G.J. Wellenberg

Viral infections and bovine mastitis: a review

This review deals with the role of viruses in the aetiology of bovine mastitis. Bovine herpesvirus 1, bovine herpesvirus 4, foot-and-mouth disease virus, and parainfluenza 3 virus have been isolated from milk from cows with clinical mastitis. Intramammary inoculations of bovine herpesvirus 1 or parainfluenza 3 virus-induced clinical mastitis, while an intramammary inoculation of foot-and-mouth disease virus resulted in necrosis of the mammary gland. Subclinical mastitis has been induced after a simultaneous intramammary and intranasal inoculation of lactating cows with bovine herpesvirus 4. Bovine leukaemia virus has been detected in mammary tissue of cows with subclinical mastitis, but whether this virus was able to induce bovine mastitis has not been reported. Bovine herpesvirus 2, vaccinia, cowpox, pseudocowpox, vesicular stomatitis, foot-and-mouth disease viruses, and bovine papillomaviruses can play an indirect role in the aetiology of bovine mastitis. These viruses can induce teat lesions, for instance in the ductus papillaris, which result in a reduction of the natural defence mechanisms of the udder and indirectly in bovine mastitis due to bacterial pathogens. Bovine herpesvirus 1, bovine viral diarrhoea virus, bovine immunodeficiency virus, and bovine leukaemia virus infections may play an indirect role in bovine mastitis, due to their immunosuppressive properties. But, more research is warranted to underline their indirect role in bovine mastitis. We conclude that viral infections can play a direct or indirect role in the aetiology of bovine mastitis; therefore, their importance in the aetiology of bovine mastitis and their economical impact needs further attention.

Isolation and characterization of porcine circovirus type 2 from pigs showing signs of post-weaning multisystemic wasting syndrome in the Netherlands

Summary Pigs with wasting syndrome were examined for macroscopic and histopathological lesions, and for porcine circovirus (PCV). Histopathological lesions were comparable to those previously documented for post‐weaning multisystemic wasting syndrome (PMWS). In addition, in seven out of ten examined PMWS‐affected pigs focal‐to‐slight mononuclear meningitis and focal cerebral mononuclear infiltrates (4 out of 10) were observed. A virus was isolated from organs and sera from pigs showing wasting syndrome. An immunoperoxidase monolayer assay and an indirect immunofluorescence assay were performed on the infected PK‐15 and Dulac cell cultures, respectively, and both assays indicated the presence of PCV type 2 (PCV2). The nested‐polymerase chain reaction (nPCR) technique, based on the use of PCV2 specific oligonucleotides, revealed specific amplified products of 481 bp. Nucleotide sequence analysis of the entire genome of the Dutch PCV isolate 24657 NL showed a homology with known nucleotide sequences of porcine PCV type 1 (PCV1) and PCV2 isolates of 77.1% and >96%, respectively. This is the first report of the isolation and characterization of PCV2 in PMWS‐affected pigs in the Netherlands.

Identification of Brachyspira hyodysenteriae and Other Pathogenic Brachyspira Species in Chickens from Laying Flocks with Diarrhea or Reduced Production or Both

ABSTRACT Cecal samples from laying chickens from 25 farms with a history of decreased egg production, diarrhea, and/or increased feed conversion ratios were examined for anaerobic intestinal spirochetes of the genus Brachyspira. Seventy-three samples positive in an immunofluorescence assay for Brachyspira species were further examined using selective anaerobic culture, followed by phenotypic analysis, species-specific PCRs (for Brachyspira hyodysenteriae, B. intermedia, and B. pilosicoli), and a Brachyspira genus-specific PCR with sequencing of the partial 16S rRNA gene products. Brachyspira cultures were obtained from all samples. Less than half of the isolates could be identified to the species level on the basis of their biochemical phenotypes, while all but four isolates (5.2%) were speciated by using PCR and sequencing of DNA extracted from the bacteria. Different Brachyspira spp. were found within a single flock and also in cultures from single chickens, emphasizing the need to obtain multiple samples when investigating outbreaks of avian intestinal spirochetosis. The most commonly detected spirochetes were the pathogenic species B. intermedia and B. pilosicoli. The presumed nonpathogenic species B. innocens, B. murdochii, and the proposed “B. pulli” also were identified. Pathogenic B. alvinipulli was present in two flocks, and this is the first confirmed report of B. alvinipulli in chickens outside the United States. Brachyspira hyodysenteriae, the agent of swine dysentery, also was identified in samples from three flocks. This is the first confirmed report of natural infection of chickens with B. hyodysenteriae. Experimental infection studies are required to assess the pathogenic potential of these B. hyodysenteriae isolates.

