Abstract: Malassezia pachydermatis isolates (n=185) from skin sites from dogs (n=30) were characterized genetically and biochemically following in vitro culture. Two regions in the chitin synthase-2 gene (chs-2) and the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA were sequenced, and the phospholipase activity of each isolate was assessed. Three chs-2 (i.e. Ac, Bc and Cc) and eight ITS-1 (i.e. AI1, AI2, AI3, AI4, BI1, CI1, CI2 and CI3) sequence types were defined for all 185 samples. The findings revealed that multiple M. pachydermatis genotypes/subgenotypes could be cultured from healthy dogs or from dogs with single or multiple, generalized skin lesions. Subgenotypes AI1 and BI1 were associated with all skin sites of dogs sampled, whereas subgenotype CI2 was mostly linked to a particular location. Isolates derived from skin lesions showed a significantly higher phospholipase activity compared with those from skin sites with no detectable lesions. Genotype B was mainly cultured from healthy skin; only four isolates (9.3%) had low phospholipase activity, whereas other genotypes/subgenotypes were predominantly associated with skin lesions and had a high phospholipase activity. The results of the present study suggest that the distribution pattern of particular genotypes or subgenotypes of M. pachydermatis on the skin of dogs relates to the affinity of the yeast to the host and to particular skin sites.

Abstract: Canine monocytic ehrlichiosis (CME) caused by Ehrlichia canis is the most known canine tick-borne disease (TBD) spread throughout the world. Preventing tick bites is a priority to reduce the risk of TBDs and it was the aim of the present study to evaluate the efficacy of a combination of imidacloprid 10% and permethrin 50% (ImPer) (Advantix; Bayer AG, Germany) in a spot-on formulation to control CME under field conditions. On January-March 2005, 845 dogs from two kennels in southern Italy (kennels of Bari (KB)- and Ginosa (KG)), with a history of tick infestation were initially tested by serology and PCR assay for E. canis infection. Data on Leishmania infantum infection were also available from a previous study carried out on the same dog population. One hundred twenty-six dogs (14.9%) presented anti-E. canis antibodies with a relative prevalence of 15.6% (n=65 dogs in KB) and 14.2% (n=61 dogs in KG). Five hundred thirty-five animals found negative both for E. canis and L. infantum infections were enrolled in three groups (Group A--treated with ImPer once a month; Group B--treated every 2 weeks; and Group C--untreated control animals) and monitored for E. canis infection by serology and PCR in November 2005 (first follow-up) and in March 2006 (second follow-up). The E. canis infection was serologically revealed, at the first and/or second follow-up, in 26 animals from Group C in KB and KG (mean incidence density rate (IDR), 13.24%) while in none of the animals from Group A (KB and KG) and only in one animal from Group B (IDR 1.13%) in KG. The final protection efficacy of ImPer ranged from 95.57% to 100% in Groups B and A. At PCR only 15 dogs from KG were positive for Rickettsiales only at the first follow-up and at the sequence analysis two (both in Group C) revealed 100% homology with E. canis sequences while 13 with Anaplasma platys. Four out of 13 A. platys PCR-positive dogs were also seropositive for E. canis at one or both follow-ups. ImPer, by virtue of its repellent and acaricidal activity against ticks, has been shown to be efficacious to prevent E. canis infection in treated dogs living under natural conditions in endemic areas.

Abstract: Helicobacter pylori is an organism widespread in humans and sometimes responsible for serious illnesses, such as gastric and duodenal ulcers, MALToma and even gastric cancer. It has been hypothesized that the infection route by H. pylori involves multiple pathways including food-borne transmission, as the microorganism has been detected from foods such as sheep and cow milk. This work reports the results of a survey conducted in order to investigate the presence of H. pylori in raw goat, sheep and cow milk produced in Southern Italy, employing a Nested Polymerase Chain Reaction (Nested-PCR) assay for the detection of the phosphoglucosamine mutase gene (glmM), as screening method followed by conventional bacteriological isolation. Out of the 400 raw milk samples examined, 139 (34.7%) resulted positive for the presence of glmM gene, but no strains were isolated. In this work H. pylori DNA has been firstly detected from 41 (25.6%) raw goat milk samples. The results deserve further investigations on the contamination source/s of the milk samples and on the major impact that it may have on consumers.

