Pathologic lesions caused by coinfection of Mycoplasma gallisepticum and H3N8 low pathogenic avian influenza virus in chickens.

Chickens were infected under experimental conditions with Mycoplasma gallisepticum and low pathogenic avian influenza (LPAI) strain A/mallard/Hungary/19616/07 (H3N8). Two groups of chickens were aerosol challenged with M. gallisepticum strain 1226. Seven days later, one of these groups and one mycoplasma-free group was challenged with LPAI H3N8 virus; one group without challenge remained as negative control. Eight days later, the birds were euthanized and examined for gross pathologic and histologic lesions. The body weight was measured, and the presence of antimycoplasma and antiviral antibodies was tested before the mycoplasma challenge, before the virus challenge, and at the end of the study to confirm both infections. Chickens in the mycoplasma-infected group developed antibodies against M. gallisepticum but not against the influenza virus. Chickens of the group infected with the influenza virus became serologically positive only against the virus, while the birds in the coinfected group developed antibodies against both agents. The LPAI H3N8 virus strain did not cause decrease in body weight and clinical signs, and macroscopic pathological lesions were not present in the chickens. The M. gallisepticum infection caused respiratory signs, airsacculitis, and peritonitis characteristic of mycoplasma infection. However, the clinical signs and pathologic lesions and the reduction in weight gain were much more significant in the group challenged with both M. gallisepticum and LPAI H3N8 virus than in the group challenged with M. gallisepticum alone.

Characterization of pseudorabies virus of wild boar origin from Europe.

Pseudorabies virus (PrV) infections appear to be more widely distributed in the European wild boar (Sus scrofa) population than assumed. In Europe, attempts to isolate and characterize the causative agents have been limited so far. We therefore collected and examined a total of 35 PrV isolates obtained from wild boar or hunting dogs in Germany, France, Spain, Italy, Slovakia and Hungary between 1993 and 2008. Restriction enzyme analysis of genomic DNA using BamHI showed that all isolates, except one, belonged to genogroup I but different subtypes were evident. For further investigations of the phylogenetic relationships, a 732-bp fragment of the glycoprotein C (gC) gene was amplified by PCR. Sequence analysis revealed about 40 variant positions within this fragment. Comparison of the nucleotide sequences supported the separation into a clade containing isolates from North-Rhine Westphalia, Rhineland-Palatinate (Germany), France and Spain (clade B) and an apparently more variable clade comprising isolates from Brandenburg, Baden-Wurttemberg, Saxony, Saxony-Anhalt (Germany), Slovakia, Hungary, Italy and France (clade A).

Efficacy of a live bovine herpesvirus type 1 marker vaccine under field conditions in three countries.

The performance of a live marker vaccine for bovine herpesvirus type 1 (bhv-1) was studied in the field in three European Union countries with different farming conditions. The progress in the eradication of the virus was followed in a large herd in Germany and one in Italy, and a major serological survey involving 147 farms was conducted in Hungary. Commercial batches of the same vaccine were used in all three studies. The herds were vaccinated according to agreed protocols and the animals' bhv-1 antibody status was determined at local institutes by using commercial glycoprotein B (gB)- and glycoprotein E (gE)-elisas. In all three studies, the seroprevalence of bhv-1 gE decreased progressively. Given the starting conditions and the long duration of the studies, reactivation events and virus circulation would have been more likely to have occurred if the herds had not been vaccinated.

The detection of an unidentified type of adenovirus in the stools of calves with weak calf syndrome by use of a commercial kit designed for the detection of human adenoviruses.

An outbreak of polyarthritis in newborn calves in a large collective dairy herd was characterized by intra-articular blood-tinged synoviae, blood tainted faeces and massive subcorneal haemorrhages. Faecal samples from eight clinical newborn cases, 10 from unrelated dairy farms and 10 faecal samples from healthy calves were examined by the Rida Quick rotavirus/adenovirus-combi test . A specific adenovirus antigen precipitin-line was seen in the reaction in all the faecal samples from the diseased calves (n = 8), while all the others (n = 20) were negative. In addition, the same positive reaction was noted when one aqueous humor and two synovial samples were tested with this kit. Several other enteropathogens were found sporadically, but no conclusive significance could be attributed to their presence. Bovine viral diarrhoea and infectious bovine rhinothracheitis viruses as well as Chlamydia spp. and Mycoplasma spp. were not involved in this episode.

