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To detect Schmallenberg virus (SBV) infections in ruminants and to perform SBV epidemiological studies a cost-effective serological test is required. For these purposes an indirect whole virus Enzyme-linked Immunosorbent Assay (ELISA) for detection of SBV specific antibodies in ruminant blood samples was developed. Schmallenberg virus antigen was produced by propagation on Vero cells, partly purified and coated onto ELISA plates. The indirect ELISA procedure included the subsequent incubation of diluted samples, protein-G-HRP conjugate and TMB substrate solution. Net Optical Densities (OD) values were calculated and expressed as a sample to positive percentage (S/P%) by comparison of the average net OD with the OD of the positive control. Validation of this assay was performed using 633 samples from SBV-free sheep, goats and cattle, and 141 samples from SBV suspect ruminants. The diagnostic specificity was 98.8%. Test results of 86 ruminant serum samples using both the SBV-ELISA and an SBV virus neutralization test (VNT), designated as the gold standard serological test for SBV, showed good correlation: at an S/P cut-off of 15% only one VNT positive sample tested negative in the SBV ELISA. The diagnostic sensitivity of the ELISA, relative to the VNT, was 98.8% (95% CI: 93.3-100.0%). The ELISA showed a high repeatability (cv=6.5%) and reproducibility (100% agreement). It was concluded that this ELISA is a suitable test method for the detection of SBV antibodies in sera from cows, sheep and, possibly, goats.

Guillain-Barr, syndrome (GBS) is a post-infectious disease in which the human peripheral nervous system is affected after infection by specific pathogenic bacteria, including Campylobacter jejuni. GBS is suggested to be provoked by molecular mimicry between sialylated lipooligosaccharide (LOS) structures on the cell envelope of these bacteria and ganglioside epitopes on the human peripheral nerves, resulting in autoimmune-driven nerve destruction. Earlier, the C. jejuni sialyltransferase (Cst-II) was found to be linked to GBS and demonstrated to be involved in the biosynthesis of the ganglioside-like LOS structures. Apart from a role in pathogenicity, we report here that Cst-II-generated ganglioside-like LOS structures confer efficient bacteriophage resistance in C. jejuni. By bioinformatic analysis, it is revealed that the presence of sialyltransferases in C. jejuni and other potential GBS-related pathogens correlated significantly with the apparent degeneration of an alternative anti-virus system: type II Clusters of Regularly Interspaced Short Palindromic Repeat and associated genes (CRISPR-Cas). Molecular analysis of the C. jejuni CRISPR-Cas system confirmed the bioinformatic investigation. CRISPR degeneration and mutations in the cas genes cas2, cas1 and csn1 were found to correlate with Cst-II sialyltransferase presence (p < 0.0001). Remarkably, type II CRISPR-Cas systems are mainly found in mammalian pathogens. To study the potential involvement of this system in pathogenicity, we inactivated the type II CRISPR-Cas marker gene csn1, which effectively reduced virulence in primarily cst-II-positive C. jejuni isolates. Our findings indicate a novel link between viral defence, virulence and GBS in a pathogenic bacterium.

Quinolone resistance is studied and reported increasingly in isolates from humans, food-producing animals and companion animals. Resistance can be caused by chromosomal mutations in topoisomerase genes, plasmid-mediated resistance genes, and active transport through efflux pumps. Cross sectional data on quinolone resistance mechanisms in non-pathogenic bacteria from healthy veal calves is limited. The purpose of this study was to determine the prevalence and characteristics of quinolone resistance mechanisms in Escherichia coli isolates from veal calves, after more than 20 years of quinolone usage in veal calves. MIC values were determined for all isolates collected as part of a national surveillance program on antimicrobial resistance in commensal bacteria in food-producing animals in The Netherlands. From the strains collected from veal calves in 2007 ( n = 175) all isolates with ciprofloxacin MIC ≥ 0.125 mg/L ( n = 25) were selected for this study, and screened for the presence of known quinolone resistance determinants. In this selection only chromosomal mutations in the topoisomerase type II and IV genes were detected. The number of mutations found per isolate correlated with an increasing ciprofloxacin MIC. No plasmid-mediated quinolone resistance genes were found. The contribution of efflux pumps varied from no contribution to a 16-fold increase in susceptibility. No correlation was found with the presence of resistance genes of other antimicrobial classes, even though all quinolone non-wild type isolates were resistant to 3 or more classes of antibiotics other than quinolones. Over twenty years of quinolone usage in veal calves in The Netherlands did not result in a widespread occurrence of plasmid-mediated quinolone resistance, limiting the transmission of quinolone resistance to clonal distribution.

BACKGROUND: Enterococcus hirae-associated endocarditis, characterized by a peak in mortality during the second week of the grow-out, and occasionally lameness, was diagnosed at Dutch broiler farms. OBJECTIVES: Field cases were studied to increase knowledge on clinical and pathological characteristics, pathogenesis and epidemiology of these infections. ANIMALS AND METHODS: In total, 1266 birds of 25 flocks from 12 farms were examined. Post-mortem examinations, bacteriology, histopathology, PCR and DNA fingerprinting was carried out. Six flocks were followed longitudinally (n = 1017 birds). RESULTS: Average mortality was 4.1% for the entire grow-out, of which 36% was attributed to endocarditis. Fibrinous thromboendocarditis of the right atrioventricular (AV) valve was found in 24% of hearts, compared to 7% and 4% with lesions of left and both AV valves, respectively. Thrombotic lesions were found in 24% (n = 432) of lungs, but only in larger branches of the Arteria pulmonalis. Occasionally, thrombi were found in the Arteria ischiadica externa and in liver and brain vessels. Enterococcus was cultured from 54% (n = 176) of heart and in 75% (n = 28), 62% (n = 106) and 31% (n = 16) of liver, bone marrow and lung samples, respectively. Further identification, using the Rapid ID Strep 32 API system and a PCR targeting mur-2 and mur-2(ed) genes was carried out on a subset of Enterococcus positive isolates (n = 65): both techniques identified the isolates as Enterococcus hirae. Pulsed-field gel electrophoresis did not indicate evidence of clonality between farms and flocks. CONCLUSIONS: The relevance of these findings for pathogenesis and epidemiology of E. hirae infections is discussed. CLINICAL IMPORTANCE. This study may facilitate diagnosis of field cases and may contribute to the design of further research and development of control measures.

