Reduction of veterinary antimicrobial use in the Netherlands. The Dutch success model.

Use of antimicrobials in animals poses a potential risk for public health as it contributes to the selection and spread of antimicrobial resistance. Although knowledge of the negative consequences of extensive antimicrobial use in humans and animals accumulated over the decades, total therapeutic antimicrobial use in farm animals in the Netherlands doubled between 1990 and 2007. A series of facts and events formed a window of opportunity to reduce antimicrobial use in farm animals. The recent discovery of significant reservoirs of antimicrobial-resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and extended spectrum beta-lactamase-producing bacteria (ESBL) in farm animals, with potential public health implications, combined with an increasing lack of confidence of the public in intensive livestock industries, and discrepancy between the very low antimicrobial use in humans and high use in animals, resulted in intensive collaboration between the government, veterinary professional organizations and important stakeholders within the livestock sector. A combination of compulsory and voluntary actions with clear reduction goals resulted in a 56% reduction in antimicrobial use in farm animals in the Netherlands between 2007 and 2012 and aims at accomplishing a 70% reduction target in 2015. This article describes and analyses the processes and actions behind this transition from an abundant antimicrobial use in farm animals towards a more prudent application of antimicrobials in farm animals in the Netherlands.

International regulations and standards for avian influenza, including the vaccine standards of the World Organisation for Animal Health.

For avian influenza the World Organisation for Animal Health (OIE) has laid down international standards on notification, trade, diagnosis, surveillance and the production and use of vaccine. These standards are science- and risk-based to ensure safe trade in poultry and poultry products without unjustified barriers. The European Union, with its 27 Member States, has in place harmonised legislation in line with OIE standards. Early detection, rapid diagnosis, notification and high quality Veterinary Services are crucial for ensuring a rapid response to avian influenza outbreaks and for swiftly reducing the risk of virus spread via trade. Depending on the situation, vaccination may also be a very important tool for disease control. The use of high quality vaccines and postvaccination monitoring are essential for the successful implementation of vaccination. Compliance with international standards is of paramount importance for protecting animal and human health in the global crisis of the highly pathogenic avian influenza of the H5N1 subtype.

Simultaneous intramammary and intranasal inoculation of lactating cows with bovine herpesvirus 4 induce subclinical mastitis.

In this study, we examined whether an experimental bovine herpesvirus 4 (BHV4) infection can induce bovine mastitis, or can enhance bovine mastitis induced by Streptococcus uberis (S. uberis). Four lactating cows were inoculated intramammarily and intranasally with BHV4, and four lactating control cows were mock-inoculated. After 14 days, two of four cows from each group were inoculated intramammarily with S. uberis. No clinical signs were recorded in cows inoculated only with BHV4, and their milk samples showed no abnormal morphology, despite the fact that BHV4 replicated in inoculated quarters. Somatic cell count increased significantly in milk from three of six BHV4-inoculated quarters, compared to the non-inoculated quarters of the same cows (within-cow) and the quarters of mock-inoculated cows (control group) on days 8, 9 and 11 post-inoculation (pi). BHV4 was isolated from nasal swabs between days 2 and 9 pi. Clinical mastitis was observed in all four cows intramammarily inoculated with S. uberis. A preceding BHV4 infection did not exacerbate the clinical mastitis induced by S. uberis. S. uberis infections appeared to trigger BHV4 replication. From one quarter of each of two cows inoculated with BHV4 and S. uberis, BHV4 was isolated, and not from quarters inoculated with BHV4 only. In conclusion, BHV4 did not induce bovine clinical mastitis after simultaneous intranasal and intramammary inoculation. However, the BHV4 infection did induce subclinical mastitis in 50% of the cows and the quarters.

Presence of European bat lyssavirus RNas in apparently healthy Rousettus aegyptiacus bats.

Apparently healthy Rousettus aegyptiacus bats were randomly chosen from a Dutch colony naturally infected with European bat lyssavirus subgenotype 1a (EBL1a). These bats were euthanised three months after the first evidence of an EBL1a infection in the colony. EBL1a genomic and antigenomic RNAs of the nucleoprotein gene were detected by nested reverse transcriptase PCR in 75% of the examined Rousettus aegyptiacus bats. The EBL1a RNAs of the nucleoprotein gene were detected mainly in brain tissues, but also in other organs. EBL1a messenger RNAs of the nucleoprotein gene and the glycoprotein gene were detected in brain tissues. The standard fluorescent antibody test revealed the presence of lyssavirus antigens in brain tissues from 7 (17.5%) Rousettus aegyptiacus bats. Furthermore, EBL1a could not be detected by virus isolation on murine neuroblastoma cells or by intracerebral inoculation of suckling mice. Neutralising antibodies directed against EBL1 were detected in 11% of the examined bats. This study shows that at least 85% of the apparently healthy Rousettus aegyptiacus bats must have been infected with EBL1a, and that these bats may survive from an EBL1a infection. Furthermore, the study supports the possibility of a long-term maintenance of EBL1a genome in Rousettus aegyptiacus bats.

