Could honey bees signal the spread of antimicrobial resistance in the environment?

The honey bee has long been known to be a bioindicator of environmental pollution and the use of antimicrobials in the beekeeping industry is strictly regulated. For these reasons, this paper was aimed to evaluate for the first time the role of Apis mellifera as a possible indicator of environmental antimicrobial resistance (AMR). The study isolated and analysed the resistance patterns of Enterobacteriaceae from a pool of honey bee guts located in five different environmental sites (ES), where different antimicrobial selective pressures were hypothesized. In all, 48 isolates were considered for identification and underwent analyses of AMR to ampicillin, amoxicillin/clavulanic acid, cefazolin, ceftazidime, tetracycline, imipenem, enrofloxacin, amikacin and trimethoprim/sulfamethoxazole. In all, 12 isolates out of 48 (25%) showed resistance to at least one antimicrobial drug. There were no significant differences between the resistance rates observed in the ESs, even if the highest percentage of resistance was found in ES4. Resistances to amoxicillin/clavulanic acid resulted significantly higher than those detected towards the other antimicrobials. Amoxicillin/clavulanic acid is not commonly used in beekeeping but it is extensively used in animals and in humans, suggesting an environmental origin of this resistance and supporting the hypothesis that honey bees could be used as indicators of AMR spread in the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, a possible role of honey bees as indicator of environmental antimicrobial resistance is hypothesized. Enterobacteriaceae were isolated from bees living in different environmental sites (ES) where different antimicrobial selective pressures were hypothesized. Even if no differences between the resistances in the five ES were observed, the resistance rates for amoxicillin/clavulanic acid, compared to other antimicrobials, were significantly higher. Since amoxicillin/clavulanic acid is not used in beekeeping but it is extensively used in animals and in humans, an environmental origin of this resistance is suggested that supports our hypothesis.

First description of Streptococcus lutetiensis from a diseased cat.

This paper describes for the first time the isolation of Streptococcus lutetiensis in a cat with intestinal lymphoma. The Streptococcus bovis group has undergone significant taxonomic changes over the past two decades and, in 2002, Poyart et al. described two distinct novel species within the genus Streptococcus: Streptococcus lutetiensis and Streptococcus pasteurianus. The bovis group streptococci include commensal species and subspecies or opportunistic pathogens of humans and animals. The cat was referred to the Veterinary Teaching Hospital, University of Bologna for chronic diarrhoea associated with fresh blood. A diagnosis of intestinal lymphoma was advanced. S. lutetiensis was accidentally isolated from the faeces of the cat and identified through MALDI-TOF and 16s rRNA sequencing. The Kirby-Bauer test revealed that the isolate was resistant to enrofloxacin, erythromycin, clindamycin, marbofloxacin and tetracycline. The detection of S. lutetiensis in cat faeces might suggest that it could be a normal inhabitant of cat intestinal tract or that it could be involved in the manifestation of intestinal diseases. Since bacteria belonging to the S. bovis group are considered emerging pathogens, additional research is required to evaluate the role of S. lutetiensis in cats and its role in the transmission of antimicrobial resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study the isolation of Streptococcus lutetiensis from a cat with intestinal lymphoma was described for the first time. An antimicrobial susceptibility test performed by means of the disc diffusion method revealed that the isolate was resistant to enrofloxacin, erythromycin, clindamycin, marbofloxacin and tetracycline. Nowadays the ecological or pathogenetic role of S. lutetiensis in the gut of animals remains unclear but, even if its role as commensal bacterium was confirmed, the presence of multi-resistant S. lutetiensis in cat gut could favour the transmission of antimicrobial resistance to other bacteria.

Daptomycin Plasma and CSF Levels in Patients with Healthcare-Associated Meningitis.

