Human respiratory syncytial virus infection in the pre-clinical calf model.

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.

Perceptions and actions of Dutch sheep farmers concerning worm infections.

Gastrointestinal (GI) nematode infections are considered among one of the toughest challenges sheep farmers face worldwide. Control still is largely based on the use of anthelmintics, but anthelmintic resistance is becoming rampant. To facilitate implementation of alternative nematode control strategies and to reduce anthelmintic usage, the purpose of this study was twofold: (i) to gain insight in common practices, knowledge gaps and perceptions of farmers regarding nematode control, and (ii) to provide foci of attention for improving parasite control practices and transfer of knowledge within the sheep husbandry. An internet-based questionnaire was made available to all sheep farmers pertaining to the year 2013, resulting in 450 entered questionnaires for analysis. The two most important nematodes mentioned, were Haemonchus contortus and, to a lesser extent, Nematodirus battus. Of all respondents, 25.6% said they did not have any worm problems. Of these, almost a third did notice clinical signs that can be related to worm infections and about three quarters did use anthelmintics. Overall, clinical symptoms mentioned by farmers matched the worm species they identified as the cause of problems. Ewes and lambs were treated up to 6 times in 2013. On average, ewes were treated 1.53 and lambs 2.05 times. Farmers who treated their ewes more often, also treated their lambs more often (P<0.001). Both ewes and lambs were frequently treated based on fixed moments such as around lambing, at weaning and before mating, rather than based on faecal egg counts. Treatments based on faecal egg counts were practiced, but on a minority of the farms (32.7%). The majority of the farms (75.6%) did not leave 2-5% of the sheep within a flock untreated. About 74% of farmers keep newly purchased animals quarantined for at least 10days, but some (13.4%) leave quarantined animals untreated nor check faecal egg counts. Of farmers who do treat their quarantined animals, just 12.6% check the efficacy of the treatment. Slightly over 40% of the respondents said they did not experience bottlenecks in parasite control. Yet, over half of these said having problems with worm infections, over half did see clinical signs related to worm infections and over three quarters used anthelmintics. Within the group of farmers experiencing difficulties in parasite control, the most often mentioned bottleneck concerned pasture management (75.8%). When asking farmers for solutions, 90% of all respondents indicated they are willing to adjust their pasture management. Farmers are also interested in other methods to reduce the risk of worm infections, such as possibilities to enhance the immune system of sheep in general (71%), to increase specific genetic resistance to worms and to apply anti-parasite forages, both about 40%. Results of this study gave the following potential foci of attention: (1) making complex scientific knowledge more accessible to farmers through simple tools and applicable in the daily farming process; (2) changing the mindset of farmers about their current worm control practices, i.e. breaking long-standing habits such as treating ewes and lambs at fixed moments rather than based on actual worm infection monitoring data; (3) demonstrating effective pasture rotation schemes on specific farms and using these in extension work; (4) making farmers more aware that checking anthelmintic efficacy is important; (5) improving quarantine procedures; (6) creating a wider array of applicable alternative control measures from which individual farmers can choose what fits them most; and finally, (7) improving mutual understanding among farmers, veterinary practitioners and parasitologists alike.

Vertical transmission of Rift Valley fever virus without detectable maternal viremia.

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.

Efficacy of three candidate Rift Valley fever vaccines in sheep.

Rift Valley fever virus (RVFV) is a mosquito-transmitted Bunyavirus that causes high morbidity and mortality among ruminants and humans. The virus is endemic to the African continent and the Arabian Peninsula and continues to spread into new areas. The explosive nature of RVF outbreaks requires that vaccines provide swift protection after a single vaccination. We recently developed several candidate vaccines and here report their efficacy in lambs within three weeks after a single vaccination. The first vaccine comprises the purified ectodomain of the Gn structural glycoprotein formulated in a water-in-oil adjuvant. The second vaccine is based on a Newcastle disease virus-based vector that produces both RVFV structural glycoproteins Gn and Gc. The third vaccine comprises a recently developed nonspreading RVFV. The latter two vaccines were administered without adjuvant. The inactivated whole virus-based vaccine produced by Onderstepoort Biological Products was used as a positive control. Five out of six mock-vaccinated lambs developed high viremia and fever and one lamb succumbed to the challenge infection. A single vaccination with each vaccine resulted in a neutralizing antibody response within three weeks after vaccination and protected lambs from viremia, pyrexia and mortality.

Rift Valley fever virus immunity provided by a paramyxovirus vaccine vector.

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 naïve populations. The RVFV structural glycoproteins Gn and Gc 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 Gc 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.

