Molecular Diversity and Genetic Relatedness of Candida albicans Isolates from Birds in Hungary.

The molecular epidemiology of Candida albicans infections in animals has been rarely studied. In this study, multilocus sequence typing was used to characterise the genetic diversity and population structure of 24 avian origin C. albicans isolates collected from different birds with candidiasis and compared to human isolates. Fourteen diploid sequence types (DSTs) including six new DSTs were determined. Cluster analysis revealed that isolates grouped into 8 clades. Bird isolates mainly belonged to minor clades and Clade 15 with DST 172 was the most common (11 isolates; 45.8%). The remaining isolates were clustered into Clade 7 (5 isolates; 20.8%), Clade 10 (4 isolates; 16.6%), Clade 8 (2 isolates; 8.3%), Clade 4 (1 isolate; 4.2%) and Clade 16 (1 isolate; 4.2%). Unweighted pair group method with arithmetic averages (UPGMA) and eBURST analyses showed that the genetic construction of avian origin C. albicans population is fairly diverse. Although species-specific lineages were not found, some degree of separation in the evolution of bird and human strains could be observed.

Virulence gene profiling and ompA sequence analysis of Pasteurella multocida and their correlation with host species.

This study describes the prevalence of capsule biosynthesis genes, LPS genotypes, virulence associated genes and the analysis of the outer membrane protein (ompA) sequence of Pasteurella multocida isolates (n = 180) from different locations in Hungary, from various host species, including humans. When combining capsular types with LPS genotypes, eight capsule - LPS genotype combinations were detected. A: L3 was the most dominant in bovine and porcine isolates, A: L1 in feline and human isolates, while D: L3 was the most common among strains from small ruminants. The P. multocida toxin encoding gene toxA was highly prevalent among small ruminant and porcine strains, while in human, feline and bovine isolates it could not be detected. Combination of the tested virulence associated genes (hgbA, nanH, hgbB, tbpA, pfhA, hsf1, hsf2, tadD, ptfA) classified our P. multocida isolates into 13 different virulence gene profiles (VGPs). These VGPs showed an association with host species. Analysis of the ompA sequence data confirmed this distribution by host species, which may indicate that host adaptation is taking place. The typing scheme used in this study may be useful in epidemiological investigations.

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.

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.

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.

Characterization of Francisella tularensis strains, comparing their carbon source utilization.

Thirteen Francisella tularensis strains were isolated from 22 seropositive brown hares (Lepus europaeus) originating from different parts of Hungary, and further two from a patas monkey (Erythrocebus patas) and vervet monkey (Chlorocebus aethiops). The isolates were identified as F. tularensis ssp. holarctica on the basis of culture, morphological and biochemical characteristics. The identification was verified by polymerase chain reaction and the sequencing of the partial 16S rRNA gene. Utilization of carbon sources of the 15 F. tularensis strains was characterized with the Biolog system. The system was able to identify the strains already after 4 h of incubation, not only after the standard 24 h. After the analysis and comparison of the metabolic profiles of our strains with the Biolog database, it was concluded that not all carbon sources indicated in the database were utilized by our isolates. The Biolog software fails to distinguish the highly virulent F. tularensis ssp. tularensis and the moderately virulent F. tularensis ssp. holarctica but the Biolog microplates can be manually read to differentiate the two subspecies based on glycerol source utilization. As all the studied strains were unable to use glycerol, they could be identified as F. tularensis ssp. holarctica. The dendrogram based on the metabolic relationship of the strains shows that the isolates are very similar to each other, which correlates with the conservative genetic character of F. tularensis ssp. holarctica.

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.

Plasmid profiles of virulent Rhodococcus equi isolates from soil environment on horse-breeding farms in Hungary.

The plasmid profiles of virulent Rhodococcus equi strains isolated on three horse-breeding farms located in different parts of Hungary were investigated. From 49 soil samples collected on the three farms, 490 R. equi isolates (10 from each sample) were obtained and tested for the presence of 15- to 17-kDa antigens (VapA) by immunoblotting and PCR. Ninety-eight VapA-positive isolates were detected from 30 of the 49 culture-positive samples with a prevalence ranging from 13.1% to 23.2%. Of the 98 virulent isolates, 70 contained an 85-kb type I plasmid, 13 contained an 87-kb type I plasmid, and 15 contained an 85-kb type III plasmid which had been uniquely isolated from soil isolates in the United States. This study demonstrates that the virulent form of R. equi is very widespread in the soil environment of these stud farms in Hungary and the plasmid pattern is different from farm to farm.

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.

Comparison of selective media for the isolation of Rhodococcus equi and description of a new selective plating medium.