Age at which cattle become faecal culture positive for Mycobacterium avium subsp. paratuberculosis (Map) can be used as a proxy parameter for age at onset of faecal shedding, which is an important parameter in the control of Map in cattle herds. To investigate the age at becoming faecal culture positive, survival analysis methods were applied. The analyses were carried out on asynchronous interval censored data of faecal culture results of samples collected from 18,979 female Holstein-Frisian cattle in 353 Dutch herds between 1996 and 2002. The data were analysed with a Weibull proportional hazards model. The results indicate that the distribution of age at onset of faecal shedding in Holstein-Frisian dairy cattle in infected herds is associated with the within-herd prevalence. In higher classes of apparent prevalence, cattle started to shed Map at younger age on average. In herds with an apparent prevalence

SUMMARYCampylobacteris a major cause of acute bacterial diarrhea in humans worldwide. This study was aimed at summarizing the current understanding of host mechanisms involved in the defense againstCampylobacterby evaluating data available from three sources: (i) epidemiological observations, (ii) observations of patients, and (iii) experimental observations including observations of animal models and human volunteer studies. Analysis of available data clearly indicates that an effective immune system is crucial for the host defense againstCampylobacterinfection. Innate, cell-mediated, and humoral immune responses are induced duringCampylobacterinfection, but the relative importance of these mechanisms in conferring protective immunity against reinfection is unclear. Frequent exposure toCampylobacterdoes lead to the induction of short-term protection against disease but most probably not against colonization. Recent progress in the development of more suitable animal models for studyingCampylobacterinfection has opened up possibilities to study the importance of innate and adaptive immunity during infection and in protection against reinfection. In addition, advances in genomics and proteomics technologies will enable more detailed molecular studies. Such studies combined with better integration of host and pathogen research driven by epidemiological findings may truly advance our understanding ofCampylobacterinfection in humans.

Recombinant form of Haemonchus contortus aminopeptidase H11, an intestinal membrane glycoprotein considered to be in its native form the most promising vaccine candidate, was produced in insect cells, characterised and tested in pilot vaccination-challenge trial on sheep. The sequence of the cloned gene, obtained by RT PCR isolated from adult worms, showed 97% identity to the highly immunogenic H11 clone, described by Graham et al., (database accession number AJ249941.1). A 1305 bp fragment of H11 was expressed in E. coli and used to raise a specific antiserum, which recognized recombinant forms of H11 and 110 kDa protein from H. contortus extract. H11 was expressed by baculovirus recombinants in insect cells in full length and as a fusion protein with H. contortus glutathione S-transferase (GST). The baculovirus produced recombinant antigens were used without adjuvants to immunize sheep, which resulted in 30% (full length H11) and 20% (GST-H11) reduction of worm burden. These animal experiments indicated that, although the protection induced by in vitro produced protein is lower than in case of H11 isolated from worms, recombinant forms of aminopeptidase may be considered as antigens for the control of haemonchosis.

Transmission of an infectious agent can be quantified from experimental data using the transient-state (TS) algorithm. The TS algorithm is based on the stochastic SIR model and provides a time-dependent probability distribution over the number of infected individuals during an epidemic, with no need for the experiment to end in final-size (e.g., where no more infections can occur). Because of numerical limitations, the application of the TS algorithm is limited to populations with only a few individuals. We investigated the error of using the easily applicable, time-independent final-size (FS) algorithm knowing that the FS situation was not reached. We conclude that the methods based on the FS algorithm: (i) underestimate R(0), (ii) are liberal when testing H(0):R(0)1 against H(1):R(0)1, (iii) are conservative when testing H(0):R(0)1 against H(1):R(0)1, and (iv) are conservative when testing H(0):R(control)=R(treatment) against H(1):R(control)R(treatment). Furthermore, a new method is presented to find a difference in transmission between two treatment groups (MaxDiff test). The MaxDiff test is compared to tests based on FS and TS algorithms. The TS test and the MaxDiff test were most powerful (approximately equally powerful) in finding a difference, whereas the FS test was less powerful (especially, when both R(control) and R(treatment) are1).

The Gram-negative bacterium Campylobacter is the leading cause of bacterial entero-colitis in humans and is associated with the occurrence of life-threatening auto-immune based neurological disorders. Chickens, which are often heavily colonized with Campylobacter without signs of pathology, are considered the most important source for human infection. Although vaccination is a well established and effective method to combat various microbes in poultry, a commercial vaccine against Campylobacter has not yet been developed. For the development of such a vaccine, three main challenges can be identified: (1) the identification of novel cross-protection-inducing antigens, (2) the induction of a rapid, potent immune response, and (3) the development of novel adjuvants to further stimulate immunity against Campylobacter. The rapidly emerging knowledge of the biology of Campylobacter in combination with the recent advances in the fields of molecular vaccinology and immunology provide the required setting for the development of an effective vaccine against Campylobacter in poultry.

Campylobacter bacteria are an important cause of foodborne infections. We estimated the potential costs and benefits of a large number of possible interventions to decrease human exposure to Campylobacter by consumption of chicken meat, which accounts for 20-40% of all cases of human campylobacteriosis in the Netherlands. For this purpose, a farm-to-fork risk assessment model was combined with economic analysis and epidemiological data. Reduction of contamination at broiler farms could be efficient in theory. However, it is unclear which hygienic measures need to be taken and the costs can be very high. The experimental treatment of colonized broiler flocks with bacteriophages has proven to be effective and could also be cost efficient, if confirmed in practice. Since a major decrease of infections at the broiler farm is not expected in the short term, additional measures in the processing plant were also considered. At this moment, guaranteed Campylobacter-free chicken meat at the retail level is not realistic. The most promising interventions in the processing plant are limiting fecal leakage during processing and separation of contaminated and noncontaminated flocks (scheduling), followed by decontamination of the contaminated flock. New (faster and more sensitive) test methods to detect Campylobacter colonization in broilers flocks are a prerequisite for successful scheduling scenarios. Other methods to decrease the contamination of meat of colonized flocks such as freezing and heat treatment are more expensive and/or less effective than chemical decontamination.

The organisation of animal populations in social groupings may play a crucial role in the transmission of any infectious disease that requires close contact. The objective of this study was to quantify the contact structure of part of the Heck cattle population in a Dutch nature reserve and its hypothetical effect on the transmission of bovine herpesvirus 1 (BHV1). The contact structure was quantified by observing the number of different animals with whom contact was made (i.e. the number of contactees) within a fixed time period. Two types of behaviour sampling methods, namely focal sampling and scan sampling were used to observe the contact structure. In this study only those contacts between individuals were observed that were assumed to be a proxy measure of an at-risk event for BHV1-infection. Two reproduction ratios (R), i.e. the average number of new cases caused by a typical infected individual, were estimated, one for the observed contact structure and another for a random mixing contact structure. The two reproduction ratios were then compared to study the hypothetical effect on BHV1 transmission. The overall number of contactees was highest during summer and lowest during winter-spring. The contact structure of the homogeneous population did differ significantly from a random mixing contact structure, resulting in that the variation in the number of contactees was higher than under random mixing. Bulls, young bulls and cows had the highest number of contactees during, respectively, summer, autumn and winter-spring. From the analysis of the contingency tables it was clear that contacts between animal types did not occur at random during summer and autumn. For example, during summer more contacts than expected occurred between bulls and cows. This heterogeneity at animal type level was taken into account in the calculation for R, which resulted for the observed contact structure in higher estimates for R than for the homogeneous population. When looking at heterogeneity at individual level it was found that during summer almost all individuals were observed together direct or indirect in the same group except for certain bull groups. During autumn and winter-spring almost all individuals were seen together in the same group when considering a long contact period of 14 days but the groups were fallen apart in smaller groups and solitary individuals for a short contact period of 5 days. It could be concluded that based on the observed contact structure transmission would be favoured most during summer.

