Biomedical subjects

G Baljer

Publications and source records attributed to G Baljer.

At least 19 recordsLinked to original sources

PubMed2003

[Diagnosis of Lawsonia intracellularis using the polymerase chain reaction (PCR) in pigs with and without diarrhea and other animal species].

Lawsonia (L.) intracellularis, an obligately intracellular bacterium, causes proliferative enteropathy (PE) in swine and, occasionally, in other animals. To determine the spread of the agent among German pig herds pooled fecal samples of five animals each of clinically normal Hessian pig herds collected between november 1998 and february 1999 as well as feces (n = 1684) from individual animals representing 648 herds, sent to our laboratory by veterinarians from all parts of Germany, were tested for L. intracellularis using the polymerase chain reaction (PCR). In addition, fecal samples from diarrhoic foals (n = 46), dogs (n = 57), cats (n = 50), calves (n = 37), hedge hogs (n = 9), seals (n = 8) and one giraffe were also studied. DNA was extracted from feces using high concentrations of chaotropic salt and diatomaceous earth. For PCR, primers flanking a 279 bp fragment of L. intracellularis DNA were used (JONES, G. F., WARD, G. E., MURTAUGH, M. P., LINN, G. (1993), J. Clin. Microbiol. 31, 2611-2615). Amplificates were separated by agarose gel electrophoresis and visualized under UV-light. L. intracellularis was found in 26 (12.8%) samples from 21 (30.0%) of the Hessian pig herds without symptoms of diarrhoea. In feces of pigs with diarrhoea (n = 1684) the agent was present in 431 (25.6%) samples originating from 224 (34.6%) herds. Of the other animal species studied, L. intracellularis was detected in feces of 4 (7.0%) dogs, 2 (5.4%) calves, 3 (33.3%) hedge hogs and in the sample of the giraffe. The remaining species were all tested negative.

Animals↗

PubMed2002

Bovine lymphocytes express functional receptors for Escherichia coli Shiga toxin 1.

Interactions of Shiga toxins (Stxs) and immune cells contribute to the pathogenesis of diseases due to Stx-producing Escherichia coli (STEC) infections in humans and facilitate the persistence of infection in asymptomatically infected cattle. Our recent findings that bovine B and T lymphocytes express Gb(3)/CD77, the human Stx-receptor, prompted us to determine whether the bovine homologue also mediates binding and internalization of Stx1. In fact, Stx1 holotoxin and recombinant B subunit (rStxB1) bound to stimulated bovine peripheral blood mononuclear cells, especially to those subpopulations (B cells, BoCD8(+) T cells) that are highly sensitive to Stx1. Competition and HPTLC-binding studies confirmed that Stx1 binds to bovine Gb(3), but different receptor isoforms with varying affinities for rStxB1 were expressed during the course of lymphocyte activation. At least one of these isoforms mediated toxin uptake. An anti-StxB1 mouse monoclonal antibody, used as a model for bovine serum antibodies specific for Stx1, modulated rather than generally prevented rStxB1 binding to and internalization by the receptors. The presence of functional Stx1-receptors on bovine lymphocytes explains the immunomodulatory effect of Stx1 observed in cattle at a molecular level. Furthermore, expression of such receptors by bovine but not human T cells enlightens the background for the differential outcome of STEC infections in cattle and man, i.e., persistent infection and development of disease, respectively.

Animals↗

PubMed2001

Globotriaosylceramide (Gb(3)/CD77) is synthesized and surface expressed by bovine lymphocytes upon activation in vitro.

