Human Mozdok leptospirosis first diagnosed by serum agglutinin-absorption tests in the Slovak Republic.

INTRODUCTION: The serovar Mozdok related leptospirosis in humans were not yet feasibly diagnosed using merely the standard micro-agglutination test (MAT) what was perhaps due to the impossibility to distinguish them from illnesses that are caused by Leptospira strains belonging to other serovars of the serogroup Pomona. On the contrary, leptospires of the Mozdok serovar were cultured from rodents and domestic animals world-wide including Central Europe where only Leptospira strains of the serovars Pomona and Mozdok are known to be present till now. STUDY OBJECTIVE: The aim of the study was to discover if leptospires of Mozdok serovar may cause human leptospirosis that remained hidden till now among infections diagnosed merely by MAT as Pomona illnesses. MATERIAL AND METHODS: The reference Leptospira strains of Pomona and Mozdok serovars (Pomona and 5621), as well as three endemic, and in some tests only two strains of human and pig origin (Simon, S-23, Pöstényi), and two strains of rodent provenance - Apodemus agrarius (M-210/98 and M-71/01) were used for this purpose. First, the endemic strains were assigned to one of the afore-mentioned two serovars by agglutinin cross-absorption tests performed using rabbit immune sera, monoclonal antibodies and random amplified polymorphic DNA methods. Afterwards, twenty-one sera of patients with a Pomona leptospirosis confirmed by MAT were examined by agglutinin absorption test (AAT). RESULTS: Based on the results of the mentioned laboratory method used, the endemic Leptospira strains of human and pig origin could be affiliated to the serovar Pomona, while those of rodent origin were classified as serovar Mozdok strains. Out of the 21 patients sera, an illness caused by the serovar Mozdok strains was found out in 13 cases and a disease caused by serovar Pomona strains in 8 cases. Their differentiation was made on the strength of the following results of AATs: All strains from the serovar Mozdok have completely absorbed antibodies (anti-Pomona and anti-Mozdok) from the tested sera, however following the absorption of these sera with the Pomona strains, high levels of residual antibodies reacting in MAT with the Mozdok strains have still persisted. In this way, it was possible to prove the Mozdok infection in thirteen patients. On the contrary, following the absorption of the sera with the strains of the serovar Pomona, a complete absorption of all antibodies (anti-Pomona and anti-Mozdok) was achieved in seven cases using the strain Simon, and in one case with the strain S-23, whereas after absorption using the Pomona strain, the residual antibodies were still present in all sera, and also in the majority of them when they were absorbed using the strains S-23 and Pöstényi. In this context, the Pomona infection was determined in the case of eight patients. Hence it follows that not all strains of the Pomona serovar were suitable for the AATs. CONCLUSION: The presence of the human Mozdok leptospirosis was confirmed for the first time by the use of the agglutinin absorption test. A clear correlation between the habitat areas of the A. agrarius and the patients who were infected with the strains of the Mozdok serovar was determined.

DNA relatedness and serotyping of Leptospira strains.

BACKGROUND: In a previous study (5), the constructed phylogenetic tree for leptospires belonging to 12 different serovars common in Central Europe made the prediction of serovar from knowing the genotype and vice versa possible. OBJECTIVE: The study was aimed at investigation of the usefulness of such procedure to distinguish in between at present to us available and worldwide accepted reference strains of pathogenic Leptospira serovars. MATERIAL AND METHODS: One hundred and seventy seven Leptospira strains representing different serovars were tested. DNA fingerprints of these strains were performed, digitally captured and as described earlier of those phylogenetic tree using different fingerprinting software was constructed using UPGMA clustering method with band matching by the Dice coefficient (5). RESULTS: At this tree, 145 of 177 Leptospira strains tested each took a unique position, and the remaining 32 strains were distributed at 15 different positions (each of 14 positions taken by two different strains and one position taken by four strains). CONCLUSION: The constructed phylogenetic tree likely to be very useful in prediction of Leptospira serovar in most cases of an infection so the saving time and being helpful in serovar identification of the pathogenic agent (Fig. 1, Ref. 9).