10 p.-4 fig.-1 tab., A novel vaccine against porcine parvovirus (PPV), composed of recombinant virus-like particles (PPV-VLPs) produced with the baculovirus expression vector system (BEVS) at industrial scale, was tested for its immunogenicity and protective potency. A formulation of submicrogram amounts of PPV-VLPs in a water-in-mineral oil adjuvant evoked high serum antibody titres in both guinea pigs, used as reference model, and target species, pigs. A single immunisation with 0.7microg of this antigen yielded complete foetal protection against PPV infection after challenge with a virulent strain of this virus. Furthermore, also in the presence of mild adjuvants the protective action of these PPV-VLPs is excellent. This recombinant subunit vaccine overcomes some of the drawbacks of classical PPV vaccines., The authors acknowledge and appreciate the financial support received for the project from the European Commission (BIO4-CT98-0215) and for a grant to L.M. from Fundaçao para a Ciência e Tecnologia – Portugal (PRAXIS ˆXXI/BD/16136/98)

Potential risk factors for clinical signs of post-weaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS) in pigs in the Netherlands were investigated in a matched case-control study using a questionnaire (personal interview). Eighty-two pig farmers were questioned about management, hygiene, husbandry systems, disease history, and preventive health care. In this study, 30 pig herds with (cases) and 30 pig herds without (controls) characteristic clinical signs of PMWS were compared. For PDNS, 11 pig herds with (cases) and 11 pig herds without (controls) characteristic clinical signs of PDNS were compared. Univariate analysis (P

In the present study, cross-protection to bovine herpesvirus type 5 (BHV-5) induced by bovine herpesvirus type 1 (BHV-1) vaccination was examined following inoculation of rabbits and calves with a glycoprotein E (gE)-negative BHV-1 vaccine and subsequent challenge with BHV-5. Rabbits (n=5) and calves (n=8) were vaccinated [five rabbits intranasally (IN), four calves IN and four intramuscularly (IM)] with 7.1 log(10)median tissue culture infective dose (TCID(50)) of the BHV-1 vaccine. Rabbits and calves were challenged IN [rabbits 2 weeks post-vaccination (pv); calves 5 weeks pv] with 9.1log(10)TCID(50) of BHV-5. Two out of five vaccinated rabbits died after challenge with typical BHV-5 disease, as did 3/5 non-vaccinated controls. In calves, 4/8 vaccinated animals displayed mild signs of disease, whereas 6/6 non-vaccinated controls developed signs of disease, so severe that 2/6 had to be killed. Besides, nasal virus shedding post-challenge was not reduced by vaccination. At necropsy, on day 21 post-challenge, typical BHV-5 lesions were evident in brain tissues of both vaccinated and non-vaccinated calves. Dexametasone administration at 180 days post-infection did not reactivate clinical signs despite BHV-5 shedding in nasal secretions of both vaccinated and non-vaccinated calves. These results show that the BHV-1 vaccine evaluated here did not confer protection to BHV-5 in rabbits. In calves, BHV-1 vaccination did confer some protection to BHV-5 induced clinical disease, but it did not prevent infection and had no effect on nasal virus shedding or on the development of encephalitic lesions.

This study compares the immune responses and protection induced by intra-typic heterologous vaccination with that induced by homologous vaccination against challenge with foot-and-mouth disease virus (FMDV). Humoral and cell-mediated immune responses and protection against challenge with FMDV O Taiwan were examined in a non-vaccinated group, a group vaccinated with O Taiwan FMD vaccine and a group vaccinated with O Manisa FMD vaccine. Five pigs from each group were challenged with FMDV type O Taiwan 14 days after vaccination and five other pigs were contact-exposed to the inoculated pigs. Both homologous and heterologous vaccination protected against challenge with FMDV O Taiwan at 2 weeks after vaccination. In the heterologous vaccinated group, cross-neutralizing antibody titres against O Taiwan could be detected although the ratio 'r(1)' was 0.4, which was significantly smaller than the critical r-value. Cell-mediated immune responses were detected after both homologous and heterologous vaccination. Virus-induced in vitro lymphocyte (cross-) proliferation and production of both a Th1-type (IFN-gamma) and a Th2-type (IL-10) cytokine response were demonstrated in cultures of peripheral blood mononuclear cells (PBMC). The findings show that heterologous (emergency) vaccination can prevent clinical disease and shedding of virus. The induction of the cell-mediated immune responses after (heterologous) vaccination needs more research but data on these responses might provide additional tools for both vaccine choice and vaccine development.

DNA vaccines are capable of priming the immune system of neonates in the presence of maternal antibodies. However, it is still not clear whether the extent of priming and protection against challenge infections induced by a DNA vaccine in maternally immune newborns is better than that induced by conventional vaccines. To study this, we used the pseudorabies virus (PRV) infection model in the natural host, the pig. We compared the efficacy of a DNA vaccine with the efficacy of a conventional modified live vaccine (MLV) and an inactivated vaccine (IV) in maternally immune newborn piglets. We measured the priming of the immune response and the degree of protection against challenge infection for all vaccine types. We vaccinated piglets with or without maternal immunity twice, at the age of 5 and 9 weeks, and we assessed protection by challenge infection with virulent PRV at the age of 15 weeks. Vaccination with DNA or conventional vaccines induced both humoral and cell-mediated immune responses in maternally immune animals. DNA vaccination seemed not to suffer from suppression by maternal immunity and resulted in similar or stronger immune responses in maternally immune piglets as compared in naïve piglets. In contrast, vaccination with conventional vaccines resulted in weaker immune responses in maternally immune piglets than in naïve piglets. Moreover, DNA vaccination provided better protection against challenge infection in maternally immune piglets than in naive piglets, whereas vaccination with conventional vaccines did not.

The aim of the study was: i) to specifically target tumor tissue by Newcastle disease virus (NDV) with oncolytic properties, ii) to improve the delivery system for systemic application of NDV via a bispecific adapter protein and iii) to investigate anti-tumor activity and side-effects. We selected two oncolytic virus strains, one native and the other recombinant, which showed multicyclic replication patterns in tumor cells. In order to reduce normal cell binding, they were modified by preincubation with a recombinant bispecific protein which blocks the viral native cell binding site and introduces a new binding site for a tumor-associated target (in this study, the interleukin-2-receptor, IL-2R). After intravenous transfer to mice, uptake of modified NDV in liver, spleen, kidney and lung was greatly reduced in comparison to unmodified NDV as determined by RRT-PCR of viral M gene copies. In IL-2R+ tumor bearing mice, the same assay revealed a high replication efficiency of the modified virus in the tumor tissue. Tumor therapy experiments showed that the side-effects induced by systemic application were greatly reduced by the adapter protein and that the anti-tumor effects were mostly undiminished. The demonstration of significant systemic anti-tumor activity of this viral vector suggests potential for augmentation by inclusion of one or more therapeutic genes.