Neutral glycosphingolipids (GSLs) are considered activation markers on human lymphocytes, which are fundamental for studying the immune system. For cattle, only a limited number of activation markers has yet been identified. We recently showed that Shiga toxin 1, known to use globotriaosylceramide (Gb(3) syn. CD77) as a cellular receptor, depresses proliferation of activated bovine lymphocytes [Infect. Immunol. 67 (1999b) 2209]. In order to confirm the expression of Gb(3)/CD77 on bovine lymphocytes, we flowcytometrically examined a bovine B-lymphoma cell line (BL-3) and bovine peripheral blood mononuclear cells (PBMC) before and after mitogenic stimulation and biochemically characterized neutral GSLs extracted from PBMC. CD77 was detected on the surface of BL-3 cells and cultured PBMC essentially after mitogenic stimulation. Although expressed by all PBMC subpopulations identified, the portion of CD7+ cells was highest for BoCD8+ cells, followed by B-cells and BoCD4+ cells at day 4 of cultivation. Ceramide trihexoside of stimulated PBMC was structurally determined as Gal(alpha1-4)Gal(1-4)Glc(1-1)ceramide (Gb(3)). Biochemically, Gb(3) was also detected within unstimulated PBMC which contained ceramide monohexoside (CMH) and Gb(3) in a ratio of about 4:1. However, stimulation induced an increase of CMH and Gb(3) by a factor of 2.5 and 10, respectively, implicating that bovine lymphocytes regulate surface expression of Gb(3)/CD77 predominantly by quantitative changes in the Gb(3) metabolism. This report presents Gb(3)/CD77 as the first GSL identified on bovine immune cells and highly recommends this activation dependent antigen as a useful tool to investigate lymphocyte activation within the bovine immune system.

Animals↗

PubMed2001

Prevalence of enteropathogens in suckling and weaned piglets with diarrhoea in southern Germany.

Faecal samples from suckling (n = 205) and weaned piglets (n = 82) with diarrhoea from 24 farms in Southern Germany were examined for shedding of important metazoic parasitic, viral and bacterial pathogens using culture, microscopic and electronmicroscopic methods. Escherichia coli isolates were tested further for the enterotoxin genes est-Ia and elt-I by colony blot hybridization. Isospora suis was diagnosed in 26.9% and Cryptosporidium parvum in 1.4% of the piglets investigated. The proportion of coronavirus-positive animals was 13.4% and 4% were positive for rotavirus. It was found that 17.6% of the animals were infected with enterotoxigenic E. coli (ETEC; 10.1% ETEC-ST-Ia and 8.6% ETEC-LT-I, respectively). The occurrence of the pathogens was significantly associated with the age of the animals examined (P < 0.001). Isospora suis was predominantly isolated from suckling piglets (in the second and third week of life), while in weaned piglets (fourth week of life) rotavirus and ETEC were most prevalent. On 22 of the 24 piglet production farms examined at least one of the investigated pathogens was detected. Coronavirus was diagnosed in 66.7%, I. suis in 62.5%, rotavirus in 20.8% and C. parvum in 8.3% of the farms. These results underline the fact that despite the hygienic, technical and immune preventive efforts during the last years, enteropathogens are still common in German piglet production units.

Animals↗

PubMed2001

The AIDA autotransporter system is associated with F18 and stx2e in Escherichia coli isolates from pigs diagnosed with edema disease and postweaning diarrhea.

Pathogenic Escherichia coli strains are known to cause edema disease (ED) and postweaning diarrhea (PWD) in piglets. Although the exact mechanisms of pathogenicity that lead to ED-PWD remain to be elucidated, E. coli-borne Shiga-like toxin and adhesion-mediating virulence factors such as F18 adhesin or F4 fimbriae are believed to play a central role in ED-PWD. In light of these observations we investigated whether another E. coli adhesin, the plasmid-encoded AIDA (adhesin involved in diffuse adherence) might also be present in ED-PWD-causing E. coli isolates. For rapid screening for the AIDA system in large numbers of isolates, a multiplex PCR method along with a duplex Western blot procedure was developed. When screening 104 strains obtained from pigs with or without ED-PWD, we observed a high prevalence of the AIDA operon in porcine E. coli isolates, with over 25% of all strains being AIDA positive, and we could demonstrate a significant association of the intact AIDA gene (orfB) with ED-PWD, while defects in orfB were associated with the absence of disease. Although our data hint toward a contribution of AIDA to ED-PWD, further studies will be necessary since the presence of the AIDA genes was also associated with the presence of the Shiga-like toxin and F18 adhesin genes, two reported virulence factors for ED-PWD.

Adhesins, Escherichia coli↗

PubMed2001

Naturally occurring Clostridium perfringens nontoxic alpha-toxin variant as a potential vaccine candidate against alpha-toxin-associated diseases.

Clostridium perfringens mutant strain 121A/91 shows neither enzymatic (phospholipase C) nor hemolytic activity. Nevertheless, the cpa gene and the corresponding alpha-toxin variant are detectable. Vaccination with this genetically constructed alpha-toxin variant, rAT121/91, induces antibodies capable of significantly reducing activities induced by wild-type toxin. Thus, rAT121/91 could be a useful vaccine candidate.

