Fatal combined infection with canine distemper virus and orthopoxvirus in a group of Asian marmots (Marmota caudata).

A fatal combined infection with canine distemper virus (CDV) and orthopoxvirus (OPXV) in Asian marmots (Marmota caudata) is reported in this article. A total of 7 Asian marmots from a small zoological garden in Switzerland were found dead in hibernation during a routine check in the winter of 2011. The marmots died in February 2011. No clinical signs of disease were observed at any time. The viruses were detected in all individuals for which the tissues were available (n = 3). Detection of the viruses was performed by reverse transcription polymerase chain reaction. The most consistent gross lesion was a neck and thorax edema. A necrotizing pharyngitis and a multifocal necrotizing pneumonia were observed histologically. Numerous large intracytoplasmic eosinophilic inclusions were seen in the epithelial cells of the pharynx, of the airways, and in the skin keratinocytes. Brain lesions were limited to mild multifocal gliosis. Phylogenetic analysis revealed that the marmot CDV strain was closely related to the clusters of CDVs detected in Switzerland in wild carnivores during a local outbreak in 2002 and the 2009-2010 nationwide epidemic, suggesting a spillover of this virus from wildlife. The OPXV was most closely related to a strain of cowpoxvirus, a poxvirus species considered endemic in Europe. This is the first reported instance of CDV infection in a rodent species and of a combined CDV and OPXV infection.

Expression of interleukin 4, interleukin 4 splice variants and interferon gamma mRNA in calves experimentally infected with Fasciola hepatica.

Interleukin 4 (IL-4) is expected to play a dominant role in the development of T helper (Th) 2 cells. Th2 immune responses with expression of relatively large amounts of interleukin 4 (IL-4) but little interferon gamma (IFN-gamma) are characteristic for chronic helminth infections. But no information is available about IL4 expression during early Fasciola hepatica (F. hepatica) infections in cattle. Therefore, we investigated F. hepatica specific IL-4 and IFN-gamma mRNA expression in peripheral blood mononuclear cells (PBMCs) from calves experimentally infected with F. hepatica. Cells were collected prior to infection and on post-inoculation days (PIDs) 10, 28 and 70. Interestingly, PBMCs responded to stimulation with F. hepatica secretory-excretory products (FhSEP) already on PID 10 and expressed high amounts of IL-4 but not of IFN-gamma mRNA suggesting that F. hepatica induced a Th2 biased early immune response which was not restricted to the site of infection. Later in infection IL-4 mRNA expression decreased whereas IFN-gamma mRNA expression increased slightly. Isolated lymph node cells (LNCs) stimulated with FhSEP and, even more importantly, non-stimulated LN tissue samples indicated highly polarized Th2 type immune responses in the draining (hepatic) lymph node, but not in the retropharyngeal lymph node. During preliminary experiments, two splice variants of bovine IL-4 mRNA, boIL-4delta2 and boIL-4delta3, were detected. Since a human IL-4delta2 was assumed to act as competitive inhibitor of IL-4, it was important to know whether expression of these splice variants of bovine IL-4 have a regulatory function during an immune response to infection with F. hepatica. Indeed, IL-4 splice variants could be detected in a number of samples, but quantitative analysis did not yield any clue to their function. Therefore, the significance of bovine IL-4 splice variants remains to be determined.

Interferon-gamma and interleukin-4 mRNA expression by peripheral blood mononuclear cells from pregnant and non-pregnant cattle seropositive for bovine viral diarrhea virus.

The acceptance of the fetal allograft by pregnant women and mice seems to be associated with a shift from a Th 1 dominated to a Th 2 dominated immune response to certain infectious agents. The goal of this study was to examine cytokine expression in peripheral blood mononuclear cells (PBMCs) from cattle immune to bovine viral diarrhea virus (BVDV) to determine whether pregnancy also has an influence on the type of immune response in this species. Forty-six heifers and cows between 14 months and 13 years of age were included in this study. Twenty-four were seropositive and 22 seronegative for BVDV. Eleven of the seropositive animals and 11 of the seronegative animals were in the eighth month of gestation, the remaining animals were virgin heifers. PBMC from these animals were analyzed for Interferon (IFN)-gamma and Interleukin (IL)-4 mRNA expression by real-time RT-PCR after stimulation with a non-cytopathic strain of BVDV. Additionally, an ELISA was performed to measure IFN-gamma in the supernatants of stimulated cell cultures. In BVDV seropositive animals, IFN-gamma mRNA levels were significantly higher than in BVDV seronegative animals and there was a significant positive correlation between the changes in IFN-gamma and IL-4 mRNA expression. There was, however, no significant difference in IFN-gamma and IL-4 mRNA levels between pregnant and non-pregnant animals. These results are inconsistent with BVDV inducing a Th1 or Th2 biased immune response. Furthermore, a shift in the cytokine pattern during bovine pregnancy was not evident.