To investigate tumor-selective viral replication, we compared several tumorigenic human cell lines to nontumorigenic human cells from the blood for the sensitivity to become infected by a recombinant lentogenic strain of Newcastle Disease Virus (NDV) with incorporated transgene EGFP (NDFL-EGFP). Although fluorescence signals in nontumorigenic cells were only weak or missing completely, a massive and long-lasting transgene-expression was observed in all tumor cell lines. The majority of tumor cells (50-95%) could be infected, and viral replication was associated with an increase in the cell surface density of viral antigens. To clarify the underlying mechanism of the observed difference in virus susceptibility we examined the kinetics of interferon-induced antiviral enzymes because NDV is a strong type-I interferon inducer. This analysis revealed several defects of tumor cells in their antiviral defence responses: They showed no response to UV-inactivated NDV, whereas nontumorigenic cells reacted with induction of high-levels of the antiviral enzymes PKR and MxA. Upon coincubation with live NDV, tumor cells showed a delayed response in the increased expression of the antiviral enzymes in comparison with PBMC. In nontumorigenic cells the replication cycle of NDV stopped after the production of positive-strand RNA, while tumor cells continued in the replication cycle and copied viral genomes 10-50 hr after infection. These results can explain the tumor selective replication behavior of this interesting antineoplastic virus.

Campylobacter fetus can be divided into the subspecies C. fetus subsp. fetus and C. fetus subsp. venerealis. C. fetus subsp. fetus causes sporadic infections in humans and abortion in cattle and sheep and has been isolated from a variety of sites in different hosts. C. fetus subsp. venerealis is host restricted, being isolated mainly from the genital tracts of cattle, and is the causative agent of bovine genital campylobacteriosis. Despite differences in niche preference, microbiological subspecies differentiation has proven difficult. Different typing methods divided C. fetus isolates into different subgroups, depending on the methods used. The relative value of these methods can be assessed by the evolutionary relationship of isolates belonging to the genus; therefore, we developed a multilocus sequence typing (MLST) scheme for C. fetus . This scheme was applied to 140 C. fetus isolates previously typed by amplified fragment length polymorphism (AFLP) analysis. A total of 14 different sequence types (STs) were identified, and these exhibited low levels of inter-ST genetic diversity, with only 22 variable sites in 3,312 nucleotides. These MLST data indicate that C. fetus is genetically homogeneous compared to the homogeneity of other Campylobacter species. The two C. fetus subspecies were extremely closely related genetically, but ST-4 was associated only with C. fetus subsp. venerealis , which represents a “bovine” clone. The C. fetus subsp. fetus isolates studied were more diverse in terms of their STs, and the STs correlated with epidemiological relationships. Congruence was observed among C. fetus subspecies, sap type, and ST; therefore, MLST confirms that mammalian C. fetus is genetically stable, probably as result of the introduction of a single ancestral clone into a mammalian niche.

Bovine herpesvirus 1 (BHV1) is endemically present in a cattle population that lives in a nature reserve in the Netherlands. Red deer ( Cervus elaphus ), living in the same nature reserve, can come into contact with the BHV1-infected cattle and could then become infected with BHV1. For the eradication of BHV1 in cattle, it is, therefore, important to know whether red deer alone can play a role in the transmission of BHV1. For that reason, we quantified the transmission of BHV1 among farmed red deer under experimental conditions. Two groups of ten animals were formed. In each group, five of these animals were inoculated with BHV1 and the other five served as contact animals. Three inoculated animals in each transmission experiment became infected and none of the contact animals became infected. The one-sided 95% confidence interval for R [0.0–0.94] showed that limited transmission might occur among red deer. Based on these results, we would expect only minor outbreaks of BHV1 to occur in red deer populations. We concluded that BHV1 will probably not survive longer than a few decades (several times the mean deer lifetime) in red deer populations. Consequently, it is not necessary for the eradication of BHV1 in cattle to eradicate BHV1 in red deer populations as well.

Vaccination of pigs againstClassical swine fever virus(CSFV) by using live-virus vaccines induces early protection before detectable humoral immune responses. Immunological analyses indicate that this is associated with T-cell activation, underlining the importance of targeting cytotoxic T-lymphocyte (CTL) responses for vaccine improvement. Antigen-presenting cells (APCs) transfected with mRNA encoding structural protein E2 or non-structural viral proteins NS3–NS4A were used to identify viral genes encoding CTL epitopes. Monocyte-derived dendritic cells (DCs) and fibrocytes served as the APCs.In vitrotranslation of the mRNA and microscopic analysis of transfected cells demonstrated that E2 and NS3–NS4A could be identified. APCs transfected with either of the mRNA molecules restimulated CSFV-specific T cells to produce gamma interferon and specific cytotoxic activity against CSFV-infected target cells. The presence of CTL epitopes on E2 was confirmed by using d/d-haplotype MAX cells expressing E2 constitutively as target cells in d/d-haplotype CTL assays. A potent CTL activity against E2 was detected early (1–3 weeks) after CSFV challenge. This work corroborates the existence of CTL epitopes within the non-structural protein domain NS3–NS4A of CSFV. Furthermore, epitopes on the E2 protein can also now be classified as targets for CTLs, having important implications for vaccine design, especially subunit vaccines. As for the use of mRNA-transfected APCs, this represents a simple and efficient method to identify viral genes encoding CTL epitopes in outbred populations.

Brown rot (Ralstonia solanacearum) comprises a major threat to the Dutch potato production chain. Eradication of the disease has not been achieved thus far, due to insufficient knowledge of the relative importance of possible risk factors with respect to brown rot prevalence and dispersal in the potato production chain. To study the relationship between brown rot infections in potatoes and possible risk factors, we evaluated two epidemiological models, i.e. a compartmental state-variable model, and a spatial individual-based model (IBM). Our approaches differ from most existing ecological applications of the two modelling techniques in that they focus on disease epidemiology within the industrially defined dynamics of the brown rot pathogen in the potato production chain. The state-variable model proved useful for obtaining insight into the basic principles of brown rot dispersal. It showed that the dynamics of the fraction of infected seed lots in the total potato lot population forms the key to a general understanding of brown rot epidemics. However, this model was unable to reflect the large fluctuation in yearly number of infections that is inherent to brown rot epidemics. To give a more detailed and realistic representation of the fraction of infected seed lots, a conceptual IBM was developed. As in this IBM a specific location is assigned to each individual potato lot, it becomes straightforward to include spatial heterogeneities based on detailed data on the potato production sector. In contrast to the state-variable model, the IBM enables us to study the effects of specific brown rot control policies in spatially defined areas. Moreover, the inherent high level of detail makes the IBM a convenient technique for policy application. The IBM will be further developed and extended to a bio-economic model for application in brown rot control strategy analysis.

The capacity of farm animals to produce cytokines could be an important determinant of robustness and health. From research in rodents and humans it appears that the production and the balance of T helper 1 (Th1) and T helper 2 (Th2)-type cytokines influences susceptibility to autoimmune and infectious diseases. It is known that pigs show a large variation in many immune response parameters. So far the extent of individual variation in the production of Th1- and Th2-type cytokines in commercial outbred pigs has not been reported. In the current experiment we determined mRNA expression, as well as protein production of cytokines in 32 pigs from eight litters. From each litter two male and two female pigs were tested at 2, 5 and 8 weeks of age. Two Th1-type cytokines, interleukin (IL)-2 and interferon (IFN)-gamma, and two Th2-type cytokines, IL-4 and IL-10, were measured after phytohaemagglutinin (PHA)-stimulation of blood mononuclear cells. Cytokine production and the Th1/Th2-ratio were highly variable. The variation in cytokine protein production was moderately consistent across ages, i.e. pigs that produced high levels of cytokine at 2 weeks of age tended to do so as well at 5 and 8 weeks of age. Cytokine production tended to increase with age, and gilts and boars differed in their IL-2/IL-4 ratio. Unexpectedly, age, gender and litter effects often differed for mRNA and protein production data. We hypothesize that cytokine production is a consistent trait in pigs, especially at the protein production level. Future investigations in more animals and across a wider age range are necessary.