Amino Acid Substitution↗

PubMed2001

[Prevalence of the Salmonella plasmid virulence gene "spvD" in Salmonella strains from animals].

Strains of Salmonella isolated from animals in Germany (n = 878) were analysed for the presence of the spvD gene ("Salmonella plasmid virulence gene D") by DNA-DNA hybridization. The spvD gene was only detected in strains of serovars Typhimurium (93.3%), Enteritidis (97.1%), and Dublin (100%) as well as in two rough strains of Salmonella enterica. Salmonella isolates from mammals carried the gene more frequently (cattle 94.0%, horses 92.6%, pigs 73.7%) than those from birds (33.3%) or reptiles (4.5%). Due to its high prevalence in epidemiologically relevant salmonellae, the virulence factor spvD may represent a sensitive and specific target in various serovars for diagnostic or immunization strategies.

Animals↗

PubMed2000

Sequencing and characterization of the cryptic plasmid QpRS from Coxiella burnetii.

Plasmid QpRS from Coxiella burnetii, isolate Priscilla Q177, phase I, has been completely sequenced. DNA for sequencing was amplified with "extra-long" (XL) PCR. The size of the QpRS plasmid sequence was determined to be 39,280 bp, with a G + C content of 39.3%. Putative proteins associated with replication and recombination were identified. The sequence of QpRS plasmid was analyzed for shared and unique regions among C. burnetii plasmids.

Base Sequence↗

PubMed2000

Enterohemorrhagic Escherichia coli (EHEC) strains of serogroup O118 display three distinctive clonal groups of EHEC pathogens.

A recent case report of a child infected with enterohemorrhagic Escherichia coli (EHEC) of serotype O118:H16 in Bavaria, in association with the isolation of a bovine O118 strain on the same farm (A. Weber, H. Klie, H. Richter, P. Gallien, M. Timm, and K. W. Perlberg, Berl. Muench. Tieraerztl. Wochenschr. 110:211-213, 1997), prompted us to investigate the relationship between bovine and human strains of serogroup O118. A total of 29 human O118 E. coli strains from Europe (21), Canada (4), and Peru (4) were compared by virulence typing and macrorestriction analysis with 7 bovine O118 EHEC strains isolated in Bavaria. Twenty-five of the human strains were characterized as EHEC. By serotyping and determination of the virulence-associated factors Shiga toxin (stx1 stx2 stx2 variants), intimin (eae), and EHEC hemolysin (Hly(EHEC)), three distinctive groups of O118 human pathogens were identified. Most of the strains belonged to serotype O118:H16, displaying the virulence traits Stx1, intimin, Hly(EHEC), and EspP/PssA (group 1). In addition, we identified strains of serotype O118:H12 (Stx2d only; group 2) and of serotype O118:H30 (Stx2 and intimin; group 3). Macrorestriction analysis with BlnI and XbaI revealed that all strains with a single O118 serotype profile (O118:H12, O118:H16, and O118:H30) belonged to one clonal cluster, irrespective of their origin. Group 1 strains clustered in the same clonal group as the bovine O118:H16 strains. Moreover, four pairs of strains of different origins and indistinguishable by all other methods applied were identified as group 1 strains. Our data support the direct transmission of an EHEC O118:H16 strain from a calf to a 2-year-old boy in the above-mentioned case report. Since bovine and human O118:H16 strains represent the same clones, they must be considered zoonotic EHEC pathogens. In contrast, EHEC strains of serotypes O118:H12 and O118:H30 have been isolated only from humans, indicating a reservoir for certain human O118 EHEC strains other than bovines.

Adhesins, Bacterial↗

PubMed1999

Phenotypical characterization of peripheral blood leucocytes in the newborn calf.

