Occurrence of virulence markers in species of Yersinia isolated from animals in Nigeria

Fourteen strains of Yersinia species isolated from apparently healthy pigs and cattle in Nigeria were screened for four virulence markers using six test systems. These were two in vitro assays, namely, calcium dependency and autoagglutination, both at 37 degrees C, the Serény test in guinea-pigs and the detection of heat-stable enterotoxin (ST) by the rabbit ileal loop test, the ligated intestine test in pigs and the infant mouse system. Seven of the 14 strains of Yersinia were positive for one or more of these tests. Six of nine strains of Y. enterocolitica and one of four Y. intermedia were positive in one or more tests. The only strain of Y. frederiksenii isolated was negative in all six test systems. All three strains of Y. enterocolitica, serotype 0:8 and the only serotype 0:3 isolated were positive in one or more tests. However, only two of five strains of Y. enterocolitica serotype 0:12, 26, the most frequently encountered, were positive. A good correlation was observed between test results of calcium dependency, autoagglutination and Serény assays. The results indicate that cattle and pigs have the potential to transmit virulent strains of Y. enterocolitica to human beings in Nigeria.     

Competitive exclusion of Yersinia enterocolitica biotype 4, serotype O:3 by Yersinia enterocolitica biotype 1A, serotype O:6,30 in tissue culture and in pigs

AIMS: To study the adhesion properties of a biotype 4, serotype O:3 (human pathogenic) strain of Yersinia enterocolitica and to determine if adhesion in vitro and colonisation in vivo can be prevented by competition with a biotype 1A, serotype O:6,30 (non-pathogenic) strain. To study interaction between Y. enterocolitica biotype 4, serotype O:3 and cultured epithelial cells using the synthetic tripeptide arginine-glycine-aspartic acid (RGD). METHODS: The human intestinal epithelial (HEp-2) cell line was used for in vitro studies. Inocula of Y. enterocolitica biotype 4, serotype O:3 radiolabelled using tritium were incubated with HEp-2 cells and RGD tripeptide, or with Y. enterocolitica biotype 1A, serotype O:6,30 sequentially or concurrently, then washed and lysed, and radioactivity measured to determine the effect of RGD on adhesion, and competitive exclusion of pathogenic by non-pathogenic bacteria. For in vivo studies, two groups of 5-week-old piglets (n=5/group) were sequentially inoculated orally with 5 x 10(9) colony forming units (cfu) of either a non-pathogenic biotype 1A, serotype O:6,30 strain of Y. enterocolitica followed by a pathogenic biotype 4, serotype O:3 strain, or vice versa. Pigs were monitored for carriage of strains using bacterial culture and a multiplex polymerase chain reaction (PCR). RESULTS: The RGD tripeptide significantly inhibited adherence of the pathogenic Y. enterocolitica strain to cultured epithelial cells, suggesting that adhesion involved the RGD tripeptide sequence. The non-pathogenic biotype 1A, serotype O:6,30 strain of Y. enterocolitica prevented adhesion of the pathogenic strain to cells in vitro when allowed to adhere first. Pathogenic Y. enterocolitica was consistently isolated from rectal swabs from 80-100% of pigs on all sampling occasions but not from oral swabs after 14 days in pigs first inoculated with the non-pathogenic strain or at 26 days in pigs first inoculated with the pathogenic strain. CONCLUSIONS: A non-pathogenic strain of Y. enterocolitica reduced adhesion of a human pathogenic strain in vitro but not in vivo.     

Immunostimulating effect of culture filtrates of Yersinia enterocolitica

Application of sterile culturing supernatant of Yersinia (Y.) enterocolitica (tested serovars were 03 and 08) caused significantly accelerated transplant rejection in mice of various inbreeding strains. Action correlated with dosage (r = -0.92). C57B16 mice were tested for their pregnancy rates in response to the same filtrate (serovar 03), with 5.5 live births per mating being recorded from 47 control matings but only 4.4 from 45 experimental matings (alpha less than 0.0025). The mean gestation period of experimental animals was extended by five percent over that of simultaneous controls (alpha = 0.25). Particular reference is made in this paper to Vesikari et al. (1987) who found Y. enterocolitica to function as interleukin-1 inductor via lipopolysaccharide. The active substance tested in this context, however, proved to be thermolabile, with 30 minutes of heating to 56 degrees C eliminating the action tested before. Y. enterocolitica infections were frequently found to coincide with rheumatoid arthritis, and evidence has been produced to the unspecific stimulating effect of Y. enterocolitica culture filtrates (testing being applied to serovar 03, biovar 4 and serovar 08, biovar 2). It is against the background of these aspects that chronic and other infections by Y. enterocolitica are considered to be of substantive relevance to the etiopathogenesis of autoimmune diseases, above all rheumatoid arthritis.     