The prevalence of gastrointestinal parasites in suckling piglets, weaners, fattening pigs and sows was studied from November 2001 to October 2002 on 16 free-range farms (FRF), 11 organic farms (OF) and 9 conventional farms (CF) by means of faecal examinations of composite samples. Each farm was visited four times with a 3-month interval. Infections with coccidia were found on 43.8% of the FRF, 90.9% of the OF and 66.7% of the CF. Sows had the highest prevalence, particularly on FRF (87.5%) and OF (80%). Ascaris suum was present on 50% of the FRF, 72.7% of the OF and 11.1% of the CF, whereas fattening pigs on FRF (42.9%) and OF (54.5%) had the highest prevalence. Oesophagostomum spp. were observed on 25% of the FRF, 27.2% of the OF and 22.2% of the CF. The infection was most prevalent in the sows on all farm types: 37.5% on FRF, 30% on OF and 22.2% on CF.Trichuris suis was found on 37.5% of the FRF, 36.4% of the OF and 11.1% of the CF. Again, this infection was most prevalent in the sows, particularly on the FRF (50%) and OF (30%). No other gastrointestinal parasite species were found and no clinical signs were observed. No seasonal trends could be distinguished. In many cases, when an age group on a farm was positive for a certain parasite, it remained so during the whole study. This indicates that this parasite was really 'endemic' on that farm. The results of this study demonstrate that the prevalence of helminth infections of pigs on farms with outdoor facilities is higher than in pigs on conventional farms.

The pestivirus glycoprotein E(rns), a ribonuclease, is expressed on the surface of virions and in infected cells as a disulfide-linked homodimer. E(rns) is involved in the infection process and its RNase activity is probably involved in viral replication and pathogenesis. The most C-terminal cysteine residue forms an intermolecular disulfide bond with another E(rns) monomer, resulting in an E(rns) dimer. To study the function of dimerisation of E(rns) for viral replication, the cysteine residue at amino acid position 438 was mutated into a serine residue. The mutated C438S gene was cloned into a vector containing an infectious cDNA copy of the CSFV C-strain genome. Using reverse genetics, a mutant virus was generated that only expressed monomeric E(rns), confirming that Cys 438 is essential for homo-dimerization. Characterization of this mutant virus and of a baculovirus-expressed C438S mutant protein indicated that the loss of the dimeric state of E(rns) reduced the affinity of binding of virions and E(rns) to heparan sulphate (HS), the receptor for E(rns) on the cell surface of SK6 cells. This suggests that interaction of virus-bound E(rns) homodimers with membrane associated HS may be a joined action of the two HS-binding domains (one in each monomer) present in the homodimer.

Despite the recognized stability of rabies virus, differences among isolates from different species have been found. This work was carried out with the aim to identify antigenic and genomic differences in Brazilian rabies virus isolates and to verify whether such alterations would bear any relationship with the different hosts for the virus in nature. For that, 79 Brazilian rabies viruses isolated from different host species and from distinct regions within Brazil were submitted to antigenic characterization with a panel of 11 monoclonal antibodies (Mabs) directed to lyssavirus antigens and to genomic analyses by the reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of the N gene followed by restriction endonuclease analysis (REA). In addition, the nucleotide sequences of part of the N gene (225 bp) of seven isolates, taken as representative of the majority of the viruses under study, were determined. The analyses with the Mabs and RT-PCR/REA allowed the identification of two major groups of variants, the first formed by most isolates of cattle and bats and the second formed by viruses of dog origin. Partial sequencing of the N gene confirmed the similarity among isolates from cattle origin and those of vampire bats. However, viruses from non-haematophagous bats exhibited consistent differences from those of vampire bat isolates. Such findings suggest that the variants have evolved fairly stable modifications, which are not altered after passage in a dead-end host of a distinct species. No association could be established between antigenic or genomic alterations and geographic distribution of the isolates, which suggests that evolution of the virus has been directed to adaptation to the host species.

We constructed a stochastic bio-economic model to determine the optimal cost-efficient surveillance program for bovine tuberculosis. The surveillance programs differed in combinations of one or more detection methods and/or sampling frequency. Stochastic input variables in the epidemiological module described the dynamics of infection and the probability of detection. By means of an efficiency frontier, the trade-off between the expected cost and the epidemiological risk parameter relating to the outbreak size was evaluated. The surveillance scheme based on visual inspection of lesions on carcasses at slaughter was optimal given the current prevalence of the disease in the Netherlands if the objective was to minimise the expected costs. However, the efficient set also included two other schemes: slaughterhouse inspection in combination with GAMMA-interferon testing of blood samples and slaughterhouse inspection in combination with two-stage tuberculin testing. The choice ultimately will depend on the risk attitude of the decision-maker; a more-stringent surveillance scheme will be enforced if the expected outbreak size is to be constrained. In future scenarios, ELISA testing of bulk-tank milk in combination with the current slaughterhouse inspection procedure would outperform the surveillance scheme of solely slaughterhouse inspection if ELISA testing of bulk-tank milk becomes feasible.

A multi-sulfonamide biosensor immunoassay (BIA), based on a previously developed mutant antibody (A.3.5) in an optical biosensor (Biacore 3000), was applied to analyse the serum and plasma samples obtained from the broilers treated with sulfamethoxazole and sulfadiazine. The assay was fast (5 min per sample), the sample preparation easy (dilution in antibody containing buffer only) and an equal sensitivity for the two sulfonamides was obtained with limits of detection (LOD) in serum and plasma below 10 ng ml −1 . The concentrations found with the BIA in serum and plasma of the treated broilers were comparable and higher than the concentrations found in the tissue by LC–MS/MS. The average serum/tissue ratios for sulfamethoxazole were 6.2 (leg meat), 2.5 (liver) and 1.3 (skin + fat) and for sulfadiazine 8.7 (leg meat), 3.1 (liver) and 2.2 (skin + fat). To predict the concentrations of the two sulfonamides below the maximum residue limit (MRL) of 100 ng g −1 in the tissue with the highest level (skin + fat), the proposed action level of the multi-sulfonamide BIA in serum is 130 ng ml −1 . A later developed mutant antibody (M.3.4), with a better sensitivity towards more sulfonamides, was applied during a survey. Serum samples ( n = 300) of broilers from 30 different flocks were found negative. Concentrations between −1 (sulfamethoxazole equivalents) were found in the serum samples of one flock ( n = 160) with an average of 25 ± 21 ng ml −1 . The sulfonamide identified by LC–MS/MS in these samples was sulfamethoxazole.

