Dairy farm management practices and the risk of contamination of tank milk from Clostridium spp. and Paenibacillus spp. spores in silage, total mixed ration, dairy cow feces, and raw milk.

The occurrence of Paenibacillus and Clostridium spores in silage is of great concern for dairy producers because their spores can contaminate milk and damage processed milk and semi-hard cheeses. Spoiled silage is considered to be the main contamination source of the total mixed ration (TMR), feces of dairy cows, and consequently bulk tank milk via the contamination of cow teats by dirt during milking. The presence of an anaerobic and facultative anaerobic sporeformer population in different matrices (soil, corn silage, other feeds, TMR, feces, and milk) and its transmission pathway has been studied on 49 dairy farms by coupling plate count data with 16S-DNA identification. The different matrices have shown a high variability in the anaerobic and facultative anaerobic spore count, with the highest values being found in the aerobically deteriorated areas of corn silages. Clostridium tyrobutyricum, Paenibacillus macerans, and Paenibacillus thermophilus were detected in all the matrices. The TMR spore count was influenced by the amount of spoiled corn silage in the TMR and by the care taken when cleaning the spoiled silage before feed-out. Most of the farms that prevent the presence of visible moldy silage in the silo and carefully clean to remove molded spots were able to maintain their TMR spore counts below 4.0 log spores/g. When a level of 4.5 log spores/g of TMR was exceeded, the feces presented a greater contamination than 3.0 log spores/g. Moreover, the higher the number of spores in the feces was, the higher the number of spores in the milk. Most of the farms that presented a feces contamination greater than 5.0 log spores/g had a higher milk spore contamination than 1,000 spores/L. Careful animal cleaning and good milking practices have been found to be essential to maintain low levels of contamination in bulk tank milk, but it has emerged that only by coupling these practices with a correct silage management and cleaning during TMR preparation can the contamination of milk by spores be kept at a low level. It has been found that aerobically deteriorated silage has a great capacity to contaminate TMR and consequently to increase the risk of milk spore contamination, even when routine milking practices are adopted correctly.

Detection, identification, and typing of Listeria species from baled silages fed to dairy cows.

Anaerobiosis, critical for successful ensilage, constitutes a challenge in baled silages. The loss of complete anaerobiosis causes aerobic deterioration and silages undergo dry matter and nutrient losses, pathogen growth, and mycotoxin production. Silage may represent an ideal substrate for Listeria monocytogenes, a pathogen of primary concern in several cheeses. The aim of this research was to investigate the occurrence of Listeria in baled silage fed to cows producing milk for a protected designation of origin cheese, and to characterize isolates by repetitive sequence-based PCR. Listeria spp. were detected in 21 silages and L. monocytogenes in 6 out of 80 of the analyzed silages; 67% of positives were found in molded zones. Results of the PCR typing showed genotypic homogeneity: 72.9 and 78.8% similarity between strains of Listeria spp. (n=56) and L. monocytogenes (n=24), respectively. Identical profiles were recovered in molded and nonmolded areas, indicating that contamination may have occurred during production. The application of PCR allowed the unambiguous identification of Listeria isolated from baled silages, and repetitive sequence-based PCR allowed a rapid and effective typing of isolates. Results disclose the potential of the systematic typing of Listeria in primary production, which is needed for the understanding of its transmission pathways.

Differential expression of living mammary epithelial cell subpopulations in milk during lactation in dairy cows.

Epithelial cells are shed into milk during lactation, and although they generally reflect the cellular characteristics of terminally differentiated luminal cells, previously the detection of more primitive cells was described in human milk where a cell population of epithelial lineage was detected expressing markers typical of progenitor cells. In this investigation, we report the development of flow cytometry analysis to allow multiparametric assessment of mammary epithelial cells observed in milk. Cells collected from milk samples of 10 healthy dairy cows were directly analyzed for 6 different markers: CD45, CD49f, cytokeratin 14, cytokeratin 18, presence of nucleus, and cell viability. Milk samples were collected in 3 different periods of lactation: early lactation (EL=d 0-30), mid-lactation (ML=d 90-120), and late lactation (LL=210-250). Here we identify the differential expression of precursor or differentiated cell markers (or both) in mammary epithelial cells present in bovine milk. Myoepithelial cells, as indicated by cells staining positively for cytokeratin 14(+)/cytokeratin 18(-), were observed to increase from EL to LL with a high correlation with nuclear staining inferring potential proliferative activity. Furthermore, a significant increase in CD49f(+) and cytokeratin 14(+)/cytokeratin 18(+) positive cells was observed in LL. This assay is a sensitive approach for evaluating the variations in the frequency and features of living epithelial cells, whose reciprocal balance may be significant in understanding mammary gland cellular function throughout lactation. These observations suggest that mammary epithelial cell immunophenotypes could be investigated as biomarkers for mammary gland function in dairy cows.