Isolation, serotypes, and virulence-associated properties of Yersinia enterocolitica from the tonsils of slaughter hogs.

The objectives of this study were to determine the carriage rate of Yersinia enterocolitica in the tonsils of slaughter hogs, and to characterize them with regard to phenotypic and virulence-associated properties. Of 202 pigs examined from an abattoir in Prince Edward Island, 85 were culture positive for Y. enterocolitica. Sixty-seven percent of isolates belonged to serotype O:3, and 20% were serotype O:5. All isolates produced urease and 95% of O:3 isolates showed virulence-associated characters of autoagglutination at 37 degrees C and lack of fermentation of esculin and salicin. All isolates were tested for crystal violet binding, calcium dependency, and virulence plasmids. Eight isolates (5 belonging to serotype O:3, 2 belonging to O:5,27, and 1 belonging to O:7,8) were tested in addition for the production of heat-stable enterotoxin (ST), and iron-chelating siderophores. Of the 57 O:3 isolates, 93% were positive for crystal violet binding and calcium dependency and 98% possessed a 40-45 MDa plasmid. Four of the 5 O:3 isolates tested for ST related to Escherichia coli STa in a commercial enzyme immunoassay were positive. Six of the 8 isolates belonging to 3 different serotypes produced large orange halos around the colonies on a chrome-azurol-s agar assay medium, for siderophores. Antimicrobial susceptibility tests of all 85 isolates against 16 drugs showed 100% susceptibility against 12 drugs, including trimethoprim-sulfamethoxazole and tetracycline.     

Prevalence of Yersinia enterocolitica in goat herds from Northern Germany

The prevalence of human pathogenic Yersinia enterocolitica isolates in livestock farming is of paramount interest. Raw goat milk has been proposed as a source of human yersiniosis; however, no data on the prevalence of human strains of Y. enterocolitica in goat herds are available. Therefore, fecal samples (n = 575) were collected from 24 goat herds from Lower Saxony, northern Germany. Pre-enrichment in peptone, sorbitol and bile salts broth was followed by plating on cefsuloidin irgasan novobiocin agar. Yersinia enterocolitica was isolated from 17 (3%) samples of five (21%) goat herds. All isolates were biovar 1A, but represented various serovars. PCR assays targeting Yersinia adhesin (yad) gene and the yopT gene, both associated with pathogenicity, produced no amplification products. Therefore, the isolates can be regarded as opportunistic apathogenic bacteria. Consequently, milk, cheese or meat from goats should not be considered as an important source for human yersiniosis.     

Overshoot and high equilibrium in body temperature responses of rats to ambient heat: relation to thermoregulatory ability and improvement in estimation of survival time in a hot environment.

Physiological significance of body temperature responses to ambient heat (BTRAH) with high overshoot or a high equilibrium phase was studied in relation to thermoregulatory ability. Subsequently, an attempt was made to predict survival time (ST) by measuring the period required to attain a colonic temperature (Tco) of 42.0 degrees C (t42.0), whereas ST hitherto having been calculated on the basis of a period until Tco attains 42.5 degrees C (t42.5). Tco of rat was monitored continuously with a Cu-Co thermocouple during exposure to an ambient temperature (Ta) of 42.5 degrees C. The lower the overshoot temperature (Tos) or the equilibrium temperature (Teq), the longer the rats survived in a hot environment. The present findings further suggest that these two types of BTRAH are immature variations of the typical triphasic BTRAH which is characteristic of heat resistant individuals. A new regression line of ST (Y) as a function of the t42.0 (X) was obtained for most rats as follows: Y = 0.963X + 43.85 (male); and Y = 0.973X + 39.10 (female). This equation enabled to calculate ST without thermal death. However, the former approach based on t42.5 must be applied yet in small number of rats which showed the irregular BTRAHs with Tos or Teq higher than 42.0 degrees C. Delayed influence on survival and fecundity at a Ta of 24 degrees C were not found during one year following this hyperthermia up to 42.5 degrees C.     