In order to improve the isolation rate of Rhodococcus equi from animals and soil, the efficacy of four previously described selective media (CAZ-NB, M3T, NANAT and TINSDALE) and that of four other media (NC, PNP, TCP and TVP) composed by us was compared and evaluated. Two selective plating media proved to be the best for the isolation of R. equi from contaminated samples. One of them was CAZ-NB containing ceftazidime, novobiocin and cycloheximide, while the other was the newly composed TCP containing trimethoprim, cefoperazone, polymyxin B, cycloheximide and potassium tellurite as selective components. These two media allowed the growth of at least 62-72% of R. equi present in the artificially contaminated samples, and the inhibition of unwanted contaminant bacteria and fungi was satisfactory with both media. TCP medium proved to be superior to CAZ-NB since the colony morphology of R. equi was much more characteristic (shiny, smooth, black colonies 3-5 mm in diameter) on it, and it inhibited the unwanted contaminant bacterial and fungal flora more effectively, especially in the case of faecal and soil samples. Therefore, TCP is recommended as a new, highly selective plating medium for the isolation of R. equi from contaminated samples.

Characterization of virulence plasmid types in Rhodococcus equi isolates from foals, pigs, humans and soil in Hungary.

Rhodococcus equi isolates (204) obtained from foals (lung abscesses, lymph nodes, nasal discharge, rectal swabs) bred in 15 studs located throughout Hungary, isolates from soil samples, lymph nodes of pigs and from lesions of human patients were examined to determine genotypic diversity of virulence-associated plasmids. Isolates were tested for the presence of 15-17 kDa virulence-associated protein antigen (VapA) and 20k Da (VapB) genes by polymerase chain reaction (PCR). Plasmid DNAs were isolated and analysed by digestion with restriction endonucleases for estimation of size and comparison of polymorphisms. Of 146 clinical isolates from foals in 15 studs, 129 (88.3%) gave positive results for the VapA gene, showing a 564 bp product of the expected size in the PCR amplification. Of the 129 clinical isolates from foals, 123 contained an 85 kb type I plasmid and the remaining six contained an 87 kb type I plasmid. Of 48 soil isolates from two horse studs, 26 (54.2%) were positive for VapA gene and contained an 85 kb type I plasmid. Of three pig isolates, one was positive for VapA gene and contained an 85 kb type I plasmid, and the remaining two were positive for the VapB gene, showing a 827 bp product of the expected size in the PCR amplification and were R. equi of intermediate virulence which contained a 95 kb type S5 plasmid. Of the seven human isolates, five were positive for VapB gene by PCR, these were R. equi of intermediate virulence, which contained a 95 kb type S5 plasmid. These results revealed that virulent R. equi strains harbouring a virulence plasmid of 85 kb type I or 87 kb type I, which have been found in clinical isolates from Europe and North and South America, are widespread in Hungary. Furthermore, same intermediately virulence plasmid type was found in both human and pig isolates.

Characterisation of some Ornithobacterium rhinotracheale strains and examination of their transmission via eggs.

The biochemical characteristics and antibiotic susceptibility of 12 Ornithobacterium rhinotracheale strains isolated from chickens and turkeys suffering from respiratory clinical signs and the survival of some isolates on egg-shell and within chicken eggs during hatching were examined. All O. rhinotracheale strains showed typical biochemical characteristics. Among the 16 drugs examined, penicillin G, ampicillin (MICs ranging from < or = 0.06 microgram/ml to 1 microgram/ml), ceftazidim (with MICs from < or = 0.06 microgram/ml to 0.12 microgram/ml), erythromycin, tylosin, tilmicosin (with some exceptions MICs ranged from < or = 0.06 microgram/ml to 1 microgram/ml) and tiamulin (MICs varied from < or = 0.06 microgram/ml to 2 micrograms/ml) were the most effective. Lincomycin, oxytetracycline and enrofloxacin also gave good inhibitions, but with most strains in a higher concentration (MICs ranged in most cases from 2 micrograms/ml to 8 micrograms/ml). The other antibiotics inhibited the growth of O. rhinotracheale only in very high concentrations (colistin) or not at all (apramycin, spectinomycin, polymyxin B). At 37 degrees C, O. rhinotracheale did not survive on egg-shell for more than 24 hours, while upon inoculation into embryonated chicken eggs it killed embryos by the ninth day, and from the 14th day post-inoculation no O. rhinotracheale could be cultured from the eggs at all. These results suggest that O. rhinotracheale is not transmitted via eggs during hatching.

Rapid immunohistochemical detection of Rhodococcus equi in impression smears from affected foals on postmortem examination.

The first objective of this study was to develop an immunohistochemical procedure for rapid detection of Rhodococcus equi in impression smears from affected organs of foals on postmortem examination. The second aim was to demonstrate whether R. equi can be detected in smears of tracheal exudates collected from the same foals using an immunohistochemical method. Impression smears and cryostat and paraffin-embedded sections were made from the lungs and mediastinal lymph nodes of three foals (A, B and C) that had died of respiratory disease caused by R. equi, and also from the caudal mesenteric lymph node of foal A. Impression smears were made from the tracheal exudates of all foals. An affinity purified rabbit IgG was used for the immunohistochemical demonstration of R. equi. This antibody reacted with serotype 1 of R. equi in Ouchterlony's immunodiffusion and in the passive haemagglutination test, but not with other serotypes or with Streptococcus equi ssp. equi or Staphylococcus aureus, and failed to give an immunohistochemical reaction with Mycobacterium bovis or M. paratuberculosis. The immunohistochemical method proved to be of identical sensitivity to bacterial culture; moreover, from the lungs and mediastinal lymph nodes of one foal, R. equi could only be detected by this method. R. equi was demonstrated in smears of the tracheal exudates of all three foals. The results of this study indicate that the immunohistochemical method may be used for the rapid detection of R. equi in impression smears from the affected organs, especially abscesses, obtained postmortem, and possibly as a tool for diagnosing R. equi pneumonia in live foals by examining smears of tracheal aspirates.