Utility of phylogenetic studies in the identification of Leptospira strains.

A phylogenetic tree, which distinguishes between the serovars and serogroups of leptospires common in Central Europe was constructed using an established RAPD procedure together with digital reading and evaluation (using different computer software programs) of the generated amplified DNA patterns. The application of this procedure has revealed a consistent correspondence between serogroup and genotype (position in constructed tree) in 69 cases, and serovar and genotype in 72 cases, of wild strains of leptospires. There was an agreement between serovar and genotype in cases of strains of Grippotyphosa, Pomona, Mozdok, Arborea and Sorexjalna as well as between serogroup and genotype in cases of Australis, Bataviae and Sejroe. With the procedure used in this study, it was not possible to distinguish between reference strains of serovars Jalna, Bratislava and Lora (all serogroup Australis) as well as between serovars Icterohaemorrhagiae and Copenhageni (both of serogroup Icterohaemorrhagiae). In contrast to this, wild strains belonging to serogroup Sejroe were distributed between Polonica, Istrica, Saxkoebing and Sejroe serovars. Endemic strains of leptospires tested, were also distinguishable.

Mycobacterium avium subsp. paratuberculosis infection in cattle in Austria, diagnosis with culture, PCR and ELISA.

Serum samples from healthy, infected (n=11) and diseased (n=2) cattle as well as positive (n=17) and negative (n=41) reference sera were tested for antibodies to Mycobacterium avium subsp. paratuberculosis with two ELISA-methods (A-ELISA, Allied Monitors, Fayette, USA; H-ELISA, Institute of Microbiology and Animal Diseases, Veterinary University Hannover). Fecal samples of these animals were examined by PCR and culture. Also field serum samples found to be positive (n=664) or inconclusive (n=1589) by A-ELISA during a survey done on 11028 cattle of 2757 farms at different districts in Austria were retested with H-ELISA (Gasteiner et al., 1999). In both ELISA-methods total agreement between antibody detection and shedding of M. avium subsp. paratuberculosis (PCR, culture) in cases of diseased animals during the testing period was found. In subclinically infected animals H-ELISA showed a better correlation with the results of PCR and fecal culture. Reference serum samples of culturally negative cattle were negative in 98% by H-ELISA and in 82% by A-ELISA, and those of positive animals were positive in 59% by H-ELISA and in 82% by A-ELISA. The 664 A-ELISA positive field serum samples were positive in 20.5%, inconclusive in 32.5% and negative in 47% by H-ELISA. A-ELISA inconclusive sera gave positive reactions by H-ELISA in 5.2%, negative in 74.8% and inconclusive results in 20%. The highest prevalence of antibodies (7.9% by A- and 2.2% by H-ELISA) against M. avium subsp. paratuberculosis were found in cattle at the age of six and seven years. Seropositive animals were found at all tested ages. The A-ELISA gave two to three times more positive reactors than the H-ELISA. Also both tests showed the highest prevalence of reagents among Holstein Friesian (6.2% by A-ELISA, 2.5% by H-ELISA) followed by other cattle breeds. Seropositive cattle were observed in all districts of Austria in 3. 3-7.1% and in 0.5-1.8% of herds according to A- and H-ELISA, respectively.

Characterization of intestinal cnf1+ Escherichia coli from weaned pigs.

Escherichia coli isolated from 204 cases of porcine postweaning diarrhoea were tested by PCR for the genes of cytotoxic necrotic factors (CNF) and of cytolethal dystending toxin (CDT). Selected strains were also examined by PCR for the presence of papC-, sfa-, f17-, f18-, and afa-specific sequences encoding P, S, F17, F18 fimbriae and afimbrial adhesins. A 5.9% (12/204) of the strains had cnf1 gene, and two of them had cdt gene as well. Further six cdt+ strains were detected which were cnf-negative. Most of the cnf1+ strains belonged to serogroups O2, O6, O8, O54 characteristic of necrotoxic E. coli (NTEC) of humans. All the cnf1+ strains possessed the genes for P or S fimbriae or both, but were negative for F4, F17, or F18 or afimbrial adhesins. Results suggest that these enteric isolates may have entero- and/or uropathogenic significance in weaned pigs, and may have zoonotic potential for humans.

