Salmonella enterica serovar Typhimurium and Enteritidis infection of pigs and cytokine signalling in palatine tonsils.

Pigs are considered as one of the major sources of zoonotic strains of Salmonella enterica for humans. Out of many S. enterica serovars, S. Typhimurium dominates in pigs, however, in several countries in Central Europe, S. Enteritidis is also quite frequent in pig herds. In this study we therefore compared the colonisation of pigs with S. Typhimurium and S. Enteritidis. We found that 3 weeks after infection S. Enteritidis 147 colonised the intestinal tract in higher quantities but was shed in faeces in lower quantities than S. Typhimurium 17C10. In a second experiment we found out that S. Enteritidis 147 and its SPI-1 and SPI-4 mutants increased proinflammatory cytokine (IL-1ß and IL-8) signalling in the ileum 5 days post infection. On the other hand, independent of SPI-1 or SPI-4, S. Enteritidis 147 suppressed expression of IL-18, MCP1, TLR2, CD86, IL-7, IL-10 and IL-15 in the palatine tonsils. The suppression of cytokine signalling may facilitate the initial colonisation of the palatine tonsils by Salmonella. Moreover, immune suppression may also influence pig resistance to opportunistic pathogens and Salmonella infection in pigs thus may become an issue not only in terms of pork contamination but also in terms of affecting the immunological status of pig herds.

Coated fatty acids alter virulence properties of Salmonella Typhimurium and decrease intestinal colonization of pigs.

Salmonella Typhimurium infections in pigs are a major source of human foodborne salmonellosis. To reduce the number of infected pigs, acidification of feed or drinking water is a common practice. The aim of the present study was to determine whether some frequently used short- (SCFA) and medium-chain fatty acids (MCFA) are able to alter virulence gene expression and to decrease Salmonella Typhimurium colonization and shedding in pigs using well established and controlled in vitro and in vivo assays. Minimal inhibitory concentrations (MIC) of 4 SCFA (formic acid, acetic acid, propionic acid and butyric acid) and 2 MCFA (caproic and caprylic acid) were determined using 54 porcine Salmonella Typhimurium field strains. MIC values increased at increasing pH-values and were two to eight times lower for MCFA than for SCFA. Expression of virulence gene fimA was significantly lower when bacteria were grown in LB-broth supplemented with sub-MIC concentrations of caproic or caprylic acid (2 mM). Expression of hilA and invasion in porcine intestinal epithelial cells was significantly lower when bacteria were grown in LB-broth containing sub-MIC concentrations of butyric acid or propionic acid (10 mM) and caproic or caprylic acid (2 mM). When given as feed supplement to pigs experimentally infected with Salmonella Typhimurium, coated butyric acid decreased the levels of faecal shedding and intestinal colonization, but had no influence on the colonization of tonsils, spleen and liver. Uncoated fatty acids, however, did not influence fecal shedding, intestinal or tonsillar colonization in pigs. In conclusion, supplementing feed with certain coated fatty acids, such as butyric acid, may help to reduce the Salmonella load in pigs.

Cytokine response of porcine cell lines to Salmonella enterica serovar typhimurium and its hilA and ssrA mutants.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular bacterium which can infect and colonize pigs. After contact with enterocytes and macrophages, S. Typhimurium induces production of cytokines thus triggering the innate immune response. In this study we evaluated the cytokine response of two porcine cell lines, IPI-2I and 3D4/31, of epithelial or macrophage origins, respectively, to the wild-type S. Typhimurium and its hilA and ssrA mutants. We observed that the 3D4/31 cell line essentially did not respond to S. Typhimurium infection when a medium with foetal calf serum was used. However when the 3D4 cell line was incubated overnight in the presence of porcine serum, it efficiently responded to the wild-type strain and the ssrA mutant but not to the noninvasive hilA mutant as measured by mRNA quantification of TNF-alpha, IL-8 and GM-CSF by the real-time RT-PCR. In IPI-2I, all the cytokines were also induced by the wild-type S. Typhimurium and the ssrA mutant although the induction of TNF-alpha was lower than that induced by the wild-type strain. The hilA mutant was unable to induce any of the cytokines tested. The ssrA mutant can therefore be considered as more suitable for further vaccine development as the stimulation of innate immune response is important for animal protection against a challenge with virulent strains.

Medium-chain fatty acids decrease colonization and invasion through hilA suppression shortly after infection of chickens with Salmonella enterica serovar Enteritidis.