[Outbreak of bovine virus diarrhea on Dutch dairy farms induced by a bovine herpesvirus 1 marker vaccine contaminated with bovine virus diarrhea virus type 2]. / Uitbraak van bovine virus diarree op Nederlandse rundveebedrijven na vaccinatie met een met BVDV type 2 gecontamineerd BHV1 markervaccin.

On 23 February 1999, the Dutch Animal Health Service advised all Dutch veterinary practices to postpone vaccination against bovine herpesvirus 1 (BHV1) immediately. The day before severe disease problems were diagnosed on four dairy farms after vaccination with the same batch of BHV1 marker vaccine. Using monoclonal antibodies, bovine virus diarrhoea virus (BVDV) type 2 was found in the vaccine batch. This paper describes an outbreak of BVDV type 2 infection caused by the use of a batch of modified live BHV1 marker vaccine contaminated with BDVD. Sources of information used were reports of farm visits, minutes of meetings, laboratory results, and oral communications from the people involved. The first symptoms of disease were observed on average six days after vaccination. Morbidity was high on 11 of the 12 farms. On five farms more than 70% of the animals became ill, while on one farm no symptoms could be detected. During the first week after vaccination, feed intake and milk production decreased. During the second week, some animals became clinically diseased having nasal discharge, fever, and diarrhoea. At the end of the second week and at the start of the third week, the number of diseased animals increased rapidly, the symptoms became more severe, and some animals died. Mortality varied among herds. Necropsy most often revealed erosions and ulcers of the mucosa of the digestive tract. In addition, degeneration of the liver, hyperaemia of the abomasum, and swollen mesenterial lymph nodes and swollen spleen were found. On 11 of the 12 farms all animals were culled between 32 and 68 days after vaccination after an agreement was reached with the manufacturer of the vaccine. This was the third outbreak of BVD in cattle after administration of a contaminated vaccine in the Netherlands. The possibilities to prevent contamination of a vaccine as a consequence of infection of fetal calf serum with BVDV are discussed. Improvement of controls to prevent contamination before and during vaccine production, and improvement of the monitoring of side-effects is necessary.

[Detection of bovine virus diarrhea virus in a live bovine herpes virus 1 marker vaccine]. / Detectie van bovine virus diarree virus in een levend bovine herpesvirus 1 markervaccin.

In February 1999, 12 Dutch herds were vaccinated with a live bovine herpesvirus 1 vaccine from which bovine virus diarrhea virus (BVDV) could be isolated. All vaccine batches that were on the Dutch market and that had not yet reached the expiry date were tested for BVDV. In total, seven of 82 batches tested were found positive. Batch numbers TX3607, VB3914, VB3915, VB4046, TW3391, and TV3294 were positive for BVDV type 1, and batch number WG4622 was positive for BVDV type 2. This latter batch induced clinical signs of BVDV in an animal experiment with susceptible animals.

[Vaccination of calves with contaminated batches of bovine herpes virus 1 vaccines did not result in infection with bovine virus diarrhea virus]. / Vaccinatie van kalveren met bovine herpesvirus 1 vaccins afkomstig van gecontamineerde charges leidde niet tot besmetting met bovine virus diarree virus.

