Tibor Magyar

Role of the Dermonecrotic Toxin of Bordetella bronchiseptica in the Pathogenesis of Respiratory Disease in Swine

ABSTRACT Bordetella bronchiseptica is one of the etiologic agents causing atrophic rhinitis and pneumonia in swine. It produces several purported virulence factors, including the dermonecrotic toxin (DNT), which has been implicated in the turbinate atrophy seen in cases of atrophic rhinitis. The purpose of these experiments was to clarify the role of this toxin in respiratory disease by comparing the pathogenicity in swine of two isogenic dnt mutants to their virulent DNT+ parent strains. Two separate experiments were performed, one with each of the mutant-parent pairs. One-week-old cesarean-derived, colostrum-deprived pigs were inoculated intranasally with the parent strain, the dnt mutant strain, or phosphate-buffered saline. Weekly nasal washes were performed to monitor colonization of the nasal cavity, and the pigs were euthanized 4 weeks after inoculation to determine colonization of tissues and to examine the respiratory tract for pathology. There was evidence that colonization of the upper respiratory tract, but not the lower respiratory tract, was slightly greater for the parent strains than for the dnt mutants. Moderate turbinate atrophy and bronchopneumonia were found in most pigs given the parent strains, while there was no turbinate atrophy or pneumonia in pigs challenged with the dnt mutant strains. Therefore, production of DNT by B. bronchiseptica is necessary to produce the lesions of turbinate atrophy and bronchopneumonia in pigs infected with this organism.

The live attenuated bovine viral diarrhea virus components of a multi-valent vaccine confer protection against fetal infection.

Fetal infection with bovine virus diarrhea virus (BVDV) causes severe economic loss and virus spread in cattle. This study investigated the ability of modified live BVDV I and II components of a commercially available modified live virus (MLV) vaccine (Breed-Back FP 10, Boehringer Ingelheim Vetmedica Inc.) to prevent fetal infection and abortion, and therefore the birth of persistently infected animals. Heifers immunized with vaccine 4-8 weeks before insemination showed no adverse effects. All vaccinated animals had seroconverted to BVDV 4 weeks after immunization. Pregnant heifers were divided into two vaccination and two control groups and challenged with type I or II BVDV on days 60-90 of gestation. Seroconversion, clinical signs, immunosuppression, viremia, mortality, abortion rate, and fetal infection were studied. Post-challenge, 6/11 (type I challenged) and 8/11 (type II challenged) vaccinated heifers were free from clinical signs of BVD. Post-challenge clinical signs noted in the vaccinated groups were mild to moderate, while all unvaccinated controls had clinical signs ranging from moderate to severe. Viremia was not detected post-challenge in any of the vaccinated heifers. However, 100% of the controls were BVDV viremic on at least 1 day post-challenge. One of 22 vaccinated heifers had transient leukopenia, whereas 2/8 and 6/7 unvaccinated heifers in control groups I and II, respectively, had transient leukopenia. Type II BVDV infection led to abortion or death in 86% of unvaccinated heifers. The corresponding vaccinated group showed no deaths or abortions. All control group fetuses were infected with BVDV. The test vaccine gave 91% (type I BVDV challenged) and 100% (type II BVDV challenged) protection from fetal infection. This vaccine is safe and effective against fetal infection, abortion (type II BVDV) and the birth of persistently infected animals.

Antimicrobial susceptibility of Francisella tularensis subsp. holarctica strains from Hungary, Central Europe

