Search

In the veal industry in The Netherlands, each year around 1.2 million “white” veal calves are produced on around 1100 farms. Bovine respiratory disease (BRD) causes serious health issues in these calves, also resulting in high usage of antimicrobials. To reduce antimicrobial usage, a more targeted treatment regime is needed, for which it is necessary to identify the causative agent. This study aimed at determining associations between pathogens and clinical disease, between prevalence of pathogens and BRD outbreaks, and BRD and performance. A cohort study was conducted involving ten veal farms, in which calf respiratory health was evaluated for the first 12 weeks. Whenever there was an outbreak of BRD, as determined by the farm veterinary surgeon, samples were taken from diseased and control calves through broncho-alveolar lavage. From these samples a broad spectrum of micro-organisms were isolated. Performance data were also collected. A total of 23 outbreaks happened during the 12 week study period, mostly in the first six weeks. BRD associated pathogens found were: BHV1, BPI3V, BRSV, BVDV, Pasteurella multocida, Mannheimia haemolytica, Trueperella pyogenes, Histophilus somni, Mycoplasma bovis, Mycoplasma bovirhinis and Mycoplasma dispar. For most BRD associated pathogens, there was no clear association between presence or prevalence of the micro-organisms and clinical issues. Only T. pyogenes (7.4% in healthy, 14.6% in diseased calves, p 0.013), M. bovis (37.6% and 63.2% respectively, p 0.001) and BVDV (9.9% and 16.9% respectively, p 0.03) were found more often in diseased animals. BPI3V was found in a few early outbreaks, which might suggest involvement in early outbreaks. It appears to be difficult to associate specific pathogens to outbreaks at the species level. BRD is the major reason for treatment with antimicrobials. More specific knowledge about the association between pathogens and health/disease could help to reduce antimicrobial use.

Human respiratory syncytial virus (hRSV) is the most important respiratory pathogen in young children worldwide. Experimental modelling of hRSV disease by bovine RSV (bRSV) infection in calves provides an important tool for developing new strategies for prevention and treatment. Depending on the scientific hypothesis under investigation, this cognate host-virus model might have the disadvantage of using a highly related but not genetically identical virus. In this study, we aim to describe viral kinetics and (clinical) disease characteristics in calves inoculated with hRSV. Our results show that hRSV infects the upper and, to a lesser extent, the lower respiratory tract of calves. Infection causes upper airway clinical disease symptoms and neutrophilic infiltration of the lower airways. We conclude that a hRSV model in calves may aid future research involving distinct scientific questions related to hRSV disease in children.

Water is one of our greatest but finite resources and as such should be treated with special care. The framework for Community action in the field of water policy by the European Commission (Directive 2000/60/EC) states that water should be treated as heritage which needs to be protected. Water can vary in salinity, nutrient levels and levels of contamination (microbiological and/or chemical). For food production as well as for nature conservation, water plays a crucial role. In order to protect our fresh water sources from depletion, we need to reduce, reuse and recycle water in the most efficient way. Safeguard the future of our food supply and to prevent water from running out, the role of water in a circular food system needs to be made transparent. This report is innovative as it aims to open the dialogue about water’s role in a circular food system. Which types of waters could be identified from a circular perspective and to what extend do these interact with each other? The subdivision into green, blue and grey found in literature is enlightening, but falls short of giving a complete circular perspective. These three colours of water often represent a footprint approach related to one product (e.g. to produce one kilogram coffee requires x litres of fresh water). In this report a first exploration towards circular use is made to include nature in the analysis in addition to food production analysis. In order to broaden the dialogue of the representation of the world’s potential circular use of water, we have added six colours.