The aim of the experiment was to study whether bovine herpesvirus 1 (BHV1) marker vaccine batches known to be contaminated with bovine virus diarrhoea virus (BVDV) type 1 could cause BVD in cattle. For this purpose, four groups of cattle were used. The first group (n = 4 calves, the positive control group), was vaccinated with vaccine from a batch contaminated with BVDV type 2. The second group (n = 4 calves, the negative control group), was vaccinated with vaccine from a batch that was not contaminated with BVDV. The third group (n = 39 calves), was vaccinated with a vaccine from one of four batches contaminated with BVDV type 1 (seronegative experimental group). The fourth group (n = 6 seropositive heifers), was vaccinated with a vaccine from one of three batches known to be contaminated with BVDV type 1. All cattle were vaccinated with an overdose of the BHV1 marker vaccine. At the start of the experiment, all calves except those from group 4 were seronegative for BVDV and BHV1. The calves from group 4 had antibodies against BVDV, were BVDV-free and seronegative to BHV1. After vaccination, the positive control calves became severely ill, had fever for several days, and BVDV was isolated from nasal swabs and white blood cells. In addition, these calves produced antibodies to BVDV and BHV1. No difference in clinical scores of the other groups was seen, nor were BVDV or BVDV-specific antibody responses detected in these calves; however, they did produce antibodies against BHV1. The remainder of each vaccine vial used was examined for the presence of infectious BVDV in cell culture. From none of the vials was BVDV isolated after three subsequent passages. This indicates that BVDV was either absent from the vials or was present in too low an amount to be isolated. Thus vaccination of calves with vaccines from BHV1 marker vaccine batches contaminated with BVDV type 1 did not result in BVDV infections.

[The effect of a high dose bovine herpes virus 1 marker vaccine in pregnant heifers: virological, bacteriological, immunological, and pathological findings]. / De effecten van vaccinatie met een hoge dosis BHV1-markervaccin in hoogdrachtige vaarzen: virologische, bacteriologische, immunologische en pathologische bevindingen.

To determine a possible relationship between the compulsory vaccination against bovine herpesvirus 1 (BHV1) and cattle wasting disease, the effects of BHV1 vaccination on heifers were investigated. Twenty heifers in the third trimester of pregnancy were randomly allotted to a vaccine and a control group. The vaccine group was vaccinated twice with a 50-fold dose of BHV1 vaccine and the control group was inoculated with the diluent. The experiment was performed double blind. After vaccination, the cows were examined daily and condition scores were determined weekly. Blood, milk, and faeces samples were collected weekly for virological, bacteriological, and immunological investigation. The heifers were euthanized either 9 or 13 weeks after the first inoculation and pathological, virological, and bacteriological examination was performed. No differences were detected between the vaccine group and the control group. No concurrent infections were detected and there were no indications of immunosuppression after vaccination. No relationship between the BHV1 vaccination and wasting disease in cattle was detected.

Airborne transmission of BHV1, BRSV, and BVDV among cattle is possible under experimental conditions.

To control the diseases caused by bovine herpesvirus 1 (BHV1), bovine respiratory syncytial virus (BRSV), and bovine virus diarrhoea virus (BVDV), it is crucial to know their modes of transmission. The purpose of this study was to determine whether these viruses can be transmitted by air to a substantial extent. Calves were housed in two separate isolation stables in which a unidirectional airflow was maintained through a tube in the wall. In one stable, three of the five calves were experimentally infected with BHV1 and later with BRSV. In the BVDV experiment, two calves persistently infected with BVDV (PI-calves) instead of experimentally infected calves, were used as the source of the virus. In all the calves infections were monitored using virus and antibody detection. Results showed that all the three viruses were transmitted by air. BHV1 spread to sentinel calves in the adjacent stable within three days, and BRSV within nine days, and BVDV spread to sentinel calves probably within one week. Although airborne transmission is possibly not the main route of transmission, these findings will have consequences for disease prevention and regulations in control programmes.

An experimental multivalent bovine virus diarrhea virus E2 subunit vaccine and two experimental conventionally inactivated vaccines induce partial fetal protection in sheep.

The primary aim of a bovine virus diarrhea virus (BVDV) vaccine is to prevent transplacental transmission of virus. We studied the efficacy of two experimental conventionally inactivated vaccines, based on BVDV strain Singer and containing a different antigen amount, against three antigenically different BVDV strains in a vaccination-challenge experiment in sheep. We also studied the efficacy of an experimental multivalent E2 subunit vaccine against four antigenically different BVDV strains. The vaccine contained the glycoproteins E2 of BVDV strains that belong to antigenic groups IA, IB and II. All three vaccines induced neutralizing antibodies against all challenge strains. Only the conventional vaccine that contained the highest antigen amount induced complete protection against homologous challenge. Neither of the conventional vaccines provided complete protection against heterologous challenge. The multivalent subunit vaccine induced partial protection against the homologous challenge strains. However, the immune response did inhibit virus replication in ewes, as shown by the results of the virus titrations.

Vaccination of cattle against bovine viral diarrhoea.