Pseudomonas aeruginosa in raw and pasteurized milk

Eighteen strains of Pseudomonas aeruginosa, i.e. 8.87%, were isolated during a year from 203 samples of raw milk. Two Pseudomonas aeruginosa strains, i.e. 4%, were isolated from 50 samples of pasteurized milk. The strains were isolated using propagation techniques in meat-peptone broth with malachite green and on selective media--on centrimide agar (CEM) and on Pseudomonas F agar. All the isolated strains produced protease, whereas lipase was produced by only five strains. The strains were devitalized when exposed to pasteurization temperatures (72 degrees C) for 20 seconds. At cold store temperatures (4 degrees C), Pseudomonas aeruginosa strain cells propagated on average by two orders, inhibitory effects of low temperatures were recorded only with one strain. Inhibitory effects of milk cultures (cream, yogurt) on Pseudomonas aeruginosa were observed; their effects were more clear-cut at the temperature of 4 degrees C. The strains were markedly susceptible to gentamycin.     

The application of PCR to the identification of selected virulence markers of Yersinia genus

The subject of this study was thirty nine strains of Yersinia enterocolitica, isolated from faeces of humans who showed symptoms typical of intestinal yersiniosis, and seventy strains of Y enterocolitica, four strains of Y. pseudotuberculosis, and one strain of Y. kristensenii from healthy pigs. In the population tested the following serogroups appeared: O3, O9, O2, O5. A PCR was used to detect the presence of pathogenic chromosomal markers, such as myfA and inv genes of the tested Yersinia species. Among Y. enterocolitica strains isolated from humans and belonging to serogroup O3 (thirty four strains) and serogroup O9 (five strains) thirty three Y. enterocolitica O3 strains and four Y. enterocolifica O9 strains, gave a positive reaction to the nmyfA gene, yielding a fragment of 280 base pairs (bp). Among seventy Y. enterocolitica strains isolated from pigs forty strains belonging to serogroup O3 and fifteen strains belonging to serogroup O9 gave a positive reaction to the myfA gene. The presence of 390 bp amplified products, corresponding to the inv gene fragment, was detected in PCR products of three Y pseudotlluberculosis strains from pigs and only in one Y. enterocolitica O3 strain from humans, which had no myfA gene. The results obtained show that the myfA gene is only present in the strains that belong to pathogenic serotypes of Y. enterocolitica. The myfA gene prevailed in the Y. enterocolitica O3 and O9 strains from humans but was less common in the Y. enterocolitica O3 and O9 strains from pigs.     

The activity of chosen bacteriophages on Yersinia enterocolitica strains

The aim of the present study was to evaluate the lytic activity of three bacteriophages on Yersinia enterocolitica strains isolated from humans and pigs. The Y. enterocolitica strains tested belonged to 0:3, 0:9 and 0:2 serogroups. The ZD5 phage was obtained from a water sample, but remaining phages were obtained from the lysogenic Y. frederiksenii 7291 and Y. enterocolitica 8684 strains. All the Y. enterocolitica strains tested which belonged to 0:9 serogroup did not show any susceptibility to the bacteriophages used. The bacteriophages tested showed different lytic activity on the Y. enterocolitica 0:3 strains investigated. The phage susceptibility of Y. enterocolitica 0:3 strains revealed 9 different phage patterns. ZD5 phage showed the highest lytic activity, because it produced confluent lysis of the most Y. enterocolitica 0:3 strains tested. The Y. enterocolitica 0:2 strains isolated from pigs showed the similar phage susceptibility. The Y. kristensenii and Y. pseudotuberculosis strains tested were not sensitive to the bacteriophages used.     