Human respiratory syncytial virus (RSV) is the most important cause of severe lower respiratory tract disease (LRTD) in young children worldwide. Extensive neutrophil accumulation in the lungs and occlusion of small airways by DNA-rich mucus plugs are characteristic features of severe RSV-LRTD. Activated neutrophils can release neutrophil extracellular traps (NETs), extracellular networks of DNA covered with antimicrobial proteins, as part of the first-line defence against pathogens. NETs can trap and eliminate microbes; however, abundant NET formation may also contribute to airway occlusion. In this study, we investigated whether NETs are induced by RSV and explored their potential anti-viral effect in vitro. Second, we studied NET formation in vivo during severe RSV-LRTD in infants and bovine RSV-LRTD in calves, by examining bronchoalveolar lavage fluid and lung tissue sections, respectively. NETs were visualized in lung cytology and tissue samples by DNA and immunostaining, using antibodies against citrullinated histone H3, elastase and myeloperoxidase. RSV was able to induce NET formation by human neutrophils in vitro. Furthermore, NETs were able to capture RSV, thereby precluding binding of viral particles to target cells and preventing infection. Evidence for the formation of NETs in the airways and lungs was confirmed in children with severe RSV-LRTD. Detailed histopathological examination of calves with RSV-LRTD showed extensive NET formation in dense plugs occluding the airways, either with or without captured viral antigen. Together, these results suggest that, although NETs trap viral particles, their exaggerated formation during severe RSV-LRTD contributes to airway obstruction.

Respiratory syncytial virus (RSV) infection is characterised by airway obstruction with mucus plugs, containing DNA networks in the form of neutrophil extracellular traps (NETs). We investigated the effect of dornase alfa on histopathological NETs-induced airway obstruction and viral load in an age-relevant calf model of severe bovine RSV disease. As compared with the control animals, dornase alfa treatment resulted in a strong reduction of NETs-induced airway obstruction. Viral load in the lower respiratory tract was not different between the two groups. We conclude that NETs form a relevant target for treatment of airway obstruction in severe RSV disease.

Background Pasteurella multocida, Mannheimia haemolytica, Histophilus somni and Trueperella pyogenes are four bacterial agents commonly associated with bovine respiratory disease (BRD). In this study a bacterial multiplex real-time PCR (the RespoCheck PCR) was evaluated for the detection in bronchoalveolar lavage fluid (BALF) of these four bacterial agents. Results The analytical sensitivity of the multiplex real-time PCR assay determined on purified DNA and on bacterial cells of the four target pathogens was one to ten fg DNA/assay and 4 × 10−1 to 2 × 100 CFU/assay. The analytical specificity of the test was, as evaluated on a collection of 118 bacterial isolates, 98.3% for M. haemolytica and 100% for the other three target bacteria. A set of 160 BALF samples of calves originating from ten different herds with health problems related to BRD was examined with bacteriological methods and with the RespoCheck PCR. Using bacteriological examination as the gold standard, the diagnostic sensitivities and specificities of the four bacterial agents were respectively between 0.72 and 1.00 and between 0.70 and 0.99. Kappa values for agreement between results of bacteriological examination and PCRs were low for H. somni (0.17), moderate for P. multocida (0.52) and M. haemolytica (0.57), and good for T. pyogenes (0.79). The low and moderate kappa values seemed to be related to limitations of the bacteriological examination, this was especially the case for H. somni. Conclusion It was concluded that the RespoCheck PCR assay is a valuable diagnostic tool for the simultaneous detection of the four bacterial agents in BALF of calves.

