachyzoites and results of comparison of tachyzoite immunodominant proteins revealed that they were identical to those of N. hughesi, a species described recently from a naturally infected horse. The isolate recovered from the naturally infected horse in the present study (designated NA1) is thought to be an isolate of N. hughesi, although confirmation of this awaits additional molecular characterisation. These results provide some additional evidence that N. hughesi is a valid species and that Neospora infections in horses may occur in widely separated geographic regions of the United States.

Scrapie infectivity and proteinase K-resistant prion protein in sheep placenta, brain, spleen, and lymph node: implications for transmission and antemortem diagnosis.

Probable transmission of bovine spongiform encephalopathy to humans has focused intense interest on all of the transmissible spongiform encephalopathies (TSEs) and how they spread. In all TSEs, an abnormal disease-associated, proteinase K-resistant protein referred to as PrP-res or PrPsc accumulates in brain. In some species, PrP-res accumulates in other tissues as well. Sheep placenta, brain, spleen, and lymph node were analyzed in detail for PrP-res and infectivity. Both were detected in all brain and spleen samples and in placenta and lymph nodes of 80% of the scrapie-infected sheep. A perfect correlation was observed between infectivity and PrP-res detection. These results substantiate the probability that placenta plays an important role in natural transmission of scrapie, suggest that analysis of placenta for PrP-res could be the basis for an antemortem test for sheep scrapie, and show that PrP-res, scrapie infectivity, and scrapie disease are closely associated.