Detection, seroprevalence and antimicrobial resistance of Yersinia enterocolitica and Yersinia pseudotuberculosis in pig tonsils in Northern Italy.

Yersiniosis is the third most common reported zoonoses in Europe, with Y. enterocolitica and Y. pseudotuberculosis responsible for 98.66% and 0.94% of the confirmed human cases in 2013. From June 2013 to October 2014, 201 pigs at slaughter belonging to 67 batches were tested for Y. enterocolitica and Y. pseudotuberculosis in tonsils. Diaphragm muscle samples were tested for antibodies against Yersinia by a commercially available ELISA test. Y. enterocolitica 4/O:3 was detected in 55/201 pig tonsils (27.4%; 95% CI 23.1-37.1). The positive pigs came from 38/67 batches (56.7%) and were reared in 36/61 farms (59.0%). There was no statistical difference between farrow-to-finish and finishing farms. The mean count of Y. enterocolitica was 3.56±0.85log10CFU/g with a minimum of 2.0log10CFU/g and a maximum of 4.78log10CFU/g. Y. pseudotuberculosis was isolated from 4/201 pig tonsils (2.0%; 95% CI 0.0-4.5). Three isolates belonged to serotype O:3 and one to serotype O:1. The positive pigs belonged to 4/67 batches (6.0%) and came from finishing farms only. Y. pseudotuberculosis could be enumerated in one sample only (4.27log10CFU/g). The ELISA test demonstrated that 56.1% of the meat juice samples were positive for Yersinia antibodies. Serological positivity was found in 67.9% (36/53) of the Y. enterocolitica- and 75.0% (3/4) of the Y. pseudotuberculosis positive pigs. A significant association was found between serological results and the presence of Y. enterocolitica in tonsils (OR=1.97, p=0.044). All the Y. enterocolitica 4/O:3 isolates were susceptible to amoxicillin-clavulanic acid, gentamicin, ceftazidime, ertapenem and meropenem, 94.5% to cefotaxime, 89.1% to kanamycin and 78.2% to tetracycline. The highest resistance rates were observed for ampicillin (100%), sulphonamides (98.2%) and streptomycin (78.2%). Y. pseudotuberculosis strains were sensitive to all the antimicrobials tested, i.e. amoxicillin, amoxicillin/clavulanic acid, azithromycin, cephalothin, cefoxitin, ceftriaxone, ciprofloxacin, nalidixic acid, sulphonamide, tetracycline and ticarcillin. The study shows that Italian fattening pigs are frequently infected with human pathogenic Y. enterocolitica 4/O:3. Although the isolation rate is slightly lower than in other European countries, the serological test demonstrates that the infection is widespread among pig population. In fact, seroprevalence is similar to other EU countries. The detection of Y. pseudotuberculosis serotypes O:1 and O:3 in pig tonsils is of concern. Since tonsils may represent a contamination source for pig meat at slaughter, further studies regarding human infections by both microbial species are strongly recommended.

Supportive relationships within ongoing families: cross-lagged effects between components of support and adjustment in parents and young adult children.

Italy is the first country in which the phenomenon of cohabitation of parents and young adult children was examined. From the earliest studies, it seemed clear that the transition to adulthood occurs within the family of origin: indeed, the successful outcome of this transition depends on the quality of family relationships. Using the Social Relations Model, this study examines the importance of the components of support within family relationships during the transition of young adults from university to job contexts (Kenny & La Voie, 1984). The cross-lagged influence among the components of perceived support and the adjustment of family members has also been investigated. Findings show that family components of support are significant for perception in both parents and young adults. Furthermore, cross-lagged models reveal different results for parents than for young adults. Discussion of results regarding the transition to adulthood and family theory is provided.

Isolation of Mycobacterium caprae (Lechtal genotype) from red deer (Cervus elaphus) in Italy.