Clinically silent rabies infection in (zoo) bats

An outbreak of tetanus was observed in lambs from a flock of 600 ewes in the Sabzevar district of the Khorasan province in Iran during the winter of 1996. Over a 35-day period, 19 lambs showed signs of tetanus and 18 of them died. The flock (without lambs) grazed on a poor pasture and was supplemented with alfalfa at night. In this flock ear tagging of lambs was performed seven to eight days after birth. All the lambs were vaccinated against enterotoxaemia at 12 to 20 days of age. Tetanus in these lambs was observed at 15 to 38 days of age. The clinical signs exhibited initially were mild stiffness, especially in the forelimbs, and asymmetrical positioning of the ears. These symptoms progressed to severe stiffness and rigidity of the neck and limbs. Other symptoms included tremor, hypersensitivity to touching, lockjaw, dilation of nostrils, and recumbency with extension of the limbs in the terminal stages. All affected lambs, with the exception of one, died within five to 14 days of the appearance of the clinical signs. Haematological parameters (tested in three lambs) and serum calcium and magnesium levels (tested in two lambs) were within normal ranges. Cerebrospinal fluid was taken from the cisterna magna in three lambs and examined for the presence of aerobic and anaerobic bacteria; the results of the cultures were negative. Seven lambs were submitted for postmortem examination. There were no wounds of any significance on the animals other than at the ear tagging sites. Large size, locally made, plastic tags had inadvertently been applied too close to the base of the ears and the point of insertion of all of the tags was covered by a blood clot. Samples from these sites taken from five affected lambs were cultured anaerobically and a pure growth of C tetani was observed in two. Histopathological examination of the brain tissues did not reveal any lesions. Following diagnosis of the disease, the ear tagging was stopped in the newborn lambs and no new cases of tetanus were seen in the flock during 1996. The diagnosis in this outbreak was made on clinical grounds and laboratory findings. There was evidence that the lesions at the

Detection of bovine herpesvirus 4 glycoprotein B and thymidine kinase DNA by PCR assays in bovine milk.

A polymerase chain reaction (PCR) assay was developed to detect bovine herpesvirus 4 (BHV4) glycoprotein B (gB) DNA, and a nested-PCR assay was modified for the detection of BHV4 thymidine kinase (TK) DNA in bovine milk samples. To identify false-negative PCR results, internal control templates were constructed, added to milk samples, and co-amplified with viral DNA using the same primers for both templates. Specificity, sensitivity, and reproducibility of the two PCR assays were examined. In both PCR assays, all 31 BHV4 strains examined were scored positive, whereas 14 unrelated viruses scored negative. Sensitivity studies showed that two-ten copies of BHV4 DNA were detectable by the gB-PCR, while one-three copies could be detected by the TK-PCR. For the detection of BHV4 in milk samples, the gB-PCR amplification was found to be ten-times, and the TK-PCR was found to be 55-times more sensitive than virus isolation. BHV4 DNA was detected by gB-PCR and TK-PCR in 93 and 95%, respectively, of 61 milk samples collected from cows infected intramammarily with BHV4, while only 61% were positive by virus isolation. Four out of 48 cows with clinical mastitis were positive for BHV4-gB and BHV4-TK DNA, whereas no BHV4 DNA was detected in milk from control cows. Considerable agreement was seen between the results of the two PCR assays, and both methods were considered as rapid and reliable tests for the screening of BHV4 DNA in bovine milk. The less laborious gB-PCR might be the recommended test of choice for screening large amounts of milk samples for the presence of BHV4.

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

Summary. 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 nucleo-protein 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 neuro-blastoma 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.

Antibodies against bovine herpesvirus (BHV) 5 may be differentiated from antibodies against BHV1 in a BHV1 glycoprotein E blocking ELISA

We examined whether antibodies against bovine herpesvirus (BHV) 5 cross-react with BHV1 antigens and whether they could interfere with BHV1 eradication programmes. Six calves were experimentally infected with different doses of BHV5 strain N569; homologous antibodies were first detectable on day 11 post infection; they cross-reacted in a BHV1 virus neutralisation test, in a BHV1-glycoprotein (g)-B blocking ELISA and in a BHV1-gE ELISA, but not in a BHV1-gE blocking ELISA. This study indicates that, in ongoing BHV1 eradication programmes, based on vaccines that lack gE, BHV5 infections may not lead to false-positive serological reactions in case cattle are tested for BHV1-gE antibodies by the BHV1-gE blocking ELISA; antibodies against BHV5 may be differentiated from antibodies against BHV1. The BHV1-gE blocking ELISA may, therefore, offer opportunities for the serological differentiation between BHV1 and BHV5 infections.