BACKGROUND: There are currently few data concerning the cerebrospinal fluid (CSF) penetration of daptomycin in patients with healthcare-associated meningitis. This study aims (1) to better characterize the pharmacokinetics of daptomycin in humans during a 7-day intravenous (IV) therapy course, and (2) to study the penetration of daptomycin in the CSF after IV infusion at the dose of 10 mg/kg. RESULTS: In this prospective observational study, we enrolled nine patients with an implanted external ventricular drainage and a diagnosis of a healthcare-associated meningitis. Daptomycin was administered at 10 mg/kg for a maximum of 7 days. The pharmacokinetic of daptomycin was studied using a two-compartment population/pharmacokinetic (POP/PK) model and by means of a nonlinear mixed effects modeling approach. A large inter-individual variability in plasma area under the curve (Range: 574.7-1366.3 h mg/L), paralleled by high-peak plasma concentration (Cmax) (all values > 60 mg/L), was noted. The inter-individual variability of CSF-AUC although significant (range: 1.17-6.81 h mg/L) was narrower than previously reported and with a late occurrence of CSF-Cmax (range: 6.04-9.54 h). The terminal half-life between plasma and CSF was similar. tmax values in CSF did not show a high inter-individual variability, and the fluctuations of predicted CSF concentrations were minimal. The mean value for daptomycin penetration obtained from our model was 0.45%. CONCLUSIONS: Our POP/PK model was able to describe the pharmacokinetics of daptomycin in both plasma and CSF, showing that daptomycin (up to 7 days at 10 mg/kg) has minimal penetration into central nervous system. Furthermore, the observed variability of AUC, tmax and predicted concentration in CSF was lower than what previously reported in the literature. Based on the present findings, it is unlikely that daptomycin could reach CSF concentrations high enough to have clinical efficacy; this should be tested in future studies.

Occurrence of putative virulence genes on Arcobacter butzleri isolated from three different environmental sites throughout the dairy chain.

AIMS: This comparative study investigated the occurrence of cadF, cj1349, ciaB, pldA, tlyA, hecA, hecB, mviN, irgA and IroE genes in 212 Arcobacter butzleri isolated from three different environmental sites linked to the dairy chain (farms, industrial and artisanal dairy plants) located in three Italian regions (Lombardy, Emilia-Romagna and Calabria). METHODS AND RESULTS: According to the presence of these genes, different pathotypes (P-types) were determined. The main genes detected were ciaB, mviN, tlyA, cj1349, pldA and cadF, while the least common genes were iroE, hecA, hecB and irgA. TlyA, irgA, hecA, hecB and iroE, which were significantly more frequent in isolates recovered in industrial dairy plants. Twelve P-types were detected. The occurrence of the most frequently detected P-types (P-types 1, 2, 3 and 5) differed significantly (P < 0·001) in relation to both the environmental site and geographical area of isolation. The highest diversity in P-types was observed in industrial dairy plants and in the Calabria region. CONCLUSIONS: The results of this study show a correlation between the occurrence of putative virulence genes and virulence genotype variability depending on the environmental site and geographical origin of the isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study provides insights into the similar distribution of putative virulence genes in a dairy chain and other sources' isolates and also into a geographical distribution of some P-types. We have shown that industrial dairy plants may represent an environmental site favouring a selection of the isolates with a higher pathogenetic pattern.

Short communication: Investigation into Mycobacterium avium ssp. paratuberculosis in pasteurized milk in Italy.

This study investigated the presence of viable Mycobacterium avium ssp. paratuberculosis (MAP) in pasteurized milk produced by Italian industrial dairy plants to verify the prediction of a previously performed risk assessment. The study analyzed 160 one-liter bottles of pasteurized milk from 2 dairy plants located in 2 different regions. Traditional cultural protocols were applied to 500mL of pasteurized milk for each sample. The investigation focused also on the pasteurization parameters and data on the microbiological characteristics of raw milk (total bacterial count) and pasteurized milk (Enterobacteriaceae and Listeria monocytogenes). No sample was positive for MAP, the pasteurization parameters complied with European Union legislation, and the microbiological analysis of raw and pasteurized milk showed good microbiological quality. The results show that a 7-log (or >7) reduction could be a plausible value for commercial pasteurization. The combination of hygiene practices at farm level and commercial pasteurization yield very low or absent levels of MAP contamination in pasteurized milk, suggesting that pasteurized milk is not a significant source of human exposure to MAP in the dairies investigated.

Multilocus sequence typing of Arcobacter butzleri isolates collected from dairy plants and their products, and comparison with their PFGE types.

