Retrospective study on the distribution of Dirofilaria immitis in dogs in Hungary.

Europe has experienced the spreading of vector-borne helminths including heartworms (Dirofilaria immitis) from the Mediterranean countries towards the northern ones in the past decades. Recently, the establishment of D. immitis was confirmed in Hungary on the basis of period prevalence studies involving dogs, red foxes (Vulpes vulpes) and golden jackals (Canis aureus). The aim of our retrospective study was to describe the spatial distribution of the parasite and the time course of spreading of D. immitis in Hungary. Necropsy records of 2622 dogs received at our laboratories from 2001 to 2015 were reviewed for heartworm infections. The locality of origin of animals was recorded in a geographic information system database and compared to the results of the period prevalence study involving wild canids. Autochthonous heartworm infection was detected in 27 dogs. The time course analysis indicates that the parasite established in Hungary in 2007. As temperature is one of the most important determinants of the distribution of D. immitis, the climate of the Great Hungarian Plain is the most suitable region for the establishment of D. immitis in Hungary. Our studies revealed that the Great Hungarian Plain became a D. immitis endemic region for 2015. Nevertheless, sporadic cases in wild canids and dogs also occur in other regions of the country.

Acute tracheal oedema and haemorrhage with fibrinonecrotic tracheitis in pigs--a porcine counterpart of bovine honker syndrome?

Cases of acute tracheal oedema and haemorrhage with fibrinonecrotic tracheitis have been described in Hungarian pig herds. Clinical signs and gross and microscopical tracheal lesions bore resemblance to those of bovine 'honker syndrome'. Diagnostic examination of affected tracheas and corresponding lungs revealed the presence of a variety of agents; however, in some cases tracheal lesions developed without detectable pathogens or associated pulmonary pathology. In line with the bovine condition, this suggests the possibility of cough-induced tracheal damage as an initiating factor for this tracheal change in swine.

Dirofilaria immitis: an emerging parasite in dogs, red foxes and golden jackals in Hungary.

Hungary was not considered to be a heartworm (Dirofilaria immitis) endemic country until 2007, when the first autochthonous canine infection was described. Herein we report additional autochthonous heartworm infections in two dogs (Canis familiaris), twenty red foxes (Vulpes vulpes) (n=534; prevalence: 3.7%; 95% CI=2.4-5.7%) and two golden jackals (Canis aureus) (n=27; prevalence: 7.4%; 95% CI=2.1-23.4%) coming from eight counties. The identification of the parasite was based on morphology, morphometrics and amplification of 12S rDNA followed by sequencing in all cases. Our results indicate that Hungary became a D. immitis endemic country in the past decade. The prevalence and intensity of heartworm infection in wild canids is similar to or lower than that observed in the Mediterranean countries of Europe (3.7-7.4% vs. 0.4-12.7% and 1.5 vs. 2.9-4.4 worms/animal). These findings are in line with the results of the recently developed climate based forecast model to predict the establishment of D. immitis in Hungary.

Association of porcine circovirus type 2 with vascular lesions in porcine pneumonia.

The aim of this study was to evaluate the vasculature in porcine circovirus type 2-infected (PCV2-infected) lungs and to identify the PCV2 subtypes involved in porcine pneumonia. Pulmonary samples from 140 pigs, 2 weeks to 7 months of age, from 36 Hungarian commercial herds with clinical signs of respiratory disease were examined for the presence of respiratory pathogens, with bacterial culture, pathologic evaluation, and immunohistochemistry for PCV2, porcine reproductive respiratory syndrome virus, and swine influenza virus. PCV2 was the most commonly identified pathogen (49 cases) among the 74 of 140 cases (53%) with respiratory pathogens. PCV2 was detected immunohistochemically in the wall of 13% to 100% of pulmonary vessels (mean, 89%) in 38 of 49 cases (78%). Detection of PCV2 antigen was positively correlated with the presence of vascular lesions (P < .001, odds ratio [OR]: 159.54). Other pathogens capable of vascular injury in swine were found in 29 of 49 of the PCV2-positive cases (59%). The probability of detecting vascular lesions in PCV2-infected lung was higher than in infection with porcine reproductive respiratory syndrome virus (P < .002, OR: 14.63), Pasteurella multocida infection (P < .001, OR: 5.75), or Streptococcus spp. infection (not significant, OR: 1.45). Sequence analysis of open reading frame 2 amplicons was possible in 6 PCV2-positive cases, from which 5 cases proved to be PCV2b subtype and 1 case, PCV2a subtype. In conclusion, PCV2 antigen was commonly colocalized with pulmonary vascular lesions in pneumonia in Hungarian swine, and PCV2b was the dominant subtype.

