Study on the epidemiology of equine arteritis virus infection with different diagnostic techniques by investigating 96 cases of equine abortion in Hungary

The occurrence of equine arteritis virus (EAV) induced equine abortions was studied with different laboratory methods during a 3-year period. Tissue samples from 96 aborted equine foetuses or newborn foals were collected from 57 farms located in different parts of Hungary. Virus isolation, polymerase chain reaction (PCR), immunohistochemistry and serology were used for the detection of EAV infection. The overall seroprevalence of EAV infection in mares was 65%. EAV induced abortion was diagnosed in eight (8.3%) cases from six (10.5%) herds. Abortion was sporadic in all herds except for one, where epidemic abortion happened. Fetal serology in six (75%) cases, the virus isolation in one (12.5%) case whereas PCR in all of the four investigated cases were positive. The virus could be observed with immunohistochemistry in seven (87.5%) cases mostly in the spleen followed by other organs and the allantochorion. In conclusion, PCR and immunohistochemistry seem to be the most sensitive and useful tests for the diagnosis of EAV induced equine abortion.     

Proliferative and necrotising pneumonia and severe vascular lesions in pigs naturally infected with porcine circovirus type 2

Severe disease induced by porcine circovirus type 2 (PCV2) was observed in three pigs originating from a large herd affected by respiratory and digestive signs as well as wasting. Proliferative and necrotising pneumonia (PNP) was diagnosed in two animals, while severe acute interstitial pneumonia characterised by the presence of abundant hyaline membrane in the alveoli and fibrin in the bronchioles was found in one pig. In all cases, large amounts of PCV2 antigen were found in each tissue sample collected from the lungs and mediastinal lymph nodes. Neither porcine reproductive and respiratory syndrome virus (PRRSV) nor swine influenza virus (SIV) was detected, and no bacteria could be cultured in any of the cases. Vascular lesions, e.g. degeneration of endothelial cells, perivascular and intramural oedema, fibrinoid necrosis, vasculitis, perivasculitis, and vascular thrombi were observed in all cases, associated with the presence of PCV2 antigen. The viral antigen was present in the intravascular mononuclear cells, endothelial cells, myocytes and infiltrating inflammatory cells in lymph and blood vessels. In one case, obliterating thrombi in the lymph and blood vessels were directly connected to areas of tissue necrosis and were associated with abundant PCV2 antigen. The results further suggest the causative role of PCV2 infection in PNP, and the importance of the vascular system in the pathogenesis of PCV2-associated diseases of swine.     

Application of anti-BCG antibody for rapid immunohistochemical detection of bacteria, fungi and protozoa in formalin-fixed paraffin-embedded tissue samples

The applicability of an anti-Mycobacterium bovis (BCG) antibody-based immunohistochemistry (IHC) procedure was investigated using everyday veterinary pathological samples collected from 13 different animal species. Fifty-one formalin-fixed and paraffin-embedded tissue samples were selected for this study. Forty, 4 and 7 tissue samples contained different species of bacteria, fungi and protozoa, respectively. Three serial sections were prepared in each case. Two sections were pre-treated with enzyme and heat, respectively, while the last section was not pre-treated. In seven cases the sensitivity of histochemical staining (HSM), IHC and bacteriological culture were compared. Heating of the sections in a microwave oven was the most effective method in the case of almost all pathogens used. Strong or moderate positive reactions were observed for 26 bacterial species, all fungal and 2 protozoal species, while weak reactions occurred for 2 bacterial and 1 protozoal species. Only 4 protozoal and 12 bacterial species, including Leptospira and all the five Mycoplasma species examined, showed no reaction in this test. IHC had almost the same sensitivity as bacteriological culture and was more sensitive than HSM. The IHC method presented here should be preferred to HSM as a general screening tool in cases where pathological lesions suspicious for infections are evident and no microorganism can be cultured in vitro or only formalin-fixed tissue samples are available for the laboratory examination.     