Background In this study we evaluated the RespoCheck Mycoplasma triplex real-time PCR for the detection in bronchoalveolar lavage fluid (BALF) of Mycoplasma (M.) dispar, M. bovis and M. bovirhinis, all three associated with bovine respiratory disease (BRD). Primers and probes of the RespoCheck Mycoplasma triplex real-time PCR are based on the V3/V4 region of the 16S rRNA gene of the three Mycoplasma species. Results The analytical sensitivity of the RespoCheck triplex real-time PCR was, as determined by spiking experiments of the Mycoplasma strains in Phosphate Buffered Saline, 300 colony forming units (cfu)/mL for M. dispar, and 30 cfu/mL for M. bovis or M. bovirhinis. The analytical sensitivity of the RespoCheck Mycoplasma triplex real-time PCRwas, as determined on purified DNA, 10 fg DNA per assay for M. dispar and 100 fg fo rM. bovis and M. bovirhinis. The analytical specificity of the RespoCheck Mycoplasma triplex real-time PCR was, as determined by testing Mycoplasmas strains (n = 17) and other bacterial strains (n = 107), 100, 98.2 and 99.1% for M. bovis, M. dispar and M. bovirhinis respectively. The RespoCheck Mycoplasma triplex real-time PCR was compared with the PCR/DGGE analysis for M. bovis, M. dispar and M. bovirhinis respectively by testing 44 BALF samples from calves. Conclusion In conclusion, the RespoCheck PCR assay can be a valuable tool for timely and accurate detection of three Mycoplasma species associated with in bovine respiratory disease.

Rift Valley fever virus (RVFV) is an emerging mosquito-borne virus causing significant morbidity and mortality in livestock and humans. Rift Valley fever is endemic in Africa, but also outside this continent outbreaks have been reported. Here we report the evaluation of two vaccine candidates based on the viral Gn and Gc envelope glycoproteins, both produced in a Drosophila insect cell expression system. Virus-like particles (VLPs) were generated by merely expressing the Gn and Gc glycoproteins. In addition, a soluble form of the Gn ectodomain was expressed and affinity-purified from the insect cell culture supernatant. Both vaccine candidates fully protected mice from a lethal challenge with RVFV. Importantly, absence of the nucleocapsid protein in either vaccine candidate facilitates the differentiation between infected and vaccinated animals using a commercial recombinant nucleocapsid protein-based indirect ELISA.

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)

Veterinary vaccines are usually tested for the absence of contaminants. However, the quality control does not always imply that vaccines are not contaminated as, for example, illustrated by the bovine herpes virus 1 (BHV1) vaccine used in The Netherlands in 1999 that contained a small amount of bovine viral diarrhoea virus (BVDV1). Thousands of cows were vaccinated with BHV1 vaccine batches, and the question arose as to whether these small amounts of BVDV1, most likely not detected with in vitro tests, could have infected cattle. More in general, the question was whether the outcome of the in vitro tests, i.e. the in vitro infectivity, was indicative for the infectivity for cattle, i.e. the in vivo infectivity. We therefore carried out in vitro experiments to determine the sensitivity of a BVDV1 isolation assay. In addition, we performed two animal experiments, in which we estimated the lowest dose needed to infect calves with BVDV L We extrapolated the experimental in vitro and in vivo results from a tissue culture infectious dose (TCID50) to a cattle infectious dose (CID50). We observed a partial response in the calves inoculated with this dose: four out of six calves turned out to be infected. In the tissue culture test, all 20 samples tested negative. The response in vivo, however, was not significantly higher than the in vitro response, which implies that no difference in susceptibility was observed between the animal test and the tissue culture test. Based on the results in our experiments, some cattle may have been infected with BVDV1 after the application of the contaminated BHV1 vaccine during the vaccination campaign. The question remains that how many cattle received contaminated vaccine, and became infected with BVDV1. (C) 2004 Elsevier B.V. All rights reserved.

Rift Valley fever virus (RVFV) is a zoonotic bunyavirus that causes abortions in domesticated ruminants. Sheep breeds exotic to endemic areas are reportedly the most susceptible to RVFV infection. Within the scope of a risk assessment program of The Netherlands, we investigated the susceptibility of a native breed of gestating sheep to RVFV infection. Ewes were infected experimentally during the first, second, or third trimester of gestation. Mortality was high among ewes that developed viremia. Four of 11 inoculated ewes, however, did not develop detectable viremia nor clinical signs and did not seroconvert for immunoglobulin G (IgG) or IgM antibodies. Surprisingly, these ewes were found to contain viral RNA in maternal and fetal organs, and the presence of live virus in fetal organs was demonstrated by virus isolation. We demonstrate that RVFV can be transmitted vertically in the absence of detectable maternal viremia.