Brucellosis of the European brown hare (Lepus europaeus).

The European brown hare (Lepus europaeus) is an important reservoir of Brucella suis biovar 2 and also of the life-threatening zoonotic agent Francisella tularensis. Since both bacteria can produce similar gross pathological lesions in this species, laboratory tests are necessary for the final diagnosis. The aim of the present study was to develop an immunohistochemical method for the detection of B. suis infection and to describe the pathological and histological lesions caused by B. suis in European brown hares. Hyperimmune serum for immunohistochemistry (IHC) was produced by subcutaneous infection of mice with 2 × 10(9) colony forming units of live B. suis biovar 2, injected four times at 1-week intervals. The antiserum did not react with F. tularensis or Yersinia pseudotuberculosis in IHC and displayed only weak cross-reaction with B. canis. Numerous, yellow-white necrotic foci (0.1-0.5 cm diameter) were found in the spleen of five B. suis-infected female European brown hares and also in the lung, uterus, kidney or liver of four of these cases. Microscopically, the foci comprised single or coalescing granulomas with a central necrotic area. Both bacterial isolation and IHC gave positive results for B. suis infection in these animals. B. suis antigens were found as granular or amorphous extracellular material in the necrotic centre of several granulomas. IHC appears to be a suitable complementary diagnostic method for the detection of B. suis infection in the European brown hare.

Comparative histopathology and immunohistochemistry of QX-like, Massachusetts and 793/B serotypes of infectious bronchitis virus infection in chickens.

The aim of this study was to compare experimentally the pathogenicity and tissue distribution of the recently emerged QX-like strain of infectious bronchitis virus (IBV) with the widespread M41 and 793/B serotypes of the virus. Histopathological and immunohistochemical methods were employed to define the main sites of virus replication. One-day-old specific pathogen free chickens were inoculated with five different QX-like strains, or with the M41 and 793/B IBV strains and monitored for 42 days post-infection. Tracheal lesions developed in all infected birds, confirming the ability of all of the tested strains to induce respiratory disease. Replication of the isolates in the alimentary tract was detected, but the infection did not cause significant gut lesions. Four of the five QX-like IBV strains induced severe kidney lesions. Dilation of the oviduct with accumulation of serum-like fluid in the lumen of this structure, reported previously from field cases of QX-like IBV infection, was observed following experimental infection with all of the five QX-like strains. Microscopical and immunohistochemical examination of the affected oviducts did not help to elucidate the pathogenesis of this lesion.

Tularemia of European Brown Hare (Lepus europaeus): a pathological, histopathological, and immunohistochemical study.

The European brown hare (Lepus europaeus) plays an important role in the ecology of tularemia, and it may serve as a significant source of human infection. The aim of the present study was to examine the lesions induced by Francisella tularensis in 50 cases of naturally infected seropositive European brown hares. Gross pathological examination revealed scant to numerous grayish-white foci with diameters of 0.1 to 1.0 cm in single organs (24 cases) or multiple organs (20 cases) in 44 of 50 cases (88%). These lesions proved to be areas of granulomatous inflammation, frequently encompassing necrosis. F tularensis antigen was detected with immunohistochemistry in 46 of 50 cases (92%), whereas F tularensis ssp holarctica was isolated by culture and identified by polymerase chain reaction from 35 of 50 cases (70%). Infection by the respiratory route is suggested by the presence of the tissue lesions in thoracic organs of 44 of 50 cases (88%). These results emphasize the importance of the European brown hare as a reservoir of tularemia.

An investigation of the aetiological role of bovine herpesvirus 4 in bovine endometritis.

Uteri from 31 infertile cattle were examined for the presence of bovine herpesvirus 4 (BoHV-4) by nested polymerase chain reaction (PCR). Samples were also tested for bacteria, including chlamydiae and Mycoplasma bovis. BoHV-4 was detected by PCR in 27/31 (87.1%) samples, but the presence and amount of viral DNA was not correlated with histological and bacteriological findings. Arcanobacterium pyogenes, Histophilus somni and Pasteurella multocida were isolated from five cows with endometritis. Chlamydiae were detected in four cases (12.9%), but only two of these had endometritis. The study does not support a role for BoHV-4 as primary agent in bovine endometritis.