Much interest exists presently in development of vectors for gene therapy of tumors based on RNA viruses because these viruses replicate in the cytoplasm and do not integrate into DNA. The negative stranded paramyxovirus, Newcastle Disease Virus (NDV) from chicken has the additional advantages of preferential replication in tumor cells and of oncolytic and immunostimulatory properties. We here describe the bispecific fusion protein alphaHN-IL-2 which binds to NDV, inhibits its normal cell binding property and introduces a new binding specificity for the interleukin-2 receptor (IL-2R). We demonstrate selective gene transfer to tumor cells expressing IL-2R via the bispecific fusion protein when using recombinant NDV carrying as marker gene the enhanced green fluorescence protein (NDFL-EGFP). Hemadsorption (HA) and neuraminidase activities (NA) of the HN protein of NDV were shown to be blocked by alphaHN-IL-2 simultaneously and the absence of HA-activity of modified NDV was confirmed in vivo. Retargeted virus-binding to IL-2R positive tumor cells was not sufficient for the process of cellular infection. It required in addition membrane fusion via the viral F-protein. By modification of recombinant NDV with a bispecific molecule, our results demonstrate a novel and safe strategy for selective gene transfer to targeted tumor cells

Herpesviruses can remain dormant in once-infected hosts and, upon reactivation, cause such hosts to become infectious. This phenomenon of latency and reactivation may enable herpesviruses to persist for a long time in small host populations. To quantify the effect of reactivation on persistence, the time to extinction of bovine herpesvirus type 1 (BHV-1) in small cattle populations was calculated. For realistic parameter values the mean time to extinction is already more than 100 years in a population of 10 animals. In a population of 20 animals the time to extinction is approximately 2000 years. The effects of vaccination on persistence were also studied, revealing that continued vaccination of the whole population could result in much faster eradication. For instance, in an isolated herd of 20 animals BHV-1 could be eradicated in 44 years.

The herd sensitivity (HSe) and herd specificity (Hsp) of clinical diagnosis of an infection with classical swine fever (CSF) virus during veterinary inspection of breeding sows in a herd was evaluated. Data gathered from visits to herds during the CSF outbreak in 1997¿1998 in The Netherlands were used for the analysis. Herds were visited one or more times by the same or by different veterinarians. On the basis of the veterinarians¿ reports, each visit was coded as 0 (negative clinical diagnosis) or 1 (positive clinical diagnosis). The HSe for clinical diagnosis of CSF was modelled as a function of days elapsed since introduction of the virus. The moment of introduction of the CSF virus in the CSF-positive herds was unknown, so for each herd, a probability distribution for the unknown number of days since introduction was derived from serum samples collected at depopulation. The information from the reports of the veterinarians and from the test results of the serum samples at depopulation was combined in a Bayesian analysis. Data from CSF-negative herds were analysed to estimate HSp of clinical diagnosis of CSF. The HSe of clinical diagnosis was 0.5 at 37 days after virus introduction (95% CI: 31, 45) and reached 0.9 at 47 days after virus introduction (95% CI: 41, 54). The estimated herd specificity was 0.72 (95% CI: 0.64, 0.79). Dependence of HSe and HSp on characteristics of the veterinarians and the herds also was studied. Specialisation of the veterinarian significantly, although not markedly, affected the HSe.

Pseudorabies or Aujeszky's disease (AD), caused by pseudorabies virus (PRV) is a major concern in swine production. In the state of Rio Grande do Sul, Brazil, AD was only detected in 1954, in cattle. In 2003 two outbreaks of encephalitis occurred on the northern region of the state, close to the border with the state of Santa Catarina. Pseudorabies virus (PRV) was isolated from distinct farms within the region and subjected to antigenic and genomic analyses. These isolates were compared with prototype strains NIA-3 and NP. Antigenic characterization with a panel of monoclonal antibodies (Mabs) directed to viral glycoproteins (gB, gC, gD and gE-,) was performed by an imunoperoxidase monolayer assay (IPMA) on infected cell monolayers. Genomic characterization was carried out by restriction enzyme analysis (REA) of the whole DNA viral genome with Bam HI. The antigenic profile of the eight isolates from Rio Grande do Sul as well as strains NIA-3 and NP were similar. REA analysis revealed that all isolates from Rio Grande do Sul displayed a genomic type II arrangement, a genotype often found in other outbreaks of AD previously reported in other Brazilian states. The results obtained suggest that the eight isolates examined here were similar.

Mycoplasma gallisepticum (MG) and M. synoviae (MS) are the cause of considerable economic losses in the poultry industry. Molecular differentiation of avian Mycoplasma strains may be helpful in tracing infections and in the evaluation of implemented intervention strategies. Amplified Fragment Length Polymorphism (AFLP) has shown to be a powerful typing technique but the application for poultry Mycoplasma strains is very limited. The aim of this study was to evaluate the reproducibility and discriminatory power of AFLP HindIII/HhaI and AFLP BglII/Mfel for the inter- and intraspecies differentiation of avian mycoplasmas and to compare these test characteristics with digitalized Random Amplified Polymorphic DNA (RAPD) analysis. The reproducibility of RAPD, AFLP HindIII/HhaI and AFLP BglII/Mfel was 50-100, 97-98 and 86-99%, respectively. RAPD and both AFLP enzyme combinations were able to differentiate between five avian Mycoplasma species. For AFLP, five MG and four MS clusters could be identified. The phylogenetic tree for both enzyme combinations was comparable. For RAPD, four MG clusters could be identified. For MS, however, due to the poor reproducibility of the RAPD technique, no clear genogroups could be identified. On basis of the results of this study it can be concluded that AFLP is a powerful technique for the genotyping of avian mycoplasmas and that, although AFLP HindIII/HhaI generated patterns with less fragments, the final results showed homologous results.

Aims: To determine whether an association exists between partial depopulation of a flock and increased Campylobacter colonization in that flock. Methods and Results: Data from 1737 flocks of two Dutch integrators were used. Flocks that experienced partial depopulation were defined as 'exposed' and those that did not as 'nonexposed'. Multivariable modelling was accomplished with, in addition to 'exposure', the independent variables 'age of broilers at slaughter' and 'season' to adjust for possible confounding. The response variable was 'Campylobacter colonization'. The odds ratio (OR) for partial depopulation for integrator A was 0·8 [95% CI (0·4, 1·8)]; for integrator B the OR = 0·8 [95% CI (0·5, 1·3)]. Age and season were confounders: the difference in Campylobacter status between exposed and nonexposed flocks of integrator A could be explained by both variables; for integrator B, only season was associated with Campylobacter status. Conclusions: We found no significant association between partial depopulation and an increased risk of Campylobacter colonization among broiler flocks at final depopulation. Significance and Impact of the Study: This study demonstrates that Campylobacter colonization in a broiler flock is not influenced by the partial depopulation of that flock

Bovine genital campylobacteriosis caused by Campylobacter fetus subsp. venerealis (Cfv) is a genital infection that threatens the cattle industry. Detection and identification of Cfv are key factors in control programmes. Trade regulations should be based on scientifically and internationally accepted methods of detection and identification of Cfv. Such methods are described in the World Health (OIE) Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. A study was conducted to determine which methods are in use in OIE Member Countries and to get an overview of new or improved tests. A questionnaire was sent to OIE Member Countries, and 26 out of 166 were returned. Globally, a diversity of methods for the detection and identification of Cfv are in use. The authors conclude that there is a lack of harmonisation that may have consequences for the description of the health status of countries and may lead to disputes with respect to trade regulations