Abstract: Little precise information is available on the systematics, genetics, ecology and epidemiology of yeasts of the genus Malassezia from different animal species. In the present study, one hundred and four isolates of Malassezia (lipid dependent or non-lipid dependent) from dogs were characterized by their chitin synthase 2 gene (CHS2), and the large subunit (LSU) and the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA sequences, and compared genetically with well-defined reference strains of Malassezia pachydermatis and heterologous species, including Malassezia furfur and Candida albicans. For each locus examined, three main sequence types (i.e. A, B and C) represented all of the 104 isolates, which were designated as genotypes A, B and C, respectively. A fourth, minor sequence type was also defined for the ITS-1. The nucleotide differences among genotypes was consistent with the magnitudes of intraspecific variability reported in previous studies. The genetic analysis of the sequence data sets (for individual loci) showed that all Malassezia genotypes clustered (with moderate to strong support) with the reference sequences of M. pachydermatis to the exclusion of the outgroups M. furfur and C. albicans. The present study reveals that multiple genetic variants of M. pachydermatis occur on dogs. The multilocus approach employed herein provides a foundation for future investigations of M. pachydermatis from other animals and humans, and their ecology and epidemiology.

Abstract: Eleven cattle farms, 8 layer farms, 7 broiler farms and 30 broiler meat samples were investigated in south-eastern Italy throughout 2003 to evaluate the prevalence, the molecular type and antimicrobial resistance of thermophilic Campylobacters. A total of 398 samples were analysed. One Campylobacter isolate for each positive faecal swab and three isolates per positive broiler meat sample were selected for further analysis. Multiplex PCR was performed for species-level identification and PCR-RFLP of the flagellin A gene for genotyping. Resistance to 14 antimicrobials was studied in 188 Campylobacter isolates. Prevalence of campylobacters was high both on farms (100%) and in food samples (73%). On 4/11 cattle farms and on 10/15 poultry farms more than one species was isolated. The presence of more than one genotype was found on 8/11 cattle farms, on 10/15 poultry farms and in 8/22 Campylobacter-positive food samples. High rates of resistance to quinolone were observed: 9/31 (29%) C. jejuni bovine isolates, 4/22 (18%) C. jejuni poultry isolates, and 14/26 (54%) C. coli poultry isolates. Resistance to sulphamethoxazole-trimethoprim was also observed frequently: 18/26 (69%) of the avian C. coli strains, 25/31 (80%) of the C. jejuni strains isolated from poultry and 15/22 (68%) of those isolated from cattle were resistant. There was a significant difference between the rate of resistance to macrolides of C. coli and C. jejuni isolated in poultry, which amounted to 23% and 3%, respectively. This study provided data on the prevalence and antimicrobial resistance of thermophilic campylobacters in south-eastern Italy and confirmed that flaA-typing is an efficient tool to study the epidemiology of Campylobacter strains in short-term investigations.

Abstract: Staphylococcus aureus is considered to be one of the leading causes of food-borne illnesses. Milk, dairy products and meats are often contaminated with enterotoxigenic strains of this bacterium. Foodstuff contamination may occur directly from infected food-producing animals or may result from poor hygiene during production processes, or the retail and storage of foods, since humans may carry the microorganism. The number of S. aureus strains that exhibits antimicrobial-resistance properties has increased, together with the potential risk of transmitting the same properties to the human microflora via foods or inducing infections hard to be treated. This paper reports the results of a 3-year survey (2003-2005) on the occurrence of S. aureus in meat and dairy products. Of 1634 samples examined, 209 (12.8%) were contaminated with S. aureus. A total of 125 enterotoxigenic S. aureus strains were biotyped and their antimicrobial resistance pattern tested. Most of the isolated strains produced SED (33.6%), followed by SEA (18.4%), SEC (15.2%), SEB (6.4%) and belonged mainly to the Human ecovar (50.4%), followed by Ovine (23.2%), Non-Host-Specific (17.6%), Bovine (7.2%) and Poultry-like (1.6%) ecovars. Finally, the 68.8% analysed strains showed antimicrobial resistance properties at least at one of antibiotics tested. Human biotype showed antimicrobial resistance at more than one antibiotic than the other biotypes (p<0.05). The results provided evidence that the presence of enterotoxigenic and antimicrobial resistant strains of S. aureus has become remarkably widespread in foods. This calls for better control of sources of food contamination and of the spread of antimicrobial-resistance organisms.

