Stefan Roels

Scrapie case similar to Nor98 diagnosed in Belgium via active surveillance

SCRAPIE is a fatal transmissible spongiform encephalopathy (TSE) caused by prions. Other diseases caused by prions include Creutzfeldt-Jakob disease in human beings and bovine spongiform encephalopathy (BSE) in cattle. Typical features of these diseases are a long incubation period and the gradual vacuolation of brain neurons and neuropil. The pathogenesis of the diseases is believed to be due to the conversion of the normal protease-sensitive prion protein, PrPc, into a partly protease-resistant isoform, PrPSc, which accumulates progressively in the central nervous system of affected animals (Prusiner 1982, Bueler and others 1993). Several (classical) scrapie strains have been described based on lesion profiling in mice (Wood and others 1997, Ligios and others 2002). Since April 2002, all sheep older than 18 months in Belgium have been tested with a rapid test (BSE Platelia test; Bio-Rad) through the active TSE surveillance programme (Pastoret and others 2001, Roels and others 2002) (EC regulation 999/2001). The rapid test is used for the diagnosis of BSE in bovine brain tissues (Moynagh and Schimmel 1999), but is also suitable for the diagnosis of scrapie in sheep, as the monoclonal antibodies used recognise the ovine PrP (Andreoletti and others 2000). This short communication describes a case of scrapie which was similar to Nor98, an unusual case of scrapie first detected in Norway (Benestad and others 2003). Up until the end of 2003, more than 5000 sheep had been tested for scrapie, and six primary outbreaks were detected. Five of the outbreaks showed a classical scrapie lesion profile, but one sheep showed unusual features. The ewe was apparently healthy and presented for slaughter. According to the active epidemiosurveillance protocol, only part of the medulla oblongata around the region of the obex was removed. The sample repeatedly tested positive with the rapid test (Debecker and others 2000). Histopathological investigation revealed no vacuolar lesions either in the neurons or in the neuropil in the region of the obex. There was no detectable PrPSc as revealed by immunohistochemistry of the obex region and tonsils using polyclonal R524-7. The detection of scrapie-associated fibrils (SAFs) was also negative. PrPSc analysis using Western blotting (Bio Rad protocol) with antibodies 12F10 and SAF60 (Fig 1) and BAR226 and SAF60, gave a clear positive result, showing a PrPSc glycoprofile with a strong lower band at approximately 12 kDa, compared with a classical scrapie glycoprofile (Fig 1). The glycoprofile of the present case was confirmed by the National Veterinary Institute, Oslo. Under the terms of the surveillance protocol, the whole flock was culled and the brains of all the animals older than 18 months were examined, but no other animal in the flock tested positive with the rapid test. The affected sheep’s PrP genotype was A136R154Q171 homozygous; this was determined by denaturing gradient gel electrophoresis (Bossers and others 1996). The present case has unusual characteristics: only one of the 55 animals in the flock was affected; no lesions were present in the brainstem (obex) compared with the lesion profiles of classical scrapie cases (Wood and others 1997, Ligios and others 2002); there was no PrPSc immunolabelling in the area of the obex; and the PrPSc glycoprofile differed clearly from the glycoprofiles found in isolates of classical scrapie strains and the BSE strain, and was not distinguishable from the Nor98 glycoprofile. All these features corresponded very well with those reported of the unusual Nor98 strain detected in Norway (Benestad and others 1999, 2002, 2003, Bratberg and others 2003). This raises questions concerning the scrapie active epidemiosurveillance protocol because, from a diagnostic point of view, the positive results obtained with a rapid test require confirmation by standard methods such as histopathological examination and the immunohistochemical detection of PrPSc at the level of the obex. In the present case, the confirmation tests (histology, immunohistochemistry and detections of SAFs), which are officially recognised in Belgium, were all negative. Only Western blotting was positive for scrapie; thus, the diagnosis of scrapie could have been overlooked. This may be of significance for future sampling in scrapie surveillance programmes and confirmation tests.

Decision support tools for clinical diagnosis of disease in cows with suspected bovine spongiform encephalopathy.

ABSTRACT Reporting of clinically suspected cattle is currently the most common method for detecting cases of bovine spongiform encephalopathy (BSE). Improvement of clinical diagnosis and decision-making remains crucial. A comparison of clinical patterns, consisting of 25 signs, was made between all 30 BSE cases, confirmed in Belgium before October 2002, and 272 suspected cases that were subsequently determined to be histologically, immunohistochemically, and scrapie-associated-fiber negative. Seasonality in reporting suspected cases was observed, with more cases being reported during wintertime when animals were kept indoors. The median duration of illness was 30 days. The 10 most relevant signs of BSE were kicking in the milking parlor, hypersensitivity to touch and/or sound, head shyness, panic-stricken response, reluctance to enter in the milking parlor, abnormal ear movement or carriage, increased alertness behavior, reduced milk yield, teeth grinding, and temperament change. Ataxia did not appear to be a specific sign of BSE. A classification and regression tree was constructed by using the following four features: age of the animal, year of birth, number of relevant BSE signs noted, and number of clinical signs, typical for listeriosis, noted. The model had a sensitivity of 100% and a specificity of 85%. This approach allows the use of an interactive decision-support tool, based entirely on odds ratios, a statistic independent of disease prevalence.

