Aspects of clinical features, diagnosis, notification and tracing back referring to Trichinella outbreaks in north Rhine-Westphalia, Germany, 1998.

52 cases of human trichinellosis were notified from 11 towns in North Rhine-Westphalia from November 1998 to March 1999. After non-typical symptoms in the enteral phase, fever, muscular ache, headache, oedema, disorder of vision and rash occurred in the parenteral phase. Trichinellosis was serologically confirmed by ELISA, IFAT or western blot. Raw sausage and minced meat produced from raw pork could be determined as probable source of infection with 44 and eight notified cases, respectively. Whereas questionable raw sausage was not available for examination, frozen minced meat from the second outbreak could be secured in households of infected people. Larvae were isolated from minced meat and were identified by PCR as Trichinella spiralis. Tracing back to the source of infection was difficult because of the long time between clinical symptoms, laboratory diagnosis and notification as well as complex trade routes for pork and its products. Trichinella cases emphasize the necessity to meet the prescribed slaughter inspection and to guarantee a reliable prove of origin for meat products especially in view of specific consumer habits, i.e. the consumption of raw meat.

[Increasing number of Salmonella paratyphi B isolates from slaughtered poultry sent in to the national Salmonella reference laboratory]. / Gehäufte Einsendungen von Salmonella Paratyphi B-Isolaten aus Schlachtgeflügel an das Nationale Referenzlabor für Salmonellen.

In the last years the number of isolations of Salmonella enterica subspecies enterica serovar paratyphi B (S. paratyphi B) sent to the national salmonella reference laboratory of Germany has increased steadily. Most of the isolates originated from fowl or poultry products. The bacteriological, serological and biochemical properties of the isolates were investigated. Special emphasis was given to the utilization of d-tartrate which subgroups the serovar. All of them belonged to the d-tartrate positive variant, which is generally considered less virulent for humans and was formerly called S. java. The performance of various tests is compared and in addition the possibility of the spread within the production line is discussed.

Characterization of shiga toxin-producing Escherichia coli strains isolated from calves in Poland.

Fecal samples from 67 3-5-months-old calves with diarrhea were screened for the presence of shiga toxin-producing Escherichia coli (STEC). Several accessory virulence factors genes were also tested. Among 192 E. coli isolates tested, 15 (7.6%) were found to harbour the shiga toxin 1 or 2 (stx1 or stx2) genes. The stx2-carrying samples were further subtyped by PCR for the stx2c, stx2d, and stx2e toxin variants. It was shown that stx2-positive bacteria mainly possessed the stx2c shiga toxin type gene. The enterohemolysin (hlyA) and intimin (eae) genes were found in seven (46.7%) STEC strains whereas the cytotoxic necrotizin factor 1 and 2 or the P fimbrial genes were detected in two isolates only. This study confirmed that calves are a reservoir of STEC strains (with all pathogenicity genes) that may be virulent for humans.

Rapid and specific differentiation of enterotoxin-producing Escherichia coli strains from other gram-negative enteric bacteria using multiplex PCR.

Enterotoxigenic Escherichia coli (ETEC) may produce heat-labile (LT) and heat-stable (STI or STII) enterotoxins. Differentiation between ETEC and other pathogenic and non-pathogenic E. coli as well as other Gram-negative bacteria responsible for induction of diarrhoea, requires isolation, biochemical identification and determination of toxins (or their genes--elt, estI, estII). A multiplex polymerase chain reaction (PCR) system for the rapid and specific detection of enterotoxin-gene-positive E. coli was developed. The primers described by other authors, specific for the universal stress protein A (UspA) of E. coli and enterotoxin genes were used and allowed a simultaneous amplification of the E. coli-specific uspA and the respective toxin genes. The specificity of this multiplex PCR system was confirmed by testing ETEC, non-ETEC and other non-E. coli bacteria. The specific 884 bp uspA gene and 280 bp (eltI), 166 bp (estI) or 278 bp (estII) amplification products were generated with the respective ETEC strains whereas no amplification was detected with non-E. coli bacteria. The multiplex PCR developed allowed the rapid and specific identification of enterotoxin-producing E. coli colonies directly grown from faecal samples of pigs with diarrhoea. The test may be used as a method for the determination of ETEC among other pathogenic groups of E. coli and other Gram-negative enteric isolates.