Expression of inducible nitric oxide synthase in spontaneous bovine bronchopneumonia.

The expression of inducible nitric oxide synthase (iNOS), major histocompatibility class II molecules (MHC-II), CD68, and the calcium-binding proteins S100A8 and S100A9 (also called MRP8 and MRP14, respectively) was assessed in lung tissues from cattle that succumbed to pneumonia. Expression patterns of these markers were related to the types of lung lesion. iNOS expression was only observed in lungs infected with Arcanobacterium pyogenes or Pasteurella haemolytica but not in lungs from cattle with subacute chronic interstitial pneumonia and acute interstitial pneumonia due to Escherichia coli infection. High levels of iNOS were expressed by cells (probably leukocytes) surrounding necrotic foci. Occasionally, iNOS was expressed by intraalveolar macrophages in viable parenchyma, by leukocytes within the airways, and by some chondrocytes in the supporting cartilage of bronchi. Cells expressing MHC-II were distributed relatively evenly throughout areas of inflammation and did not display any clear association with necrotic foci. Cell types expressing MHC-II included type II alveolar epithelial cells, spindle-shaped cells of the interstitium, cells in bronchus-associated lymphoid tissue, and leukocytes in lymph and blood vessels but largely excluded iNOS-positive cells. Likewise, CD68-positive cells were rarely positive for iNOS and were not confined to the areas surrounding necrotic tissue. As with MHC-II and CD68, there was little if any coexpression of iNOS and either of the S100 proteins tested. Thus, in cattle with necrotizing bronchopneumonia, iNOS-expressing cells were largely restricted to the cellular zone surrounding necrotic areas.

Non-suppurative myocarditis in piglets associated with porcine parvovirus infection.

The involvement of porcine parvovirus (PPV) in the aetiology of non-suppurative myocarditis in sucking piglets was investigated by a polymerase chain reaction (PCR), designed to assess the presence of viral genome in formalin-fixed paraffin wax-embedded tissue of diseased animals. Myocardium and lung of stillborn piglets with a confirmed PPV infection were used to set up the PCR amplification method. Subsequently, 20 myocardia with inflammatory lesions were examined in parallel with 20 myocardia without lesions, from age-matched control piglets. Tissues were first tested for the presence and the integrity of porcine DNA by amplifying a sequence encoding the highly conserved nuclear protein histone H4. Tissue from 15 out of 20 animals with myocarditis contained amplifiable histone H4 DNA and in 12 of the 15 histone H4-positive samples, PPV DNA was detected. It proved possible to amplify histone H4 DNA in all 20 negative controls (without myocarditis), and PPV DNA was detected in three cases. In-situ hybridization with a digoxigenin-labelled probe homologous to PPV was performed in four PCR-positive cases of non-suppurative myocarditis. In two animals several positively stained nuclei were observed in the myocardium, within or close to the mild inflammatory cellular infiltrates. These results strongly suggest that PPV can cause non-suppurative myocarditis in sucking piglets.

Diagnosis of fetal infection with porcine parvovirus by in situ hybridization.

In situ hybridization (ISH) for the diagnosis of fetal infection with porcine parvovirus (PPV) was compared with immune electron microscopy (IEM) and serology by immunofluorescence (IF) for its sensitivity and its applicability in a routine diagnostic laboratory. The technique was applied to the examination of sections of formalin-fixed paraffin-embedded tissues from 68 fetuses. Fifty-three of these fetuses were diagnosed serologically since they had a crown rump length of more than 17 cm, i.e. they were mature enough to mount a humoral immune response; 38 were positive and 15 negative. Eleven out of 15 smaller fetuses examined for the presence of viral antigen by immune electron microscopy (IEM) were positive and 4 were negative. Heart and lung were found to be the most suitable organs for in situ hybridization. In situ hybridization yielded a positive result in 8 of the 11 IEM positive fetuses and in 33 of the 38 serologically positive fetuses. No signal was detected in any of the 4 IEM or the 13 serologically negative fetuses. Expenses for IEM were estimated to be 179% of the expenses for ISH. Expenses for serology by IF on the other hand were 67% of the expenses for ISH. From this it was concluded that the most efficient way to diagnose a fetal infection with PPV was serology by IF, if possible with samples from several fetuses and that the other techniques, IEM or ISH, ought to be reserved for those cases where no immunocompetent fetuses were available for diagnosis.

An immunohistochemical study of the distribution of border disease virus in persistently infected sheep.