Pretreatment of guinea-pigs with iron or desferal influences the course of Yersinia enterocolitica infections

The effects of iron excess and desferrioxamine in pretreated guinea-pigs on the immune response (production of Yops) and on the histological changes in infections with Yersinia enterocolitica 0:3 and Y. enterocolitica 0:8 were investigated. The prior overload of the guinea pigs with Dextrofer or treatment with Desferal increased the pathogenic activity of Y. entercolitica 0:3 and led to a generalized infection. Immunoblot analysis showed that in conditions of iron overload the expression of outer membrane proteins (Yops) of Y. enterocolitica 0:8 was blocked. This was accompanied by weak changes in the tissues. The iron limited conditions stimulated production of a low molecular weight protein (17 kDa) on day 6 and easier proliferation of the bacterium. This in vivo study intends to show that in Y. enterocolitica infections a leading role is played not only by iron itself but also by the bacterial strain.     

Fecal immunoglobulin A (IgA) antibodies to Yersinia enterocolitica in mice orally vaccinated and infected

Fecal immunoglobulin-A (IgA) antibodies to Yersinia enterocolitica serovar O3 strain were detected in the mice orally immunized with formalin-killed organisms. Y. enterocolitica O3 organisms were inhibited to colonize in the intestines of the mice producing fecal IgA. The fecal IgA antibodies were detected in the mice orally infected with the bacteria. When IgA was produced in the mice infected, they ceased shedding the organisms in their feces.     

Colonization control of lactose-fermenting Salmonella typhimurium in young broiler chickens by use of dietary lactose

Inclusion of lactose in the diets of chickens has been determined to reduce cecal colonization with Salmonella typhimurium. We hypothesized, therefore, that dietary lactose may be a practical means for reducing the prevalence of Salmonella contamination of chicken products. Because some strains of Salmonella are atypical and ferment lactose, we investigated the effects of dietary lactose on cecal colonization with lactose-fermenting S typhimurium. Broiler chicks were inoculated intracloacally with Lac+ S typhimurium selected for resistance to novobiocin and rifampicin. The chicks also were inoculated orally with certain anaerobes that do not effectively inhibit colonization by S typhimurium, but do appear essential for lactose mediated inhibition of cecal colonization. Control chicks were not given dietary lactose, and chicks in the experimental group were fed a diet containing 7% lactose. Enumeration of Lac+ S typhimurium in cecal contents revealed dietary lactose to be effective at controlling this organism. Control was correlated with changes in cecal pH and increases in undissociated volatile fatty acids, especially propionic acid.     

Experimental Yersinia enterocolitica placentitis in sheep.

A strain of Yersinia enterocolitica of O serogroup 6,30 isolated from the liver of an aborted ovine fetus was inoculated intravenously into a group of pregnant ewes at about 90 days gestation and produced placentitis with abortion or delivery of infected lambs about 50 days later. Y. enterocolitica of the same serogroup was recovered from the necrotic placental cotyledons and most other fetal tissues and could be isolated from vaginal discharges of the ewes for a least 2 weeks after abortion. Histological changes were consistent with an acute bacterial necrotizing placentitis and systemic infection of the fetus. Subsequent pregnancies in the ewes proceeded to term without evidence of infection.     

Isolation and characteristics of nutritional variants of streptococci of animal origin

Four strains of nutritionally variant streptococci (NVS) were isolated from the milk of mastitic cows and one strain from the lungs of a laboratory Norway rat which died from suppurative pneumonia. In primary cultivation NVS grew aerobically and anaerobically within 48-hour incubation at a temperature of 37 degrees C as minute nonhemolytic satellite colonies around a previously overlaid S. aureus strain or around other gram-positive and gram-negative bacteria. In the first subcultures NVS were growing in nutrient media enriched with 10% bovine serum and 5% staphylococcal filtrate, or 0.02% to 0.002% pyridoxal hydrochloride. All isolates did not grow in presence of 10%, 40% bile, and 6.5% of sodium chloride, neither did they grow at a temperature of 45 degrees C, they did not hydrolyze sodium hippurate, esculin, arginine, they did not produce levane and dextran from saccharose, they produced acid from mannitol, sorbitol, inulin, lactose, raffinose, trehalose, glucose, saccharose and maltose. Two strains produced acid from xylose and four strains from salicin. The strains isolated from mastitis did not have different biochemical properties from those isolated from a laboratory Norway rat with pneumonia. All strains of NVS were sensitive to chloramphenicol, ampicillin, gentamycin, lincomycin and cephalothin, four strains were sensitive to erythromycin and tyrosine, two to penicillin and one to streptomycin, oxytetracycline, chlortetracycline and novobiocin. All strains were resistant to neomycin, tetracycline, oxacillin and sulphonamides. The antigen prepared from the isolated strains by the method of Fuller did not react with any streptococcal group serum A-Z.     