AIMS: The present study aimed to determine, by multilocus sequence type (MLST), the heterogeneity level of Arcobacter butzleri isolates and to compare MLST and pulsed-field gel electrophoresis (PFGE) in terms of discriminatory power (DI) as well as unidirectional and bi-directional concordance. METHODS AND RESULTS: Arcobacter butzleri isolates (N = 133) from dairy products and environmental samples, collected from dairy plants, were characterized by MLST and PFGE with SacII and classified in 29 sequence types (STs), 47 PFGE and 62 type strains (TS). Among the 119 alleles, 19 were previously unreported and the same for all the STs but two. A significant linkage disequilibrium was detected when the complete ST data set was analysed The DIs of MLST, PFGE and their combination were 0·937, 0·953 and 0·965 respectively. The adjusted Wallace coefficients between MLST and PFGE as well as PFGE and MLST were 0·535 and 0·720 respectively; the adjusted Rand coefficient was 0·612. CONCLUSIONS: The A. butzleri studied population showed recombination to some degree. PFGE showed a DI higher than MLST. Both methods presented good concordance. The TS analysis seems to show persistence of the same strain on time and possible cross-contaminations between food and environmental sites. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides insights in the A. butzleri population found in raw milk, cheese, and dairy production plants. The data suggest that MLST and PFGE genotypes correlate reasonably well, although their combination results in optimal resolution.

Presence of Campylobacter and Arcobacter species in in-line milk filters of farms authorized to produce and sell raw milk and of a water buffalo dairy farm in Italy.

The objectives of this study were to investigate the presence of Campylobacter spp. and Arcobacter spp. in dairy herds authorized for the production and sale of raw milk and in a water buffalo dairy farm, and to test the antimicrobial susceptibility of the isolates. A total of 196 in-line milk filters were collected from 14 dairy farms (13 bovine and 1 water buffalo) for detection of Campylobacter spp. and Arcobacter spp. by microbiological culture. For each farm investigated, 1 isolate for each Campylobacter and Arcobacter species isolated was tested using the Etest method (AB Biodisk, Solna, Sweden) to evaluate the susceptibility to ciprofloxacin, tetracycline, chloramphenicol, ampicillin, erythromycin, and gentamicin. A total of 52 isolates were detected in 49 milk filters in 12 farms (85.7%) out of 14 and the isolates were identified as Campylobacter jejuni (6), Campylobacter hyointestinalis ssp. hyointestinalis (8), Campylobacter concisus (1), Campylobacter fetus ssp. fetus (1), Arcobacter butzleri (22), and Arcobacter cryaerophilus (14). The small number of isolates tested for antimicrobial susceptibility precludes any epidemiological consideration but highlights that all Campylobacter isolates were susceptible to macrolides, which are the first-choice drugs for the treatment of campylobacteriosis, and that resistance to fluoroquinolones and tetracycline was detected; for Arcobacter isolates, resistance to ampicillin and chloramphenicol was detected. The sale of raw milk for human consumption by self-service automatic vending machines has been allowed in Italy since 2004 and the presence of C. jejuni in in-line milk filters confirms that raw milk consumption is a significant risk factor for human infection. The high occurrence of emerging Campylobacter spp. and Arcobacter spp. discovered in dairy farms authorized for production and sale of raw milk represents an emerging hazard for human health.

Isolation of glucosidase and phospholipase positive Bacillus circulans on ALOA medium.

AIM: To report the growth of glucosidase and phospholipase positive bacteria on agar Listeria according to Ottaviani and Agosti (ALOA) different from Listeria monocytogenes, Listeria ivanovii and Bacillus cereus. METHODS AND RESULTS: Raw water-buffalo milk was analysed according to EN ISO 11290. Streaking of Fraser broth on ALOA resulted in green colonies with an opaque halo after 48 h at 30°C. Colonies were transferred onto Tryptone soya yeast extract agar and showed cultural characteristics atypical for L. monocytogenes. Results of confirmation tests according to EN ISO 11290 method: negative haemolysis test, weak positive camp test in correspondence with Staphylococcus aureus, no fermentation of rhamnose, fermentation of xylose. Gram staining showed tapered, curved, Gram-positive rods with subterminal to terminal ellipsoidal spores, 0.5-0.7 µm diameter 4-5 µm. API 50CH CHB kit (99.9% percentage of identification) and the sequence of the 833 bp PCR product (portion of 16S rRNA, generic primers 1492-r; p27-f) showed 97% identity with Bacillus circulans ATCC 4513 (GenBank AY724690). CONCLUSIONS: Some B. circulans strains can grow on ALOA, according to ISO 11290, confirmation tests readily differentiate B. circulans from L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The different morphology of the colonies must be kept in mind to select true L. monocytogenes for confirmation test and to avoid overestimation of L. monocytogenes count.