During tuberculosis (TB) surveillance, 53 hunted red deer (Cervus elaphus) were collected to determine whether TB was present in free-ranging animals from an Italian alpine area. Samples (lungs, liver, intestine, and lymph nodes) were cultured and analyzed by real-time PCR assay carried out directly on tissue. Mycobacterium caprae was isolated from small granulomatous, tuberculosis-like lesions in the liver of a 12-yr-old female. Identification of suspect colonies was done by PCR restriction fragment length polymorphism analysis of the gyrb gene, and genotyping was performed by spoligotyping and mycobacterial interspersed repetitive unit variable number tandem repeat analysis. The isolated strain was genetically identical to strains isolated in the study area in 2001 from dairy cows imported from Austria and in 2010 from an indigenous cow. The genotype, called "Lechtal," is the most frequently detected in the TB outbreaks in Austria and Germany. The possibility that red deer act as a maintenance host of M. caprae between TB outbreaks could be not excluded. Despite the high red deer population density, the detection of only one infected red deer could suggest that the wildlife management measures applied in the study area (prohibition of artificial feeding and secure removal of offal from hunted animals) may reduce the risk of TB spreading.

Virulence-associated genes in avian pathogenic Escherichia coli from laying hens in Apulia, Southern Italy.

1. Escherichia coli isolated from lesions (Avian Pathogenic E. coli - APEC) of layer hens affected by colibacillosis and from intestinal contents of clinically-healthy birds (Avian Faecal E. coli - AFEC) were serotyped. All the isolates were investigated for the presence of virulence genes to determine which genes were more closely related to those from lesions. 2. A number of different serogroups were detected, O78 being predominant among the isolates from colibacillosis. 3. E. coli isolated from lesions were not linked to a specific pathotype (set of common virulence genes). 4. The presence of the virulence genes, with the exception of astA, was associated more generally with APEC strains. 5. Statistically, genes such as cva/cvi, tsh, iss, irp2 and iucD were more related to isolates from colibacillosis. 6. It is suggested that the detection of these genes in a rapid and inexpensive test for field practitioners could provide useful information about the potential virulence of E. coli isolated in commercial layer flocks.

Detection, semiquantitative enumeration, and antimicrobial susceptibility of Yersinia enterocolitica in pork and chicken meats in Italy.

Yersinia enterocolitica is recognized as an etiological agent of gastroenteritis, lymphadenitis, and chronic sequelae. During 2006 and 2007, 205 samples (125 pork and 80 chicken meats) were collected in Italy and tested for detection and most-probable-number (MPN) enumeration of Y. enterocolitica organisms. The microorganism was isolated from 45 samples (21.9%): 19 (15.2%) pork samples and 26 (32.5%) chicken samples. Y. enterocolitica MPN contamination levels were low, ranging from 0.30 to 1.50/g. Most (94.4%) Y. enterocolitica strains were biotype 1A (serotypes O:3; O:5; O:6,30; O:6,30-6,31; O:7,8-8-8,19; O:8; O:9; O:25,35; O:36; and O nontypeable), and 5.6% of the isolates were bioserotype 2/O:9. All isolates were tested for yadA, ail, inv, ystA, and ystB virulence sequences. The yadA gene was detected in two strains (3.7%) isolated from chicken samples: one Y. enterocolitica 2/O:9 yadA+ ail+ ystA+, and one Y. enterocolitica 1A/O:7,8-8-8,19 yadA+ inv+ ystB+. Two (3.7%) 2/O:9 strains, isolated from pork products, were ail+ ystA+. Most biotype 1A strains were ystB+ (84.3%) and inv+ (39.2%). All strains were sensitive to cefotaxime, ciprofloxacin, chloramphenicol, nalidixic acid, streptomycin, sulfonamide, tetracycline, trimethoprim, and trimethoprim-sulfamethoxazole. Resistance to gentamicin and aztreonam was observed in 1.9% of the isolates. High levels of resistance were detected toward amoxicillin-clavulanic acid (27.8%), ampicillin (75.9%), and erythromycin (100%). The authors hypothesize that Y. enterocolitica pathogenic biotypes are rather uncommon in foods when compared with their isolation rates from animal sources and that chicken meat could be contaminated as well as pig meat and its derived products.

Expression of major histocompatibility complex class II antigens in porcine leptospiral nephritis.