The present study was undertaken to establish reference values for the composition of blood leucocyte populations in neonatal calves by differential leucocyte counts and immunophenotyping. Neonatal calves 1 h post partum (p.p.) were found to have a very high absolute number of granulocytes while the number of peripheral blood mononuclear cells was lower than in calves aged 3-9 weeks. The relative numbers of T cell subpopulations were similar in newborn and older calves, but newborn calves had lower percentages of B cells and MHC class II positive cells. Within the first 4 h of life the relative numbers of CD2+, CD6+, and CD8+ T cells declined in colostrum-fed as well as in colostrum-deprived calves. In contrast, the percentage of MHC class II positive cells and monocytes increased from 1 h to 4 h p.p. particularly in colostrum-fed calves. Although there is some evidence for immaturity of lymphocytes in neonatal calves, the immune system of these animals seems to be fully present at birth.

Animals↗

PubMed1999

Shiga toxin 1 from Escherichia coli blocks activation and proliferation of bovine lymphocyte subpopulations in vitro.

Shiga toxin-producing Escherichia coli (STEC) is widespread in the cattle population, but the clinical significance of Shiga toxins (Stx's) for the bovine species remains obscure. Since Stx's exert immunomodulating effects in other species, we examined the effect of purified Stx1 on a bovine B lymphoma cell line (BL-3) and peripheral blood mononuclear cells (PBMC) isolated from adult bovine blood by viability assays and flow cytometry analysis. Stx1 markedly induced apoptosis in stimulated BL-3 cells. The susceptibility of this B-cell-derived cell line was induced only by either lipopolysaccharide (LPS) or pokeweed mitogen, while cultures stimulated with T-cell mitogens were unaffected by the toxin. In contrast, Stx1 did not induce cellular death-neither apoptosis nor necrosis-in primary cultures of PBMC but hindered the mitogen-induced increase in metabolic activity. The influence of Stx1 on single PBMC subpopulations varied with the type of mitogenic stimulus applied. Stimulation with phytohemagglutinin P particularly induced the proliferation of bovine CD8-expressing (BoCD8(+)) cells, and this proliferative response was blocked by Stx1. On the other hand, Stx1 reduced the portion of viable B cells in the presence of LPS. Modulation of activation marker expression (BoCD25 and BoCD71) by Stx1 indicated that the toxin hindered the proliferation of cells by blocking their activation. In conclusion, we assume that Stx1 contributes to the pathogenesis of STEC-associated diarrhea in calves by suppressing the mucosa-associated immune response. The usefulness of cattle as a model in which to study Stx-induced immunomodulation is discussed.

Animals↗

PubMed1999

[Animals as a source of infections for humans--diseases caused by EHEC].

EHEC (enterohaemorrhagic E. coli) bacteria are new, only since 1982 recognized zoonotic pathogens. EHEC differ from E. coli intestinal commensales by the fact that they are lysogenic infected with bacteriophages, which carry the genetic information for the production of shigatoxins (Stx type 1 and/or 2). Due to the obligatory released Stx EHEC are classified also among the Shigatoxin producing E. coli (STEC). EHEC are capable of causing a Hemorrhagic Colitis and some sequelae of diseases such as the haemolytic uraemic syndrome. Due to their virulence factors they can be divided into typical and non-typical EHEC. Typical EHEC possess a pathogenicity island (Locus of Enterocyte Effacement) harboring genes, which apart from the characteristic necrotic activity of Stx enable the pathogens to closely attach to the epithelial cells of the intestinal mucosa and to destruct the microvilli. Additionally a so-called virulence plasmid codes for the production of a haemolysin, a peroxidase-katalase, an enterotoxin as well as a serine protease. EHEC are one of the world-wide most important causes of foodborne infections. Depending upon the country, most of the incidences in 1998 varied between 1 to 3 cases per 100,000 inhabitants. Since EHEC are only notifiable in a few countries, one must count however on substantially higher numbers. In Germany the estimated incidence is about 13 cases per 100,000 inhabitants. Since the first EHEC outbreaks were recognized in humans, studies investigating the prevalence of EHEC within animals were repeatedly performed. From the outset one assumed that cattle are a possible reservoir. Actually EHEC were isolated from fecal samples world-wide (typical and non-typical EHEC) from a large percentage of cattle (> 50%). Besides EHEC were isolated sporadically from fecal samples of other animals and healthy humans. The EHEC bacteria are shed by infected humans and animals, in particular by infected ruminants. They are spread over manure, slurry, sewage etc. Humans can get infected directly by contact with infected persons or animals or indirectly by contaminated food, water etc. The clinical outcome within humans appears as aqueous to bloody diarrhea. Beyond that approximately 5 to 10% of the patients develop the haemolytic uraemic syndrome. In contrast to humans, animals are mostly infected clinically inapparent. The therapy is based upon a symptomatic treatment. At present in man the control of EHEC infections concentrates on a particularly strict hand hygiene after the contact with infected humans and animals (above all ruminants). Since EHEC are heat sensitive, the prophylaxis by sufficient heating of risk food (raw milk, ground beef) is of special importance. In veterinary medicine above all EHEC infections must be controlled in ruminants, which are the primary reservoir. Due to the wide spread of EHEC in the ruminant population it is not realistic to demand an EHEC free cattle stock. Since EHEC are spread only via fecal excretion, at present it is most important to reduce the fecal shedding and to avoid fecal contamination of food of animal origin. In detail prophylactic hygienic measures concerning the farm management, the feeding hygiene, the food hygiene, the meat hygiene as well as the food hygiene are available.

