Characterization of Listeria monocytogenes from encephalitis cases of small ruminants from different geographical regions, in Greece.

AIMS: The aim of this study was to evaluate the genetic diversity and resistance phenotypes of Listeria monocytogenes strains isolated from clinical encephalitis cases, and compare this population to isolates derived from tank milk of healthy animals. METHODS AND RESULTS: A total of 57 L. monocytogenes strains isolated from ruminant's listeriosis cases (n = 31) and from tank milk of healthy ruminants (n = 26) were characterized by species PCR, molecular serotyping, PCR detection of virulence genes, pulsed-field gel electrophoresis and antimicrobial susceptibility testing. All strains possessed inlA, inlC, inlJ, plcA, actA, hlyA and iap virulence-associated genes while serotyping analysis revealed that they were mainly assigned into IVb group. Genotyping revealed 50 pulsotypes among the 57 strains assigned into seven clusters while indistinguishable pulsotypes between clinical and milk strains were not identified. Resistance of L. monocytogenes isolates to 14-16 antimicrobial agents tested was observed and 23 antimicrobial resistance profiles (ARPs) were defined while no apparent predominant ARP type was observed among isolates. CONCLUSIONS: Small ruminants are exposed to a broad range of antimicrobial-resistant as well as genetically diverse strains of L. monocytogenes carrying virulence-associated genes but not all of them associated with the disease. Pulsed-field gel electrophoresis analysis suggests that pulsotypes associated with encephalitis are found in farms only in association with listeriosis. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings are valuable in understanding the ecology of this important food-borne pathogen and creating awareness for the emerging antimicrobial resistance.

Culture phenotypes and molecular characterization of Mycobacterium avium subsp. paratuberculosis isolates from small ruminants.

In this study the suitability of different solid media was investigated for the isolation of Mycobacterium avium subsp. paratuberculosis (Map) in order to identify the optimum single or combination of media to permit the isolation of all strain types from small ruminants. A subset of these Map strains was then further characterized by molecular typing methods to assess the genetic diversity of Map strains in the study area (Northern Greece). Map strains were isolated from tissues and faeces of infected goats (n=52) and sheep (n=8) and were analysed for polymorphisms in IS1311 to classify the strain type as Type C or S. The study found that M7H11 supplemented with mycobactin j, OADC and new born calf serum (M7H11+Mj) is the best single choice of medium for the primary isolation of Map of both Type C and S from small ruminants. The combination of M7H11+Mj and Herrolds egg yolk medium supplemented with mycobactin j and sodium pyruvate allowed the detection of all Map isolates in this study. Nineteen Map isolates were characterised by pulsed-field gel electrophoresis and the isolates demonstrated significant genetic diversity. Twelve different SnaBI and 16 distinct SpeI profiles were detected of which 25 have not been described previously and are new profiles. The combination of both enzyme profiles gave 13 different multiplex profiles. Ten different multiplex profiles were detected in goats and three in sheep. One ovine isolate gave the same multiplex profile as a caprine isolate and two different profiles were found within a single goat herd.

Experimental infection of pregnant ewes with enteric and abortion-source Chlamydophila abortus.

Two groups of pregnant ewes were experimentally infected oronasally in midpregnancy. A faecal and an abortion-source isolate of Chlamydophila abortus were used. They were derived from a healthy ewe from a flock with no history of abortion, and from an aborted foetus in a farm with enzootic abortion. As assessed by modified Ziehl-Neelsen (MZN) staining, egg culture, antigen ELISA, the Clearview test and immunohistochemistry, inoculation resulted in placental and/or foetal infection in all ewes. Histopathology revealed placentitis in two and four ewes inoculated with the enteric or abortion-source isolate, respectively, in addition, these samples were immunohistochemically positive for chlamydial antigen. All six ewes infected with the enteric isolate and five of seven ewes infected with the abortion-source isolate showed evidence for a serological response by an indirect ELISA or CFT. Neither chlamydiae nor lesions were detected in the placentae and lambs of the uninfected control ewes, which remained seronegative. Our results suggest that enteric C. abortus can be associated with placental and foetal lesions in sheep.

Evaluation of two assays, MSRV and RV, for the isolation of Salmonella spp. from wastewater samples and broiler chickens.

The Rappaport Vassiliadis (RV) and the Modified Semi-solid Rappaport Vassiliadis (MSRV) methods were evaluated in 130 municipal wastewater samples and 30 flocks of broilers. Analysis of the results showed that 71 Salmonella serotypes were isolated in wastewater samples. The positivity of the MSRV method was 33% and of the RV method, 45%. The sensitivity was 95% and 83% with the MSRV and RV, respectively. The concordance between the two methods was 89% (k = 0785). One hundred serotypes were isolated from broiler internal organs and 50 from intestinal samples. For internal organs, the positivity of MSRV was 56% and of RV, 45%. For intestinal samples, the respective percentages were 28 and 22. For internal organs, the sensitivity was 100% with MSRV and 81% with RV, whereas for intestinal samples, the sensitivity was 100% with MSRV and 80% with RV. The specificity was 100% in all cases. The concordance between the two methods was 89% (k = 0790) for internal organs and 94% (k = 0851) for intestinal samples.