Hepatitis E virus (HEV) genotypes 3 and 4 are a cause of human hepatitis and swine are considered the main reservoir. To study the HEV prevalence and characterize circulating HEV strains, fecal samples from swine in the Netherlands and Belgium were tested by RT-PCR. HEV prevalence in swine was 7-15%. The Dutch strains were characterized as genotype 3, subgroups 3a, 3c and 3f, closely related to sequences found in humans and swine earlier. The HEV strains found in Belgium belonged to genotypes 3f and 4b. The HEV genotype 4 strain was the first ever reported in swine in Europe and an experimental infection in pigs was performed to isolate the virus. The genotype 4 strain readily infected piglets and caused fever and virus shedding. Since HEV4 infections have been reported to run a more severe clinical course in humans this observation may have public health implications.

The severity of respiratory syncytial virus (RSV) infections appears to differ with age in both humans and bovines. A primary RSV infection in naïve infants and in young calves runs a more severe course when they are 1–6 months old than in their first month of life. The relative lack of clinical signs in the first month of age may be due to high levels of maternally derived neutralizing antibodies or low exposure to infectious virus. This study examined whether age-dependent differences in the pathogenesis of bovine RSV (bRSV) between neonatal and young calves may be due to differences in age-dependent immunocompetence. To study the effect of age and immune parameters on bRSV disease in neonatal and young calves, neonatal (1-day-old) calves without maternally derived antibodies were infected experimentally with bRSV and the severity of disease and immune responses were evaluated in comparison with disease in similar 6-week-old infected calves. Neonatal calves had more extensive virus replication and lung consolidation, but lower pro-inflammatory [in particular tumour necrosis factor alpha (TNF-{alpha})] responses, specific humoral immune responses, lung neutrophilic infiltration and clinical signs of disease than 6-week-old calves. The lack of correlation between virus replication and clinical signs suggests an important role of pro-inflammatory cytokines, in particular TNF-{alpha}, in the disease. The capacity to produce pro-inflammatory TNF-{alpha} appeared to increase with age, and may explain the age-dependent differences in RSV pathogenesis.

Rift Valley fever virus (RVFV) causes recurrent large outbreaks among humans and livestock. Although the virus is currently confined to the African continent and the Arabian Peninsula, there is a growing concern for RVFV incursions into countries with immunologically naive populations. The RVFV structural glycoproteins Gn and Cc are preferred targets in the development of subunit vaccines that can be used to control future outbreaks. We here report the production of Gn and Cc by a recombinant vaccine strain of the avian paramyxovirus Newcastle disease virus (NDV) and demonstrate that intramuscular vaccination with this experimental NDV-based vector vaccine provides complete protection in mice. We also demonstrate that a single intramuscular vaccination of lambs, the main target species of RVFV, is sufficient to elicit a neutralizing antibody response. (C) 2010 Elsevier Ltd. All rights reserved.

Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in infants and calves. Bovine RSV (bRSV) is a natural pathogen for cattle, and bRSV infection in calves shares many features with the human infection. Thus, bRSV infection in cattle provides the ideal setting to evaluate the safety and efficacy of novel RSV vaccine strategies. Here, we have evaluated the efficacy and safety of modified vaccinia virus Ankara (rMVA)-based vaccine candidates, expressing the bovine RSV-F protein, either or not in combination with the G protein, in colostrums-deprived SPF calves born by caesarean section. Vaccination induced bRSV-specific IgG and CD8 T cell responses. Importantly, no IgE responses were detected. After bRSV challenge, rMVA vaccinated calves experienced less severe symptoms of lower respiratory tract disease compared to the mock-immunized control group. Immunized animals showed reduced pulmonary virus loads, and no eosinophilic infiltration or enhanced respiratory distress. In conclusion, candidate rMVA/bRSV vaccines induced protective and safe immune responses in calves.