[Detection of Mycobacterium avium subspecies paratuberculosis (M. p.) by the polymerase chain reaction (PCR) and culture experiments in animals in Austria]. / Nachweis von Mycobacterium avium subspecies paratuberculosis (M. p.) mittels Polymerase-Kettenreaktion (PCR) und Kulturversuch (KV) bei Tieren in Osterreich.

The polymerase chain reaction and cultural method was applied to detect Mycobacterium avium subspecies paratuberculosis (M. p.) in fecal samples of 165 suspected and of 35 diseased cattle, 18 intestinal tissues and 14 lymph nodes of diseased ruminants as well as organ material (n = 10) were also tested. An agreement (+/+, -/-) in the results between both methods was found in 89.7% of all cases examined. 12.7% of samples of suspected and 9.1% of diseased animals were only positive by PCR. In cases of intestinal lymph node and tissue as well as udder tissue of diseased cattle a total agreement between PCR and culture was observed. Lymph node of lung of one diseased cow was positive only in PCR.

Phage restriction and the presence of small plasmids in Salmonella enteritidis.

Between 1990-1994, a total of 16,505 S. enteritidis strains of human, animal and food origin were phage-typed, using the Hungarian scheme and the changes of incidence of the dominant phage types were monitored. The incidence of PT1 (corresponding to Ward's PT1 was very high between 1990 and 1992 (67.9-71.0% of the total S. enteritidis isolates), later, it decreased. The prevalence of PT6 (corresponding to Ward's PT4) was rare until 1992, then it gradually increased. The phage type and plasmid content of 78 Salmonella enteritidis strains were determined. Small plasmids were present in 59% of the isolates, together with a serotype-specific (38 MDa) plasmid. A correlation was found between the presence of the small plasmid and phage restriction to two phages used for subdividing the Hungarian phage types 1 (PT1) and 6 (PT6) of S. enteritidis (corresponding to PT1 and PT4 in Ward's typing scheme, respectively).

[Clinical and microbiological investigations of atrophic rhinitis of Upper Austrian swine herds]. / Klinische und mikrobiologische Untersuchungen über die Rhinitis atrophicans in oberösterreichischen Schweinebeständen.

The aim of this study was to determine the distribution of the progressive and non-progressive atrophic rhinitis of Upper Austrian swine herds. Further on the resistance pattern of the pathogens involved to chemotherapeutics was tested. In the period of May 1993 to June 1996 a total of 56 Upper Austrian swine herds were examined and on the occasion of the animal herd health management 997 nasal swab-samples of young pigs taken. The area of this investigation included 14 Upper Austrian districts and the herds examined were divided into 3 types. Type 1 were swine herds of the swine breeding association (SZV), type 2 piglet producing farms (FP) and type 3 closed swine herds (GB). Sucking- and weaning piglets aged from 4 to 10 weeks were selected for these examinations. On the average 10 nasal swab-samples (2 swabs per animal) per herd were taken, microbiologically examined and the toxin by means of ELISA-tests determined. In terms of resistance testing (antibiogram) 20 different chemotherapeutics, which consisted of 11 different groups of drugs, were used. As a result of 997 nasal swab-samples examined, 304 (30.5%) P. m., 111 (11.1%) tox. P. m. and 35 (3.5%) B. b. were isolated. 50% of the Upper Austrian swine herds showed PAR by means of microbiological examination and ELISA-tests as well. The resistance pattern of P. m. and B. b. exhibited significant differences. Penicillin and lincospectin were highly resistant concerning isolates of B. b., but were highly sensitive for P. m. Enrofloxacin turned out as the most effective drug to the P. m.-toxin-negative- and B. b.-strains tested, because no resistance was observed. Finally efficient PAR control programmes of swine herds belonging to the Upper Austrian swine herd health service are described.

