Development of a multiplex PCR for rapid detection of verocytotoxin-producing Escherichia coli O26 in raw milk and ground beef.

Verocytotoxin-producing Escherichia coli (VTEC) O26 is an emergent pathotype that has caused an increasing number of sporadic cases and outbreaks of gastroenteritis, hemorrhagic colitis, and hemolytic uremic syndrome in the United States and Europe. Many cases are associated with the consumption of milk and undercooked or fermented meats. The stx(2) strains of VTEC O26 seem to be more likely to cause human infections than isolates expressing only stx(1). The isolation and identification of VTEC O26 from foods is labor intensive and time-consuming. We developed a multiplex PCR (M-PCR) assay for the identification and characterization of E. coli O26 VTEC and its detection in raw milk and ground beef. The method is based on the amplification of the wzx, stx(1), and stx(2) genes for the simultaneous detection of the O26 antigen and verocytotoxin types 1 and 2. This M-PCR assay had a sensitivity of 10(8) CFU/ml when applied to a bacterial suspension and of 10(6) CFU/ml or g when applied to both inoculated milk and minced beef samples. This M-PCR assay also was highly specific, and results were consistently negative for negative controls (nonpathogenic E. coli strains, uninoculated milk and beef samples, and samples inoculated with the nontarget microorganisms). This method could be used for the rapid detection of E. coli O26 VTEC from foods and for the rapid identification and characterization of clinical and environmental isolates.

Escherichia coli O26 in minced beef: prevalence, characterization and antimicrobial resistance pattern.

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.

Detection of Chlamydophila abortus in sheep and goat flocks in southern Italy by PCR using four different primer sets.

An epidemiological survey was performed to detect the presence of Chlamydophila (C.) abortus and other members of the order Chlamydiales in ovine and caprine flocks with a history of abortion in southern Italy. Four pairs of primers were compared to evaluate their ability to detect Chlamydiales using purified DNA preparations and tissue samples from aborted foetuses with suspected chlamydial infections. As expected, amplification of DNA of the reference strain C. abortus using primer pairs U23F/23Sigr, 16SF2/23R, CTU/CTL and CpsiA/CpsiB produced fragments of about 600 bp, 585 bp, 1000 bp and 300 bp, respectively. The detection limits of the four PCR tests performed on serial DNA dilutions of the C. abortus reference strain were of 10 pg, 0.1 pg, 0.1 pg and 1 fg of DNA, respectively. The most sensitive amplification of DNA extracted from the organ tissues was obtained with primer pairs CpsiA/CpsiB, which detected Chlamydophila spp. DNA in all infected tissue samples. Only C. abortus was identified during the survey. The presence of this agent was confirmed in 3 out of 27 ovine and caprine flocks included in the survey suggesting that abortion due to C. abortus is uncommon in southern Italy.

Effects of bivalent bluetongue virus serotypes 2 and 9 vaccine on reproductive performance of cattle: a case study in Calabria, Italy.

Following the occurrence of bluetongue (BT) in Italy in the summer and autumn of 2000, the Italian Ministry of Health decreed (in May 2001) that all sheep, goats and cattle in infected, and in neighbouring areas, be vaccinated. The principal aim of the vaccination campaign was to create a resistant animal population, thereby reducing overall virus circulation. Accordingly, during 2002, the live-attenuated bivalent vaccine against BT virus (BTV) serotypes 2 and 9, produced by Onderstepoort Biological Products in South Africa, was administered in Calabria. A large herd of cattle (over 900 animals, including 390 cows) was monitored for four months after vaccination to establish whether any abnormalities (such as stillbirth and placental retention) occurred during parturition. During the study, 111 cows (89 vaccinated and 22 unvaccinated) gave birth; in 26 cows, abnormalities were observed but no association was found to occur between the vaccination of cows against BT and the birthing abnormalities observed (Pearson's chi-square=0.517, P>0.05). The animals were divided into four groups according to the number of days between vaccination and parturition (less or equal to 31 days, 31-60 days, 61-90 days, more than 90 days); again no link was found between vaccination and the abnormalities observed.

HBcAg identification in the placental cytotypes of symptom-free HBsAg-carrier mothers: a study with the immunoperoxidase method.

In this study the presence of HBcAg in placental tissue of symptom-free HBsAg-carrier mothers has been determined by the immunoperoxidase technique. The presence of HBcAg in the major placental cytotypes was mostly limited to the trophoblastic lineage. Moreover the activated macrophages with their strong cytoplasmic reaction to the antigen phagocytosis play a remarkable role in the immune response. Because there are continuous channels that link the human amnion through the trophoblastic lineage, including the endovascular trophoblast with maternal decidual vessels, a functional exchange system may be considered valid. This would influence the transmission of infection.

Immunocytochemical HBsAg evidence in placentas of asymptomatic carrier mothers.

We report three chronic asymptomatic HBsAg carrier mothers. We studied their placentas via application of the immunohistochemical method to identify HBsAg by peroxidase-antiperoxidase techniques. All three placental specimens showed a positive HBsAg reaction. Due to the strong cytoplasmatic reactivity of the cytotypes, we deduced their active participation in the infection, proving that virosis occurs via transplacental circulation.