The most common source of Salmonella infections in humans is food of poultry origin. Salmonella enterica serovar Enteritidis has a particular affinity for the contamination of the egg supply. In this study, the medium-chain fatty acids (MCFA), caproic, caprylic, and capric acid, were evaluated for the control of Salmonella serovar Enteritidis in chickens. All MCFA were growth inhibiting at low concentrations in vitro, with caproic acid being the most potent. Contact of Salmonella serovar Enteritidis with low concentrations of MCFA decreased invasion in the intestinal epithelial cell line T84. By using transcriptional fusions between the promoter of the regulatory gene of the Salmonella pathogenicity island I, hilA, and luxCDABE genes, it was shown that all MCFA decreased the expression of hilA, a key regulator related to the invasive capacity of Salmonella. The addition of caproic acid (3 g/kg of feed) to the feed of chicks led to a significant decrease in the level of colonization of ceca and internal organs by Salmonella serovar Enteritidis at 3 days after infection of 5-day-old chicks. These results suggest that MCFA have a synergistic ability to suppress the expression of the genes required for invasion and to reduce the numbers of bacteria in vivo. Thus, MCFA are potentially useful products for reducing the level of colonization of chicks and could ultimately aid in the reduction of the number of contaminated eggs in the food supply.

Respiratory symptoms, bronchitis and asthma in children of Central and Eastern Europe.

The multicentre Central European Study of Air Pollution and Respiratory Health (CESAR) aimed to measure the respiratory health of schoolchildren using a standardised questionnaire in six countries of Central and Eastern Europe (CEE), allowing comparisons within this region and with other European countries. A cross-sectional study was conducted in 25 urban areas of Bulgaria, Czech Republic, Hungary, Poland, Romania, and Slovakia in 1996. Parents of 21,743 schoolchildren of age 7-11 yrs completed a questionnaire based on items from the World Health Organization and International Study of Asthma and Allergies in Childhood questions on cough and wheeze symptoms, as well as on diagnoses by doctors. Life-time prevalence of bronchitis was 55.9%, asthma 3.9%, and asthmatic, spastic or obstructive bronchitis 12.3%. In CEE countries the prevalence of bronchitis is higher and prevalence of asthma appears lower than in Western Europe. However, if asthma is defined as a diagnosis of either asthma or asthmatic, spastic or obstructive bronchitis, then its prevalence is comparable to Western Europe, or higher. In this region, within-country variation for most respiratory parameters is less than between-country variation. Between-country comparisons in doctors' diagnoses appear dependent on the choice of definition of asthma. Europe-wide comparisons in prevalence of respiratory symptoms and diagnosis are reported in this study. Some of the East-West difference in asthma prevalence may be attributable to differences in diagnostic practice.

BIOMarkers for occupational diesel exhaust exposure monitoring (BIOMODEM)--a study in underground mining.

Methods for the assessment of exposures to diesel exhaust were evaluated, including various biomarkers of internal exposure and early biological effects. The impact of possible biomarkers of susceptibility was also explored. Underground workers (drivers of diesel-powered excavators) at an oil shale mine in Estonia were compared with surface workers. Personal exposures to particle-associated 1-nitropyrene (NP) were some eight times higher underground than on the surface. Underground miners were also occupationally exposed to benzene and polycyclic aromatic hydrocarbons, as indicated by excretion of urinary metabolites of benzene and pyrene. In addition, increased O(6)-alkylguanine DNA adducts were detected in the white blood cells of underground workers, suggesting higher exposure to nitroso-compounds. However, no differences between underground and surface workers were observed in the levels of other bulky DNA adducts determined by 32P-postlabelling, or in DNA damage. The study indicated that smoking, diet and residential indoor air pollution are important non-occupational factors to consider when interpreting biomonitoring results.

Five new species of coccidia (Apicomplexa: Eimeriidae) from Madagascan chameleons (Sauria: Chamaeleonidae).

Coprological examination of 19 Madagascan chameleons of the genera Furcifer and Brookesia revealed the presence of five new coccidian species. Isospora brygooi n. sp. from Furcifer pardalis has spherical to subspherical oöcysts with a slightly pitted wall, 20.7 (17-24.5) x 19.3 (16-23) microm and broadly ellipsoidal sporocysts, 12.2 (11.5-13) x 8.1 (8-8.5) microm, with Stieda and substieda bodies. Oöcysts of Eimeria glawi n. sp. from Furcifer pardalis are cylindrical to ellipsoidal, 27.7 (26-29.5) x 18.4 (17-19) microm, with ellipsoidal sporocysts, 7.3 (6.5-8) x 5.2 (5-5.5) microm. E. vencesi n. sp. described from F. pardalis has spherical to subspherical oöcysts, 14.3 (13-15.5) x 13.0 (12-13) microm, with small granules, one to three globular polar granules and ellipsoidal sporocysts, 7.3 (6.5-8) x 5.2 (5-5.5) microm. E. worthi n. sp., described from Furcifer oustaleti has spherical oöcysts, 17.9 (17.5-19.0) x 15.0 (14.5-16.0) microm without a polar granule and ellipsoidal to cylindroidal sporocysts, 8.2 (7.0-9.5) x 5.8 (5.0-6.5) microm. Oöcysts of E. brookesiae n. sp. from Brookesia decaryi are cylindrical, 25.6 (23-27) x 15.0 (13-16) microm with ellipsoidal sporocysts, 10.1 (9-11) x 6.9 (6-7) microm. Endogenous development of E. vencesi is confined to the intestine, while that of E. glawi occurs in the gall-bladder.

