An observational study of the presence and variability of the microbiota composition of goat herd milk related to mainstream and artisanal farm management.

Goat milk is produced on mainstream and artisanal farms. It was expected that the farm management may influence the microbial population of the milk. Therefore, we investigated the bacterial content and microbiota composition of raw milk in relation to Dutch goat farm management. After amplicon sequencing we analyzed the taxa at phylum and genus levels, and used the relative values enabling to provide information about the variation among the different samples. On ten farms our results indicated that the number of bacterial colony forming units and microbiota composition of the milk, directly after milking was variable among farms and not related to the farm management system. At the phylum level the phyla Firmicutes, Actinobacteria, Proteobacteria, and to a minor extend Bacteriodota were the dominant phyla in the raw goat milk, together usually comprising 90% of the total bacterial phyla. The most dominant genera were Staphylococcus, Pseudomonas, Lactococcus, Microbacteria, Acinetobacteria, and Corinebacteria. The number of bacterial phyla and genera does not differ between the mainstream and artisanal farms, although the Shannon index may be numerically higher in the mainstream farms as compared to artisanal farms. In addition, the variability is higher among artisanal farms, which may be due to less standardization of the management. The milk microbiota composition differed among farms. Repeated sampling of a farm showed that this changed over time. The lactic acid producing bacteria showed a similar pattern. Variable microbiota richness amount and diversity of microorganisms were present in different farming systems. We concluded that farm-specific management and sampling moment were the major determining factors for the milk microbiota composition.

Respiratory pathogens in veal calves: Inventory of circulating pathogens.

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.

Local dornase alfa treatment reduces NETs-induced airway obstruction during severe RSV infection.

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.

Evaluation of a multiplex real-time PCR for detection of four bacterial agents commonly associated with bovine respiratory disease in bronchoalveolar lavage fluid.

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.

First isolation of hepatitis E virus genotype 4 in Europe through swine surveillance in the Netherlands and Belgium.

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.

Parenteral vaccination of mammalian livestock with Newcastle disease virus-based vector vaccines offers optimal efficacy and safety.

Newcastle disease virus (NDV) is an avian virus that is being evaluated as a vaccine vector for the delivery of foreign genes in mammals. The use of NDV as a vaccine vector in these species offers two major advantages. First, NDV is highly attenuated in mammals, rendering its use inherently safe. Second, mammals lack pre-existing NDV immunity, which minimizes the risk of vaccination failure. NDV-vector vaccines are generally administered to mammals via the respiratory route. We recently showed that intramuscular vaccination with NDV-based Rift Valley fever virus (RVFV) vaccines provides complete protection in mice and induces neutralizing antibodies in sheep and cattle, the main target species of RVFV. Here, we discuss the use of NDV as a vaccine vector for applications in mammalian livestock with an emphasis on the vaccination route. We also report the results of novel experiments that underscore our notion that vaccination via a parenteral route is more effective than immunization via the respiratory route.

Age-dependent differences in the pathogenesis of bovine respiratory syncytial virus infections related to the development of natural immunocompetence.

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-α)] 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-α, in the disease. The capacity to produce pro-inflammatory TNF-α appeared to increase with age, and may explain the age-dependent differences in RSV pathogenesis.

Vaccination with recombinant modified vaccinia virus Ankara expressing bovine respiratory syncytial virus (bRSV) proteins protects calves against RSV challenge.

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.

Efficacy of a modified live intranasal bovine respiratory syncytial virus vaccine in 3-week-old calves experimentally challenged with BRSV.

Two experimental bovine respiratory syncytial virus (BRSV) challenge studies were undertaken to evaluate the efficacy of a single intranasal dose of a bivalent modified live vaccine containing BRSV in 3-week-old calves. In the first study, vaccine efficacy was evaluated in colostrum deprived (maternal antibody negative) calves 5, 10 and 21 days after vaccination. Nasal shedding of BRSV was significantly reduced in vaccinated calves challenged 10 or 21 days after vaccination. Virus excretion titres were also reduced in vaccinates challenged 5 days after vaccination but reduction in duration of shedding and total amount of virus shed were not statistically significant. Clinical disease after challenge in this study was mild. In the second study, vaccine efficacy was assessed in calves with maternal antibodies against BRSV by challenge 66 days post-vaccination. Vaccination significantly reduced nasal shedding after challenge and the severity of clinical disease was also reduced.

A novel recombinant virus-like particle vaccine for prevention of porcine parvovirus-induced reproductive failure.

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.

Kinetics of antiviral CD8 T cell responses during primary and post-vaccination secondary bovine respiratory syncytial virus infection.

We have measured antiviral CD8 T cells responses in bovine respiratory syncytial virus (bRSV) infected calves that had been immunized with either formalin-inactivated (FI) or live-attenuated (L) bRSV, with evidence of immunopathology following challenge of calves vaccinated with FI-bRSV. In all cases, bRSV infection induced potent pulmonary CD8 T cell responses. The kinetics of the post-challenge response in L-bRSV immunized animals was accelerated compared to the FI-bRSV and PBS groups, suggesting that only the L-bRSV vaccine, and not the FI-bRSV vaccine, had primed memory T cells. The differences between primary and post-vaccination secondary infection were very minor, in terms of the proliferation status of pulmonary CD8 T cells. Functional IFN-gamma+ CD8 responses were slightly higher in the FI-bRSV vaccinated animals. Furthermore, the existence of strong IFN-gamma+ CD8 responses in FI-bRSV vaccinated animals after challenge suggests (i) that these IFN-gamma+ responses in FI-bRSV immunized animals do not protect against immunopathology, and (ii) that Th-2 biased responses during bRSV challenge after vaccination with FI-bRSV have a limited impact on the CD8 responses in the bronchoalveolar lavage fluid. Thus, several response patterns (Th-l/Th-2) seem to co-exist during bRSV infection.

Comparison of the sensitivity of in vitro and in vivo tests for detection of the presence of a bovine viral diarrhoea virus type 1 strain.

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 BVDV1. 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.

Vaccine-induced immunopathology during bovine respiratory syncytial virus infection: exploring the parameters of pathogenesis.

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.

Alpha4-integrin (CD49d) expression on bovine peripheral blood neutrophils is related to inflammation of the respiratory system.

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.