Identification of Yersinia enterocolitica in minced meat: a comparative analysis of API 20E, Yersinia identification kit and a 16S rRNA-based PCR method

The isolation and identification of Yersinia enterocolitica from minced meat on CIN agar medium is still one of the major problems in food microbiology because of the low selectivity of cefsulodin-irgasan-novobiocin (CIN) agar. A total of 198 minced meat samples were collected from commercial establishments (butcher shops and supermarkets) in seven German cities in order to investigate the sensitivity and specificity of three identification techniques suitable for the differentiation of Y. enterocolitica within the rich background flora on CIN agar plates. As expected isolation of Y. enterocolitica from minced meat on CIN agar medium after 72 h enrichment in peptone, sorbitol and bile salts (PSB) broth was difficult because all plates were abundantly covered with numerous 'typical'Yersinia-like colonies of bull's eye appearance as well as with atypical colonies. Based on the phenotype of the colonies it was possible to detect colonies showing Yersinia-like growth on CIN agar in 52 samples (26%). For identification of Y. enterocolitica the API 20E system (bioMerieux, Nürtingen, Germany), the Yersinia identification kit (Merlin, Bornheim-Hersel, Germany) and a 16S rRNA based PCR assay were compared. Only in one sample (0.5%) a Y. enterocolitica strain was detected by all methods. Of the three identification systems tested for routine laboratory diagnostics the API 20E system was found to be the most suitable tool to identify Y. enterocolitica colonies within the rich background flora from minced meat samples on CIN agar plates.     

Killing of Streptococcus uberis by bovine neutrophils following growth in chemically defined media

Following growth in a chemically defined medium (CDM), five strains of Streptococcus uberis were tested for their ability to survive killing by bovine neutrophils. Strains 0140J, ST10, EF20 and C221 were easily killed, whereas strain C197C was highly resistant. The ability of strain 0140J to resist phagocytosis and killing was increased by supplementation of the growth medium with milk whey, casaminoacids, casein, or, to a lesser extent, bovine serum. Supplementation of the growth medium with yeast extract or bovine serum albumin did not affect the resistance of this strain. Following growth in CDM supplemented with casein, strains ST10 and C221, like strain 0140J, were significantly more resistant to killing by neutrophils. The resistance of strains EF20 and C197C was unaffected by the addition of casein to the medium; strain EF20 remained susceptible and strain C197C highly resistant to killing. The effect of supplementing the growth media with components other than casein was only studied for strain 0140J. Decapsulation of strains C197C, ST10 and 0140J, grown in CDM + casein, with type-X hyaluronidase did not significantly affect their ability to survive in the presence of bovine neutrophils.     

The effect of aging of milk samples on the accuracy of infrared analysis of milk composition]