Mucocele of the appendix. Two case reports.

The authors present two cases of mucocele of the appendix and discuss them in relation to the literature and the clinical features of this disease. They clarify the definition of mucocele as an intraluminal accumulation of mucus in the appendix, and concentrate on the observable pathological processes, agreeing on the higher frequency of mucinous cystadenoma and the possibility that mucocele can develop into peritoneal pseudomyxoma or degenerate into cystadenocarcinoma. They also note that most diagnoses are made intra-operatively during appendectomy, and that, in cases suspected preoperatively, thorough investigation with imaging techniques is very important in order to plan the best treatment.

[A complication in biliary surgery: the biloma]. / Una complicanza degli interventi sulle vie biliari: il biloma.

Authors stress that complications in laparotomic and laparoscopic extrahepatic biliary surgery aren't exceptional. Lesions could be due to section or to stenosis of biliary duct. We can distinguish: intraoperative lesions, which consist in a intraperitoneal bile groan that needs an immediate treatment; lesions that could be found in postoperative period and in any case many days after surgery. Among this complications there is biloma, i.e. a localized uncapsulated extraductal bile collection. Authors refer about their experience and describe clinical findings of this complication. They conclude affirming how biloma treatment doesn't require always resurgery or CT scan drain. Main biliary tract endoscopic decompression could be often useful.

Comparison of Lactococcus garvieae strains isolated in northern Italy from dairy products and fishes through molecular typing.

AIMS: To investigate the genetic relatedness between Lactococcus garvieae strains isolated from fish and dairy samples collected in northern Italy, using random-amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR), Sau-PCR and amplified fragment length polymorphism (AFLP). METHODS AND RESULTS: Eighty-one isolates from bovine and caprine dairy products (n = 53) and from diseased rainbow trouts and other fishes (n = 28) were examined. All methods showed a typeability of 100%, repeatability ranging from 84.4% to 97.5% and discriminatory powers from 0.798 to 0.986. Dairy and fish strains revealed a low genetic relatedness as they are often grouped into distinct clusters. RAPD analysis discriminated 52 genotypes when primer M13 was used, whereas with primer P5 only 27 genotypes were identified. When Sau-PCR was performed, 13 genotypes were detected while AFLP analysis allowed the differentiation of 32 genotypes. CONCLUSION: L. garvieae strains isolated from dairy samples are generally not related to those collected from fish lactococcosis outbreaks. SIGNIFICANCE AND IMPACT OF THE STUDY: L. garvieae strains exhibit a genetic diversity related to the specific animal host they colonize. RAPD M13 fingerprinting proved to be a molecular tool for comparing isolates, whereas Sau-PCR and AFLP analyses were useful techniques to investigate the distribution of L. garvieae populations in the environment.

Characterization of an immunodominant epitope of small ruminant lentivirus (SRLV) nucleoprotein.

To extend and complete the epitope mapping of gag-encoded structural proteins, the immunodiagnostic potential of nucleoprotein (NP) of two different small ruminant lentivirus (SRLV) genotypes were antigenically characterized. Respective recombinant counterparts were generated and used in an enzyme-linked immunosorbent assay (ELISA) format to test a panel of sera from infected flocks. Results clearly indicate that a single linear epitope located within the C-terminal is partially cross-reactive among different SRLV genotypes and may complement multiple epitope ELISA for serological diagnosis of infection. However, in contrast to matrix and capsid antigen epitopes, which drive a genotype-specific immunoresponse, a moderate degree of variation was identified in NP independently of the genotype to which it belongs.

Anisakis spp. larvae in different kinds of ready to eat products made of anchovies (Engraulis encrasicolus) sold in Italian supermarkets.