The comparison of two different extenders for the improvement of large‐scale sperm cryopreservation in common carp (Cyprinus carpio)

In our study, a traditionally used (Grayling, already used in cyprinid species) and a newly tested (Pike) extender was tested to avoid sperm agglutination phenomenon following thawing during carp sperm cryopreservation. A large‐scale (elevated volume of sperm) freezing method in a controlled‐rate freezer using 5 ml straw and 10 ml cryotube was also systematically established. In all experiments, the sperm cryopreserved in using Grayling extender (except only one sample) showed an agglutination phenomenon (damaged and intact cells adhered to each other) after thawing where Pike extender resulted the regular cell suspension. No significant difference was observed between the two cryopreserved groups (Pike and Grayling extender) in all motility parameters using the 0.5 ml straw and the polystyrene box. Similarly, motility parameters did not show a significant difference in the two frozen groups with the 5 ml straw, also in the polystyrene box. A significantly higher progressive motility (pMOT, Grayling: 54% ± 8%, Pike: 37% ± 5%), straight line velocity (VSL, Grayling: 50 ± 5 µm/s, Pike: 39 ± 4 µm/s) and beat cross frequency (BCF, Grayling: 20 ± 1 Hz, Pike: 17 ± 1 Hz) was observed in the case of the grayling extender by the 5 ml straw cryopreserved in a controlled‐rate freezer (CRF) compare to the pike extender. A significantly higher VSL (Grayling: 45 ± 3 µm/s, Pike: 38 ± 4 µm/s) was observed by the grayling extender using the 10 ml cryotube than with the pike extender. Despite the randomly occurring differences in a few parameters, our new controlled freezing method using the newly tested Pike extender, the 5 ml straw or the 10 ml cryotube can be a good solution for the preservation of elevated volume of carp sperm.     

Detection of Bovine herpesvirus 4 DNA in aborted bovine fetuses

The presence of Bovine herpesvirus 4 (BoHV-4) was investigated by several methods in 24 aborted bovine fetuses. Polymerase chain reaction (PCR) and in situ DNA hybridization proved the presence of BoHV-4 DNA in 7 (29%) of the fetuses. The BoHV-4 genome was detected in the cytoplasm of splenic lymphocytes and monocytes, and sometimes in renal tubular epithelial cells or hepatic Kupffer cells, in all 7 PCR-positive fetuses. However, BoHV-4-specific monoclonal antibody failed to detect viral antigen in the formalin-fixed, paraffin-embedded tissue samples. No bacterial pathogens were found in the tissues of the BoHV-4-positive fetuses. Fungi were detected in 1 sample, and antibody to bovine viral diarrhea virus was detected in another. These results indicate that BoHV-4 could play a role in reproductive disorders of cattle, including abortion.     

No indication of strict host associations in a widespread mycoparasite: grapevine powdery mildew (Erysiphe necator) is attacked by phylogenetically distant Ampelomyces strains in the field

Pycnidial fungi belonging to the genus Ampelomyces are common intracellular mycoparasites of powdery mildews worldwide. Some strains have already been developed as commercial biocontrol agents (BCAs) of Erysiphe necator and other powdery mildew species infecting important crops. One of the basic, and still debated, questions concerning the tritrophic relationships between host plants, powdery mildew fungi, and Ampelomyces mycoparasites is whether Ampelomyces strains isolated from certain species of the Erysiphales are narrowly specialized to their original mycohosts or are generalist mycoparasites of many powdery mildew fungi. This is also important for the use of Ampelomyces strains as BCAs. To understand this relationship, the nuclear ribosomal DNA internal transcribed spacer (ITS) and partial actin gene (act1) sequences of 55 Ampelomyces strains from E. necator were analyzed together with those of 47 strains isolated from other powdery mildew species. These phylogenetic analyses distinguished five major clades and strains from E. necator that were present in all but one clade. This work was supplemented with the selection of nine inter-simple sequence repeat (ISSR) markers for strain-specific identification of Ampelomyces mycoparasites to monitor the environmental fate of strains applied as BCAs. The genetic distances among strains calculated based on ISSR patterns have also highlighted the genetic diversity of Ampelomyces mycoparasites naturally occurring in grapevine powdery mildew. Overall, this work showed that Ampelomyces strains isolated from E. necator are genetically diverse and there is no indication of strict mycohost associations in these strains. However, these results cannot rule out a certain degree of quantitative association between at least some of the Ampelomyces lineages identified in this work and their original mycohosts.     