The study was aimed to quantify infections with nematodes of the genus Amidostomum in wild ducks of north-western Poland. The birds examined (282 individuals re-presenting 16 species) belonged to three, ecologically different, tribes: the Anatini, Aythyini and Mergini. The 923 individuals of Amidostomum found (A. acutum, A. orientale, and Amidostomum sp.) accounted for 29.1 % of all the nematodes identified in the ducks and occurred in 57.8 % of the birds at an average intensity of 5.7 per infested duck. Of all the duck groups examined, the heaviest exposure was found in the adult male Mergini (an average infestation intensity of 15.1 and 12.3 parasites per infested duck and examined duck, respectively)

Faeces of 484 horses were sampled twice with an interval of 6 weeks while anthelmintic therapy was halted. Faecal eggs counts revealed that 267 (55.2%) horses had consistently low numbers of eggs per gram faeces (EPG) (EPG 100). Horses with consistently high EPGs were more often mares with access to pasture, aged less than 6 or more than 23 years, that were dewormed at intervals longer than 6 months, and were treated for the last time more than 3 months before the start of the study. Horses with consistently low EPGs were more often male horses with no or limited access to pasture, that were dewormed at maximally 6-month intervals, and were aged between 6 and 23 years. The results are an indication that some horses have consistently low EPGs and perhaps could be used as non-treated animals in a selective anthelmintic treatment scheme aimed at the prevention of the development of anthelmintic resistance.

The objective of this study was to develop quantitative real-time polymerase chain reaction (ReTi-PCR) tests for the detection of five economically important viruses in swine semen namely, pseudorabies virus (PRV), classical swine fever virus (CSFV), foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), and porcine reproductive and respiratory syndrome virus (PRRSV). Each ReTi-PCR test was validated for specificity, analytical sensitivity (detection limits), and experimental infection studies were performed to compare the conventional virus isolation methods with the newly developed ReTi-PCR tests. All five developed ReTi-PCR tests are very rapid compared to virus isolation, highly specific, and even more sensitive (lower detection limits) than conventional virus isolation methods for the detection of mentioned viruses in semen. In semen of experimentally infected boars, viruses were detected much earlier after infection and more frequently by ReTi-PCR tests than by virus isolations. The high throughput of these rapid ReTi-PCR tests makes it possible to screen large number of semen samples for the presence of viruses prior to insemination. This is a substantial advantage, in particular for boar semen the quality of which deteriorates quickly after storage. In general, the newly developed ReTi-PCR tests are valuable tools for the early, reliable and rapid detection of five economically important viruses, namely PRV, CSFV, FMDV, SVDV, and PRRSV in boar semen. These ReTi-PCR tests will improve the control of viral diseases transmitted via semen. (C) 2004 Elsevier B.V. All rights reserved.

1. The aim of the experiments was to evaluate whether selected probiotic lactobacillus strains have different immunomodulating effects in layer- and meat-type strain chickens. 2. Humoral and cellular specific and non-specific immune responses were studied by experiments on cellular proliferation, entry and survival of Salmonella bacteria in gut and spleen leukocytes, immunoglobulin isotypes and specific immunoglobulin titres. 3. The effects of two different feeding regimes (short and continuous feeding) and doses for administration of lactobacilli were studied. 4. The lactobacillus strains that were evaluated showed modulating effects on the immune system of layer- and meat-type chickens. 5. In meat-type strain chickens the lactobacilli had a stimulating effect when the chickens were young (up to 3 weeks) and the dose was relatively high, whereas in layer- type chickens a lower effective dose and discontinuous administration was also effective. 6. Immunoprobiotic lactobacilli can have a positive effect on humoral and cellular immune responses in layer- and meat-type strain chickens, but the lactobacillus strain to be used, the age of the animals and effective dose of lactobacilli to be administered need to be optimised.

Cathepsin L (CL)-like proteases are important candidate vaccine antigens for protection against helminth infections. We previously identified an immunogenic 32 kDa protein specifically present in newly excysted juveniles (NEJs) of Fasciola hepatica. Here we show by N-terminal protein sequencing that this protein represents a CL-like protease still containing the propeptide. Two cDNAs encoding this CL were subsequently isolated from NEJs by RT-PCR. The predicted amino acid sequences of these cDNAs showed approximately 70% sequence homology to both CL1 and CL2 sequences isolated from adult stage F. hepatica and are, therefore, referred to as CL3. The CL3 clones encoded asparagine at position PI of the propeptide cleavage site, suggesting a dependence on asparaginyl endopeptidases for maturation. Recombinant expression of a CL3 cDNA in Saccharomyces cerevisiae resulted in secretion of the proenzyme form. The propeptide of CL-like proteins was predicted to contain important B-cell epitopes. To determine the contribution of the propeptide to protective immunity, rats were vaccinated with Keyhole Limpet Haemocyanin-conjugated synthetic peptides encoding these putative B-cell epitopes derived from the CL1 or CL3 sequence. A subsequent challenge infection resulted in a significant (P

The phase II detoxification system glutathione transferase (GST) is associated with the establishment of parasitic nematode infections within the gastrointestinal environment of the mammalian host. We report the functional analysis of a GST from an important worldwide parasitic nematode of small ruminants,Haemonchus contortus. This GST shows limited activity with a range of classical GST substrates but effectively binds hematin. The high-affinity binding site for hematin was not present in the GST showing the most identity, CE07055 from the free-living nematodeCaenorhabditis elegans. This finding suggests that the high-affinity binding of hematin may represent a parasite adaptation to blood or tissue feeding from the host.

The human pathogen Campylobacter jejuni is a highly motile organism that carries a flagellum on each pole. The flagellar motility is regarded as an important trait in C. jejuni colonization of the intestinal tract, however, the knowledge of the regulation of this important colonization factor is rudimentary. We demonstrate by phosphorylation assays that the sensor FlgS and the response regulator FlgR form a two-component system that is on the top of the Campylobacter flagellum hierarchy. Phosphorylated FlgR is needed to activate RpoN-dependent genes of which the products form the hook-basal body filament complex. By real-time reverse transcriptase-PCR we identified that FlgS, FlgR, RpoN, and FliA belong to the early flagellar genes and are regulated by sigma70. FliD and the putative anti-sigma-factor FlgM are regulated by a sigma54- and sigma28-dependent promoters. Activation of the fla regulon is growth phase-dependent, a 100-fold rpoN mRNA reduction is seen in the early stationary phase compared with the early logarithmic phase. Whereas flaB transcription decreases, flaA transcription increases in early stationary phase. Our data show that the C. jejuni flagellar hierarchy largely differs from that of other bacteria. Phenotypical analysis revealed that unflagellated C. jejuni mutants grow three times faster in broth medium compared with wild-type bacteria. In vivo the C. jejuni flagella are needed to pass the gastrointestinal tract of chickens, but not to colonize the ceaca of the chicken.