Class II major histocompatibility complex (MHCII) is required for the presentation of antigens to CD4 helper T cells. During nephritis, not only primary antigen presenting cells such as histiocytes and lymphocytes, but also cytokine-stimulated tubular epithelial cells express MHCII. Leptospirosis in fattening pigs is characterized by several degrees of nephritis, from absence of lesions to severe multifocal tubulo-interstitial inflammation. Renal tissue from 20 8-month-old pigs with spontaneous nephritis and 6 control pigs without renal lesions were investigated for leptospirosis by indirect immunohistochemistry (IHC) and polymerase chain reaction (PCR). IHC for MHCII also was performed on renal samples. Serum samples were tested for different serovars of Leptospira interrogans. Control pigs were free of interstitial nephritis and negative for leptospirosis by all tests. In pigs with nephritis, serology was positive for serovar Pomona in 19/20 pigs. In 16 of these 19 pigs, leptospiral renal infection was confirmed by PCR and/or indirect IHC. Nephritic lesions were classified histologically into perivascular lymphocytic (4 pigs), lymphofollicular (6 pigs), lymphohistiocytic (8 pigs), and neutrophilic (2 pigs) pattern. MHCII expression by histiocytes and lymphocytes was observed in all lesions. Prominent MHCII expression in regenerating tubular epithelium was observed in lymphofollicular and lymphohistiocytic nephritis. No tubular colocalization between leptospiral and MHCII antigen was observed. Results suggest that during leptospiral nephritis, MHCII contributes to the intensity of the inflammatory response. Furthermore de novo MHCII expression in regenerating tubules may play a role in the defence mechanism against leptospiral tubular colonization.

Isolation of Salmonella strains from reptile faeces and comparison of different culture media.

AIMS: To provide information on epidemiology and isolation of Salmonella strains from reptiles. METHODS AND RESULTS: Ninety-one samples collected from reptiles of the zoo of Rome or belonging to private owners were analysed using a standard protocol for isolation of Salmonella from food. Salmonella strains were tested for susceptibility to 15 antimicrobics by a disc-agar diffusion method. Forty-six samples (50.5%) were positive for Salmonella. Of the 22 strains serotyped, 17 belonged to Salmonella enterica subsp. I, four to the subsp. IIIa and one strain resulted untypeable. Rappaport-Vassiliadis broth (RVB) allowed to recover more Salmonella strains when bacterial growth in buffered peptone water (BPW) was scarce, while selenite cystine broth (SCB) was more efficient, whereas growth in BPW was abundant. The maximum isolation score was obtained by plating onto xylose lysine desoxycholate agar (XLD). The strains exhibited resistance at high percentages to colistin sulphate (58.7%), sulphamethoxazole (55.5%), streptomycin (32.6%), tetracycline (19.6%), ampicillin (17.4%) and nalidixic acid (13.1%). CONCLUSIONS: A high prevalence of Salmonella in reptiles was observed. For isolation, the choice of the enrichment broth depending on the degree of growth in BPW followed by plating onto XLD may be suggested. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides epidemiological data on the prevalence of Salmonella and laboratory protocols useful for isolation of Salmonella from faeces of reptiles.

Use of a macrophage cell line for rapid detection of Mycobacterium bovis in diagnostic samples.

Mycobacterium bovis isolation on bacteriological media from suspected cases of bovine tuberculosis (TB) demands laborious and time-consuming procedures. Even polymerase chain reaction (PCR) and radiometric analyses are secondary procedures and not alternatives to bacteriological procedures. Therefore, there is a need to develop new techniques aimed at rapid M. bovis detection in diagnostic samples. The human macrophage cell line THP-1 was thus investigated in experiments of M. bovis propagation and isolation from reference lymph node suspensions. THP-1 cells were shown to support a high-titered propagation within 48h of minute amounts of both M. bovis BCG and fully pathogenic M. bovis strain 503. A semi-nested PCR for TB-complex-specific insertion sequence IS6110 revealed M. bovis infection in THP-1 cells. The same was true of a flow cytometry (FC) assay for expression of M. bovis chaperonin 10 in infected cells. The reduced time for isolation and identification of M. bovis (48-72h) and the consistency of the test results make the use of macrophage cell cultures attractive and cost-effective for veterinary laboratories involved in TB surveillance.

Diagnosis of Mycobacterium bovis infection in calves sensitized by mycobacteria of the avium/intracellulare group.