Natural case of bovine herpesvirus 1 meningoencephalitis in an adult cow

Mim) and thinner (width 0-25 to 0-3 urm) than the cells of the other Brachyspira species, and have four to six fewer axial filaments (Stanton and others 1997, Trivett-Moore and others 1998). Stanton and others (1997) found it difficult to distinguish between the cells ofB hyodysenteriae, Brachyspira intermedia and Brachyspira murdochii, based on cell dimensions, cell morphology and numbers of axial filaments. The histological changes of Brachyspira species infection in the cow in the present study were broadly similar to those described in swine dysentery and porcine colonic spirochaetosis, with the exception of the previously described end-on attachment of B pilosicoli to the surface epithelium (Taylor and others 1980, Thomson and others 1996). Immunohistochemically, the organisms were stained positively by polyclonal antisera against B hyodysenteriae and B pilosicoli, but because of the low specificity of those antisera, immunohistochemical examination can only be used to identify Brachyspira species. In swine dysentery, the lesions are restricted to the colon, caecum, and rectum, but they are most constant and severe in the spiral colon; however, the caecum and the proximal colonic mucosa were severely affected with the spirochaetes in this case. These differences in the lesions and organism predilections among studies were partly attributed to the differences in the organism species and host species. Taking these results and the pathology of swine dysentery, this dairy cow case is the counterpart of swine dysentery. Since attempts to isolate the spirochaetes in this case failed, the exact identity of the spirochaetes is still unknown. Further studies with different media and culture conditions will be necessary to try and isolate spirochaetes from cattle with dysentery.

M2e-displaying virus-like particles with associated RNA promote T helper 1 type adaptive immunity against influenza A.

The ectodomain of influenza A matrix protein 2 (M2e) is a candidate for a universal influenza A vaccine. We used recombinant Hepatitis B core antigen to produce virus-like particles presenting M2e (M2e-VLPs). We produced the VLPs with and without entrapped nucleic acids and compared their immunogenicity and protective efficacy. Immunization of BALB/c mice with M2e-VLPs containing nucleic acids induced a stronger, Th1-biased antibody response compared to particles lacking nucleic acids. The former also induced a stronger M2e-specific CD4+ T cell response, as determined by ELISPOT. Mice vaccinated with alum-adjuvanted M2e-VLPs containing the nucleic acid-binding domain were better protected against influenza A virus challenge than mice vaccinated with similar particles lacking this domain, as deduced from the loss in body weight following challenge with X47 (H3N2) or PR/8 virus. Challenge of mice that had been immunized with M2e-VLPs with or without nucleic acids displayed significantly lower mortality, morbidity and lung virus titers than control-immunized groups. We conclude that nucleic acids present in M2e-VLPs correlate with improved immune protection.

DNA Immunization with Plasmids Encoding Fusion and Nucleocapsid Proteins of Bovine Respiratory Syncytial Virus Induces a Strong Cell-Mediated Immunity and Protects Calves against Challenge

ABSTRACT Respiratory syncytial viruses (RSV) are one of the most important respiratory pathogens of humans and cattle, and there is currently no safe and effective vaccine prophylaxis. In this study, we designed two codon-optimized plasmids encoding the bovine RSV fusion (F) and nucleocapsid (N) proteins and assessed their immunogenicity in young calves. Two administrations of both plasmids elicited low antibody levels but primed a strong cell-mediated immunity characterized by lymphoproliferative response and gamma interferon production in vitro and in vivo. Interestingly, this strong cellular response drastically reduced viral replication, clinical signs, and pulmonary lesions after a highly virulent challenge. Moreover, calves that were further vaccinated with a killed-virus vaccine developed high levels of neutralizing antibody and were fully protected following challenge. These results indicate that DNA vaccination could be a promising alternative to the classical vaccines against RSV in cattle and could therefore open perspectives for vaccinating young infants.

Vaccination of mice using the West Nile virus E-protein in a DNA prime-protein boost strategy stimulates cell-mediated immunity and protects mice against a lethal challenge.

West Nile virus (WNV) is a mosquito-borne flavivirus that is endemic in Africa, the Middle East, Europe and the United States. There is currently no antiviral treatment or human vaccine available to treat or prevent WNV infection. DNA plasmid-based vaccines represent a new approach for controlling infectious diseases. In rodents, DNA vaccines have been shown to induce B cell and cytotoxic T cell responses and protect against a wide range of infections. In this study, we formulated a plasmid DNA vector expressing the ectodomain of the E-protein of WNV into nanoparticles by using linear polyethyleneimine (lPEI) covalently bound to mannose and examined the potential of this vaccine to protect against lethal WNV infection in mice. Mice were immunized twice (prime – boost regime) with the WNV DNA vaccine formulated with lPEI-mannose using different administration routes (intramuscular, intradermal and topical). In parallel a heterologous boost with purified recombinant WNV envelope (E) protein was evaluated. While no significant E-protein specific humoral response was generated after DNA immunization, protein boosting of DNA-primed mice resulted in a marked increase in total neutralizing antibody titer. In addition, E-specific IL-4 T-cell immune responses were detected by ELISPOT after protein boost and CD8+ specific IFN-γ expression was observed by flow cytometry. Challenge experiments using the heterologous immunization regime revealed protective immunity to homologous and virulent WNV infection.