Characterisation of a recently isolated lyssavirus in frugivorous zoo bats

Summary. In July 1997 a lyssavirus was isolated in Denmark from a colony of Egyptian flying foxes (Rousettus aegyptiacus) originating from a Dutch zoo. Sequencing of a 400 nucleotides coding region of the nucleoprotein and of a major part of the G-protein ectodomain encoding region of the newly isolated virus, revealed a very high similarity with European Bat Lyssavirus subtype 1a (EBL-1a). For characterisation of the recently isolated lyssavirus in frugivorous zoo bats, 16 frugivorous bats (Rousettus aegyptiacus) of the same colony and 80 mice were experimentally infected with the Rousettus isolate or with a well defined EBL-1a strain isolated from a Dutch insectivorous bat (Eptesicus serotinus). Inoculation viruses were titrated in mice to determine LD50‘s of both isolates. Clinical signs of inoculated bats were recorded during 6 weeks. After showing neurological signs or at the end of the experimental infection all animals were euthanized. During the experimental infection sera and various tissues of inoculated bats were collected. Immunoassays, mouse inoculation tests (MIT) and polymerase chain reaction (PCR) were employed for detection of lyssavirus specific antibodies, antigen or RNA. Five bats inoculated with the Rousettus isolate and 2 bats inoculated with the Eptesicus isolate showed neurological signs. The remaining 9 bats survived and cleared the virus; at least under the detection limit of the used assays. Despite a much higher pathogenicity of the Rousettus isolate observed in mice, LD25’s in bats were quite the same for the 2 isolates. The pathogenicity of both isolates suggested that like many other mammals, Rousettus aegyptiacus bats could be victims of lyssavirus infection besides reservoir hosts of infectious EBL1a. There was no significant difference in detecting the different lyssavirus isolates in Rousettus aegyptiacus bats. An employed immunoperoxidase staining (IP) method was very useful for sensitive detection and localization of lyssavirus antigen in histologic preparates.

Bovine herpesvirus 4 in bovine clinical mastitis

CLINICAL mastitis has the largest economic impact on the dairy cattle industry. Despite intensive bacteriological research, 20 to 35 per cent of clinical cases of bovine mastitis have an unknown aetiology (Miltenburg and others 1996, Barkema and others 1998). Although viral infections have occasionally been associated with bovine mastitis (Siegler and others 1984, Yoshikawa and others 1997), it is generally considered that viruses do not play a role in the aetiology of bovine mastitis (Watts 1988, Radostits and others 1994). This study was undertaken to gain more insight into the possible role of viruses in bovine clinical mastitis, due to the high percentage of unknown causes of clinical mastitis. In a case/control study, serum and milk samples were collected from 58 dairy cows with clinical mastitis in 10 different Dutch herds at the acute (day 0) and the convalescent phase (day 21) of the disease. Serum and milk samples were also taken from 58 healthy, matched control cows. The control cows were from the same herd, did not show mastitis symptoms, were of the same age as the mastitis cows and were in the same stage of lactation as the mastitis cows. Milk samples from the matched control cows were collected on the

Schmallenberg virus antibodies in bovine and ovine foetuses

Since December 2011, an epizootic of congenital malformations, designated as arthrogryposis-hydranencephaly syndrome (AHS), has been seen in ruminants in several countries in Western Europe (Van den Brom and others 2012). The disease has been associated with a new orthobunyavirus, named Schmallenberg virus (SBV) (Hoffmann and others 2012). After SBV was detected in brain tissue from malformed lambs by PCR on December 15, 2011, the Dutch Ministry of Economic Affairs, Agriculture and Innovation made congenital malformations in ruminants notifiable. Farmers and veterinarians are obliged to notify and submit malformed newborns to the Animal Health Service (GD-Deventer) for autopsy examination. Until May 15, 2012, over 600 lambs and over 1200 calves had been examined, and many of them showed AHS which is likely to be caused by an intrauterine infection with SBV. Brain tissue samples of all malformed newborns were tested by PCR at the Central Veterinary Institute (CVI). However, SBV could only be confirmed in 22 per cent of the lambs and 19 per cent of the calves that were investigated until May 15, 2012. Initially, a real-time PCR protocol targeting the L-segment was used. Since February 17, 2012, a real-time PCR protocol targeting the S-segment was used, since the latter PCR demonstrated a higher analytical and diagnostic sensitivity as indicated by the Friedrich Loeffler Institute in Germany and Dijkman and others (2012). Both protocols were kindly provided by the Friedrich Loeffler Institute in Germany. Persistent infection in Akabane virus-infected foetuses has not been reported, and the virus cannot be easily isolated from affected full-term calves or lambs because of the presence of neutralising (NT) …