Three seronegative sheep persistently infected with Border disease virus and six seropositive, non-viraemic sheep were examined for the cellular distribution of the agent. These animals originated from a closed flock which had been kept in an isolation facility for 5 years. They were killed and immediately necropsied. There were no gross abnormalities other than reduced body weight of the persistently infected sheep. Two samples of each major organ were collected. The first sample was fixed by immersion in formalin and processed for histological examination, which showed no lesions unequivocally attributable to the viral infection. The second sample was snap-frozen for immunohistochemical examination. This revealed viral antigen in all organs of the persistently infected, but in none of the seropositive animals. The infected cells included smooth muscle cells of hollow organs and blood vessels, epithelial cells of the alimentary tract and urogenital organs, lymphocytes in lymphoid organs, endocrine cells, neurons and glial cell.

[The infectious causes of abortion and stillbirth in swine in Switzerland]. / Untersuchungen zu infektiösen Ursachen für Aborte und Totgeburten beim Schwein in der Schweiz.

Fetuses and placentae of 171 cases of porcine abortion, stillbirth and mummification were examined for pathological lesions, bacterial infections and PPV (porcine parvovirus) infection. Furthermore IgG (immunoglobulin G) levels were determined in fetal body fluids. Selected maternal sera were tested for antibodies against Leptospira, Aujeszky's disease and hog cholera. PPV infection was diagnosed in 29.2% of all cases. Bacterial abortion was diagnosed in 8.2%. Indications for an infectious agent were demonstrated in about 10% of the cases. The etiology of the abortion could not be identified in 52%. Inflammatory alterations in association with the isolation of bacteria consisted of neutrophilic infiltration and necrosis and were of particular value to differentiate bacterial contamination from infection. Infiltrations of mononuclear leucocytes in brain, leptomeninx, kidney or myocard were observed in about 50% of the fetuses with PPV infection. IgG levels were consistently elevated in fetuses serologically positive for PPV, but also in two fetuses, where no infectious agent could be identified.

Specific diagnosis of parvovirus enteritis in dogs and cats by in situ hybridization.

In a retrospective study, the fixed intestines of 10 dogs and 10 cats with intestinal lesions characteristic of parvovirus infection were assayed for the presence of parvovirus by in situ hybridization and immunohistochemistry. Parvoviral nucleic acid was localized by in situ hybridization in intestinal tissue in all 10 dogs and in nine of the 10 cats, whereas antigen was detected only in seven of 10 canine and eight of 10 feline intestines by immunohistochemistry. We conclude that an aetiological diagnosis can be established with a high degree of certainty by routine histology. Demonstration of the infectious agent by in situ hybridization, however, proves to be a valuable specific tool which allows an exact cellular localization of parvovirus in formalin-fixed, paraffin wax-embedded tissue sections.

Association of virulent and avirulent strains of bluetongue virus serotype 11 with premature births of late-term bovine fetuses.

Two strains of bluetongue virus serotype 11 (BTV 11), UC-2 avirulent and UC-8 neurovirulent in newborn mice, were inoculated into late-term bovine fetuses to investigate whether infection with these two BTV strains in late gestation would produce congenital infection and pathological changes. Fetuses were inoculated by intramuscular injection through the uterine wall at 243 days gestation and recovered after spontaneous delivery. In calves inoculated with UC-8, births occurred 15 to 27 days before expected parturition, resulting in small, weak calves. These calves had a mild encephalitis and were unthrifty at birth. Calves inoculated with UC-2 appeared healthy when born 7 to 11 days prior to expected parturition. No lesions were found in these calves at necropsy. All calves seroconverted by the time of birth. Viraemia was present in the calves inoculated with UC-8 and in one calf inoculated with UC-2. Plasma cortisol concentrations were prematurely elevated, particularly in the calves inoculated with UC-8, indicating that they were stressed by the infection. The elevated cortisol, associated with an active congenital infection caused by bluetongue virus serotype 11 strain UC-8, is capable of causing premature delivery of low birth-weight, weak calves.

Infection of bovine fetuses at 120 days' gestation with virulent and avirulent strains of bluetongue virus serotype 11.

Bluetongue virus infection in sheep and cattle during fetal development causes neuropathology. Two strains of bluetongue virus serotype 11 designated as UC-2 and UC-8 have different virulence patterns in newborn mice. These viruses have distinctly different electropherotype patterns on polyacrylamide gel electrophoresis indicating a genetic difference in these two viruses of the same serotype. Four bovine fetuses each were inoculated intramuscularly with either UC-2 or UC-8, and one fetus was inoculated with placebo. The inoculation was made intramuscularly through the uterine wall at 120 days' gestation, and the bovine fetuses were recovered by cesarean section 12 or 20 days after inoculation. Fetal blood was collected for virus isolation and serology. Virus was reisolated from brain, blood, lung and liver. Both strains, UC-2 and UC-8, cause severe lesions in the 120 day fetuses. The encephalomalacic lesions occurred earlier and were more severe in fetuses inoculated with UC-8 as compared to those inoculated with UC-2. The subtle differences observed in the fetuses inoculated with the two different strains suggest that there is a difference in pathogenic potential of the two viruses. These differences do not appear to be completely dependent upon the host species.