[Blood serum and milk serum levels of sodium benzylpenicillin after intravenous regional antibiosis in surgery of the claw area in cattle]. / Blutserum- und Milchserumspiegel nach intravenöser Stauungsantibiose mit Na-Benzylpenicillin bei Eingriffen im Zehenbereich des Rindes.

An intravenous regional antibiosis using 10 Mega I.U. Sodium-Benzylpenicillin was carried out during surgery in 14 dairy cows affected with spontaneous occurring digital diseases. Blood serum and milk serum levels were determined over a period of 48 hours by means of a micro agar diffusion method using Sarcina lutea Stamm BGA Berlin as a test germ. Even before opening of the rubber tourniquet, penicillin traces were detectable in the blood serum of 6 cows. Maximal concentrations ranging from 0.43 mcg/ml to 8.63 mcg/ml were determined after 45 minutes to 4 hours. 24 hours after intravenous regional antibiosis no levels above the limit of detectability were determined. Significant lower maximal concentrations were found in claw amputation cases as compared with claw preserving surgical interventions. Milk serum levels showed delayed and lower maximal concentrations, ranging between 2.09 and 0.09 mcg/ml. In 5 animals no positive levels were detected. After 48 hours, penicillin levels were below the limit of detectability in the milk of all examined animals.

[The effect of husbandry, management and stall climate on lung changes in swine]. / Der Einfluss von Haltung, Management und Stallklima auf die Lungenveränderungen bei Schweinen.

21 swine fattening units (17 closed and 4 open swine herds), their pigs suffering from chronic respiratory disease since a long time were investigated concerning fattening period, morbidity, mortality, lung lesions by slaughtered swine, microorganisms involved, husbandry and management conditions, stall climate (indoor temperature, relative air humidity, contents of NH3, CO2, H2S, air microorganisms and dust). We estimated annual fattening periods of 137.4 +/- 6.8 days, morbidity of 49.1 +/- 12.2% and mortality of 6.8 +/- 3.7%. About 55-96% of lungs of slaughtered pigs showed pneumonic lesions in different degrees. Bacteriological examination of such lesions revealed up to 9 and not less than 3 different bacteria species per unit. Continuous producing systems were used by 19 units, only 2 units were managed by all in all out system. Pigs of 70 kg weight have air spaces of 1.7-3.1 m3, indoor temperature were between 21-26 C degrees and relative air humidity between 83.9 +/- 13.7%. Concentration of NH3 was found between 3-35 ppm, of CO2 between 500-3500 ppm, the numerical content of bacteria was within 900-1620 cfu (colony forming units) and of fungi between 2-80 cfu per litre air. There were also E. coli with and without haemolysis, Klebsiella pneumoniae, beta-haemolyzing streptococci and staphylococci in stall air. 2 swine houses gave 3-5, 15 houses 12-50 and 4 houses more than 50 cfu/cm2 blood agar plates exposed for 1 minute to the stable air (sedimentation method to judge stall dust). Animals of the later mentioned 4 houses showed the highest incidence of respiratory disorders (> 50% of pigs).(ABSTRACT TRUNCATED AT 250 WORDS)

[Occurrence and drug resistance of bacteria pathogenic to the lungs from autopsy material of swine]. / Vorkommen und Resistenz gegen Chemotherapeutika von lungenpathogenen Bakterien aus Sektionsmaterial beim Schwein.