Abstract: Helicobacter pylori (H. pylori) is a very important bacterial pathogen of humans which may cause gastrointestinal illnesses ranging from gastric and duodenal ulcers to neoplastic diseases such as MALToma and gastric cancer. Transmission via contaminated food is still uncertain but several authors believe this can realistically occur and milk may act as a vehicle of infection. This paper reports the results of H. pylori survival trials in pasteurized and ultrahigh temperature (UHT) milks artificially contaminated and aerobically stored at 4 degrees C. The results obtained showed that the four strains used in this study (H. pylori nat 18-19-20 and H. pylori ATCC 43504), had a progressive reduction in bacterial load with a median survival of 9 days in pasteurized milk and 12 days in UHT milk, with approximate average of initial inoculum of 10(5) and 10(6)cfu/ml, respectively. These findings are very important to clarify the route of transmission of H. pylori to humans via food and for implementation of a correct risk analysis for food safety purposes.

Abstract: In December 2005, equine influenza virus infection was confirmed as the cause of clinical respiratory disease in vaccinated horses in Apulia, Italy. The infected horses had been vaccinated with a vaccine that contained strains representatives from both the European (A/eq/Suffolk/89) and American (A/eq/Newmarket/1/93) H3N8 influenza virus lineages, and the H7N7 strain A/eq/Praga/56. Genetic characterization of the hemagglutinin (HA) and neuraminidase (NA) genes of the virus from the outbreak, indicated that the isolate (A/eq/Bari/2005) was an H3N8 strain closely related to recent representatives (Kentucky/5/02-like) of the American sub-lineage Florida, that was introduced in Italy through movement of infected horses from a large outbreak described in 2003 in United Kingdom. Strain A/eq/Bari/2005 displayed 9 amino acid changes in the HA1 subunit protein with respect to the reference American strain A/eq/Newmarket/1/93 contained in the vaccine. Four changes were localized in the antigenic regions C-D and likely accounted for the vaccine failure.

Abstract: Dicrocoelium dendriticum (Rudolphi, 1819) and Dicrocoelium hospes (Looss, 1907) are recognised to affect the liver of domestic and wild ruminants. A third species, Dicrocoelium orientalis which was described from musk deer in the Baikal region of the former Soviet Union and re-named to Dicrocoelium chinensis (Sudarikov and Ryjikov, 1951) Tang and Tang, 1978 was isolated from other species of deer in Asian countries and from mouflon and roe deer in Europe. Scant information is available for D. chinensis, including the range of species that act as definitive and intermediate hosts. To provide morphological and molecular evidences differentiating D. chinensis versus D. dendriticum, 239 Dicrocoelium spp. specimens were collected from sheep, cattle and sika deer from different localities in Austria, Germany and Italy. Specimens were morphologically identified based on the testes orientation, overall size, and level of maximum body width and other morphometric measurements. From this sample, 10 specimens of D. chinensis and 25 of D. dendriticum from different hosts and geographical localities were characterized molecularly through sequencing of partial 18S rDNA (approximately 1400 bp) and ITS-2 (including the 5.8S and 28S flanking regions; approximately 600 bp). Interspecific differences between D. dendriticum and D. chinensis of 0.14% and 3.8% were recorded in 18S rRNA and ITS-2 sequences, respectively. Phylogenetic analyses via Bayesian inference were conducted using sequences of ITS-2 (276 bp) and partial 28S (221 bp) of the above species of Dicrocoelium together with 20 species belonging to the Xiphidiata within the Plagiorchiida available in GenBank. Both gene regions were strongly concordant in differentiating the Dicrocoeliidae, Gorgoderidae and Plagiorchiidae and were in agreement with their current classification. Morphological and molecular characterization clearly differentiate D. dendriticum and D. chinensis as two distinct digeneans infecting ruminants. The implications on the separate status of D. chinensis on the etiology, biology and diagnosis of dicrocoeliosis are discussed.