[Methods for checking up screening tests for detection of shiga toxin producing Escherichia coli (STEC) using PCR]. / Kontrollmöglichkeiten für Screeningtests zum Nachweis von Shigatoxin-produzierenden Escherichia coli (STEC) mittels PCR.

Methods for making check ups of STEC-Screening-PCRs by using internal and external systems are given. A control-DNA with identical primer binding sites in relation to the target-DNA (stx-genes) was produced and by HPLC-chromatography quantified. It was used in an internal system. But we had to use too high concentrations of this DNA for making the check up in each sample. A false negative result could be shown in samples with a low stx content as a result of this coamplification process. Therefore we used an external system. A control-DNA with heterologous primer binding sites in relation to the stx-screening-PCR and the special primers were given in each vessel. The optimized system was used for making investigations of some faecal samples from cattle.

Salmonella in slaughter pigs of German origin: an epidemiological study.

The Salmonella prevalence in slaughter pigs of German origin was determined in seven abattoirs located in different regions of the country between February and June 1996. A total of 11,942 pigs delivered to the abattoirs in 752 batches, most of them comprised of pigs from individual finishing farms, was investigated by the bacteriological examination of faecal and gut lymph node samples, as well as of surface swabs taken from the carcasses. Salmonellae were isolated from 3.7% of the faecal samples, 3.3% of the lymph nodes and 4.7% of the surface swabs. The estimated overall prevalence of Salmonellae was 6.2% in the slaughter pigs, ranging between 1.9% and 12% in individual abattoirs. In the samples taken from carcasses, the estimated prevalence of Salmonellae reached 10.3%. 648 out of 752 batches could be included in a statistical analysis. No Salmonellae were detected in nearly 70 percent of the batches included in this analysis (n = 648). High Salmonella prevalences of more than 50 percent positive animals were detected only in 13 batches (2.0%). A statistically significant influence of the duration of the transport of slaughter pigs to the abattoirs or the waiting period in the abattoirs prior to slaughter could not be detected.

[Detection and prevalence of verotoxin-producing Escherichia coli (VTEC) in raw sausage]. / Untersuchungen zum Nachweis und Vorkommen von Verotoxin-bildenden Escherichia coli (VTEC) in Rohwurst.

We investigated 158 samples of shortly ripened raw sausages bought in supermarkets of Dessau within 4 month. In 14 (8.8%) samples Verotoxin-producing E. coli were detected. 13 VT-positive samples were found in the group of easily spread raw sausages. The 14 isolates belonged to 6 different O-serotypes. 4 VT1-, 8 VT2- and 2 VT1/VT2-producers were found. 4 isolates belonged to serogroups which were already described in WHO tables and associated with EHEC infections in human beings. One strain of serogroup O22: H8, isolated from a "Teewurst", possessed the complete virulence gene combination of EHEC (eae, hlyA, stx). The detection procedure, already successfully used for detection and isolation of VTEC from raw milk, soft cheese and raw minced beef showed a sensitivity of approximately 10 CfU/25 g of raw sausages. It has to be considered that VTEC are frequently (8.8%) present in shortly ripened raw sausages. The group of easily spread raw sausages has a higher VTEC-contamination rate than firm raw sausages. Raw sausages, especially easy to spread types, belong to the risk foods for EHEC-infections in human beings.

The use of polymerase chain reaction for determination of virulence factors of Escherichia coli strains isolated from pigs in Poland.

E. coli strains isolated from pigs with postweaning diarrhea or edema disease were tested by phenotypic and genotypic methods for the presence of virulence antigens and genes, respectively. The slide agglutination and ELISA analyses were used for determination of F4, F5, F6, F17, and F41 fimbriae whereas the prevalence of fimbrial fedA and toxin eltI, estI, estII, stx1, stx2 and stx2e genes were recorded by the means of PCR. Only F4 antigen (ac variant) was found in strains of the serogroup O149:K91 isolated from pigs with diarrhea. PCR analyses showed that the fedA gene encoding F18 fimbriae was present in 61.9% of strains isolated from pigs with diarrhea and in 84.2% of strains isolated from pigs with edema disease. The eltI genes encoding heat-labile toxin I (LTI) were present only in 9 out of 21 strains recovered from pigs with diarrhea. Shiga toxin 2 variant (stx2e) genes were found in six isolates from edema disease and also in one strain from diarrhea. The PCR test used in the study was a sensitive and valuable method for determination of virulence factors of E. coli strains.