OBJECTIVES Determining the in vitro susceptibility to 11 antibiotics of Francisella tularensis subsp. holarctica strains belonging to the phylogenetic group B.13, from different areas of Hungary. METHODS Twenty-nine F. tularensis strains isolated between 2003 and 2010 from free-ranging European brown hares (Lepus europaeus) and a captive patas monkey (Erythrocebus patas) were collected from different parts of Hungary and examined for antibiotic susceptibility with commercially available MIC test strips on modified Francis agar plates; values were interpreted according to CLSI breakpoints. RESULTS The strains were susceptible to aminoglycosides (MIC(90) values: gentamicin, 0.75 mg/L; and streptomycin, 6.0 mg/L), tetracyclines (MIC(90) values: tetracycline, 0.5 mg/L; and doxycycline, 1.0 mg/L), quinolones (MIC(90) values: ciprofloxacin, 0.047 mg/L; and levofloxacin, 0.023 mg/L) and chloramphenicol (MIC(90) value: 1.5 mg/L), i.e. antibiotics commonly used in therapy. Tigecycline (MIC(90) value: 0.19 mg/L) and rifampicin (MIC(90) value: 1.0 mg/L) were also active against F. tularensis strains, while resistance to erythromycin (MIC(90) value: >256 mg/L) and linezolid (MIC(90) value: 32 mg/L) was observed in all strains. CONCLUSIONS Based on the results, quinolones are recommended as first choice therapy for F. tularensis infection. The in vitro susceptibility of the strains to tigecycline may encourage the application of this antibiotic as well. The similar antibiotic susceptibilities of the Hungarian strains belonging to different subclades of phylogenetic group B.13 indicates that strains from other Central and Eastern European countries belonging to this group might also have the same susceptibility profile.

Prevalence of Coxiella burnetii in Hungary: screening of dairy cows, sheep, commercial milk samples, and ticks.

Q fever is an important zoonotic disease caused by Coxiella burnetii. There are few reliable data about C. burnetii infection available. The aim of this study was to assess the importance and potential infectious sources of Q fever in Hungary. A total of 215 milk samples (10 individual samples from each herd and 1 bulk tank milk sample from each cattle herd), and 400 serum samples (20 from each herd) were tested from 15 dairy cattle herds and 5 sheep flocks located in different parts of Hungary. The study found 19.3% (58/300) and 38.0% (57/150) seropositivity in cattle, and 0% (0/100) and 6.0% (3/50) seropositivity in sheep, by complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA), respectively. C. burnetii DNA was detected by IS1111 element-based TaqMan real-time polymerase chain reaction (PCR) in 8.7% (13/150) of individual dairy cow milk samples, 4.0% (2/50) of individual sheep milk samples, and 66.7% (10/15) of dairy bulk tank milk samples. Samples taken from nine different commercially-available pasteurized cow milk products from different Hungarian producers were also tested for the presence of C. burnetii DNA, and eight of these samples were found to be positive (88.9%). The real-time PCR examination of 5402 ixodid ticks collected from different parts of the country yielded negative results. Knowledge of the true prevalence of Q fever is crucial for policymakers involved in evidence-based decision making.

Antimicrobial susceptibility of Pasteurella multocida isolated from swine and poultry

Pasteurella multocida causes infectious diseases in a wide range of animal species. Antimicrobial therapy is still an effective tool for treatment. Generally, P. multocida isolates are susceptible to most of the widely used commercial antimicrobial agents but their excessive and unjustified use accelerates the emergence of resistant strains. We defined the antimicrobial sensitivity pattern of 56 P. multocida strains isolated from poultry (20) and swine [16 P. multocida toxin (PMT) positive and 20 PMT negative] to 16 widely applied antibiotics (apramycin, cefquinome, chloramphenicol, colistin, doxycycline, enrofloxacin, erythromycin, florfenicol, flumequine, neomycin, oxolinic acid, penicillin, trimethoprim potentiated sulphamethoxazole, sulphonamide compounds, tetracycline, tulathromycin) by the disk diffusion method. The majority of the strains was susceptible to most of the antimicrobial agents tested. However, the resistance to sulphonamides, tetracyclines, first-generation quinolones and aminoglycosides was remarkable, and thus the use of these compounds for the treatment of infection caused by P. multocida is not recommended. On the other hand, the antimicrobial activity of the classical penicillin, the newer macrolide (tulathromycin), the third-generation fluoroquinolone (enrofloxacin) and the fourth-generation cephalosporin (cefquinome) proved to be satisfactory against this bacterium.