In a case-control study, the role of porcine circovirus 2 (PCV2) and putative co-factors in the development of porcine dermatitis and nephropathy syndrome (PDNS) were investigated. Pigs with and without PDNS were examined for macroscopic lesions and histopathology. In addition, organs and tissues were collected at necropsy and examined for the presence of fibrinous deposits (immune complexes), CD8+ cells, and for the presence of bacterial and viral infections. Results from PDNS cases were compared with those of three control groups comprising pigs without clinical signs of PDNS and selected from; (1) the same compartment as PDNS cases, (2) another compartment but in the same PDNS herd, and (3) a control herd without any history of PDNS or post-weaning multisystemic wasting syndrome. Macroscopic and histopathological lesions found in PDNS cases were comparable to those previously documented for PDNS e.g. skin lesions and renal lesions representing glomerulonephritis associated with fibrinous deposits and to a lesser extent with interstitial nephritis. PCV2 was detected by PCR in 100% of the PDNS cases, mainly in lymph nodes and tonsils, and in 63% of the control pigs from PDNS free herds. Virus isolation did not reveal infectious PCV2 in all cases. In PDNS affected pigs the PCV2 serum antibody titres were consistently extremely high and the mean PCV2 antibody titre in PDNS pigs was significantly higher than the mean PCV2 antibody titres in pigs from all 3 control groups. Immunohistochemical investigation of kidneys from PDNS affected pigs revealed an increased accumulation of IgG1 + IgG2 and IgM, the complement factors C1q and C3, but also an increase of CD8+ cells. The amounts of IgA and the complement factor C5 in kidneys of PDNS pigs were only slightly increased as compared to control pigs. This study demonstrates that PCV2 infections can result in extremely high PCV2 antibody titres and that PCV2 is a candidate as primary agent in the development of PDNS. The causative physiological basis for PDNS may be the excessive levels of PCV2 antibodies.

Classification of pig carcasses in the European Community (EC) is based on the lean meat percentage of the carcasses. The lean meat percentage is predicted from instrumental carcass measurements, such as fat and muscle depth measurements, obtained in the slaughterline. The prediction formula for an instrument is derived from the data of a dissection experiment. When the relationship between percentage lean and instrumental carcass measurements differs between subpopulations, such as sexes or breeds, accuracy of prediction may differ between these subpopulations. In particular for some subpopulations predicted lean meat percentages may be systematically too low and for other subpopulations systematically too high. Producers or buyers that largely specialize in subpopulations where the percentage lean is underestimated, are put at a financial disadvantage.The aim of this paper is to gain insight, on the basis of real data, into the effects of differences between subpopulations on the accuracy of the predicted percentage lean meat of pig carcasses. A simulation study was performed based on data from dissection trials in The Netherlands, comprising gilts and castrated males, and trials in Spain, comprising different genetic types. The possible gain in accuracy, i.e. reduction of prediction bias and mean squared prediction error, by the use of separate prediction formulae for (some of) the subpopulations was determined.We concluded that marked bias in the predicted percentage lean meat may occur between subpopulations when a single overall prediction formula is employed. Systematic differences in predicted percentage lean between subpopulations that are overestimated and underestimated may exceed 4% and for selected values of instrumental measurements may run up to 6%. Bias between subpopulations may be eliminated, and prediction accuracy may be markedly improved, when separate prediction formulae are used. With the use of separate formulae the root mean squared prediction error may be reduced by 13 to 26% of the expected value when a single prediction formula is used for all pig carcasses.These are substantial reductions on a national scale. This suggests that there will be a commercial interest in the use of separate prediction formulae for different subpopulations. In the near future, when the use of implants becomes more reliable, subpopulations will be recognized automatically in the slaughterline and use of different prediction formulae will become practically feasible. Some possible consequences for the EC regulations and national safeguards for quality of prediction formulae are discussed.

How much of its resources should an individual invest in a costly immune system? In this article, we apply an evolutionarily stable strategy analysis to an epidemic model to answer this question. On the one hand, an investment in immune function confers protection to infectious agents by reducing host susceptibility, pathogen virulence, or the length of the infectious period. On the other hand, an immune system is costly since it absorbs resources that otherwise might be invested in increasing the host's fertility or longevity. In addition, an active immune system may be able to clear pathogens efficiently but at the same time may result in immunopathology. By means of a reproductive value approach, we show how to compare the costs and benefits of an immune system systematically and how to derive the evolutionarily stable level of immune function. We then apply these methods to various plausible scenarios. The analysis reveals that the relationship between the life span of an organism and the optimal level of investment in immune function is less straight-forward than one might expect. First, the prevalence of infection is reduced to the lowest possible level only under special circumstances. Second, members of a long-lived species do not necessarily have to invest more in immune function than those of a short-lived species. in fact, the opposite may be true. Third, the outcome of evolution can be contingent on the initial conditions. Depending on its initial investment strategy, a population may evolve to a state where very much or almost nothing is invested in a costly immune system.

In Maribor, Slovenië, werd van 11-15 februari 2004 het 13e internationale symposium over kreupelheidsproblemen bij herkauwers gehouden, waarvan de 10 sessies hier worden samengevat

The aim of our study was to evaluate the relative importance of antibody and T cell-mediated immunity in protection against pseudorabies virus (suid herpes virus type 1) infection in pigs. We induced different levels of immune responses by using: (1) a modified live vaccine; (2) the same modified live vaccine with an oil-in-water (o/w) adjuvant; (3) an inactivated vaccine; and (4) the same inactivated vaccine with an o/w adjuvant. Subsequently, we challenged pigs with virulent pseudorabies virus (PRV). We demonstrated that best-protected pigs stood out by maintaining strong T cell-mediated immune (CMI) responses after challenge. Of the immune parameters tested, protection against virus shedding was correlated best with the magnitude of the IFN-gamma-response of in vitro re-stimulated peripheral blood mononuclear cells (PBMC) with an additional role for PRV-specific IgG2 antibodies. The use of an o/w adjuvant resulted in higher antibody and CMI responses, in particular with an increased frequency of memory T helper blast cells of in vitro re-stimulated PBMC. However, this adjuvant-induced enhancement of the immune response had a limited additional effect on the efficacy of inactivated vaccines. This study suggests a major contribution of the CMI response in early protection against PRV infection and that PRV-induced IFN-gamma responses may serve as a suitable indicator for assessing the immune status of vaccinated pigs. (C) 2004 Elsevier B.V. All rights reserved.

Faecal samples from 224 dogs from 23 animal shelters in the Netherlands were examined for endoparasites. In total 20.5% of the faecal sample were positive for helminth and/or protozoa infections. Eggs of Toxocara canis were found in 8.5% of the faecal samples. Other endoparasites found were Toxascaris leonina (0.5%), Trichuris vulpis (4.9%), Uncinaria stenocephala (2.2%), Dipylidium caninum (1.3%), Taenia spp. (0.5%), Cystoisospora canis (1.3%), and C. ohioensis (1.3%). Dogs younger than 1 year and stray dogs showed the highest prevalence of infection. T. vulpis was found more often in dogs from shelters with a high cleaning frequency.