Comparison of methods for the diagnosis of equine herpesvirus type 1 infection.

The objective of the investigations was to study the occurrence of the equine herpesvirus type 1 (EHV-1) infection in aborted equine fetuses and in newborn foals and to compare the sensitivity of virus isolation, immunohistochemistry and histology in 101 cases and of fetal serology in 68 cases in the diagnosis of the infection. Out of the 93 aborted equine fetuses and 8 weak foals, 15 (14.9%) (14 fetuses and 1 foal) proved to be EHV-1 infected by immunohistochemical and 13 (12.9%) by virological investigation. Characteristic microscopic changes were seen in several organs in all cases, while intranuclear inclusion bodies could be found only in 25 (35.2%) of the 71 virus-positive tissue samples. Four (5.9%) cases proved to be positive by fetal serological investigation, but none of these cases showed any EHV-1 specific lesions and in none of these cases could the virus be detected by virus isolation or by immunohistochemistry. According to the results, fetal serology does not seem to be a useful test in virus-positive cases, while the immunohistochemical method seems to be a reliable and a slightly more sensitive method than virus isolation in the diagnosis of EHV-1 infection.

Pathogenicity of a bovine viral diarrhoea virus strain in pregnant sows: short communication.

The biological properties of bovine viral diarrhoea virus (BVDV) strain Oregon C24V were studied after intranasal and subcutaneous infection of pregnant sows. This virus strain is widely used in Hungary for immunising cattle against bovine viral diarrhoea (BVD). Based upon the results of the clinical, gross pathological, histopathological and virological examinations it can be established that the given strain caused asymptomatic infection and serological conversion in sows that were in the second third of gestation. The virus caused clinically apparent disease in some of the piglets born at term, which indicates that it had crossed the placenta. More than half (57%) of the live-born piglets died within 60 days of birth. The sows and their progeny did not shed the virus. BVDV infection has great differential diagnostic importance in pigs, as classical swine fever (CSF) virus strains of reduced virulence cause similar clinical symptoms and gross and histopathological changes.

Use of valnemulin in the control of Mycoplasma bovis infection under field conditions.

In a blind trial, alternate calves in six consecutive production batches of calves (total 70), on a farm with a high incidence of respiratory and reproductive disease, were allocated to treatment with either valnemulin or a placebo premix added to the milk from four days of age. The calves were weighed at the beginning and end of a 21-day period of medication. Blood samples and nasal swabs were taken and examined for the presence of Mycoplasma and Pasteurella species, and antibodies to viral agents. Clinical condition, rectal temperature, respiratory and other signs and refusals of milk were recorded daily. Dead calves were examined postmortem. The calves medicated with valnemulin gained weight more quickly, had fewer cases of Mycoplasma infection and fewer respiratory signs, and required fewer treatments with antibiotics than those in the placebo group.

The role of egg drop syndrome virus in acute respiratory disease of goslings.

An outbreak of severe acute respiratory disease characterized by tracheitis and bronchitis was observed in young goslings on a large-scale goose farm in Hungary. Histological examination revealed amphophilic intranuclear inclusion bodies in the superficial epithelial cells of the trachea and bronchi. Adenovirus-like particles were detected by electron microscopy, and the virus isolated from the trachea and the lungs was identified as egg drop syndrome (EDS) virus by serological and genomic examination. The clinical and pathological signs were reproduced by intratracheal administration of the virus isolate to 1-day-old goslings free of EDS antibodies. The presence of EDS virus DNA in different organs of the naturally and experimentally infected goslings was detected by polymerase chain reaction. This is the first report on the involvement of EDS virus in severe respiratory disease of geese.

Clinical study of the disease of calves associated with Mycoplasma bovis infection.