Trends in genotype frequency resulting from breeding for resistance to classical scrapie in Belgium (2006~2011)

In sheep, susceptibility to scrapie is mainly determined by codons 136, 154, and 171 of the PRNP gene. Five haplotypes are usually present (ARR, ARQ, ARH, AHQ, and VRQ). The ARR haplotype confers the greatest resistance to classical scrapie while VRQ renders animals most susceptible. In 2004, the European Union implemented a breeding program that promotes selection of the ARR haplotype while reducing the incidence of VRQ. From 2006 to 2011 in Belgium, frequency for the ARR/ARR genotypes increased from 38.3% to 63.8% (n = 6,437), the ARQ haplotype diminished from 21.1% to 12.9%, and the VRQ haplotype decreased from 2.0% to 1.7%. The status of codon 141, a determinant for atypical scrapie, was also evaluated. Out of 27 different breeds (n = 5,163), nine were abundant. The ARR/ARR frequency increased in eight of these nine major breeds. The selection program has had a major impact on the ARR haplotype frequency in Belgium. However, the occurrence of atypical scrapie represents a critical point for this program that warrants the continuous monitoring of scrapie. Additionally, genotype frequencies among the breeds varied greatly. Texel, a breed that is common in Belgium, can still be selected for due to its average ARR frequency.

Tick-Borne Encephalitis Virus Seropositive Dog Detected in Belgium: Screening of the Canine Population as Sentinels for Public Health

Tick-borne encephalitis virus (TBEV) is an important emerging tick-borne viral infection of humans and dogs in Europe. Currently, TBEV surveillance is virtually nonexistent in Belgium, which is considered nonendemic. A commercial enzyme-linked immunosorbent assay (ELISA) was adapted for the detection of TBEV-specific IgG-antibodies in canine sera. Serum samples of Belgian dogs were obtained from three diagnostic laboratories from Northern (n=688) and Southern Belgium (n=192). ELISA-positive and borderline samples were subjected to a TBEV rapid fluorescent focus inhibition confirmation test. One dog was confirmed TBEV seropositive. Several ELISA-positive and borderline sera underwent seroneutralization and hemagglutinin inhibition tests to rule out West Nile and Louping Ill viruses, but tested negative. The clinical history of the seropositive dog could not explain beyond doubt where and when TBEV infection was acquired. Further surveillance is necessary to determine whether this dog remains a single travel-related case or whether it represents an early warning of a possible future emergence of TBEV.

Sero-epidemiological study of the presence of hantaviruses in domestic dogs and cats from Belgium

Hantaviruses are worldwide rodent-borne pathogens infecting humans and other animals mainly through inhalation of aerosols contaminated with rodent excreta. Few data are available on hantavirus serology and geographical distribution in dogs and cats. We therefore screened sera from pet dogs (N=410) and cats (N=124) in two regions of Belgium, using IgG ELISA and IFA. We analysed the effect of the owners address as well as pet gender and age on hantavirus status. Hantavirus antibodies were found in both species with a significantly higher seroprevalence in cats than in dogs (16.9% vs. 4.9%, P=0.001). More dogs were infected in highly forested southern Belgium (harbouring more rodents) than in northern Belgium (10.5% vs. 3.0%, P=0.002). In the south, hantavirus sero-positive cats were found in more densely forested localities than sero-negatives ones were (P=0.033). These results are consistent with the ecological variations of hantavirus risks in humans.

Spatiotemporal dynamics of Puumala hantavirus in suburban reservoir rodent populations.

ABSTRACT: The transmission of pathogens to susceptible hosts is dependent on the vector population dynamics. In Europe, bank voles (Myodes glareolus) carry Puumala hantavirus, which causes nephropathia epidemica (NE) in humans. Fluctuations in bank vole populations and epidemics in humans are correlated but the main factors influencing this relationship remain unclear. In Belgium, more NE cases are reported in spring than in autumn. There is also a higher incidence of human infections during years of large vole populations. This study aimed to better understand the link between virus prevalence in the vector, vole demography, habitat quality, and human infections. Three rodent populations in different habitats bordering Brussels city, Belgium, were studied for two years. The seroprevalence in voles was influenced first by season (higher in spring), then by vole density, vole weight (a proxy for age), and capture site but not by year or sex. Moreover, voles with large maximal distance between two captures had a high probability for Puumala seropositivity. Additionally, the local vole density showed similar temporal variations as the number of NE cases in Belgium. These results showed that, while season was the main factor influencing vole seroprevalence, it was not sufficient to explain human risks. Indeed, vole density and weight, as well as the local habitat, were essential to understanding the interactions in these host-pathogen dynamics. This can, in turn, be of importance for assessing the human risks.