This brief review describes types and quality (efficacy and safety) of bovine viral diarrhoea virus (BVDV) vaccines that are in the market or under development. Both conventional live and killed vaccines are available. The primary aim of vaccination is to prevent congenital infection, but the few vaccines tested are not highly efficacious in this respect, as shown in vaccination-challenge experiments. Vaccination to prevent severe postnatal infections may be indicated when virulent BVDV strains are prevalent. Live BVDV vaccines have given rise to safety problems. A complication for the development of BVDV vaccines is the wide antigenic diversity among wild-type BVDV. There is ample room for improvement of both the efficacy and safety of BVDV vaccines, and it may be expected that better vaccines, among which marker vaccines, will be launched in the future.

The immune response of cattle, persistently infected with noncytopathic BVDV, after superinfection with antigenically semi-homologous cytopathic BVDV.

Cattle persistently infected (PI) with noncytopathic (ncp) bovine virus diarrhea virus (BVDV) are at risk for developing fatal mucosal disease (MD), which is considered to occur after superinfection with antigenically homologous cytopathic (cp) BVDV. In this study, we intranasally inoculated four PI-animals, that were PI with 2 ncp BVDV strains with 10(5) TCID50 antigenically closely related cp BVDV. Two PI-animals were inoculated with 10(5) TCID50 ncp BVDV and one PI-animal, with virus free cell culture medium. Two out of four PI-animals that were inoculated with cp BVDV, developed MD and were euthanized at day 17 and at day 24 after infection. Postmortem, both animals showed typical lesions of MD and cp BVDV was isolated. The other two PI-animals that were inoculated with cp BVDV did not develop MD and were euthanized at day 51. They showed ulcerations in the gastrointestinal tract, cp BVDV was isolated and neutralizing antibodies were detected. From the three PI-animals, that were inoculated with ncp BVDV or cell culture medium, cp BVDV was also isolated. Cross neutralization tests were performed and no antigenic differences could be detected between the cp strains isolated from the PI-animals. Lymphocyte subsets of these PI-animals were determined by flow cytometric analysis. Before superinfection, the percentages of gamma delta subsets were much higher in the PI-animals that did not develop MD than in nonviremic control animals and in the PI-animals that died of MD. From this study we conclude that the presence of antigenically closely related cp BVDV in PI-animals does not necessarily lead to the development of MD and that besides the antigenic relatedness between the persisting ncp BVDV and cp BVDV other factors, for instance the number of circulating gamma delta cells, might determine whether or not PI-animals develop MD.

Distribution of bovine virus diarrhoea virus in tissues and white blood cells of cattle during acute infection.

This study is performed to gain knowledge about the quantitative distribution of bovine virus diarrhoea virus (BVDV) in tissues and white blood cells (WBC) at different intervals after acute infection. Ten specific pathogen-free calves were intranasally inoculated with 10(5) 50% tissue culture infective dose of the non-cytopathic BVDV strain 4800. Twelve hours after inoculation tonsil biopsies were taken and WBC were collected daily for virus isolation and titration. Each day one calf was killed and virus isolations and titrations were performed from a range of tissues. The results indicate that BVDV first replicates in nasal mucosa and to high titers in the tonsil. The virus then appeared to spread to the regional lymph nodes and then disseminates throughout the body. The virus titers were highest in tonsil, thymus and ileum and were low in the WBC. Also after in vitro infection virus titers in WBC were very low, whereas, they were high in epithelial cells. Although the WBC might not be as important as other cells for replication of BVDV, they may play a role in the spread of the virus throughout the body.

A subunit vaccine based on glycoprotein E2 of bovine virus diarrhea virus induces fetal protection in sheep against homologous challenge.

The primary aim of a bovine virus diarrhea virus (BVDV) vaccine is to prevent transplacental transmission of virus. E2 genes of three BVDV strains, belonging to antigenic groups IA, IB and II, were expressed in insect cells. Three groups of 12 ewes were immunized twice with one of the E2 proteins. A fourth group served as a control. The ewes were served and the pregnant ewes of each vaccination group were allotted to three different challenge groups. Seven weeks after the second vaccination the ewes were challenged intranasally with one of the three BVDV strains. Three weeks later the fetuses were removed and fetal organs were collected for virus isolation. At the day of challenge all vaccinated ewes had neutralizing antibodies against the homologous BVDV strains. One E2 subunit vaccine prevented fetal infection after homologous challenge.