Experimental study on the role of Brachyspira alvinipulli in intestinal spirochaetosis of geese.

Ten one-day-old goslings were inoculated orally with a Brachyspira alvinipulli strain isolated from the large intestine of geese that had died of intestinal spirochaetosis (Group A), 10 day-old goslings were inoculated orally with a B. hyodysenteriae strain (Group B), and a third group of 10 goslings (Group C) served as uninfected control. The goslings were observed daily for clinical signs. They were sacrificed on days 7, 14, 21 and 35 days postinfection (PI), and necropsied. Segments of the large intestine were subjected to histopathological, immunohistochemical, electron microscopic (TEM, SEM) and microbiological examinations. Mortality did not occur during the experimental period. However, in both groups the caecum of the goslings killed by bleeding was slightly dilated, in its lumen there was a watery, yellowish and frothy content, and the mucous membrane was slightly swollen. By histopathological, immunohistochemical and electron microscopic examination, B. alvinipulli and B. hyodysenteriae could be detected in the caecum or colon, in the lumen of the glands and sometimes among the glandular epithelial cells in goslings of the respective groups, and could be reisolated from these organs by culturing. A mild inflammation of the intestinal mucosa was also noted. In transverse section of the brachyspirae, numerous (16-22) periplasmic flagella could be detected inside the outer sheath, also depending on the plane of section.

[Post-mortem diagnosis of leptospirosis in two dogs]. / Twee postmortaal vastgestelde gevallen van leptospirose bij de hond.

Leptospirosis was diagnosed post-mortem in a 2-year-old male Dogo Argentino and a 7-week-old male Foxhound puppy. The two cases were unrelated. Clinical symptoms were mainly confined to the gastro-intestinal tract. Pathological lesions were suggestive of acute leptospirosis. Leptospires infection was confirmed by serological (indirect IgM/Ig6 ELISA and MAT) and immunohistochemical techniques.

A case of equine abortion caused by Encephalitozoon sp.

A Lippizan mare aborted a male fetus a few days before the expected foaling date without showing any clinical sings. Focal lympho-histiocytic hepatitis in the foal and multiplex focal lympho-histiocytic villitis accompanied by villus necroses and marked hypertrophy of chorionic epithelial cells in the arcades were observed. Elongated nucleated organisms were seen in groups in vacuoles or solitarily located in the cytoplasm of the chorionic epithelial cells. The organisms were in large numbers and often extracellularly in areas of villitis and villus necroses. They were Gram-positive, stained with haematoxylin and eosin (HE), periodic acid-Schiff (PAS) and Giemsa, weakly with Warthin-Starry silver stain but not with Gömöri's methenamine-silver stain. By ultrastructural and immunohistochemical examinations, the organisms were identified as microsporidia belonging to the genus Encephalitozoon. No Encephalitozoon organisms were detected in the fetal organs. This is the first reported case of equine abortion induced by Encephalitozoon sp. in Europe. Although abortion induced by Encephalitozoon is rare, microsporidia should be considered a differential diagnosis for intracellular organisms observed in the chorionic epithelial cells of horses.

Immunohistochemical identification and pathologic findings in natural cases of equine abortion caused by leptospiral infection.

The aim of this study was to examine the utility of immunohistochemistry (IHC) in the diagnosis of leptospiral equine abortion and to compare IHC to silver staining and serology of the aborted mares. Ninety-six fetuses from 57 farms were examined using all 3 diagnostic techniques, revealing evidence of leptospiral infection in 3 fetuses (3.1%) from 3 (5.3%) different farms. A new finding in 1 of these confirmed cases of leptospiral abortion was the presence of macroscopic pinpoint grayish-white nodules that had a histologic correlate of hepatic necrosis; other histologic findings were consistent with those previously reported. IHC performed using 2 different leptospiral antisera (multivalent whole-cell rabbit antiserum and rabbit antiserum against the major outer membrane protein LipL32) yielded similar results. IHC was more sensitive (19/21 [90.5%] tissue samples) than silver staining (8/21 [38.1%] tissue samples), and more specific than serology performed using the microscopic agglutination test. The primary advantage of IHC over silver staining was the ability of IHC to identify leptospiral antigen not only as morphologically intact spiral forms.

Two cases of equine abortion caused by Rhodococcus equi.