Objective: Aim of the present study was to demonstrate the relevance of the vaccine strain of a new BVD vaccine, PregSure® BVD, for its use in Europe. Furthermore the vaccine¿s ability to protect from pregnancy losses due to an early infection with BVDV after artificial insemination was determined. Material and methods: The immune sera used in the in-vitro cross neutralisation were collected from 20 heifers three weeks after the completion of the primary vaccination schedule with PregSure® BVD (two vaccinations given 21 days apart). The BVDV cross-neutralising activity of these postvaccinal sera was tested by virus neutralisation employing a panel of different predominantly European BVDV type I and II strains. Furthermore, two fertility studies were carried out, where heifers between 14 and 39 months of age were primovaccinated with PregSure® BVD or left untreated as control, respectively. All animals had their oestrus cycles synchronised and were artificially inseminated. Four days after the initial artificial insemination and again three days later, all animals of experimental group 1 were challenged intranasally with two heterologous noncytopathic BVDV type I strains, whereas animals from group 2 received a type I and a type II BVDV strain. Sixty-nine to 72 days after the challenge, all dams were slaughtered and their foetuses collected. Differences in pregnancy rates between the vaccinated and the control group were assessed and then analysed using Fisher¿s Exact Test. Results: Heifers vaccinated with a novel inactivated BVDV vaccine containing a cytopathic BVDV type I strain 5960, were shown to have serum neutralising antibody titres between 5.5 to 12.3 log2 against all BVDV strains tested, three weeks after the completion of their primary vaccination course. In the first fertility experiment, pregnancy rates assessed 69-72 days after a double challenge with two different BVDV type I strains were 95.5% in the vaccinated group versus only 40.9% in the control group. A level of cross-protection against a severe BVDV type II challenge was shown in the second experiment with pregnancy rates of 47.6% in the vaccinated group and only 4.4% in the control group. Conclusions and clinical relevance: The broad cross neutralising activity shown in this study demonstrates the relevance of the vaccine strain 5960 for use in Europe. Furthermore as shown with significantly improved pregnancy in the two fertility experiments, PregSure® BVD vaccinated heifers are protected against fertility losses caused by acute BVDV infections.

Neutrophil migration into the airways and pulmonary tissue is a common finding in bovine respiratory syncytial virus (BRSV) infections. Although neutrophil trans-endothelial migration in general depends on beta(2)-integrins, alternative integrins such as the alpha(4)-integrins have been implicated. In this study, rolling and firm adhesion of peripheral blood neutrophils isolated from healthy and BRSV-infected calves to tumour necrosis factor (TNF)-alpha activated pulmonary endothelium was investigated under flow conditions in vitro. For neutrophils obtained from healthy animals, inhibition of the beta(2)-integrin reduced firm adhesion to 63% and inhibition of alpha(4)-integrin to 73% compared with untreated controls. Inhibition of both integrins reduced firm adhesion to 25%. Rolling velocity, which is used as a parameter for integrin involvement in neutrophil rolling, increased 1.7-fold by blocking beta(2)-integrin and was significantly augmented to 2.5-fold by blocking both alpha(4)- and beta(2)-integrins. For neutrophils obtained from BRSV-infected animals, however, rolling velocities at 10 days after infection (p.i.) were not influenced by blocking adhesion of alpha(4)- and beta(2)-integrins, indicating that these integrins did not support neutrophil rolling. In addition, the inhibition of firm adhesion by blocking both alpha(4)- and beta(2)-integrins was reduced significantly 9 days post-infection, resulting in a residual 68% neutrophil binding at 9 days p.i. Non-blocked firm adherence was not reduced, indicating that binding was achieved by other mechanisms than through alpha(4)- and beta(2)-integrins. These results demonstrate an important function for alpha(4)- and beta(2)-integrins in rolling and firm adherence of bovine neutrophils, to TNF-alpha-activated endothelium and show the dynamic use of these integrins for adhesion and migration by neutrophils in the course of BRSV infection.