The effect of a decrease (and/or fermentation) in the lactose content during milk storage under different conditions was investigated on the accuracy of the results obtained on a Milko-Scan apparatus to contribute to the present knowledge of this problem. The results were in agreement with some results cited in the literature. These wavelengths are used for infrared spectrophotometry on the above apparatus: for fat 3.48 microns, for proteins 6.46 microns and for lactose 9.60 microns. Bulk milk samples used for the tests were untreated or treated with potassium dichromate, bronopol, sodium azide and Milkofix at the temperatures of storage in darkness 20 degrees C and 4 degrees C. The differences against the reference values (measured on the first day) were determined and evaluated in milk composition and characteristics as arising during milk storage. These differences were used in form of either cumulative means of differences (Figs. 1 to 5) or individual differences (Fig. 8). In the first part significant correlation coefficients (P less than 0.001) were calculated for the relationship between the variations of lactose content and the fat and protein contents: r = -0.59 and/or -0.73 (Figs. 6 and 7). This suggests that the decrease in the lactose content by 0.10% recorded by the infrared analysis and caused by lactose decomposition is accompanied by a "seeming" increase in the fat and protein content by about 0.04%. In the second part the correlation coefficients for the fat and protein contents r = -0.96 and -0.96 (P less than 0.001; Figs. 9 and 10; Tab. II) were calculated on the basis of an observation of the lactose decrease in an untreated milk sample (20 degrees C for 28 hours). These coefficients are somewhat different from the preceding ones; this is due to the lower homogeneity of the first set where the milk samples were treated in a different way, but the coefficients confirm the same conclusions. The values of the correlation coefficients for the dependence between the development of the acquired titratable acidity (SH) and the variations of fat (F), protein (P) and lactose (L) contents were as follows: r = 0.95; 0.95; -0.99 (P less than 0.001; Figs. 12, 13; Tab. II). Thus the above-mentioned "seeming" increase in the F and P contents can be explained to the extent of 92.2% from the decrease in the L content, which also causes the increase in titratable acidity to the extent of 98.0%.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reproduction of Brucella titers in swine using experimental infection with Yersinia enterocolitica, serotypes 0:9 and 0:6

Measurable Brucella titres were produced by slow agglutination, slow agglutination at 57 degrees C (heat test), and complement fixation reaction with Brucella antigen in infection experiments with gilts in which Yersinia enterocolitica Serotypes 0:9 and 0:6 were used. Slow agglutination gave brucellosis titres up to 1:1280 and titres against Yersinia enterocolitica, Serotype 0:9, up to 1:20480. The antibody titres stayed persistent throughout the 80 days of the experiment. Yersinia enterocolitica infection was found to be transmissible between the animals. Aspects relating to the development and course of the infection as well as to pathogen detection are discussed.     

Epidemiological study of Yersinia enterocolitica in swine herds in Québec

The objectives of this study were the identification of the different contamination sources of Yersinia enterocolitica, as well as the determination of the prevalence and the distribution of the different genotypes in swine herds. The owners of 20 farms, located in the Richelieu-Yamaska region, agreed to participate in the study. Each farm was visited a minimum of 5 times between May and October 1997, and, at each visit, 20 environmental and 10 fecal samples were collected. Yersinia enterocolitica isolates were identified, serotyped, and submitted to a genetic characterization by pulsed-field gel electrophoresis. The correlation coefficient (0.61) between prevalence in environment and in feces was significant (P = 0.004). Among the 153 positive samples, 93.5% belonged to serotype 0:3. The comparison of PFGE profiles revealed that all environmental Y. enterocolitica isolates had a profile identical to that of isolates recovered in feces from the corresponding farms. Also, when the genetic profiles of isolates recovered from feces collected at the first visit were compared with the profiles of isolates obtained from the subsequent visits, the same profile was observed on every farm. We concluded that environment does not represent the main source of contamination of swine by Y. enterocolitica and that, in most instances, the same strain persists in a barn from one production lot to another.     

The pathogenicity of Yersinia enterocolitica strains isolated from various sources in four test systems.

Six tests were applied to 39 strains of Yersinia enterocolitica of various serotypes and from several sources in an attempt to relate the test to pathogenicity of the strains. The tests that were used were the pig gut loop test and the infant mouse test for heat stable enterotoxin, the Sereny and HeLa cell tests for invasiveness, inhibition of growth on magnesium oxalate agar, and the ability to cause diarrhea in infant mice. The pig gut loop test was found to be unsuitable for detection of heat stable enterotoxin but 20 strains produced heat stable enterotoxin that was detected in infant mice. None of the strains was positive in the Sereny test but 21 invaded HeLa cells. The growth of 20 strains was inhibited at 37 degrees C on magnesium oxalate agar and, in the orally-infected mice, 23 strains caused diarrhea or death. These findings indicate a discrepancy between the infant mouse test and the ligated intestine test in pigs for heat stable enterotoxin and a significant difference in Y. enterocolitica heat stable enterotoxin compared with Escherichia coli heat stable enterotoxin because the former failed to elicit a significant response in pig intestine.