Rhodococcus equi was isolated from lung, liver, spleen, and stomach content of two aborted equine fetuses of 7 and 8 months gestation from two different farms. Lesions included diffuse pyogranulomatous pneumonia with numerous Gram-positive coccobacilli within the cytoplasm of macrophages, multinucleated Langhans giant cells and neutrophils, and enhanced extramedullary hematopoiesis with megakaryocytosis within the liver and spleen. Detection of R. equi was made by bacteriology and immunohistochemistry for R. equi and VapA, the virulence factor of R. equi. R. equi and VapA were identified within the lungs of both fetuses, and its distribution correlated with lesions. Fetal lesions were similar to those observed in foals. We speculate that the fetuses contracted infection from the placenta by normal breathing movements or by swallowing of the amniotic fluid contaminated with R. equi.

Typhlocolitis associated with spirochaetes in goose flocks.

The role of Brachyspira bacteria in the aetiology of increased mortality observed in two breeder goose flocks (Flock A consisting of 1,500 and Flock B comprising 4,500 laying geese) at the end of the first egg-laying season, in the period of moulting, was studied. In Flock A 415 geese (28%) died during an 8-week period while in Flock B 834 geese (18%) died during a 12-week period. On gross pathological examination, the geese were found to have haemorrhagic-to-necrotic inflammation of the large intestine (colon and rectum) and fibrinonecrotic typhlitis accompanied by severe degeneration. Often, fibrosis of the kidneys, and in five of the geese secondary visceral urate deposition ("visceral gout") was also observed. Histopathological examination consistently demonstrated spirochaetes in the mucous membrane of the affected large intestine. This was confirmed by the results of immunohistochemical and electron microscopic examination. In addition, Trichomonas stages were also detected from the large intestine of 11 geese. On the basis of their cultural and biochemical properties, and PCR sequencing analysis, eight out of the nine spirochaete strains isolated from the geese by culture on special media under anaerobic conditions were identified as Brachyspira alvinipulli. This is the first report on the isolation of B. alvinipulli from laying geese affected with fibrinonecrotic typhlocolitis.

Epidemiological and pathological study on the causes of abortion in sheep and goats in Hungary (1998-2005).

The objective of the investigations was to study the causes of abortion in sheep and goats in Hungary during a 7.5-year period. The authors investigated 246 cases of ovine and 75 cases of caprine abortions by different diagnostic methods. An infectious origin was found in 126 cases (51.2%) of ovine and 19 cases (25%) of caprine abortions. The most important cause of ovine and caprine abortions was Chlamydophila abortus infection with a prevalence of 46% and 17%, respectively. Other infections causing sheep and goat abortions were present only in 5.2% and 8% of the cases, respectively. The results obtained by different diagnostic methods are discussed.

Study on the role of gastric Helicobacter infection in gross pathological and histological lesions of the stomach in finishing pigs.

The prevalence of gastric Helicobacter infection in finishing pigs and the influence of this infection on gastric lesions was studied. Stomachs of 89 finishing pigs from 27 randomly selected herds were sampled at the slaughterhouse. Forty cases (Group A) were selected based upon the presence of gross pathological lesions in the pars oesophagea, and further 49 cases were obtained at random (Group B). Three samples of gastric tissue (junction of pars oesophagea and pars cardiaca, fundic area, and pyloric area) were collected from each stomach for histological and immunohistochemical examination. Helicobacter antigen was detected in 76 cases (85.4%). No association was found between the presence of Helicobacter in the stomach and the occurrence of gross pathological lesions in the pars oesophagea or gastritis detected on histological examination. However, a significant association was found between the occurrence of Helicobacter in the pyloric area and the presence of erosions/ulcers in the pars oesophagea (OR: 7.01, p = 0.022) in Group B. A significant association was also evident between the presence of Helicobacter and glandular lesions (dilatation of the glands + glandular abscess + degeneration of glandular epithelial cells). In conclusion, Helicobacter infection seems to be a contributing factor to pathological changes in the stomach of finishing pigs.

High prevalence of chlamydial (Chlamydophila psittaci) infection in fetal membranes of aborted equine fetuses.