Animals↗

PubMed1999

Serum antibodies against particular antigens of Borrelia burgdorferi sensu stricto and their potential in the diagnosis of canine Lyme borreliosis.

Dog sera (n = 118) were tested for antibodies recognizing Borrelia (B.) burgdorferi sensu stricto strain B31 (ATCC 35210) antigens. In total, 18 of the dog sera gave positive results in a whole cell sonicate ELISA (WCS ELISA). These positive sera were further evaluated by immunoblot assay, utilizing a whole bacterial lysate as antigens. 94.4% (17 of 18) of the dog sera reacted with immunodominant antigens at 20-22 kDa (protein C, pC), 31 kDa (outer surface protein A, OspA), 34 kDa (outer surface protein B, OspB), 41 kDa (flagellin), 60 kDa ("common antigen"), and/or 100 kDa (presumably p100). Sera recognizing pC (20-22 kDa) and antigens > 94 kDa always detected the highest number of antigen bands, indicating the specificity of those antigens in serological diagnosis. The results clearly demonstrate that the WCS ELISA is a useful tool for testing sera of dogs for antibodies against B. burgdorferi. However, positive results should be confirmed by immunoblot, using WCS as antigen. According to the presented data, we recommend criteria for B. burgdorferi immunoblots using dog sera as follows: sera have to be considered as positive if they detect the 41 kDa flagellin, and two of the 5 immunodominant antigens, namely > 94 kDa (presumably p100), 60 kDa ("common antigen"), 34 kDa and 29-31 kDa (OspB and OspA, respectively) and 20-22 kDa (pC). If sera only recognize the 41 kDa flagellin, this result is equivocal, requiring testing a second serum sample 4 to 8 weeks later.

Animals↗

PubMed1998

Compensation of preliminary blood phagocyte immaturity in the newborn calf.

To estimate the functional maturity of the phagocytic defence in neonatal calves, we analyzed the characteristics of blood phagocytes from calves (n = 10) 1 h post partum (p.p.) and 4 h p.p. At 1 h p.p., all calves were colostrum-deprived, while 5 calves had received colostrum before the 4 h p.p. sampling. The results were compared to those obtained from 3-9-week-old calves (n = 10). Phagocytic and oxidative burst activity of polymorphonuclear leukocytes (PMNL) and monocytes were determined in whole blood and separately analyzed by flow cytometry. In neonates prior to colostrum ingestion (1 h p.p.), phagocytic activity of PMNL against non-preopsonized E. coli was lower when compared to PMNL of 3-9-week-old calves. Opsonization of bacteria with pooled plasma from adult animals only partially restituted this lower PMNL phagocytic activity, indicating that humoral as well as cellular aspects of PMNL phagocytosis are altered in neonatal calves. In contrast to PMNL, monocytes of neonates exhibited an enhanced phagocytic activity. The oxidative burst activity of PMNL, as well as of monocytes was higher in newborn calves. During the first 4 h of life, the activities of blood phagocytes changed. Colostrum ingestion was accompanied by an increase in the percentage of phagocytizing PMNL and monocytes. This increase was absent in colostrum-deprived calves. In contrast, the oxidative burst activity of phagocytes decreased with age. In monocytes, the decrease of oxidative burst activity was only observed in colostrum-fed calves. In conclusion, some blood phagocyte functions in calves were found to be immature at birth, but these functions are presumably compensated by high absolute PMNL numbers and by other the more active mechanisms.