Eight hundred and fifty-four piglets which died or were euthanized due to pneumonia or rhinitis atrophicans, were investigated during the period of 1986-1990. Of the animals, 569 showed bronchopneumonia, 218 had pleuritis, pericarditis and peritonitis, 165 had rhinitis atrophicans, 58 pleuropneumonia, and 9 animals had fibrinous pneumonia. Pasteurella multocida, Haemophilus parasuis, Bordetella bronchiseptica, Actinobacillus pleuropneumoniae and Pasteurella haemolytica were isolated in 59.1%, 29.5%, 27.8%, 3.7%, and 2.3% cases of bronchopneumonia respectively. Samples from pigs with pleuritis or rhinitis atrophicans showed Pasteurella multocida in 63.8 and 68.5%, Bordetella bronchiseptica in 28.4 and 39.4%, streptococci in 28.9 and 3.9%, Haemophilus parasuis in 25.2% and 20.6%, Actinobacillus pleuropneumoniae in 5.1 and 5.5%, and Pasteurella haemolytica in 3.2 and 3.0%, respectively Actinobacillus pleuropneumoniae was found in 51 of 58 cases of pleuropneumonia and in 5 of 9 cases of fibrinous pneumonia; 55.6% and 44.4% respectively of those forms of pneumonia were positive for Pasteurella multocida. In the agar diffusion test, 36.8-82.6% of bacterial isolates showed resistance to streptomycin, 7.7-45.5% to sulfamethoxazole-trimethoprim, 5.7-44.6% to tetracycline, 0.2-32.8% to ampicillin, 0.0-16.3% to lincospectin, 2.0-81.2% to furazolidone, 0.4-4.5% to chloramphenicol, 1.3-78.1% to penicillin and 0-0.3% to enrofloxacin.

[Legionella antibodies in domestic animals]. / Protilátky proti legionelám u domácich zvierat.

Serological examination of 420 domestic animals for the presence of antilegionella antibodies indicates their high exposure to legionellae. On examination by the microagglutination reaction with a serum dilution of 1:64 or more the highest positive values were recorded in horses which reacted with antigens of L. pneumophila 1-14 in 36.2% and with antigens of another 19 types of legionellae in 47.8%. In pigs positive values recorded in 16.2% and in 21.1%; in cattle in 3.8% and 29.5%, in sheep in 7.5% and 11.3% and laboratory rabbits were quite negative. The importance of these findings with regard to the possible role of animals in the ecology of legionellae is obscure.

Seroprevalence survey for Anaplasma marginale-infection of Austrian cattle.

A serologic survey study of 5,076 Austrian cattle farming herds was carried out in the period of December 1988 till March 1990. One animal was randomly selected from each herd and the antibody titer against Anaplasma marginale in blood serum samples was evaluated by means of the complement fixation test. The number of these tested blood samples was 3.6% of 140,081 cattle herd farms of Austria. 109 (2.1%) of the tested animals showed positive titers (1:10) against Anaplasma marginale, in relation to the 140,081 cattle herds 0.08%, 4,786 (94.3%) blood serum samples were sero-negative, 188 (3.8%) reacted anticomplementary. The highest number of antibody-positive animals of 8 tested Austrian districts could be found in Carinthia (46 = 5.7%). In Burgenland all tested sera turned out to be negative. Concerning the distribution of sero-positive animals in Austria it can be stated that a decrease of positive reactors from southern to northern region is evident. A connection between the occurrence of anaplasmosis in Italy, Yugoslavia, Switzerland and Hungary, is postulated as a result of the different systems of keeping cattle in the provinces and the regional increase of tick invasion. Possibly an intensive animal transportation is of importance due to the introduction of the disease mentioned before. The results obtained show that anaplasmosis does occur in different areas of Austria. For control of this disease in Austria it is proposed that all imported cattle should be tested serologically for antibodies against Anaplasma marginale. Other diseases in connection with anemia should be excluded by clinical, serological, blood-, as well as pathological examinations.

[Taylorella equigenitalis: cell wall proteins, gene fingerprints, plasmids, adhesion and toxicity]. / Untersuchungen an Taylorella equigenitalis: Zellwandproteine, Genomfingerprints, Plasmide, Adhäsion und Toxizität.