Abstract: Methicillin-resistant Staphylococcus aureus (MRSA) strains are a global health concern. The present study regarded 160 S. aureus strains that had been isolated from 1634 foodstuff samples of animal origin in a previous survey conducted in Italy during 2003-2005. The strains were characterized by detecting the mecA gene, the production of type A to D staphylococcal enterotoxins (SEs), and studying their resistance properties against several antibiotics; their ecological origin was determined by biotyping. Of the 160 analyzed S. aureus strains six (3.75%) were mecA positive and derived from six different samples; four isolates were from bovine milk and two from dairy products (pecorino cheese and mozzarella cheese). Two strains isolated from milk belonged to the non-host-specific biovar while the others to the ovine biovar. The strain isolated from mozzarella cheese belonged to the non-host-specific biovar and the strain isolated from pecorino cheese to the ovine biovar. All the MRSA strains isolated were enterotoxigenic; two strains synthesized SEA/SED two SED and one SEC. All the strains showed resistance to at least one of the antibiotics tested but none was resistant to glycopeptides.

Abstract: Verocytotoxin-producing Escherichia coli (VTEC) non-O157 serogroups are among the most important emerging food-borne pathogen groups. In particular, the O26 serogroup is able to cause a large spectrum of illnesses in humans which have a significant public health impact as they may range from haemorrhagic colitis (HC) to haemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). It is known that VTEC organisms are associated with animal reservoirs, i.e. ruminants, and foods of animal origin, especially undercooked meat and raw milk, are often involved in outbreaks. In this study, 250 minced beef samples collected at retail outlets in southern Italy were tested for the presence of E. coli O26 and the isolates were characterized and studied for their antimicrobial resistance properties. Three minced beef samples (1.2%) tested positive for E. coli O26; one isolate per positive sample was characterized. One isolate harboured the genes encoding for virulence factors intimin (eaeA) and enterohaemolysin (hlyA), while none presented verocytotoxin-encoding genes (stx1 and stx2) and all were negative at the verotoxicity assay. All the isolates showed resistance properties to at least four antimicrobial agents tested and two were multi-drug resistant (MDR). Although no verocytotoxin-encoding genes were found in the isolates, the presence of potentially pathogenic E. coli O26 strains in minced beef points to the need for proper hygiene during meat production to reduce the risk of food-borne illnesses and transmission of MDR organisms via foods to humans. This paper is the first report on the presence and characterization of E. coli O26 in minced beef marketed in Italy.

Abstract: Listeria monocytogenes is an important foodborne pathogen that causes gastrointestinal disorders, and, especially in immunocompromised people, serious extraintestinal diseases, such as septicemia and meningitis, as well as abortion in pregnant women. Many foods, from both plant and animal origin, have been involved in listeriosis outbreaks. This article reports the results of a 12-year survey (1993 through 2004) on the presence of L. monocytogenes in several kinds of food marketed in Italy. Of 5,788 analyzed samples, 121 (2.1%) were contaminated with L. monocytogenes. The highest prevalence was found in smoked salmon (10.6%) and in poultry meat samples (8.5%) and the lowest in red meat (0.3%). L. monocytogenes was not found in 154 samples of fresh seafood products. Fifty-two isolates were also serotyped by the agglutination method. The most common serotypes detected in the 52 strains tested were 1/2a (36.5%), followed by 1/2c (32.8%), 1/2b (13.5%), 4b (11.5%), 3a (3.8%), and 3b (1.9%). The results of the present study showed low levels of L. monocytogenes in the analyzed samples. A total of 61.5% of the 52 L. monocytogenes strains analyzed belonged to serotypes 1/2a, 1/2b, and 4b, namely the serovars that are most commonly involved in extraintestinal human listeriosis outbreaks. In the ready-to-eat samples, these three serotypes were 40.0% (1/2a), 17.1% (1/2b), and 14.3% (4b). This finding highlights the need to implement strict hygienic measures during the production, distribution, and sale of foods to reduce the risk of foodborne listeriosis in humans to an acceptable level.