Seventy-seven cases of equine abortion from 49 Hungarian farms that occurred between 1998 and 2000 were investigated for the presence of chlamydiae by immunohistochemistry, PCR and/or MZN staining. Evidence of the presence of these bacteria was obtained in 64 cases (83.1%) from 41 (83.7%) different farms. Partial ompA gene sequencing of PCR products revealed that the agent was Chlamydophila psittaci. Based on the findings of microbial diagnosis, pathology and case history, chlamydial infection was considered to be the most likely cause of abortion in at least 11 (14.3%) cases. In the remaining 53 Chlamydophila-positive cases, either other bacterial and viral agents (n = 22 or 28.6%) as well as non-infectious factors (n = 14 or 18.2%) were identified as more probable primary causes of disease, or the role of chlamydiae remained unclear because lesions in fetuses and fetal membranes were absent (n = 17 or 22.1%). When chlamydial antigen was detected in aborted equine placental tissue using immunohistochemistry it was seen only in the chorionic epithelial cells, but not in other parts of the fetal membranes nor in any of the fetal tissues. In conclusion, chlamydial infection of the genital tract should be considered a possible factor in equine reproductive disorders.

A novel subgroup among genotypes of equine arteritis virus: genetic comparison of 40 strains.

The authors determined partial nucleic sequences of the variable regions of open-reading frame (ORF5) from 151 nucleotide to 668 nucleotide and deduced amino acid sequences of 518 nucleotide respectively of 20 equine arteritis virus (EAV) isolates. About 19 Hungarian and one Austrian EAV strains were subjected to sequence analysis, the further data of 20 EAV strains: six North American and 14 European were obtained from the GenBank. Comparative sequence analysis of the Hungarian EAV strains indicated that among the three variable regions the first has been affected mostly by point mutations. Genetic comparison of the Hungarian strains with other EAV isolates from western Europe and North America (including the Bucyrus reference strain) has been performed on the aforementioned genome region. Besides the already known genetic subgroups of EAV; phylogenetic analysis revealed a novel subgroup comprising mainly Hungarian strains. Compared with the Bucyrus virus, the overall sequence divergencies of the examined Hungarian strains ranged from 81.47 to 90.73% at nucleotide and from 84.88 to 91.86% at amino acid level. Epizootiological studies have shown that the significant part of the EAV strains having been existed in Hungary before and in 2000 belong to this unique cluster (II.D) which was not indicated in former phylogenetic studies. After 2000 new EAV strains emerged in Hungary, one of them causing abortions or neonatal death. The previously dominant 'Hungarian' EAV genotypes were replaced by these new strains belonging to North American and European subgroups (I.A, I.B, II.A, II.B). The anamnesis of these cases revealed connections with persistent virus shedder stallions, those were imported to the country after 2000 or have been infected abroad. One of these Hungarian stallions became the source of abortion storms in Hungarian studs.

Equine viral arteritis in a newborn foal: parallel detection of the virus by immunohistochemistry, polymerase chain reaction and virus isolation.

A 4-days-old foal died after a short course of respiratory syndrome and fever. Large areas of the alveoli, bronchioles and bronchi were partly or completely filled by hyaline membranes. Pronounced oedema and mild interstitial pneumonia were present and, in the small muscular arteries, fibrinoid necrosis and vasculitis or perivasculitis could be seen. Vasculitis was found in several other organs, and it was most severe in the thymus. The virus was detected in the lung, kidney and spleen using virus isolation and in the lung and spleen using polymerase chain reaction. The virus was also detected in several organs and cell types using both N protein-specific monoclonal antibody and horseradish peroxidase-labelled equine arteritis virus-specific equine IgG.

Infection of two goatherds with Mycoplasma mycoides subsp. capri in Hungary, evidence of a possible faecal excretion.

During epidemic outbreaks in two goatherds clinical symptoms and deaths occurred in five (14%) of the 3-week-old goat kids in farm A, and in six (33%) of those in farm B. In the latter farm, three female goats aborted before the clinical symptoms in the kids emerged. Mycoplasma could be isolated from both healthy and sick goat kids and from female goats, which had diseased kids or had aborted. Three goat kids (one from herd A and two from herd B) were sent for post-mortem examination. In all these cases septicaemia caused by Mycoplasma was diagnosed. Based on the bacteriological examination the Mycoplasma strains proved to be Mycoplasma mycoides subsp. capri (Mmc). This was confirmed by the PCR examination. Mmc was isolated from several locations including from the rectum of one healthy female goat, and from two diseased kids. In addition, bacteria were detected in the small intestine in two of the necropsied kids by bacteriological and/or immunohistochemical methods. The finding suggests that Mmc may be transmitted via faeces in goatherds, kept under conventional conditions.