The early phase of critical illness is a progressive acidic state due to unmeasured anions.

BACKGROUND AND OBJECTIVE: Stewart's and Fencl's methods have recently been proposed to interpret acid-base disorders where traditional theory has proven inadequate. Our objectives were to evaluate: (1) the occurrence of acid-base disturbances in critically ill patients and their trend over the first 3 intensive care unit days, (2) whether Stewart's theory offers advantages over the traditional theory in the diagnosis of acid-base metabolic disturbances and (3) whether variables derived from Stewart's and Fencl's methods offer advantages over the traditional method to predict patient mortality. METHODS: A prospective cohort study in a general intensive care unit. Blood samples were analysed for arterial blood gases, electrolytes and proteins. PaCO2, pH, bicarbonate, base excess, standard base-excess, sodium, potassium, chloride, phosphorous, calcium, magnesium and lactate were measured. Anion gap, Stewart's and Fencl's variables were calculated. RESULTS: When using Stewart's method, metabolic acidosis and metabolic alkalosis were found in 92.9% and 93.4% of samples, respectively. Corresponding figures obtained with the traditional method were 15% and 18.7%. In 245 (64.5%) samples, Stewart's method revealed that metabolic acidosis and alkalosis were simultaneously present, whereas the traditional method revealed a normal acid-base status. Strong ion gap increased significantly over the first 3 intensive care unit days. Strong ion gap and lactate were independent predictors of 28-day mortality. CONCLUSIONS: Metabolic acidosis by unmeasured anions is a clinically relevant phenomenon, which is correlated with mortality. Progressive metabolic acidosis may be ongoing in the early phase of critical illness despite the absence of acidaemia.

Localized morphea after breast implant for breast cancer: A case report.

PURPOSE: Early breast cancer follow-up guidelines for patients who underwent surgery suggest a regular and accurate clinical examination of the breast area, for an early identification of cutaneous or subcutaneous breast cancer relapse. Nonetheless, breast skin lesions arising in patients treated with mastectomy for breast cancer can be caused by several diseases. A series of diagnostic hypotheses should be considered, not only focusing on cutaneous metastasis, but also on dermatologic and systemic diseases. CASE REPORT: In February 2015, a 37-year-old patient underwent a right subcutaneous mastectomy for stage IIA breast cancer. Five months after beginning adjuvant chemotherapy, she noted hyperpigmentation and thickening of the skin on the right breast. Differential diagnosis included local relapse, skin infection, lymphoma, or primary cutaneous disease, and a skin biopsy was performed. The histopathologic specimen showed full-thickness sclerosis, with features of localized morphea. Therapy with clobetasol was prescribed, with progressive resolution of the thickness. The collaboration between many professionals in a multidisciplinary team (oncologist, dermatologist, plastic surgeon, and pathologist) was crucial to achieving the diagnosis. CONCLUSION: In the literature, some articles describe correlation between connective tissue diseases and silicone breast implants, but the pathogenetic mechanisms are unknown. We report a rare case of breast morphea after positioning a silicone implant in a patient who had undergone mastectomy. This clinical report represents an interesting model of multidisciplinary management of a patient with breast cancer who developed an uncommon dermatologic disease. Further studies are needed to clarify the association between silicone implants and breast morphea.

Paediatric HUS Cases Related to the Consumption of Raw Milk Sold by Vending Machines in Italy: Quantitative Risk Assessment Based on Escherichia coli O157 Official Controls over 7 years.