Optimized ribotyping protocol applied to Hungarian Bordetella bronchiseptica isolates: identification of two novel ribotypes.

We reported previously that ribotype patterns generated with PvuII and a probe derived from the Escherichia coli rrnB gene could be used to differentiate isolates of Bordetella bronchiseptica. In the present study we report modifications made to the original ribotyping procedure that permit detection in the formerly characterized isolates of an additional 8 fragments with homology to rrnB. Ribotypes were redefined to include these fragments. Although this modification did not permit the detection of novel ribotypes from the previously characterized isolates, it did result in a more accurate reclassification of five of these isolates to other existing ribotypes. It was hypothesized that the additional fragments could form the basis for novel ribotypes in future analyses, and this was supported by the subsequent evaluation of 101 previously uncharacterized pig, rabbit, and dog B. bronchiseptica isolates from Hungary. A total of six different patterns were detected from this group, including two previously not identified that were designated ribotypes 17 and 18. The profile of ribotype 17 includes a novel fragment not associated with any other ribotype. A subset of the fragments constituting ribotype 18, essential for its differentiation from other ribotypes, is only detectable under the modified conditions reported here. Hungarian swine isolates are highly clonal, since 98.2% were identified as ribotype 3. Similarly, 83.7% of rabbit isolates from Hungary are also ribotype 3. Cluster analysis revealed that despite the existence of numerous ribotypes, B. bronchiseptica isolates display limited heterogeneity. The ability to detect additional ribotypes under the modified conditions described in this study strengthens the usefulness of ribotyping as an epidemiologic tool.

Prevalence of Francisella tularensis and Francisella-Like Endosymbionts in the Tick Population of Hungary and the Genetic Variability of Francisella-Like Agents

Several new taxa belonging to the genus Francisella have been described recently. The present study describes the prevalence of Francisella tularensis and Francisella-like endosymbionts (FLE) in ticks collected from Hungary from 2007 to 2009 and characterizes the genetic variability of FLEs. A total of 5402 Ixodid ticks (Ixodes ricinus, I. acuminatus, Dermacentor marginatus, D. reticulatus, Haemaphysalis inermis, H. concinna, H. punctata) were collected from vegetation and animal hosts and tested with conventional PCR, detecting the 16S rRNA and tul4 genes. F. tularensis ssp. holarctica was found in 2 pools of H. concinna and 1 pool of D. reticulatus, both representing minimum prevalence (calculated with 1 infected tick per pool) of 0.27% whereas the sequences of a FLE were detected in 11 pools of D. reticulatus showing a minimum prevalence of 3%. Although the tul4 gene sequence of this FLE was identical to all Hungarian and Portuguese FLEs found earlier, and the 16S rRNA sequence was also identical to the sequence of the endosymbiont of D. reticulatus described in Bulgaria, these 16S rRNA gene coding sequences differed in 2 nucleotides from the one found earlier in this tick species in Hungary. This divergence may appear to be a minor difference between the sequences, potentially even resulting from a technical failure, but it could also indicate a significant difference stemming from the conservative genetic character of Francisellaceae. Thus, it raises a question about the number of FLE variants circulating in D. reticulatus in Europe and indicates the need for further data about the FLEs described in other parts of the continent and new FLE genotyping markers.

Use of Computed Tomography and Histopathologic Review for Lung Lesions Produced by the Interaction Between Mycoplasma hyopneumoniae and Fumonisin Mycotoxins in Pigs