Actinomycosis of dogs caused by Actinomyces hordeovulneris.

Actinomyces hordeovulneris was isolated from the lesions of chronic pyogranulomatous pleuritis and pericarditis of one of three dogs showing similar symptoms. The parietal pleura and the pericardium were thickened and covered with fine short threads of angiofibroblastic tissue. About 500-1000 ml of reddish purulent exudate in the thorax of all three dogs contained large numbers of rice-grain-sized, soft, yellowish-white granules ("sulphur granules"). These granules had a central core of branching filaments of gram-positive bacteria embedded in thick granulation tissue. The parietal pleura, the mediastinal pleura and the pericardium were infiltrated mainly with neutrophils, and to a lesser extent with lymphocytes and plasma cells. A small number of eosinophils and giant cells was also observed. Large numbers of pyogranulomas embedded in the granulation tissue were composed of a core of necrotized granulation tissue, mixed with clusters of gram-positive branching bacteria, surrounded by an area of intact and degenerating neutrophils and lymphocytes. Bacteria were detected in the lesions by Brown-Brenn staining and were isolated from one of the affected animals. The isolated bacteria were identified as A. hordeovulneris. This was the first isolation of A. hordeovulneris in Hungary.

Characterisation of Rhodococcus equi strains isolated from foals and from immunocompromised human patients.

The cultural, morphological, biochemical, serological characteristics and antibiotic susceptibility of 25 Rhodococcus equi strains isolated from lungs and lung abscesses of pneumonic foals and 5 R. equi strains isolated from immuno-compromised human patients were examined. All R. equi strains showed common cultural, morphological and biochemical characteristics both with conventional tests and on the basis of their enzyme profile. The R. equi strains examined were resistant to penicillins with the exception of ampicillin, to sulphamethazine and several strains also to sulphamethoxazole-trimethoprim. All strains were susceptible to erythromycin and rifampicin. The strains isolated from humans showed somewhat higher rate of antibiotic resistance to penicillin, cefotaxime, kanamycin, streptomycin, lincomycin, and oxytetracycline. The overwhelming majority (96%) of the equine isolates belonged to serotype 1 in Prescott's serotyping system, while the human isolates could not be serotyped.

Prevention of Rhodococcus equi pneumonia of foals using two different inactivated vaccines.

Two different, inactivated, aluminium salt adsorbed vaccines, one containing a R. equi strain (serotype 1, 10(9) CFU/ml and equine herpesvirus 2 (EHV-2) (1.5 x 10(7) PFU/ml) and another containing R. equi only were used on three studfarms to determine whether the disease can be prevented by vaccination of both pregnant mares and their foals. Pregnant mares received two 3 ml doses of vaccine intramuscularly 6 and 2 weeks before parturition and their foals were vaccinated on two or three occasions at 3, 5 or 7 weeks of age. The efficacy of the vaccines was evaluated on the basis of the clinical signs, serological response (indirect haemagglutination and virus neutralisation tests) and culture of R. equi from sick or dead foals. On studs A and B where the bivalent vaccine was used, 24 and 14 foals were born respectively to the vaccinated mares but no clinical case or death occurred due to R. equi pneumonia, while out of the 10 nonvaccinated control foals (stud B) two succumbed to R. equi pneumonia and 4 other foals had to be treated with antibiotics because of fever, coughing and dyspnea. In stud C, where the vaccine containing R. equi strain alone was used, all 15 vaccinated foals remained healthy but one of the 11 control foals died of suppurative R. equi pneumonia and one foal had to be treated due to R. equi pneumonia. R. equi strains (serotype 1) were isolated from the lungs of all dead foals. The serological response was very weak to both R. equi and the EHV-2 strain. Antibody titres in the colostrum of the vaccinated mares against R. equi (in studs A and B, geometric mean 3.79 +/- 1.63 and 4.14 +/- 1.46, respectively) were practically not higher than titres in the controls (in stud B geometric mean 2.12 +/- 1.96). More antibody was present in the colostrum samples against EHV-2 (geometric mean 6.1 + 1.4 compared to 2.5 +/- 1.2). In all foals antibody levels were hardly detectable against both R. equi and EHV-2 until five weeks of age. From the fifth week, antibody levels gradually increased and by the ninth week their reached a titre of 5.5 +/- 1.8 (2.7 +/- 1.2 in the control foals) against R. equi and 5.2 +/- 1.4 against EHV-2. The favorable clinical results and the low antibody titres in the sera of the vaccinated foals during the first week of life suggest that protection probably was due to repeated vaccination of young foals rather than to vaccination of mares.