Clinical, bacteriological and serological examination of 35 calves from the age of 5 to 26 days was performed in a Holstein-Friesian dairy herd endemically infected with Mycoplasma bovis. M. bovis was isolated from 48.6% of nasal swabs taken from the calves at the age of 5 days, and from 91.4% of the same calves at the age of 26 days, indicating the gradual spread of infection. The isolation rate of Pasteurella multocida did not change much, and varied from 28.6 to 25.7%. No P. haemolytica could be detected. In addition to M. bovis and P. multocida, the herd was also infected with different viruses (including bovine viral diarrhoea virus, infectious bovine rhinotracheitis virus, bovine adenoviruses, parainfluenza-3 virus, and bovine respiratory syncytial virus) as a large proportion of the sera of newborn calves contained colostral antibodies against these viruses. In most of the newborn calves severe clinical signs (fever, depression, inappetence, hyperventilation, dyspnoea, nasal discharge and coughing) due to M. bovis infection developed. The clinical signs appeared already on the fifth day of life, and their incidence was the highest at the age of 10 to 15 days. Three calves (8.6%) died as a result of severe serofibrinous pneumonia. The surviving calves showed very poor weight gain (ranging from 1.5 to 3.5 kg) during the first two weeks of life.

Gene immunization of mice with plasmid DNA expressing rabies virus glycoprotein.

Gene immunization can be an effective vaccine strategy eliciting both humoral and cell-mediated immune responses. We constructed plasmid vectors expressing the full-length Vnukovo-32 rabies virus glycoprotein G under the control of CMV IE promoter and enhancer, adenovirus tripartite leader sequences and poly A signal of SV40. The gene vaccines were evaluated for the ability to elicit neutralizing antibodies and to protect BALB/c mice against lethal rabies virus challenge. First, mice were injected intramuscularly (i.m.) into the left hind leg and by the intradermoplantar (i.d.p.) route with equal amounts of plasmid DNA (0.25-0.1 mg). Two weeks later, immunization was boosted with an additional dose of the DNA. The immunized mice were challenged by intracerebral (i.c.) inoculation of CVS-27 (10-50 LD50) rabies virus. All mice produced anti-rabies virus neutralizing antibodies with a titre of > or = 1:45 after immunization with 0.1-0.4 mg of DNA. In challenge experiments, 83 to 91.6% protection was observed. These results confirm that a DNA vaccine could be a simple and effective solution for preventing the spread of rabies.

Analyses of restriction fragment patterns (RFPs) and pathogenicity in baby mice of equine herpesvirus 1 and 4 (EHV-1 and EHV-4) strains circulating in Danish horses.

Twenty-five strains of equine herpesvirus 1 (EHV-1) and one strain of equine herpesvirus 4 (EHV-4) isolated from material from various clinical cases in Denmark, together with reference EHV-1 and EHV-4 strains, were compared by restriction fragment pattern (RFP) analysis and inoculation of baby mice. The RFP analyses revealed that all EHV-1 strains belonged to genome type Ip. Four fetal isolates exhibited genomic characteristics that have been suggested as specific markers of the attenuated strain Rac H, widely used as a live vaccine. As the use of five vaccines against EHV-1 and EHV-4 has never been allowed in Denmark, it is assumed that Rac H derivatives have been acquired from visiting horses and thus are now circulating in the horse population. Baby mice inoculation revealed that four biotypes could be distinguished on the basis of pathogenicity. However, no strict correlation with pathogenicity in the natural host was seen.

Estimation of herd incidence of infection with bovine virus diarrhoea virus (BVDV) in herds previously without animals persistently infected with BVDV.

On a previous occasion, all animals in 9 herds had been bled for bovine virus diarrhoea virus (BVDV) and antibodies. No animals persistently infected (PI) with BVDV were detected. Three years later 10 animals in each herd were tested again. By this time 60 out of 90 previously seronegative animals had seroconverted. Seroconversions had occurred in 8 of the 9 herds corresponding to a incidence risk of herd infection of 0.52 per year. The incidence risk of seroconversions in individual animals was 0.31. Examination of young stock for antibodies and determination of antibody titer in bulk milk were good indicators for ongoing infections in the herds. The number of herd infections seemed to be higher than could be explained from purchase of PI animals.

Attempts at preventing further spread of bovine virus diarrhoea virus (BVDV) infection in 5 Danish dairy herds in which BVDV had been isolated.