Abstract: Mytilus galloprovincialis is one of the most commonly consumed of all bivalve molluscs. The consumption of raw bivalve molluscs has caused outbreaks of food poisoning due to Vibrio parahaemolyticus and Vibrio vulnificus. This paper reports the results of a survey on the presence of V. parahaemolyticus, V. vulnificus fecal coliform bacteria, Escherichia coli and Salmonella spp. in 600 M. galloprovincialis samples collected from retail outlets in the Puglia region. V. parahaemolyticus and V. vulnificus were found in 47 (7.83%) and 17 (2.83%) of the samples, respectively. One sample (0.16%) was contaminated with Salmonella spp. but no relationship was observed between vibrios and fecal coliforms and E. coli. There were no significant differences among vibrios present in bivalve molluscs during the 3-year survey.

Abstract: Brucellosis is a costly disease of water buffaloes (Bubalus bubalis). Latent infections and prolonged incubation of the pathogen limit the efficacy of programs based on the eradication of infected animals. We exploited genetic selection for disease resistance as an approach to the control of water buffalo brucellosis. We tested 231 water buffalo cows for the presence of anti-Brucella abortus antibodies (by the agglutination and complement fixation tests) and the Nramp1 genotype (by PCR-denaturing gradient gel electrophoresis). When the 231 animals (58 cases and 173 controls) were divided into infected (seropositive) and noninfected (seronegative) groups and the Nramp1 genotypes were compared, the seropositive subjects were 52 out of 167 (31%) in the Nramp1A+ (Nramp1AA or Nramp1AB) group and 6 out of 64 (9.4%) in the Nramp1A- (Nramp1BB) group (odds ratio, 4.37; 95% confidence limits, 1.87 to 10.19; chi2, 11.65 for 1 degree of freedom). Monocytes from Nramp1BB subjects displayed significantly (P < 0.01) higher levels of Nramp1 mRNA than Nramp1AA subjects and also a significantly (P < 0.01) higher ability in controlling the intracellular replication of several Brucella species in vitro. Thus, selection for the Nramp1BB genotype can become a valuable tool for the control of water buffalo brucellosis in the areas where the disease is endemic.

Abstract: Coxiella burnetii, an obligate intracellular parasite with a worldwide distribution, is the causative agent of acute and chronic Q fever in humans. Although infection is often unapparent in cattle, sheep and goats, there is increasing evidence that C. burnetii infection in these species is associated with abortion and stillbirth. This paper describes the introduction of a single-tube nested PCR protocol for the diagnosis of C. burnetii-related abortion in domestic ruminants in Italy. A total of 514 aborted foetuses from cattle (n = 138) and sheep and goat (n = 376), collected from 301 farms, were analyzed from January 2001 to March 2005. Ninety-seven of 514 (18.9%) animals tested PCR-positive, with 16/138 (11.6%) cattle and 81/376 (21.5%) sheep and goat. Eleven of 102 (10.8%) farms with reproductive disorders in cattle and 37/199 (18.6%) farms with reproductive disorders in sheep and goats were infected with C. burnetii. A greater incidence was observed in three of the seven investigated provinces (p < 0.01), with rates of infected farms of up to 23.8%. Data showed that almost all the C. burnetii-related abortions were recorded between October and April (p < 0.01). These findings suggest that Q fever in humans is largely underestimated in Italy, probably because its occurrence is obscured by flu-like symptoms in acute forms.

Abstract: Helicobacter pylori (Hp) is an organism commonly present worldwide in the human population, sometimes causing serious illnesses such as duodenal and gastric ulcers, adenocarcinoma of the stomach, and low-grade B-cell mucosa-associated lymphoid tissue lymphoma of the stomach. This article describes a multiplex-touchdown PCR method for the identification and genotyping (vacA-s1/m1, sl/m2, and s2/m2-and cagA genes) of Hp directly from sheep milk artificially contaminated with Hp strains from human gastric biopsies and with Hp ATCC 43504. The strains from humans carried sl/m2 cagA+ and s2/m2 cagA allelic combinations, while the ATCC strains carried an sl/ml cagA+ allelic combination. The technique showed a sensitivity of 15 CFU/ml for species identification and of 1,500 CFU/ml for the detection of genes encoding for VacA and CagA. It has proven to be specific and rapid, and the authors suggest that it be used as a rapid screening method to ensure that sheep milk is uncontaminated with this organism.