In this study 55 strains of Taylorella equigenitalis isolated from horses of four different studs in Austria, and a comparative strain from the Federal Republic of Germany were investigated by different methods. These investigations were carried out with the help of SDS-PAGE, immunoblotting, the analyses of genomes and by proof of plasmids. Furthermore, pathogenic mechanisms such as adhesion or the formation of toxins were investigated in vitro. On the basis of the results carried out by means of SDS-PAGE and immunoblotting all tested strains of Taylorella equigenitalis were alike, whereas by DNA analyses the strains could be divided into five groups. The comparative strain from the FRG, which clearly differed from the Austrian strains, formed one group all by itself. From three studs, which are related to each other because of an intensive exchange of horses, representatives (n = 53) of three DNA fingerprint groups were isolated. These three fingerprint patterns were very similar to each other, while the hybridisation patterns from the other two Austrian strains were very different. One of these strains, isolated from a diseased mare, could not be distinguished from the other strain isolated from a clinical healthy stallion from the same study by this method. Only 47.3% from the investigated strains showed attachment to HeLa cells, while cell extracts of all of them caused morphological changes of a varying degree of both Y1 and Vero cells. There were no connexions between these adhesion-cytotoxicity-properties and the DNA fingerprint groups as well as the studs, respectively. No plasmids were found in the Taylorella equigenitalis strains used in this study.

[Verification of the protective effect of a toxoid vaccine against edema disease of weaned piglets in an infection model]. / Uberprüfung der Schutzwirkung eines Toxoidimpfstoffes gegen die Odemkrankheit des Absetzferkels im Infektionsmodell.

105 piglets (56 vaccinated and 49 control animals) were utilized in 6 consecutive experiments. Each used litter was divided randomly into vaccine and control animals. One week prior to weaning each of the 56 piglets of the vaccine groups received 5 mg of nonpurified toxin treated with glutaraldehyd subcutaneously whereas to the remaining 49 control animals an extract of apathogenic E. coli was administered. During the first 12 hours post weaning each of the 105 piglets was challenged perorally with 10(10) cfu of edema principle toxin producing germs of E. coli serogroup O 139. 23 animals of the control groups (46.9%) and one animal of vaccine groups (1.8%) died due to the infection between days four and five post challenge. These control animals showed classical clinical symptoms as well as pathological findings typical for edema disease. In contrast, such findings as mentioned before could not be observed in the vaccinated piglets. The remaining part of the control animals and eight of those vaccinated ones exhibited edema disease symptoms. The vaccinated animals have shed the challenge strain one to three days, while the survivals of control groups shed those germs for two to six days. The vaccinated piglets showed a better growth rate than the remaining control animals. Presented data suggest that our toxoid immunizing procedure can be used successfully against edema disease of swine.

Pilus production, hemagglutination, and adhesion by porcine strains of enterotoxigenic Escherichia coli lacking K88, K99, and 987P antigens.

Three strains of enterotoxigenic Escherichia coli which adhered, colonized intensively, and caused disease in pig intestine, but which did not produce pili of the K88, K99, or 987P antigen types were designated 3P(-) ETEC. The 3P(-) ETEC caused mannose-resistant hemagglutination, adhered to porcine intestinal epithelial cells in vitro, and produced pili. However, most bacteria taken directly from the intestine of pigs infected with 3P(-) ETEC appeared to be nonpiliated. Two preparations were isolated from the 3P(-) ETEC. One (material A) contained pili, caused mannose-sensitive hemagglutination, and did not inhibit adhesion of whole bacteria to epithelial cells in vitro. The other (material B) had no demonstrable pili, caused mannose-resistant hemagglutination, and blocked ahesion of bacteria to epithelial cells in vitro. Antiserum against an acapsular mutant (K(-)) of one 3P(-) ETEC strain was absorbed to remove antibodies directed against somatic (O) antigen. The absorbed antiserum agglutinated all three 3P(-) ETEC strains grown in the K(-) form at 37 degrees C, but not when they were grown at 18 degrees C. The absorbed antiserum blocked the hemagglutinating activity of material B, but not of material A. It also reacted (via indirect immunofluorescence) with all of the 3P(-) ETEC when they were grown in pig intestine. The results were interpreted to indicate that: (i) the epithelial adhesive and mannose-resistant hemagglutinating activities of the 3P(-) ETEC strains may be mediated by an antigen contained in material B; (ii) this antigen either is not pilus associated or is associated with pili that are not demonstrable by the methods used here; (iii) the 3P(-) ETEC strains produce type 1 pili which do not mediate their adhesion to intestinal epithelium of pigs.