In this study the occurrence of visible anisakid larvae in semi-preserved anchovy products sold on the Italian market was investigated. Totally, 107 ready to eat products (33 salted-ripened, 49 in oil and 25 marinated) were sampled. Each sample was digested, then the digested material was observed under natural and UV light. Parasites were counted, collected and microscopically identified to genus level. A representative subset was molecularly identified using the cox2 gene. At least one visible Anisakis sp. larva was found in 54.2% of the total 107 products analysed and totally 1283 dead larvae were collected. Anisakis sp. larvae were found in all the 33 salted products and 1139 (88.8%) larvae were collected, with a range of 1-105 parasites per product. Larval density per gram was 0.13. Anisakis sp. larvae were found in 49.0% of the products in oil and 143 (11.1%) larvae were isolated, with a range of 0-28 and a density of 0.03. Only 1 larva was found in the 25 marinated products (4.0%, density 0.00). A highly significant difference between all the product categories in respect of number of larvae per product, frequency of products contaminated by at least one larva and larval density per gram was found. Within the subset of larvae molecularly analysed (n=122), 92 (75.4%) were identified as A. pegreffii and 30 (24.6%) as A. simplex. This study showed that semi-preserved anchovy products heavily contaminated with Anisakis spp. larvae reach the market. Beyond the negligible risk for anisakidosis, the presence of dead visible parasites may cause immediate rejection in consumers. In addition, the potential risk related to allergic reactions in sensitized individuals needs to be further assessed. In order to avoid commercialization of obviously contaminated products, fresh anchovies' batches intended for the production of such products should be accurately selected by the processing industry applying inspection methods.

Visceral larvae as a predictive index of the overall level of fish batch infection in European anchovies (Engraulis encrasicolus): A rapid procedure for Food Business Operators to assess marketability.

The European anchovy (Engraulis encrasicolus), one of the most important pelagic fish resources in the Mediterranean Sea, is frequently infected by anisakid larvae. Food Business Operators (FBOs) should use appropriate sampling plans and analytical methods to avoid commercialization of massively infected batches and reduce the risk of transmission of viable zoonotic larvae. In this study, performed at FishLab (Department of Veterinary Sciences of the University of Pisa) during 2016, an official sampling plan was associated with a digestion protocol for the inspection of anchovies. Considering that anisakid larvae are usually located in the fish visceral cavity and in the adjacent muscles (VM), this part was analyzed. In particular, we assessed the reliability of the digestion of a subsample of 150g (±30g) of VM, randomly collected from 29 specimens, in estimating the marketability of the anchovies' batch. Fifty-seven samples of 29 anchovies were collected. Each anchovy was sectioned to separate VM. All the subsamples were digested, and visible larvae counted. A high correlation between the number of larvae in VM regions and in the total batch was observed, indicating a very significant contribution of the VM region on total number of parasites. The Mean Abundance (MA) was used to assess the batch marketability according to a threshold calculated on the basis of the maximum number of nematodes tolerated per sample. Considering that the MA can be calculated only when the number of examined specimens is known, the number of visible Larvae per gram of tissue (LpG) was calculated on 150g (±30g) of VM subsamples. A LpG marketability threshold was calculated dividing the maximum number of tolerated nematodes by the average weight of a sample of 29 anchovies calculated considering data available in literature. To evaluate the diagnostic performance of the LpG threshold, the marketability of 57 batches assessed on the basis of the MA threshold was assumed as the gold standard. The proposed LpG showed very high Specificity and Sensitivity. These findings suggest that the analysis of VM is representative of the overall infestation of the batch, both when considering the absolute number of parasites and the LpG, and may represent a valid alternative to the whole anchovy digestion. In particular, the use of an automated digestive method, coupled with the aforesaid sampling plan, could allow the procedure to be used by FBOs in operational conditions.

Development of a PCR assay for the detection of animal tissues in ruminant feeds.