Neutrophil emigration from the pulmonary vasculature, is mediated by cellular adhesion molecules (CAM) expressed on the outer membranes of endothelial cells and neutrophils. Although beta(2)-integrin-dependent migration is a major mechanism of neutrophil migration, which was demonstrated by extensive invasion of neutrophils in pulmonary tissue of calves suffering from a genetic deficit in expression of beta(2)-integrins, termed bovine leukocyte adhesion deficiency (LAD), the role of alternative CAM is still unclear. We investigated whether an alternate CAM for beta(2)-integrin function, i.e. the alpha(4)-integrin, was expressed on peripheral blood neutrophils of calves. As we detected basal but significant expression, the effect of naturally acquired pulmonary infection on the expression of either integrin was determined, as an indication for its function in the migration process. In our experiments, basal expression of alpha(4)-integrins on peripheral blood neutrophils from clinically healthy calves was detected. On neutrophils of calves, experiencing field outbreaks of enzootic bronchopneumonia, higher expression of the alpha(4)-integrin was detected, which returned to normal after successful treatment of the disease. In addition, its level of expression was linearly related to plasma acute phase protein (haptoglobin) concentrations, which is a sensitive parameter for severity of respiratory inflammation. Increased expression of the alpha(4)-integrin on peripheral blood neutrophils during pulmonary inflammation indicates a role for this CAM in neutrophil migration in the lung.

The bovine and human respiratory syncytial viruses cause severe lower respiratory tract infections. Effective vaccines against the respiratory syncytial viruses have been lacking since vaccine failures in the 1960s and 1970s. In this report, we describe a bovine respiratory syncytial virus (bRSV) challenge model in which both classical bRSV respiratory infection and vaccine-enhanced immune pathology were reproduced. The classical, formalin-inactivated (FI) bRSV vaccine that has been associated with vaccine failure was efficient in inducing high antibody titers and reducing viral loads but also primed calves for a far more serious enhanced respiratory disease after a bRSV challenge, thereby mimicking the enhanced clinical situation in FI human RSV (hRSV)-immunized and hRSV-infected infants in the 1960s. We show that immunization with FI-bRSV mainly primes a Th2-like inflammatory response that is characterized by a significant eosinophilic influx in the bronchial alveolar lung fluid and lung tissues and high levels of immunoglobulin E serum antibodies. The current model may be useful in the evaluation of new bRSV candidate vaccines for potency and safety.

6 páginas, 6 figuras, 1 tabla -- PAGS nros. 45-50, Porcine parvovirus (PPV) virus-like particles (VLPs) constitute a potential vaccine for prevention of parvovirus-induced reproductive failure in gilts. Here we report the development of a large scale (25 l) production process for PPV-VLPs with baculovirus-infected insect cells. A low multiplicity of infection (MOI) strategy was efficiently applied avoiding the use of an extra baculovirus expansion step. The optimal harvest time was defined at 120 h post-infection at the MOI used, with the cell concentration at infection being 1.5×106 cells/ml. An efficient purification scheme using centrifugation, precipitation and ultrafiltration/diafiltration as stepwise unit operations was developed. The global yield of the downstream process was 68%. Baculovirus inactivation with Triton X-100 was successfully integrated into the purification scheme without an increase in the number of process stages. Immunogenicity of the PPV-VLPs tested in guinea pigs was similar to highly purified reference material produced from cells cultured in the presence of serum-containing medium. These results indicate the feasibility of industrial scale production of PPV-VLPs in the baculovirus system, safety of the product, and the potency of the product for vaccine application, The authors acknowledge and appreciate the financial support received for the project from the European Commission (BIO4-CT98-0215) and a grant to L.M. from Fundação para a Ciência e Tecnologia – Portugal (PRAXIS XXI/BD/ 16136/98)