Subdivision of the pestivirus genus based on envelope glycoprotein E2.

Conventionally, the genus Pestivirus of the family Flaviviridae has been divided into bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV), and border disease virus (BDV). To date, BDV and BVDV have been isolated from different species, whereas CSFV seems to be restricted to swine. Pestiviruses are structurally and antigenically closely related. Envelope glycoprotein E2 is the most immunogenic and most variable protein of pestiviruses. We cloned E2 genes of many different pestivirus strains, including those from a deer and a giraffe. The E2 genes were transiently expressed, characterized with monoclonal antibodies, sequenced, and compared. Based on these data, we can delineate six major groups within the Pestivirus genus. Four groups correspond to defined genotypes, whereas the two other groups could be new genotypes within the Pestivirus genus. One group comprises CSFV strains isolated from swine. A second group consists of BDV strains Moredun, L83, and X818, which have been isolated from sheep, and strain F from swine. A third group contains strain BD78 from sheep, strain 5250 from swine, and strain 178003 from cattle. On the basis of E2, these viruses are very similar to BVDV strains associated with acute severe outbreaks of bovine viral diarrhea, so-called type 2 BVDV. The fourth group consists of BVDV strains originating predominantly from cattle. This BVDV group can be divided into two subtypes or subgroups BVDV Ia and Ib: BVDV Ia contains viruses from the United States, such as like NADL and Oregon, and some others, such as 150022 and 1138 from Europe. Subgroup BVDV Ib contains strain Osloss and several Dutch isolates. The fifth and sixth "groups" could be proposed as two new genotypes and contain strains Deer and Giraffe, respectively.

Glycoprotein Erns of pestiviruses induces apoptosis in lymphocytes of several species.

Classical swine fever virus and bovine virus diarrhea virus are members of the genus pestivirus, which belongs to the family of the Flaviviridae. Recently, envelope glycoprotein Erns was identified as an RNase. RNases can express different biological actions. They have been shown to be neurotoxic, antihelminthic, and immunosuppressive. We studied the immunosuppressive properties of Erns in vitro. The glycoprotein totally inhibited concanavalin A-induced proliferation of porcine, bovine, ovine, and human lymphocytes. We then studied the direct cytotoxic effects of Erns on lymphocytes and epithelial cells in protein synthesis assays. Erns strongly inhibited the protein synthesis of lymphocytes of different species, without cell membrane damage. This suggested an apoptotic process, and indeed apoptosis of lymphocytes was detected after incubation with Erns. Pestivirus infections are characterized by leukopenia and immunosuppression. Our results suggest that Erns plays an important role in the pathogenesis of pestiviruses.

Antigenically different pestivirus strains induce congenital infection in sheep: a model for bovine virus diarrhea virus vaccine efficacy studies.

To study the efficacy and safety of bovine virus diarrhea virus (BVDV) vaccines there is need for a valid challenge model. We investigated whether sheep can be used in such a challenge model. We intranasally inoculated six groups (A-F) of seronegative sheep at day 49 of gestation with either of five antigenically different BVDV strains and one border disease virus strain. A seventh group (G) was housed for 10 days with a persistently infected calf and an eighth group (H) served as control. From each group half of the sheep were killed at 2 weeks, and half at 4 weeks after infection. For virus isolation five organs were collected from the sheep and seven from the fetuses. All sheep of groups A and H remained seronegative in the ELISA and in the serum neutralization test. At 2 and 4 weeks after infection virus was isolated from almost all fetal organs in six groups. In group A and in the control group no virus was isolated from the fetal organs. The virus distribution patterns in fetuses from sheep housed with the persistently infected calf or intranasally inoculated with the same strain were similar. We concluded that (i) antigenically different BVDV strains can induce congenital infection in sheep and that (ii) the consequences of a contact infection were similar to those after intranasal infection. In a second experiment we infected two groups of seronegative sheep with one of the strains used in the first experiment, before mating. A control group was left uninfected. The sheep were served and all sheep were challenged with antigenically homologous or heterologous BVDV at day 49 of pregnancy. Three weeks after challenge, sheep were killed and the procedure as in the first experiment was followed. None of the fetuses sheep were virus positive whereas all fetuses of the control sheep were virus positive. Hence, the immune response after BVDV infection protects fetuses against homologous and heterologous infection during pregnancy. Sheep may therefore be used in vaccination-challenge experiments to evaluate BVDV vaccine efficacy in preventing congenital infection.