A quantitative risk assessment (RA) was developed to estimate haemolytic-uremic syndrome (HUS) cases in paediatric population associated with the consumption of raw milk sold in vending machines in Italy. The historical national evolution of raw milk consumption phenomenon since 2008, when consumer interest started to grow, and after 7 years of marketing adjustment, is outlined. Exposure assessment was based on the official Shiga toxin-producing Escherichia coli O157:H7 (STEC) microbiological records of raw milk samples from vending machines monitored by the regional Veterinary Authorities from 2008 to 2014, microbial growth during storage, consumption frequency of raw milk, serving size, consumption preference and age of consumers. The differential risk considered milk handled under regulation conditions (4°C throughout all phases) and the worst time-temperature field handling conditions detected. In case of boiling milk before consumption, we assumed that the risk of HUS is fixed at zero. The model estimates clearly show that the public health significance of HUS cases due to raw milk STEC contamination depends on the current variability surrounding the risk profile of the food and the consumer behaviour has more impact than milk storage scenario. The estimated HUS cases predicted by our model are roughly in line with the effective STEC O157-associated HUS cases notified in Italy only when the proportion of consumers not boiling milk before consumption is assumed to be 1%. Raw milk consumption remains a source of E. coli O157:H7 for humans, but its overall relevance is likely to have subsided and significant caution should be exerted for temporal, geographical and consumers behaviour analysis. Health education programmes and regulatory actions are required to educate people, primarily children, on other STEC sources.

Cryptosporidium parvum: From foal to veterinary students.

This paper describes the transmission of a zoonotic subtype of Cryptosporidium parvum between two foals hospitalized in an Equine Perinatology Unit (EPU) linked to an outbreak of cryptosporidiosis in veterinary students. Fecal specimens of 36 mares (105 samples) and 28 foals (122 samples) were subjected to Ziehl-Neelsen staining, nested PCR of 18S rDNA. Two foals tested positive for Cryptosporidium; PCR restriction fragment length polymorphism (PCR-RFLP) analysis and subtyping by nested PCR of the 60kDa glycoprotein (gp60) gene revealed C. parvum subtype IIdA23G1. The introduction of Cryptosporidium into the EPU is suspected to be in a foal showing no initial clinical signs that tested positive for C. parvum during an asymptomatic phase. A second foal, hospitalized afterwards for perinatal asphyxia syndrome complicated with failure of passive transfer and sepsis, showed severe watery diarrhea after 4 days of hospitalization and was positive for the same subtype. During this period, six students attending the EPU complained of abdominal pain and diarrhea and were positive for the same subtype of C. parvum. To the authors' knowledge, this is the first description of this subtype in foals and the first report of evidence of zoonotic transmission of cryptosporidiosis from foals to human.

Epidemiological survey on Cryptosporidium in an Equine Perinatology Unit.

The present study aims to evaluate the prevalence, pattern of spread and risk factors for the transmission of cryptosporidiosis in foals and mares hospitalized in a University Equine Perinatology Unit, where a new subtype family of Cryptosporidium horse genotype was described by Caffara et al. (2013). Mares (36) and foals (37) hospitalized during the 2012 foaling season were included. Multiple sampling from each animal was performed (a total of 305 stool samples were collected). One hundred and eleven environmental samples (gauze swabs) were also collected before and after the breeding season. Fourteen foals were found positive for Cryptosporidium spp. by PCR in at least one sample; a total of 35 foal stool specimens were confirmed for the presence of the protozoa. Instead none of the stool specimens from mares were found positive. PCR-RFLP analysis shows Cryptosporidium parvum in 5 stool samples and Cryptosporidium horse genotype in 21. In 9 specimens, from 4 different foals, the profile was suggestive for a mixed infection. The subtyping at gp60 locus showed 2 strains as members of the subtype family IId and six of the subfamily IIa of C. parvum. Twenty isolates were identified as Cryptosporidium horse genotype subtype VIaA15G4. Five gauze swabs collected from the walls of the boxes where the animals were hosted out of 111 environmental samples examined were PCR positive for Cryptosporidium spp. Cryptosporidium parvum was detected in one sample collected before the foaling season, while Cryptosporidium horse genotype profile was observed in 4 wall samples collected at the end of the 2012 foaling season. The prevalence observed in foals (37.8%) was higher than that reported in other studies. These features and the diffusion of the same genotype point out as the EPU, where critically ill foals are hospitalized, can support the spread of cryptosporidiosis. Therefore, the manual tasks and the activities carried out in these facilities are of great importance, as they might favor the diffusion of the infection.