Seroprevalence of antibodies against pestiviruses in small ruminants in The Netherlands.

In this study, a serological survey was performed to determine the prevalence of pestivirus (bovine viral diarrhea virus (BVDV) and border disease virus (BDV)) infected small ruminants herds in the Netherlands. After random selection of sheep farms, a sample size was determined to detect a 5% herd prevalence. 13 out of 29 farms were tested seropositive using an ELISA which detects antibodies directed against the non structural protein 3 (NS3) of pestiviruses. This resulted in a seroprevalence for the Netherlands of 45% [0.36; 0.54]. The within farm prevalence ranged from 4 till 65%. Using a virus neutralization assay, specific anti-BDV antibodies could be detected on two farms, while on one other farm anti-BVDV antibodies were present. On four farms antibodies to both viruses could be detected, on three of these farms antibodies against both viruses were equally present. At five farms that tested positive in the NS3-ELISA we were unable to detect pestivirus neutralizing antibodies in all sera using the VN test. This resulted in an estimated prevalence using the VN for the Netherlands of 28% [0.20; 0.60]. An additional survey in sera from dairy goats revealed that 34 out of 126 farms were serological positive resulting in a seroprevalence of 27% [0.23; 0.31], with a herd prevalence of 32% ranging from 1-100%.

Restriction enzyme analysis of RT-PCR amplicons as a rapid method for detection of genetic diversity among bovine respiratory syncytial virus isolates.

Our current knowledge of antigenic variability of the bovine respiratory syncytial virus (BRSV) is quite limited and is mainly dependent on the use of monoclonal antibodies (mAb). In this study, we present not only analysis of the antigenic, but also of the genetic variability of BRSV. Using a panel of BRSV-specific mAb we distinguished five main reactivity patterns, three of which corresponded to the previously established subgroups A, B and AB. A single viral strain yielded the fourth pattern, while four viral strains did not react with any of the used mAbs forming the fifth pattern. To investigate the genetic basis for the antigenic heterogeneity of the BRS virus G protein, DNA of 11 BRSV isolates was directly sequenced. The comparison of the obtained nucleotide or amino acid sequences to those BRSV strains present in the GenBank revealed 88.1-99.4% and 77.7-98.4% similarity, respectively. These results supported the previously stated suggestion to type BRSV isolates according to their genetic relationship. In order to introduce a rapid and simple method to study the genetic variability of BRSV, we utilized the restriction enzyme analysis of RT-PCR products derived from mRNAs corresponding to the most variable region of the BRSV glycoprotein G ectodomain. Using this restriction enzyme analysis we were able to identify genetic variability among BRSV isolates. The detected non-synonymous mutations led frequently to a change in digestion pattern and were predominantly located in two mucin-like regions of the G protein gene. A correlation has been found between grouping of isolates in the phylogenetic tree and their restriction patterns clustering together isolates with the same restriction profiles. However, viruses placed distant in the tree sharing the same restriction patterns were detected supposing that phylogenetic analysis should be necessary for BRSV typing. Thus, we propose to use DNA restriction polymorphism for a rapid detection of genetic variants among BRSV isolates circulating in cattle population and as a preliminary tool for their typing.

Bovine respiratory syncytial virus infection influences the impact of alpha- and beta-integrin-mediated adhesion of peripheral blood neutrophils.

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 beta2-integrins, alternative integrins such as the alpha4-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 beta2-integrin reduced firm adhesion to 63% and inhibition of alpha4-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 beta2-integrin and was significantly augmented to 2.5-fold by blocking both alpha4- and beta2-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 alpha4- and beta2-integrins, indicating that these integrins did not support neutrophil rolling. In addition, the inhibition of firm adhesion by blocking both alpha4- and beta2-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 alpha4- and beta2-integrins. These results demonstrate an important function for alpha4- and beta2-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.

Large scale production and downstream processing of a recombinant porcine parvovirus vaccine.

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 1.5x10(6) 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.

[Case report of a granulosa-theca tumor in a cow]. / Case study van een granulosa-thecaceltumor bij het rund.

A 2 year-old cow with abnormal behaviour was observed during a farm visit. Rectal palpation of the cow revealed the presence of a mass of at least 12 cm in diameter. After further examination, it appeared that 'ovarian tumour' was the most likely differential diagnosis. In order to confirm this diagnosis, blood samples were drawn and analysed for plasma progesterone and plasma oestradiol-17 beta concentrations. Also, the gross pathology and histology of the mass were evaluated. The combination of the clinical presentation of the cow, the hormone concentrations, and the histological appearance of the mass confirmed the diagnosis ovarian tumour. The tumour was classified as granulosa-theca cell tumour.