The European Community ban on use of meat and bone meal in ruminant feed, as a consequence of the spread of bovine spongiform encephalopathy in Europe, has prompted a number of investigations about the possibility of detecting animal tissues in feedstuff. In this paper, a study on vertebrate primers, designed in the 16S rRNA gene of mitochondrial DNA, is described. These primers were able to amplify fragments that contained between 234 and 265 bp. The fragments were specific for bovine, porcine, goat, sheep, horse, rabbit, chicken, trout, and European pilchard and were confirmed by sequence analysis amplicons. The primers were used in a PCR assay applied to five samples of meat and blood meals of different species and subjected to severe rendering treatments (134.4 to 141.9 degrees C and 3.03 to 4.03 bar for 24 min). The presence of vertebrate tissues was detected in all samples. The assay proved to be rapid and sensitive (detection limit 0.0625%). It can be used as a routine method to detect animal-derived ingredients in animal feedstuff.

The microbiological conditions of carcasses from large game animals in Italy.

This study investigates the microbiological conditions of large game animal carcasses following evisceration. Carcasses of animals (N=291) hunted in the Upper Susa Valley (Italian Alps) were analysed for pH, Aerobic Viable Count (AVC), Enterobacteriaceae, Yersinia spp., Listeria monocytogenes and Salmonella spp. After shooting, evisceration occurred within 60 min in 90.7% of animals and sampling within 90 min in 88.3% of animals. Mean pH values (5.97: ruminants; 5.77: wild boar) were similar to those of regularly slaughtered domestic species. AVC values were highest in animals shot in the abdomen. Within species, AVC and Enterobacteriaceae values did not differ across different shooting-evisceration/sampling times. However, these counts exceeded 5 and 2.5 log, respectively, in 18% of wild boar and 39% of ruminants; the highest values were detected in wild boar. No pathogens were detected in any species. These results reveal inadequate hygiene in game meat handling/harvesting, implicating the need for improved practices.

Detection of Salmonella in finishing pigs on farm and at slaughter in Piedmont, Italy.

Salmonella is one of the most common causes of human gastroenteritis often associated with pork consumption. The aims of this cross-sectional study were to collect preliminary data on the presence of Salmonella enterica in pigs in Piedmont (Italy), through sampling on farm and at slaughter and to gather pilot data on serotypes and phagetypes present in the sampled area and distribution of anti-microbial resistance among isolated strains. Salmonella was detected through culture and identified with Salmonella spp. and Salmonella Typhimurium PCR; positive samples were serotyped, phagetyped and tested for antibiotic susceptibility. Positive samples (from 9% of faeces up to 29% of tonsils) were found in 64% of the herds. Salmonella spp. was retrieved also from scalding water. Most of the isolates were Salmonella Derby, Salmonella Typhimurium and Salmonella 4,5,12:i:-. The results of Salmonella Typhimurium specific PCR suggested that Salmonella 4,5,12:i:- might be unrecognized by serotyping. Anti-microbial resistance was recorded in 75-100% of the isolates. Phagetyping allowed the identification of DT104B and DT46A strains. These results set the bases for further research studies that would aim to estimate the real herd prevalence in Piedmont and the diffusion of serotypes and anti-microbial resistant strains within the same region.

Real-time subtyping via PFGE reveals potential epidemiological relatedness among human salmonellosis cases in Northern Italy.

AIM: This study aimed to assess the applicability of a combined approach of traditional and molecular epidemiology in order to detect salmonellosis outbreaks in the Piedmont region (Italy), characterized by high Salmonella prevalence. METHODS AND RESULTS: Pulsed field gel electrophoresis (PFGE) was used in real-time and in combination with clinical surveillance to assess the relatedness of salmonellosis human cases; subsequently, PFGE profiles of clinical isolates were compared with those of isolates from food items collected during the same study period to identify putative food sources of Salmonella. The real-time subtyping approach allowed the identification of an outbreak (21 isolates), which was undetected by epidemiological surveillance. CONCLUSIONS: Traditional epidemiological investigation did not allow the formulation of hypotheses on food items possibly associated with the outbreak owing mainly to patients' difficulties in remembering foods they ate, and the tendency of health-care professionals to direct patient's suspicion towards specific food items. SIGNIFICANCE AND IMPACT OF THE STUDY: This finding highlighted the value of real-time molecular subtyping in salmonellosis outbreak identification. In order to improve national epidemiological investigations implementing public health agency network and planning, information campaigns for health-care professionals are required.