Because of the frequent exposure of cattle to mycobacteria of the avium/intracellulare group, an investigation was carried out into the possible repercussions thereof on the diagnosis of bovine tuberculosis. Three calves from a bovine tuberculosis-free herd, scored avian reactors in the gamma-interferon assay for bovine tuberculosis, were sedated and inoculated endotracheally with a virulent Mycobacterium bovis strain. Then, three other avian reactors were housed with the above donor calves. Mycobacterium bovis was isolated from the nasal swabs of the three endotracheally infected, donor calves. On these samples, TB complex-specific polymerase chain reaction (PCR) tests for IS6110 were also positive, albeit with a different time kinetics. The three contact-infected calves showed clear immunological signs of infection; however, their nasal swabs were always PCR-negative and only Mycobacterium avium was isolated. In the endotracheally infected donor calves there was a rise of the gamma-interferon responses to avian and bovine purified protein derivative (PPD) tuberculins, which reached the same stable plateau levels over the whole experiment. The above effect was also observed in the contact-infected calves, even though the response to avian PPD tuberculin always remained at a higher level. By using conventional bovine and avian PPD tuberculins, the comparative intradermal test was generally positive in endotracheally infected, as opposed to contact-infected calves; a positive intradermal test for M. bovis was obtained in two contact-infected calves by different bovine PPD tuberculins based on M. bovis bacillus Calmette-Guerin (BCG) secreted or somatic antigens. It was concluded that M. bovis infection may be concealed for some time in cattle sensitized by mycobacteria of the avium/intracellulare group and that different diagnostic procedures should be adopted for such animals.

Serological survey of leptospiral infection in kennelled dogs in Italy.

Two hundred and forty-five dogs were examined serologically for the presence of antibodies against different serovars of Leptospira interrogans. The dogs belonged to five different groups: group 1 was composed of clinically healthy pet dogs referred for a regular veterinary check-up visit or for vaccination; group 2 was composed of stray dogs; and groups 3, 4 and 5 were composed of dogs maintained in three different kennels which had varying standards of hygiene. Seventy-two out of the 245 dogs examined were seropositive for leptospirosis. In group 1, there were 3-4 per cent seropositive dogs; in group 2, 30.3 per cent; in group 3, 13.8 per cent; in group 4, 38.6 per cent; and in group 5, 49.2 per cent. This study demonstrates that leptospiral infection is common in dogs housed in kennels, despite most of them being vaccinated, and that crowding of animals into unsanitary quarters is associated with a high prevalence of infection. The most common infecting serovars found were bratislava and grippotyphosa, confirming recent observations that demonstrate a significant change in the epidemiology of canine leptospirosis.

Decrease of vancomycin-resistant enterococci in poultry meat after avoparcin ban.

In Italy, 18 months after the ban of avoparcin, the percentage of poultry meat samples containing vanA gene-positive vancomycin-resistant enterococci fell from 14.6% to 8%.

Quality controls and in vitro diagnostic efficiency of bovine PPD tuberculins.

Wide heterogeneity was shown among different batches of bovine protein purified derivative (PPD) tuberculin, as regards protein content and antigenic profile. These features were also investigated in pilot preparations of Mycobacterium bovis secreted antigens and PPD tuberculins. Under controlled conditions, widely different compositions were revealed as a function of the M. bovis strain and also of the time in culture, due to the transient expression of seemingly important clusters of antigens. Furthermore, these parameters could dramatically affect the efficacy of the above preparations in in vitro assays of cell-mediated immunity on M. bovis-infected cattle. The field exposure to mycobacteria of the avium/intracellular group could also influence the readout of such assays, results being in agreement with a bystander suppression model of the response to M. bovis antigens. Due to the above, practical suggestions are put forward to improve the composition of bovine PPD tuberculins and the relevant control procedures.

Leptospira interrogans serovar sejroe infection in a group of laboratory dogs.