Retrospective study of myocardial canine parvovirus infection by in situ hybridization.

The diagnosis of myocardial canine parvovirus (CPV) infection used to depend on the presence of pathognomonic intranuclear inclusion bodies. The in situ hybridization technique, however, allowed to detect CPV specific nucleic acid in myocardial tissue where no inclusion bodies were found. Hence, we applied this technique to check formalin-fixed paraffin-embedded myocardial tissue from puppies with heart lesions for the presence of CPV. The tissues had been collected between 1977 and 1989. A biotinylated probe was used for in situ hybridization. This way CPV specific nucleic acid was detected in 3 dogs where CPV myocarditis had not been diagnosed on routinely stained slides because of the lack of intranuclear inclusion bodies. However, in spite of the application of the in situ hybridization technique no further myocardial CPV infection was detected in puppies with heart lesions from after 1979, confirming that the number of puppies with myocardial CPV infection declined after that year.

Detection of bluetongue virus in bovine fetuses using the avidin-biotin complex immunoperoxidase method.

The avidin-biotin complex immunoperoxidase technique was adapted for use in detecting bluetongue virus (BTV) antigens in BTV serotype 11-infected bovine fetuses. Fetuses were infected with BTV serotype 11 at 120 days of gestation and then removed 20 days later by Cesarean section. Blood and tissue samples were collected from each animal and used for virus isolation in embryonated chicken eggs, the immunofluorescent antibody test, and the avidin-biotin complex test. The avidin-biotin complex method successfully identified BTV antigens in both fresh and autolyzed fetal brains. Thus, the avidin-biotin complex immunoperoxidase method has potential as a possible procedure for diagnosing bluetongue disease in aborted bovine fetuses.

Neurovirulence of the UC-2 and UC-8 strains of bluetongue virus serotype 11 in newborn mice.

In vivo and in vitro experiments were done to investigate whether the difference in neurovirulence between the two strains of bluetongue virus 11, UC-2 and UC-8, is based on a different capability to gain access to the brain from the subcutaneous inoculation site or on a different tropism for neural cells. In newborn Balb/c mice subcutaneous inoculation of UC-8 at doses between 10(-0.2) plaque forming units (PFU) and 10(4.8) PFU caused a severe necrotizing encephalitis whereas UC-2 at doses of up to 10(4.4) PFU did not affect newborn Balb/c mice. However, intracranial inoculation of 10(2.4) PFU of either virus strain produced severe necrotizing encephalitis. In vitro both virus strains infected dissociated brain cell cultures similarly. Double labelling immunofluorescent staining with markers specific for neural cells did not reveal differences in the target cells for the two viruses. The difference in neurovirulence between UC-2 and UC-8, therefore, appears to be determined by the ability of UC-8 to infect the brain from a subcutaneous inoculation site.

Tropism of border disease virus for oligodendrocytes in ovine fetal brain cell cultures.

Primary dissociated ovine brain cell cultures prepared from 50- or 140-day-old fetuses were inoculated with border disease virus (BDV). The cells present in the cultures were identified, using immunofluorescence procedures and sera against various CNS cell-specific markers. These markers were glial fibrillary acidic protein, myelin basic protein, myelin-associated glycoprotein, neuron-specific enolase, neurofilament protein, and fibronectin. Double-labeling immunofluorescence techniques for visualization of BDV antigen and of the CNS cell markers were used to evaluate the pattern of individual cell susceptibility 48 hours after infection. In cultures from fetuses of both ages, about half of the infected cells were glial fibrillary acidic protein-positive astrocytes. Scattered myelin-associated glycoprotein-positive oligodendrocytes were positive for BDV antigen, but only in the infected cultures from the older fetuses. Fibronectin-positive cells were not infected with BDV. In infected and noninfected cultures, cells positive for neuron-specific enolase, myelin basic protein, or neurofilament protein were not seen. Therefore, the remaining infected cells in all the cultures were not identified by the cell-specific markers used. Results of these in vitro experiments indicate that BDV does not selectively infect oligodendrocytes, and that such a selective pattern of infection may not be the basis for the in vivo congenital hypomyelination in sheep with border disease.

Strain-dependent virulence characteristics of bluetongue virus serotype 11.

Two strains of bluetongue virus (BTV) serotype 11, UC-2 and UC-8, were identified by the electrophoretic migration pattern of their genomic RNA segments on polyacrylamide gel electrophoresis. Significant differences in virulence of these two viruses could be demonstrated by subcutaneous inoculation of newborn mice. No signs of disease were observed in mice infected with UC-2. Mice infected with UC-8 died of a severe necrotizing encephalitis, which resembled lesions in bovine and ovine foetuses infected with BTV.