Experimental transmission of Caryospora kutzeri (Apicomplexa: Eimeriidae) by rodent hosts.

Four laboratory-hatched European kestrels Falco tinnunculus L. were fed on laboratory mice and common voles Microtus arvalis Pallas previously inoculated with different doses of sporulated oocysts of Caryospora kutzeri Böer, 1982. Two kestrels that were fed infected mice shed C. kurtzeri oocysts 6 days after ingesting murine tissues. To compare direct and indirect transmissions, two of the kestrels were subsequently directly inoculated with 10(5) sporulated C. kutzeri oocysts and became patent on days 8 and 9 and shed caryosporan oocysts up to day 25 post inoculation. Additionally, four mice were inoculated with 10(6) oocysts in order to examine mouse tissues for the presence of developmental stages of C. kutzeri. No coccidian stages were found in the tissues of inoculated mice. The experiment showed that developmental stages of C. kutzeri are able to survive in mouse tissues and cause infection of suitable host after their ingestion.

SCID mice as a tool for evaluation of heteroxenous life cycle pattern of Caryospora (Apicomplexa, Eimeriidae) species.

Adult severe combined immunodeficient (SCID) mice were inoculated with oocysts of 13 different Caryospora (Protozoa, Apicomplexa) species isolated from the faeces of 10 reptilian and three raptorial bird hosts in attempt to test heteroxenous life cycle pattern. Only three reptilian isolates originated from viperid snakes, namely from Calloselasma rhodostoma, Atheris nitschei and Vipera ursinii induced lethal dermal caryosporosis in SCID mice. Neither clinical signs nor developmental stages were observed in mice infected with further nine caryosporan isolates originated from other reptilian and raptorial bird hosts. Results of this study confirmed that SCID mice represent a useful tool for evaluation of heteroxenous life cycle pattern of caryosporan coccidia and that only the Caryospora species from viperid and crotalid snakes produce dermal caryosporosis in mice

Two new species of Caryospora Léger, 1904 (Apicomplexa, Eimeriidae) from accipitrid raptors.

Two new species of Coccidia (Apicomplexa: Eimeriidae) are described from European accipitrid raptors (Falconiformes: Accipitridae). Oöcysts of Carvospora aquilae n. sp. found in faeces of the gold eagle Aquila chrysaetos are subspherical to broad ellipsoidal and measure 43 (40-49) x 37.5 (34-39) microm. Polar granule, oöcyst residuum and micropyle are absent. Each oöcyst contains one spherical to subspherical slightly polygonal sporocyst measuring 23.8 (23-25) x 23.3 (22-25) microm. Stieda and substieda bodies are absent. The sporocyst residuum is composed of numerous small granules less than 0.5 microm in diameter dispersed randomly among the sporozoites. Sporulated oöcysts of Carvospora circi n. sp. from faeces of the marsh harrier Circus aeruginosus are widely oval, measuring 24.5 (23-25) x 21.8 (21-24) microm. A polar granule, oöcyst residuum and micropyle are absent. Each oöcyst contains one spherical to subspherical sporocyst measuring 16.2 (15-17) x 15.6 (15-17) microm. A compact granular, spherical to subspherical sporocyst residuum, 10.4 (10-11) x 8.5 (7-9), was present in 76% of measured sporocysts. In 24% of sporocysts the granules of sporocyst residuum were scattered among the sporozoites.

Vipera berus and V. ammodytes (Serpentes:Viperidae) represent new host for Caryospora simplex (Apicomplexa:Eimeriidae) in Europe.

During a survey of the coccidian parasites of reptiles, caryosporan oocysts wee found in the faeces of wild and captive European viperid snakes Vipera berus (L.) and V. ammodytes (L.). Thirty two of 37 examined V. berus (86%) and 9 of 17 examined V. ammodytes (53%) specimens were found to be passing caryosporan oocysts. Morphological characters of all caryosporan isolates were identical and fitted well with the description of Caryospora simplex Léger, 1904. Experimental inoculation of severe combined immunodeficient (SCID) mice with seven isolates of C. simplex from V. berus or V. ammodytes confirmed the heteroxenous life cycle pattern, for the first time for isolates of evidently European origin. Caryosporan developmental stages were observed in the connective tissues of the nose, cheeks, ear and scrotum in all inoculated SCID mice. V. berus and V. ammodytes represent new hosts for C. simplex. The present paper represents the first widely based report on coccidian parasites of the genus Caryospora Léger in European viperids. Our findings indicate a wide distribution of C. simplex throughout the range of distribution of snakes of the genus Vipera.