Aging↗

PubMed1998

Controlled multiplex PCR of enterotoxigenic Clostridium perfringens strains in food samples.

A controlled multiplex polymerase chain reaction (PCR) for the detection of Clostridium(C.) perfringens enterotoxin gene (cpe) was established and compared with an in vitro antigenic detection method. Thecpe PCR and the classical method of electric immunodiffusion gave identical results. The predicted specific amplicon of the cpe gene was generated from all of the tested enterotoxigenic C. perfringens strains. In contrast, cultures of any other Clostridium species tested by PCR were negative (100% sensitivity, 100% specificity). Addition of an alphatoxin (plc) gene specific PCR as an in process control reaction was performed in order to prevent false negative PCR results. The PCR detection limit was 0.5 ng of genomic C. perfringens DNA per ml of bouillon culture. By contaminating minced meat with C. perfringens reference strains, the multiplex PCR was established as a tool for routine diagnostic laboratories. The detection limit was approximately 3.0x10(5)C. perfringens cells per gram meat. The results demonstrate the multiplex PCR as an easy, specific, sensitive and time saving diagnostic procedure. Application of this improved method should enhance the knowledge concerning epidemiological aspects of food borne diseases caused by enterotoxigenic C. perfringens.

Clostridium perfringens↗

PubMed1998

Detection of Borrelia burgdorferi in urine specimens from dogs by a nested polymerase chain reaction.

A nested PCR (nested flagellin PCR) carrying an internal E. coli DNA control was established and compared with an in-vitro culture method for the detection of Borrelia burgdorferi in urine specimens of dogs. The predicted specific amplicon of the flagellin gene fla was generated from all cultured strains of B. burgdorferi tested (comprising three European genospecies). In contrast, all 13 strains of seven other flagellated bacterial species were negative. The PCR detection limit yielded 20 cells of B. burgdorferi per ml of double-distilled water and approx. 250 bacteria per ml of dog urine. Using the bacterial culture method, urine specimens collected from 216 dogs in Germany were all diagnosed negative for spirochetes by in-vitro culture and dark-field microscopy. In contrast, DNA of B. burgdorferi was detected in 32 specimens (14.8%) by PCR. 31 urine specimens (14.4%) showed inhibitory activity in the PCR assay. However, 94 (44%) were inhibitory in the culture assay. The majority of the PCR-positive dogs exhibited major clinical symptoms which have not been reported in the course of B. burgdorferi infection previously, e.g. cystitis (14/32 dogs) or prostatitis (5/32 dogs). Our results indicate that the analysis of urine specimens by the nested flagellin PCR is a highly valuable procedure for the diagnosis of B. burgdorferi infections in dogs.

Animals↗

PubMed1998

Molecular characterization of Coxiella burnetii isolates.

Restriction fragment length polymorphism (RFLP) was used for the differentiation of 80 Coxiella burnetii isolates derived from animals and humans in Europe, USA, Africa and Asia. After NotI restriction of total C. burnetii DNA and pulsed field gel electrophoresis (PFGE) 20 different restriction patterns were distinguished. The index of discrimination for this typing system was 0.86. Comparison and phylogenetic analysis of the different RFLP patterns revealed evolutionary relationships among groups that corresponded to the geographical origin of the isolates. This finding was confirmed by genetic mapping. No correlation between restriction group and virulence of isolates was detected.

Animals↗

PubMed1998

PCR detection of Coxiella burnetii from different clinical specimens, especially bovine milk, on the basis of DNA preparation with a silica matrix.

For PCR detection of Coxiella burnetii in various clinical specimens we developed a sample preparation method in which silica binding of DNA was used. This method was found to be fast, easily performed with large numbers of samples, and equally sensitive for all of the specimens tested (livers, spleens, placentas, heart valves, milk, blood). The DNA preparation method described here can also be used as an initial step in any PCR-based examination of specimens. The procedure was tested with more than 600 milk samples, which were taken from 21 cows that were seropositive for C. burnetii and reportedly had fertility problems (and therefore were suspected of shedding the agent through milk intermittently or continuously). Of the 21 cows tested, 6 were shedding C. burnetii through milk. Altogether, C. burnetii DNA was detected in 6% of the samples. There was no correlation between the shedding pattern and the serological results.

Animals↗