DHPLC analysis of the MECP2 gene in Italian Rett patients.

Rett Syndrome (RTT) is an X-linked dominant neurodevelopmental disorder, which almost exclusively affects girls, with an estimated prevalence of one in 10,000-15,000 female births. Mutations in the methyl CpG binding protein 2 gene (MECP2) have been identified in roughly 75% of classical Rett girls. The vast majority of Rett cases (99%) are sporadic in origin, and are due to de novo mutations. We collected DNA samples from 50 Italian classical Rett girls, and screened the MECP2 coding region for mutations by denaturing high-performance liquid chromatography (DHPLC) and subsequent direct sequencing. DHPLC is a recently developed method for mutation screening which identifies heteroduplexes formed in DNA samples containing mismatches between wild type and mutant DNA strands, combining high sensitivity, reduced cost per run, and high throughput. In our series, 19 different de novo MECP2 mutations, eight of which were previously unreported, were found in 35 out of 50 Rett girls (70%). Seven recurrent mutations were characterized in a total of 22 unrelated cases. Initial DHPLC screening allowed the identification of 17 out of 19 different mutations (90%); after optimal conditions were established, this figure increased to 100%, with all recurrent MECP2 mutations generating a characteristic chromatographic profile. Detailed clinical data were available for 27 out of 35 mutation carrying Rett girls. Milder disease was detectable in patients carrying nonsense mutation as compared to patients carrying missense mutations, although this difference was not statistically significant (P = 0.077).

Platelet aggregation, ATP release and cytoplasmic Ca2+ movement: the effects of cloricromene.

A placebo-controlled, double-blind, randomized, cross-over study was performed in 24 healthy volunteers. 12 volunteers received Cloricromene (100mg gastroresistant capsules twice a day) for 7 days, the other volunteers received identical placebo capsules. Subsequently, after a 7-day wash-out period, at day 15, each subject received the other treatment. Blood samples were taken on days 1 and 15 (1st day of each treatment) as well as on days 7 and 21 (7th day of each treatment) before the morning drug administration and 2 and 4 hours later. Platelet aggregation and ATP secretion were studied in whole blood (WB) using ADP and collagen as stimulating agents. Ca2+ fluxes were studied in aequorin-loaded, washed platelets stimulated with ADP and collagen, while aggregation in platelet-rich plasma (PRP) was studied using PAF, ADP and adrenaline as agonists. Consistent inhibition of aggregation and release induced by both ADP and collagen was observed in WB after Cloricromene administration. Similarly, Ca2+ flux was also inhibited after drug administration. Platelet aggregation in PRP was inhibited only after 7 days of Cloricromene treatment with ADP and adrenaline as stimuli. We conclude that oral administration of Cloricromene leads to significant antiplatelet activity in healthy volunteers, in particular when platelets are studied in the presence of other blood elements.

Side-to-side weight-bearing asymmetry in subjects with low back pain.

The purpose of this project was to determine if subjects with low back pain (LBP) exhibit greater side-to-side weight-bearing (WB) asymmetry compared to healthy control subjects without LBP. This study utilized an observational double cohort design and consisted of 35 subjects with LBP and 31 healthy control subjects. Side-to-side WB asymmetry was calculated as the average of the absolute value of the difference between the right and left lower extremity from three trials. The percentage of the average side-to-side WB asymmetry relative to the total body weight was calculated to normalize expected differences in magnitude of asymmetry based on a subject's total body weight. An 11-point numeric pain rating scale was used to represent the subject's current level of pain. Patients with LBP demonstrated significantly greater normalized side-to-side WB asymmetry than healthy control subjects (8.8% vs. 3.6%, respectively, P<0.001). In patients with LBP, higher magnitudes of side-to-side WB asymmetry were significantly associated with increased pain (r=0.39, P=0.021). In conclusion patients with LBP exhibited increased side-to-side WB asymmetry compared to healthy control subjects without LBP. This asymmetry was associated with increased levels of pain. This finding is relevant for planning future studies that will attempt to provide evidence for the construct validity of manipulation by determining if side-to-side WB asymmetry normalizes after a manipulation intervention and if this improvement is associated with improvements in pain and function.