In 5 herds in which bovine virus diarrhoea virus (BVDV) had been isolated, all animals were bled for virological and serological examination. After the herd blood test, follow up blood tests were made on calves born up to 6 months later in 1 herd, 9 months later in 1 herd and up to 12 months later in 3 herds. Persistently infected animals (PI animals) were removed and after a time period a small herd sample of 10 animals that were born after removal of the PI animals were examined for BVDV antibodies. At the herd blood test a total of 21 PI animals were detected. During the follow up period another 25 PI animals were born. Among animals in the small herd samples collected after removal of the PI animals, antibody positive animals were found in the 2 herds with the shortest follow up period. In the 3 herds with a 1 year follow up period there were no antibody carriers in the herd sample. It seems possible to prevent further spread of infection with BVDV if all animals in the herds as well as animals born during the following year are examined and PI animals removed.

Immune reactions and immunopathologic changes induced in ovine fetuses and lambs by maedi-visna virus.

Seventy- to 80-day fetuses of Merino ewes were inoculated intramuscularly in utero and 2-week-old lambs of the same breed intratracheally with 10(6.3) TCID50/0.1 ml of maedi-visna virus strain K1512 isolated in Iceland. While no precipitins appeared in the serum of fetuses, such antibodies were demonstrable in the lambs from postinoculation (PI) day 30. Indirect immunofluorescence (IIF) revealed the presence of antibodies in samples from both fetuses and lambs; the detectability of these antibodies, however, varied even within a given animal during the experiment. The serologic results were inversely proportional to the kinetics of circulating immune complex (cIC) production. By the lymphocyte stimulation test (LST), the blastogenic transformation of lymphocytes as measured by 3H-TdR incorporation was 6-8% and 6-14% in the fetuses and lambs, respectively. By antibody-dependent cell-mediated cytotoxicity (ADCC) test, cytotoxic capacity (10-14%) was only demonstrable in lambs inoculated at 2 weeks of age, in the 2nd month of life. Histologic examination showed that in the lungs of both fetuses and lambs lympho-histiocytic infiltration developed from PI week 4. This was later joined by lymphoid hyperplasia in the peribronchial lymph nodes. T lymphocyte proliferation was dominant in these lesions as shown by a histochemical procedure (alpha-naphthyl-acetate-esterase, ANAE). By immunofluorescence (IF), deposited immune complexes (IC) were demonstrable in various organs (wall of cerebral ventricles, endothelium of blood vessels of the brain stem, cerebellum, lungs, kidneys). These IC may play an important role in the pathogenesis of maedi-visna.

The pathology of concurrent bovine viral diarrhoea and infectious bovine rhinotracheitis virus infection in newborn calves.

Concurrent bovine viral diarrhoea (BVD) and systemic infectious bovine rhinotracheitis (IBR) are reported from two neonatal (11 and 15 days old) calves. The diseases occurred sporadically in a large-scale herd which may have been due to the calves' heterogeneous immunobiological status. Gross pathological and histopathological examinations revealed focal interstitial pneumonia with acidophilic intranuclear inclusions in the alveolar epithelial cells and necrotic foci in the liver with a few intranuclear inclusions in the hepatocytes. There were subserous haemorrhages in the forestomachs and intestine, necrotic changes in the rumen, enteritis, lymphocytic necrosis in the Peyer's patches, and fibrinoid necrosis in the wall of some of the neighbouring blood vessels. BVD virus was demonstrated by immunofluorescence (IF), whereas IBR virus by electron microscopy, immunofluorescence and virus isolation.

Testicular lesions in rams infected by maedi/visna virus.

Pathological lesions were observed in the testicles of 5 out of 7 rams found to be infected by maedi virus serologically and by histopathological examination of the lungs. The interstitium of the testicles was infiltrated with lymphocytes, histiocytes and plasma cells. The infiltration was mainly perivascular and of varying severity, and was accompanied by fibrosis. The seminiferous tubules neighbouring the severely affected parts were atrophied and, as a result, in circumscribed areas of the testicles disturbances of spermatogenesis were observed. The epididymides were devoid of pathological changes. Studies are in progress to determine whether the testicular lesions are actually caused by maedi/visna virus. Transmission of the virus with infected rams' semen is possible.