Abstract: Staphylococcus aureus is a very common organism capable of producing several enterotoxins (SEs) that cause intoxication symptoms of varying intensity in humans when ingested through contaminated food. This paper reports the results of an investigation on the presence of Coagulase-Positive Staphylococci (CPS) and S. aureus in several food products marketed in Italy and on food contact surface swabs sampled from the food industry. A total of 11,384 samples were examined and 1971 of them (17.3%) were found to contain CPS. The assays performed on 541 CPS strains led to the identification of 537 S. aureus strains on which characterization of type A, B, C and D staphylococcal enterotoxins (SEA, SEB, SEC and SED) was performed. A total of 298 S. aureus strains (55.5%) produced one or more SEs: 33.9% of the strains produced SEC, 26.5% SEA, 20.5% SEA+SED, 13.4% SED, 2.7% SEB, 1.7% SEA+SEB, 0.7% SEC+SED and 0.3% produced SEA+SEC and SEB+SEC. The investigation highlighted that these organisms are very common and constitute a potential risk for consumers' health.

Abstract: After the Second World War, in Italy Q Fever or Coxiellosis has been shown a significant relevance, a recrudescence with an epidemic state for over ten years. Later, the infectious disease occurred as endemic since the 80s, the outbreaks were just isolated. Workflows analysis of some authors has demonstrated the spread out of the infection throughout Italian herds with a prevalence ranging from 1.2 per cent to 10 per cent. Our survey carried out throughout Campania area in cattle has shown a real positivity over 14 per cent performing the IFAT for the detection of IgG antibodies for Coxiella burnetii. Therefore, it has been so important to stress the influence of cattle farming management in stables as a real risk of Coxiellosis. For example, the Relative Risk (RR) has been registrated about 6.84 (2.18<RR<21.4) in comparison with some herds permanently housed than those kept unhoused and about 8.4 (1.8<RR<38.6) in housed herds and those permanently kept at pasture. Therefore, we have detected a seroprevalence of Q Fever about 11.8 per cent within sheep and about 6.3 per cent within goats. The investigations have focused buffaloes too, tested by several authors, especially from India, but in Italy, only Galiero has recorded significant results: the overall seroprevalence (1.2 per cent) was observed within 1012 buffaloes. Other studies have demonstrated that dogs may transmit Q Fever to humans by infected birth fluids and membranes and by urine. Because of this observation, addressed some of our search projects to assess possible relation among other pathogens, such as Leishmania infantum, Rickettsia conori and Ehrlichia canis. The results have confirmed the presence of C. burnetii (seroprevalence about 7 per cent) within tested dogs in southern Italy. There was no direct estimated relationship between C. burnetii and E. canis and R. conori, instead of significant relation has been shown for L. infantum. The investigation focused on the direct interaction between seropositivity and the age of ruminants. For example, the highest prevalence has been observed in cows and sheep ranging from 3-5 years in which the infection risk was higher than in younger herds. In accordance with the workflows serological analysis, C. burnetii is widespread in Italy among housed ruminants. PCR assay was, therefore performed to test cows with clinical signs of abortion and neonatal mortality. All the survey involved 305 animals (267 foeti and 38 stillborn) with 77 positive herds showing DNA of C. burnetii. To detect C. burnetii pathogen in milk samples, two methods have been combined: the immunomagnetic separation (IMS) and PCR assay, obtaining an increasing of sensitivity (10(-17)) more than only PCR technique (10(-8)). The overall analysis of serological and biomolecular results has demonstrated that C. burnetii is really widespread and may have detrimental effects on farm management system.

Abstract:

Abstract: A survey was conducted of Vibrio spp., Escherichia coli, fecal coliforms, and Salmonella in 644 molluscan shellfish samples marketed in the Apulia region of southern Italy. Vibrios were found in 278 samples (43%), and levels of E. coli and fecal coliforms were above the Italian legal limit in 27 and 34 samples (4 and 5%), respectively. Salmonella was not detected in any of the samples. Because the majority of the vibrio isolates were found in samples that were compliant with Italian regulations, there appears to be no relationship between the presence of microorganisms of fecal origin and the presence of vibrios potentially harmful to human health.