In vitro complement-independent activation of human neutrophils by hemodialysis membranes: role of the net electric charge.

Polymethylmethacrylate (PMMA) membranes with different net electric charges and percentage water contents (anionic 71%, neutral 70%, cationic 75%) were evaluated for their ability to stimulate plasma-free human polymorphonuclear neutrophils (PMN), and compared for potency to cuprophan (Cu), already described as being a potent trigger of PMN. The release of lysozyme, beta-glucuronidase, lactic dehydrogenase (LDH), and the generation of a platelet aggregating activity were studied in the supernatants from plasma-free human PMN incubated with different membranes. The PMN intracellular content of neutrophil cationic proteins (NCP), elastase, and cathepsin G were also studied by immunofluorescence using specific antisera on smears of PMN before and after incubation with each membrane. Only cationic, but not anionic or neutral PMMA induced a marked release of lysozyme (range 20-25% of the sonicated control, assumed as 100%), and beta-glucuronidase (40-43%), and marked depletion of the intracellular content of NCP, elastase, and cathepsin G, suggesting a degranulation process. Platelet aggregating activity was generated and referred to the release of platelet activating factor (PAF) only in the supernatants from PMN incubated with cationic, but not with anionic, or neutral PMMA membranes. These results indicate that modification of the net electric charge can per se turn PMMA, commonly recognized as inert, into a material with marked PMN activating effects, comparable to those of Cu, a highly reactive polymer.

Prevalence of Chlamydophila felis by PCR among healthy pet cats in Italy.

Conjunctival swabs were taken from 60 healthy pet cats and tested for Chlamydophila felis by PCR assays to amplify the ompA, omp2 and groEL genes. Chlamydial DNA was detected in 2 (3.3%) cats, one of which had been vaccinated against C. felis eight months before sample collection. The nucleotide and predicted amino acid sequences of three genes from two cats showed 100% identity with the same regions amplified from conjunctival swabs of cats in the same geographic area.

Studies of antithyroid antibodies in Down's syndrome.

The presence of antithyroid antibodies (antithyroglobulin and antimicrosomal) in serum of patients with Down's syndrome and their respective mothers were studied based on data from the literature, showing a possible correlation between the presence of these antibodies in the serum of mothers and the birth of mongoloid children. Antithyroid antibodies were studied in 40 patients with Down's syndrome, with ages ranging from 5 months to 21 years old, 24 males and 16 females and in the respective mothers whose ages varied from 22 to 66 years. In none of the analyzed sera of the mongoloids as well of their mothers, the authors were able to detect the antithyroid antibodies studied.

Occurrence of ε-proteobacterial species in rabbits (Oryctolagus cuniculus) reared in intensive and rural farms.

In order to investigate the occurrence of Campylobacter, Helicobacter and Arcobacter species in caecal contents of rabbits reared in intensive and rural farms, a total of 87 samples from animals belonging to 29 farms were analysed by both cultural and PCR analyses. PCR analysis directly from faecal samples detected 100% positive samples for Campylobacter genus, 3.4% for Helicobacter genus and none for Arcobacter genus. 83 out of 87 animals (95.4%) and all the 29 farms were positive for Campylobacter cuniculorum as also determined by cultural examination. Campylobacter coli and Campylobacter jejuni were isolated only from three animals reared in two rural farms. No Helicobacter and Arcobacter species were isolated. To evaluate a possible genetic variability, one strain of C. cuniculorum from each farm was analysed by Pulsed Field Gel Electrophoresis (PFGE) and Amplified Fragment Length Polymorphism (AFLP). Genotyping revealed that C. cuniculorum population is heterogeneous among the different sources and no dominant clone has spread in the investigated farms. This survey highlighted a high presence of C. cuniculorum with a high rate of intestinal colonization, low presence of C. jejuni-coli, Helicobacter spp. and any Arcobacter spp. in farmed rabbits.