[Necessity of double testing of meat juice samples from pigs using ELISA for the determination of Salmonella status in pig farms]. / Uber den Nutzen von Doppelbestimmungen bei Fleischsaftuntersuchungen mittels ELISA für die Beurteilung des Salmonellenstatus von Schweinebeständen.

According to the test protocol of the "meat juice ELISA" for detection of salmonella antibodies in pigs, all meat juice samples and serum controls are to be tested in duplicate. Results from routine investigations of repeatedly double tested meat juice and serum samples have been used to analyze the effect of double testing versus single testing with regard to the reliability of the final result. In case of an individual animal, testing of samples in duplicate increases the reliability of the results significantly, especially, if samples are retested at different occasions. In contrast, such a difference between mono and double testing of samples is not of importance when a group of animals is tested in order to determine the mean antibody rate in a herd. Here, results from double testing practically do not contribute to a higher reliability of the final result. This observation provides the possibility to reduce the costs for investigation programmes drastically.

[Detection of pas gene in shiga toxin producing Escherichia coli (STEC)]. / Bestimmung des pas-Gens in Shigatoxin-bildenden Escherichia coli (STEC).

Results of detection of the pas gene in 10 shigatoxin-producing Escherichia coli strains (STEC) isolated from foods (raw milk, certified milk, and beef) and in 18 enterohaemorrhagic Escherichia coli strains (EHEC) isolated from stool samples of patients suffering from HUS, diarrhea or from carriers without symptoms are given. All isolates showed the eae gene coding for intimin. We could make sure by using PCR that all isolates showed the pas gene, too. This gene is a factor mainly responsible for proteinsecretion and for the attacing and effacing phenomenon. A correlation of pas to other virulence factors was not given. The detection of the pas gene makes further characterization of STEC/EHEC-Isolates possible.

[Utilization of nylon membranes for the specific isolation of Escherichia coli O157 with DNA probes and checking for relationship to the shiga toxin Escherichia coli (STEC) group]. / Einsatz von Nylonmembranen zur spezifischen Isolierung von Escherichia coli O157 mittels DNA-Sonden und Prüfung auf STEC-Gruppenzugehörigkeit.

A method for specific isolation of Escherichia coli strains of serotype O157 is given. DNA-hybridization technique by using DIG-labeled specific O157 PCR-amplificates as probes is the basis. These investigations can be used for the detection and isolation of shigatoxin-producing Escherichia coli (STEC) of this serotype in combination with other tests for detection of shigatoxin genes (stx). No background is seen by using 'DIG Easy Hyb' solution and Nylon membranes for colony-and plaque-hybridization (Roche Diagnostics Boehringer Mannheim). Dark brown spots (E. coli colonies) are visible on the membranes after staining. After comparing the membrane with the masterplate it is possible to isolate the colonies from the masterplate, respectively. It is necessary to make proof that the isolates possess the rfb-genes (verification of serotype O157) and the stx-genes (test for belonging to the STEC-group). Both tests could be done by using PCR. In case of positive results other virulence factors of the isolates can be detected.

Detection of Brucella species in organs of naturally infected cattle by polymerase chain reaction.

A polymerase chain reaction (PCR) assay was used to detect Brucella species in the uterus, udder, spleen, lymph nodes, kidney and liver of three cows which had been naturally infected in an outbreak of brucellosis, and the results were compared with the results of bacteriological investigations. All 18 samples reacted positively in the PCR, but five samples had weak bands after the electrophoretic separation of PCR mixtures. No Brucella strains could be detected in these five samples by bacterial cultivation, but all the other samples gave positive results. A pre-enrichment procedure was necessary for the PCR. A PCR with DNA from eight Yersinia strains gave no amplification product.

Detection of STEC and epidemiological investigations in surrounding of a HUS patient.

After occurrence of a case of HUS infection in a 2-year-old infant from a dairy farmer's family living near Oldenburg, investigations were performed in the infant's surrounding in order to elucidate the route of infection. Since hospitalization took place at a late stage, it was not possible to isolate EHEC from the patient's stool samples. However, E. coli O157 antibody determinations in serum were positive. Since STEC of serogroup O157 were found in faeces from the 34 dairy cows of the farm, stool samples were taken from 6 members of the child's family and examined. Non-O157 STEC could be isolated from the stools of 2 family members. Determination of other virulence factors and other characteristics such as serotype, biotype and phage type showed identity of the agent for 3 isolates (2 from animals, 1 from humans). By means of pulsed-field gel electrophoresis of the restricted DNA of the isolates and by means of RAPD-PCR it was not possible to establish any differences in the band patterns. It can be assumed, therefore, that the organisms had been transmitted from animals to humans.

