Increasing number of Salmonella paratyphi B isolates from slaughtered poultry sent in to the national Salmonella reference laboratory.]

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.     

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

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 and prevalence of verotoxin-producing Escherichia coli (VTEC) in raw sausage

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.     

Methods for checking up screening tests for detection of shiga toxin producing Escherichia coli (STEC) using 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.     

The relationships of the clay mineral suites to the parent rocks of eight soil profiles in Sarawak,Malaysia

The clay mineralogy of eight selected soil profiles developed on acid igneous, pyroclastic and sedimentary rocks was investigated. The effects on the clay mineralogy of the parent-rock mineralogy and physical conditions within each soil profile were evaluated.The results indicate that the mineralogies of the parent rocks control the type of clay minerals formed in Sarawak. In the soils derived from pyroclastic and coarse-grained acid igneous rocks without muscovite, the clay-mineral suites consist almost exclusively of kaolinite and gibbsite with small amounts of goethite. In contrast, in the soils developed from fine-grained acid igneous and sedimentary rocks with muscovite, the clays contain relatively large amounts of interstratified mica-vermiculite and chlorite-vermiculite in addition to gibbsite and/or kaolinite. The presence of gibbsite in these soils depends on the presence of plagioclase feldspar in the parent rock.     

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.     

Detection of pas gene in shiga toxin producing 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.     

Humoral immune response in calves to single-dose, trickle and challenge infections with Fasciola hepatica

In cattle experimentally infected with Fasciola hepatica, parasite specific IgG1 and IgG2 responses were studied. Additionally parasite specific IgE production was assessed by the Passive Cutaneous Anaphylaxis reaction. The primary infection was administered either as a single-dose or as a trickle infection over a 4-week period. Animals were challenged 4 months later. Titres of IgG1 and IgG2 against excretory-secretory parasite products (FhESAg), and against a whole-worm extract (FhSomAg) were measured by enzyme-linked immunosorbent assay (ELISA) in relation to weight gain, serum hepatic enzyme levels, and fluke infection rate. At necropsy, the mean number of flukes recovered was similar in both infected groups. The two ELISAs specific for bovine IgG1 showed analogous sensitivity and specificity (92% and 94%). Cross-reactivity was observed towards Echinococcus granulosus, Cysticercus tenuicollis, and C. ovis but not towards C. bovis, Cooperia spp., and Ostertagia spp. FhESAg gave rise to apparently more stable specific IgG1 titres as compared to FhSomAg. Mean IgG1 titres were significantly higher in the single-dose-infected group than in the trickle-infected group during the early migratory phase of the infection (week 2 to week 4 (FhSomAg) or week 6 (FhESAg)). IgG2 values were consistently lower than IgG1 levels. The kinetic response of both isotypes yielded a similar pattern. Specific IgE antibodies were detected in cattle of both infected groups from week 2 post-primary infection (PPI) onwards. The mean serum glutamate dehydrogenase (GLDH) and gamma-glutamyl transferase (gammaGT) activities were significantly higher in the single-dose-infected group for 3 weeks around peak levels (12-14 weeks PPI and 14-16 weeks PPI for GLDH and gammaGT respectively). Western blotting revealed a major antigenic fraction in FhESAg (26-30 kDa) recognized specifically by sera from F. hepatica infected calves as early as 6-8 weeks PPI. Experimental challenge caused no statistically significant modification of any parameter (IgG1 and IgG2 titres, enzymatic activities, immunoblotting) used to monitor the course of the infection. No correlation was found between fluke size and number, and antibody titres, suggesting that IgG1 production has little protective effect against F. hepatica infection.     

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.     

Calcium-dominated organans in humic podzols from the hudson and James Bay Lowlands of Ontario (Canada)

The SEM-EDXRA analysis of cutans within five Humic Podzolic soils derived from calcareous sands and gravels revealed the presence of Ca-dominated organans. It is postulated that organic matter saturated by H and Al is translocated through the Ae-horizon and precipitated within the Bh horizon in the presence of calcareous material. Weathering of the calcareous material within the Bh horizon results in the partial replacement of the H and Al in the organans by Ca.