Gastric zygomycosis in a pig affected with postweaning multisystemic wasting syndrome--case report

A postweaning pig died in spite of antibiotic therapy showing wasting in a small herd. Postweaning multisystemic wasting syndrome (PMWS) was diagnosed on the basis of gross pathological and histological lesions and the presence of moderate amounts of porcine circovirus 2 (PCV2) antigen in tissue samples. Mycotic gastritis caused by Zygomycetes spp. was found on round areas with a diameter of 1 to 3 cm in the glandular mucosa of the stomach. Moderate amount of PCV2 viral antigen was detected almost evenly in the stomach and mostly in the macrophages. In addition, acute uraemia, revealed by an ammonia-like stink of the gastric mucosa and the presence of acute erosions on the glandular mucosa of the stomach, was observed as a consequence of PCV2-induced interstitial nephritis. Only PCV2 infection could be identified as a cause of secondary mycotic gastritis. The results further support the immunosuppressive ability of PCV2 infection in PMWS-affected pigs.     

Variation in the nrDNA ITS sequences of some powdery mildew species: do routine molecular identification procedures hide valuable information?

During the past years, nrDNA ITS sequences have supported the identification of many powdery mildew fungi because comprehensive analyses showed that differences in these sequences have always correlated with the delimitation of different species and formae speciales of the Erysiphales. Published data, obtained using direct sequencing of the PCR products, suggested that even one to five nucleotide differences in the ITS sequences delimit different, albeit closely related, species, and/or indicate differences in host range patterns. Here we show that such differences in the ITS sequences can be detected even in a single sample of a powdery mildew fungus. We sequenced the ITS region in 17 samples, representing six powdery mildew species, both directly and after cloning the PCR products. Among these, samples of O. longipes exhibited two or three, samples of O. neolycopersici three or four, those of an Oidium sp. from Chelidonium majus up to seven, and a sample of another Oidium sp. from Passiflora caerulea two different ITS types determined after cloning. No ITS nucleotide polymorphisms were found in samples of O. lycopersici and Erysiphe aquilegiae. This suggests that some powdery mildew taxa are more variable at the ITS level than others. Thus, although the ITS sequences determined by direct sequencing represent robust data useful in delimitation and phylogenetic analysis of distinct species of the Erysiphales, these need to be used with precaution, and preferably determined after cloning, especially when dealing with closely related taxa at species and sub-species levels. With this method a hitherto undetected genetic diversity of powdery mildews can be revealed.     

Effect of broth from meat of linseed‐fed cattle on glucose‐stimulated insulin release in healthy male volunteers

Polyunsaturated fatty acid consumption has been shown to improve insulin sensitivity. We studied if administration of broth with beef meat enriched with polyunsaturated fatty acids influenced glucose‐stimulated insulin release in healthy male volunteers. Broth was made either from cattles undergone dietary supplementation with lightly bruised whole linseed in addition to feeding ad libitum on grass silage (test meal) or from those fed grass silage alone (control meal). Oral glucose tolerance tests (OGTT) were performed in patients after a 6‐day period of eating 300 ml broth containing 100 g meat once a day in addition to their otherwise normal mixed nourishment. During OGTT, blood samples were taken for blood glucose level and plasma insulin immunoreactivity before and 15, 30, 60, 90, 120, and 180 min after the glucose load. Glucose‐stimulated maximum increase in plasma insulin immunoreactivity was 42 ± 6.6 and 81 ± 7.4 mU/ml (p < 0.05) after the test and the control meals, respectively. However, both fasting and postload blood glucose levels were the same after either meal period. The results suggest an insulin‐sensitizing effect of food produced from beef cattle maintained on linseed diet in healthy human volunteers.