[Verotoxin-producing E. coli (VTEC) in feces from cattle slaughtered in Germany]. / Verotoxin-bildende E. coli (VTEC) im Kot von Schlachtrindern aus Deutschland.

In man, EHEC infections may result in severe disease. Cattle and foods derived from this animal species are considered as a source of infection. The presence of VTEC being potential EHEC was studied. For analysis, feces samples were examined which had been taken from 204 heads of cattle slaughtered in various regions of Germany. VTEC could be isolated from 97 animals (47.6%). This indicates a presence of VTEC in slaughtered cattle being 5 times higher than known for Germany so far. The aeaA gene could be demonstrated in a mere 23 out of 667 VTEC isolates. The CVD 419 sequence was present in 55.3% of the VTEC isolates. Ehly was found in 61% of them. Consequently, both markers were unsuitable for the detection of VTEC in faeces samples from cattle and in foods with faecal contamination. The VTEC isolates belonged to 54 different serotypes of E. coli, VTEC 0157 have not been found so far. Some of the VTEC serovars found in this study have already been described as associated with human disease following EHEC infection. The presently available laboratory methods do not permit to exclude a risk for humans from bovine VTEC reliably. For this reason, bovine VTEC should be further on considered as potential EHEC and an infection of humans by such agents be avoided.

How to modify conditions limiting resistance in bacteria in animals and other reservoirs.

Antimicrobial agents in veterinary medicine are used for three purposes: therapy, prophylaxis, and nutrition. The major public health risk is that selection pressure leads to an increase in the pool of resistance genes. Since 1987, the nutritional use of antimicrobials in Europe has been regulated by a council directive, which demands special investigations into the potential of antimicrobials to increase rates of drug resistance. However, the prophylactic and therapeutic use of antimicrobials has sometimes led to the emergence of resistant bacteria. For example, the selective effect of the prophylactic use of gentamicin and the therapeutic use of quinolones led to the emergence of resistant salmonellae. To prevent the spread of resistant microorganisms from animals to humans, it should be recognized that antibiotics are not suitable as a compensation for poor hygiene standards or for the eradication of a pathogen from a certain environment. They should be used only by doctors or veterinarians.

[Optimization of the polymerase chain reaction (PCR) for detection and characterization of shigatoxin producing Escherichia coli (STEC) in food]. / Optimierung der Polymerasekettenreaktion (PCR) zum Nachweis und zur Charakterisierung von Shigatoxin produzierenden Escherichia coli (STEC) in Lebensmitteln.

The polymerase chain reaction (PCR) was improved to detect shigatoxin producing Escherichia coli (STEC) in milk. Numbers of colony forming units (cfu) in test samples, concentrations and types of primers, amount of MgCl2, types of thermostable DNA-Polymerase, and cycling programs were modified up to obtain the cleanest electrophoretic pattern and the highest sensitivity. Experimental conditions for further characterization of STEC-isolates by means of PCR are given by summarizing data from literature.

[Utilization of nylon membranes for specific isolation and characterization of verotoxin-producing Escherichia coli using DNA probes]. / Einsatz von Nylon Membranen zur gezielten Isolierung und Charakterisierung Verotoxin-bildender Escherichia coli mittels DNA-Sonden.

A method for specific isolation of VT(+)-strains in raw milk is given. DNA-hybridization technique with DIG-labeled PCR-amplificates as probes are the basis. No background is seen by using "DIG Easy Hyb" solution and nylon membranes for colony- and plaque-hybridization (Boehringer Mannheim GmbH). Marked colonies are visible on the membranes after detection. So it is possible to select these colonies from a masterplate. The results are available within one day (without enrichment and membrane preparation). After stripping the membranes can be used for a new hybridisation to detect another factor of virulence.

[Diagnosis of trichinellosis in living pigs using indirect ELISA]. / Intravitale Diagnostik der Trichinellose beim Schwein mit dem indirekten ELISA.