Interstitial nephritis was seen histologically in 19 (59%) out of 32 pure-breed beagle dogs (16 males and 16 females) subjected to standard safety tests. In these animals no clinical abnormalities were observed and all the tested parameters (haematology, biochemistry and urine analysis) were within the normal ranges. Leptospiral antibody titres ranging from 1 : 100 to 1 : 6400, against a serovar (hardjo) belonging to the Sejroe serogroup, were detected by the microscopic agglutination test (MAT) in the serum of the 19 dogs with interstitial nephritis. All animals without renal lesions were seronegative. Leptospiral antigen was detected immunohistochemically in the kidneys of 4 dogs; leptospires were detected in Warthin-Starry stained sections of one dog. Leptospires were isolated from the kidneys of 3 of the 4 dogs examined by bacterial culture. The isolated strains were typed as serovar sejroe by restriction endonuclease digestion and Southern blot hybridization analysis of their DNA. It was concluded that Leptospira interrogans serovar sejroe, was responsible for an asymptomatic chronic renal infection which was widespread in this group of laboratory dogs.

Detection and identification of Leptospira interrogans serovars by PCR coupled with restriction endonuclease analysis of amplified DNA.

Primers for PCR were selected from a sequenced fragment of clone pL590, which contains a repetitive element present in the genome of Leptospira interrogans serovar hardjo type hardjoprajitno (M. L. Pacciarini, M. L. Savio, S. Tagliabue, and C. Rossi, J. Clin. Microbiol. 30:1243-1249, 1992). A specific DNA fragment was amplified from the genomic DNAs of serovar hardjo type hardjoprajitno and nine serovars also belonging to L. interrogans as a consequence of the spread of the same or a closely related repetitive element within this species (Pacciarini et al., J. Clin. Microbiol. 30:1243-1249, 1992). In addition, specific amplification was obtained from two Leptospira borgpetersenii serovars (tarassovi and hardjo type hardjobovis). Negative PCR results were observed with all of the other Leptospira serovars tested, including nonpathogenic ones (serovars patoc and andamana), another spirochete (Borrelia burgdorferi), bacteria commonly found in biological samples, and swine and bovine cell lines. Direct PCR on biological samples such as kidney samples demonstrated that preliminary isolation and culture of Leptospira cells are not required for efficient detection. Furthermore, digestion of the amplified DNA with the enzymes HinfI and DdeI yielded specific polymorphic patterns, allowing discrimination among the majority of the serovars. These methods were applied to 25 field isolates of serovar pomona, leading to the conclusion that they were suitable for the simple and rapid detection of L. interrogans and for serovar identification.

Identification of Leptospira interrogans strains by monoclonal antibodies and genomic analysis.

A recombinant probe derived from a genomic library of serovar hardjo strain Hardjoprajitno, and a panel of serovar specific Monoclonal Antibodies (MAbs) were used for the characterization of 31 Leptospira isolates from cattle and swine. The two methods performed equally well in serovar identification except for the distinction of the genotypes hardjoprajitno and hardjobovis within serovar hardjo which could only be obtained by genomic analysis. The combination of immunological and genetic information was also useful to evaluate the degree of variability of Leptospira strains. The quality of the patterns and the sensitivity provided by a digoxigenin labelled probe were comparable to those obtained with a radioactive reagent.

The search for improved methods for diagnosing leptospirosis: the approach of a laboratory in Brescia, Italy.

The authors describe work in progress at the laboratory in Brescia, Italy, on the application of molecular methods to the diagnosis of leptospirosis. This work includes the following: a) Development of polymerase chain reaction (PCR) assays capable of amplifying specific deoxyribonucleic acid fragments from most Leptospira interrogans strains. b) Development of a microtitre-based assay for rapid detection of PCR-positive samples. c) Characterisation of Leptospira strains through restriction endonuclease analysis of PCR products and amplified fragment length polymorphism.

Repetitive sequences cloned from Leptospira interrogans serovar hardjo genotype hardjoprajitno and their application to serovar identification.

We selected, from a genomic library of Leptospira interrogans serovar hardjo genotype hardjoprajitno, two probes containing repetitive sequences (pL1 and pL590). The hybridization patterns of these probes to DNA isolated from a variety of Leptospira serovars were examined and their ability to detect subtle differences at the genomic organization level was established. We identified the DNA fragments within pL1 and pL590 which are sufficient to yield polymorphic hybridization patterns; these results define the upper size limit of two novel repetitive elements in the Leptospira genome. The pattern and degree of hybridization observed for the serovars tested in this work were used to divide Leptospira spp. into groups which share genetic relatedness; our conclusions are consistent with previous classifications by other authors.