Implementing sevoflurane anesthesia with small doses opioid for upper abdominal surgery. Postoperative respiratory function after either remifentanil or fentanyl.

BACKGROUND: The aim of this prospective, randomized study was to compare the effects on intraoperative cardiovascular homeostasis, recovery profile and postoperative oxygen saturation after sevoflurane anesthesia with small doses of either remifentanil or fentanyl in combination with postoperative epidural analgesia. METHODS: With Ethical Committee approval and written patient consent, 30 ASA physical status I-II patients scheduled for elective upper abdominal surgery were randomly allocated to receive sevoflurane general anesthesia implemented with small doses of either remifentanil (n = 15) or fentanyl (n = 15), followed by postoperative epidural analgesia. Remifentanil group patients received a 1 mg kg-1 bolus infused during a 60 sec period followed by a 0.15 mg kg-1 min-1 infusion; while patients of Fentanyl group were given a 3 mg kg-1 initial dose followed by 50 mg boluses as requested (according to the time to peak effect of the two drugs, the initial dose was given 5 min before induction in Fentanyl group, and 1 min before induction in Remifentanil group). Postoperatively, oxygen saturation was continuously recorded and stored on a computer during the first 12 h after surgery. SpO2 decrease < 90% for more than one minute was considered as a minor respiratory complication. RESULTS: The median sevoflurane's MAC-hour was 2.7 (1.4 - 4.9) in patients receiving remifentanil infusion and 4.1 (2.2 - 5.7) in those patients receiving fentanyl during surgery (P = 0.005). However, no differences in the recovery times were observed between the two groups. Similar pain relief was reported during coughing in the two studied groups at discharge from the recovery area and during the following study period. No major respiratory complication was observed throughout the study. Oxygen therapy was required in three patients of Fentanyl group only 20% (P = 0.22); however, 11 patients in the same group (73%) showed at least one minor respiratory complication (SpO2 < 90% for more than 1 min), with a median of 1 (range 0 - 12) episode per patient, compared with no episode in Remifentanil group (P = 0.0005). CONCLUSIONS: Implementing sevoflurane anesthesia with very small remifentanil infusion provides a safe and effective hemodynamic control reducing sevoflurane consumption during the procedure, and produces less respiratory effects postoperatively as compared with intermittent bolus administration of fentanyl.

The minimum effective dose of 0.5% hyperbaric spinal bupivacaine for cesarean section.

BACKGROUND: The aim of this prospective, blind study was to determine the minimum effective dose of hyperbaric bupivacaine required for cesarean section. METHODS: With Ethical Committee approval and written consent, 24 healthy women undergoing elective cesarean section received a combined spinal epidural anesthesia. We sought to determine the minimum effective dose of spinal bupivacaine using a staircase method. In each patient an arbitrary dose of 0.5% hyperbaric bupivacaine in relation to patient height was used. The initial dose was 0.075 mg/cm height, while the outcome of each patient's response determined the dose for the subsequent patient. When successful spinal block (sensory level = or < T4 with complete motor blockade) was achieved within 20 min from spinal injection, the dose of spinal bupivacaine for the next patient was decreased by 0.01 mg/cm height. Conversely, when successful spinal block was not observed, the dose of spinal bupivacaine for the next patient was increased by 0.01 mg/cm height. Sensory and motor blocks were evaluated every 5 min by an independent, blinded observer. If successful spinal block was not achieved within the designed period, a 5-8 ml epidural bolus of 2% lidocaine was given to achieve adequate surgical anesthesia. RESULTS: No complications were reported during the study, and all women delivered their baby uneventfully (APGAR scores 5 min after delivery ranged from 9 to 10) within 5 min from uterus incision. The duration of surgical procedure ranged from 30 to 48 minutes. The dose of hyperbaric bupivacaine providing adequate surgical anesthesia within 20 min from spinal injection in 50% of subjects was 0.036 mg/cm height (95% confidence intervals: 0.031-0.041 mg/cm height). The ED95 calculated from the probit transformation to provide effective spinal anesthesia for cesarean section was 0.06 mg/cm height. CONCLUSIONS: This prospective, blind study demonstrated that a dose as low as 0.06 mg/cm height represents the dose of intrathecal bupivacaine providing effective spinal block in 95% of women undergoing elective cesarean section.