A modified E/S-ELISA based on the procedure described by Gamble et al. (1988) was used for the diagnosis of trichinellosis in the domestic pig. The results of screening the sera of 92 pigs experimentally infected with Trichinella larvae (T. spiralis, T. nativa) with different doses, confirmed that the E/S-ELISA is suitable for the serological detection of Trichinella-specific IgG. Although the serotest shows a high sensitivity especially in the case of a low infection rate, it can not be used as an alternative for traditional meat inspection methods because of its so called diagnostic window. On the other hand, this serotest is considered to be useful for herd monitoring. False-negative results prior to completed seroconversion (the diagnostic window) were in most cases encountered up to the 4th-5th week post infection (p.i.), depending on the infectious dose used. Due to the prolonged persistence of antibodies, it was possible to demonstrate Trichinella infections by serology for a relatively long period (at least 80 weeks p.i.). Predictably, all 960 swine sera from the field tested in the E/S ELISA were negative for Trichinella. Separation and staining of the E/S antigen in SDS-PAGE revealed altogether 4 major protein bands with molecular weights of 44-67 kD. The 44 kD band showed the most intense reaction in an immunoblot using Trichinella antibodies. In contrast to the E/S-antigen, in the immunoblot using the same defined sera, the protein bands of somatic antigen caused cross reactions with non specific antibodies, which would lead to false-positive results in the ELISA.

Operant ethanol-reinforced behavior in P, NP, HAD, and LAD rats bred for high versus low ethanol preference.

These studies examined the reinforcing effects of ethanol in rats selectively bred for high versus low ethanol drinking in a two-bottle choice preference task, namely the Preferring (P), Non-Preferring (NP), High Alcohol Drinking (HAD), and Low Alcohol Drinking (LAD) rats. The results substantiate findings suggesting that genetic factors are significant in determining whether ethanol will come to serve as a reinforcer. P rats exhibited high levels of responding for ethanol compared with the water vehicle, NP and HAD rats exhibited more moderate levels of responding for ethanol, and the behavior of LAD rats suggested that ethanol served only inconsistently as a reinforcer for these rats. Overall, the results suggest the existence of distinct, biologically influenced components of ethanol drinking behavior. Preference appears to measure an inherent facilitative factor allowing animals to initiate ethanol drinking. The operant chamber paradigm appears to measure factors related to whether and to what extent ethanol will serve as a positive reinforcer following conditioned exposure to the drug. Although preferring animals generally find ethanol reinforcing there seems to be little quantitative relationship between degree of preference and whether ethanol will serve as a reinforcer. Lack of preference does not seem to be predictive of lack of reinforcement. Thus, it appears that preference for ethanol and reinforcement from ethanol are somewhat overlapping, but distinct factors that contribute to ethanol drinking. These results suggest the existence of multiple components of behavior mediated by multiple mechanisms that contribute to ethanol drinking.

[Detection of verotoxin-producing E. coli in field isolates from domestic and agricultural animals in Sachsen-Anhalt]. / Nachweis verotoxinbildender E. coli in Feldisolaten von Haus- und landwirtschaftlichen Nutztieren in Sachsen-Anhalt.

A report is given on the detection of verotoxin-producing E. coli (VTEC) strains from field isolates of healthy or ill cattle (n = 141), pigs (n = 306), sheep (n = 15), cats (n = 29) and dogs (n = 25) in the region of the new federal land Sachsen-Anhalt. 5% of the strains isolated from cattle, 32% from pigs, 20% from sheep, 4% from dogs and 0% from cats have shown VTEC. The E. coli-strains were checked for the presence of other factors of virulence, too. A good correlation (82%) was found between the colonization factor F107 and SLT 2/2v-containing strains from pigs in the region of Sachsen-Anhalt, too. Enterohemolysin was not found in SLT 2/2v-positive strains. 91% of the VTEC, isolated from pigs, produced alpha-Hemolysin. The correlation of SLT-containing strains and the production of enterohemolysin was confirmed for ruminants, only. Plasmidprofilings of VTEC from pigs showed mainly a 60 MDa or a 68 MDa plasmid or both, too. The occurrence of heat labile (LT) and in some cases of heat stable (ST) toxin was also checked, to differentiate the VTEC-strains from the enterotoxigenic E. coli strains (ETEC). These investigations showed, that VTEC produce SLT almost without exception. Correlations and conclusions on the pathogenicity for humans are discussed.