Mycoplasma hyopneumoniae has a primary role in the porcine respiratory disease complex (PRDC). The objective of this study was to determine whether fumonisin mycotoxins influence the character and/or the severity of pathological processes induced in the lungs of pigs by Mycoplasma hyopneumoniae. Four groups of pigs (n = 7/group) were used, one fed 20 ppm fumonisin B1 (FB1) from 16 days of age (group F), one only infected with M. hyopneumoniae on study day 30 (group M), and a group fed FB1 and infected with M. hyopneumoniae (group MF), along with an untreated control group (group C). Computed tomography (CT) scans of infected pigs (M and MF) on study day 44 demonstrated lesions extending to the cranial and middle or in the cranial third of the caudal lobe of the lungs. The CT images obtained on study day 58 showed similar but milder lesions in 5 animals from group M, whereas lungs from 2 pigs in group MF appeared progressively worse. The evolution of average pulmonary density calculated from combined pixel frequency values, as measured by quantitative CT, was significantly influenced by the treatment and the age of the animals. The most characteristic histopathologic lesion in FB1-treated pigs was pulmonary edema, whereas the pathomorphological changes in Mycoplasma-infected pigs were consistent with catarrhal bronchointerstitial pneumonia. FB1 aggravated the progression of infection, as demonstrated by severe illness requiring euthanasia observed in 1 pig and evidence of progressive pathology in 2 pigs (group MF) between study days 44 and 58.

Antibiotic susceptibility profiles of Mycoplasma bovis strains isolated from cattle in Hungary, Central Europe

BackgroundMycoplasma bovis is a worldwide pathogen, causative agent of pneumonia, mastitis, arthritis, and a variety of other symptoms in cattle. The economic losses due to mycoplasma pneumonia could be reduced by antibiotic treatment. The aim of the present study was to determine the in vitro susceptibility of M. bovis strains isolated from cattle in Hungary to eleven antibiotics.ResultsMinimal inhibitory concentration (MIC) values of 35 M. bovis strains collected from different parts of Hungary between 2010 and 2013 were determined by the microbroth dilution method. Strains with high MIC values were found in the case of all applied antibiotics. The most effective antibiotics tested in vitro were fluoroquinolones (MIC90 danofloxacin 0.312 μg/ml, enrofloxacin 0.312 μg/ml, marbofloxacin 0.625 μg/ml). Our results confirm the observations of increasing MIC values to antibiotics commonly used in the therapy of mycoplasma infections, primarily to tetracyclines; tetracycline (MIC90 16 μg/ml) and oxytetracycline (MIC90≥64 μg/ml) and macrolides; tylosin (MIC90≥128 μg/ml) and tilmicosin (MIC90≥128 μg/ml). The growth of many M. bovis strains was not inhibited by gentamicin (MIC90 8 μg/ml), spectinomycin (MIC90≥256 μg/ml), florfenicol (MIC90 8 μg/ml) or lincomycin (MIC90≥64 μg/ml).ConclusionsOur results emphasize the necessity of periodic testing for antibiotic susceptibility in this geographic region. Based on our in vitro examinations, fluoroquinolones could be the most effective drugs for the therapy of M. bovis infections in Hungary. However, current antimicrobial use policies have to be taken into account to avoid further antibiotic resistance development and to reserve fluoroquinolones for the treatment of severe infections which have responded poorly to other classes of antimicrobials.

Characterization of the ptfA gene of avian Pasteurella multocida strains by allele-specific polymerase chain reaction.

Pasteurella multocida is the causative agent of fowl cholera in domesticated and wild birds. The disease outcome is affected by various host- and pathogen-specific determinants. Several putative virulence factors have been proposed to play a key role in this interaction, including the ptfA gene, the products of which assemble to form type 4 fimbriae on the bacterial surface. One way to understand more precisely how ptfA contributes to pathogenesis is to gather molecular features of this gene in circulating avian P. multocida strains. Therefore, molecular characterization of the ptfA gene of P. multocida strains isolated from domestic poultry was performed using the combination of nucleotide sequence analysis and a newly developed allele-specific polymerase chain reaction assay. Two major ptfA alleles were identified among 31 strains, representing various serogroups and somatic serotypes. It was noteworthy that allele specificity and case severity of a subset of strains correlated with the available gross pathology data. Therefore, the acquisition of comprehensive clinical and epidemiological data together with molecular characteristics of individual strains will help to design and implement adequate preventive and intervention strategies.