Retrospective study of tumors from cattle slaughtered in Lombardy (Italy): preliminary evaluation on the establishment of a bovine cancer registry.

Forty­one tumors were detected in a population of 1,649,003 cattle slaughtered in 4 abattoirs in Lombardy over a 5­year period, for an overall prevalence of 2.5 tumors per 100,000 cattle. Tumors were classified according to the WHO histological classification of tumors of domestic animals. Alimentary and hemopoietic systems were commonly affected with 9 cases each. Other affected sites were the respiratory (n = 3), urinary (n = 2), endocrine (n = 2), musculoskeletal (n = 2), nervous (n = 1), and cardiovascular (n = 1) systems. The peritoneum was affected by 6 cases, while the primary location of 3 tumors of the connective tissues and 3 metastatic carcinomas was unidentified. Liver tumors and mesotheliomas, for which environmental risk factors are well­known in humans, were common, as well as tumors typically encountered in pediatric human patients (tumors of mesenchymal tissues, pulmonary blastomas and nephroblastomas). These findings suggest the useful role of bovines as sentinel and model for human carcinogenesis. Our study indicates that the establishment of a bovine cancer registry in Lombardy is feasible considering its potential contribution to understanding the role of environmental risk factors in the genesis of tumors in animals and humans.

Study of the diversity of 16S-23S rDNA internal transcribed spacer (ITS) typing of Escherichia coli strains isolated from various biotopes in Tunisia.

We investigated the 16S-23S rRNA intergenic spacer region (ISR)-PCR and the phylogenetic PCR analyses of 150 Escherichia coli isolates as tools to explore their diversity, according to their sampling origins, and their relative dominance in these sampling sources. These genetic markers are used to explore phylogenetic and genetic relationships of these 150 E. coli isolates recovered from different environmental sources (water, food, animal, human and vegetables). These isolates are tested for their biochemical pattern and later genotyped through the 16S-23S rRNA intergenic spacer PCR amplification and their polymorphism investigation of PCR-amplified 16S-23S rDNA ITS. The main results of the pattern band profile revealed one to four DNA fragments. Distributing 150 E. coli isolates according to their ITS and using RS-PCR, revealed four genotypes and four subtypes. The DNA fragment size ranged from 450 to 550 bp. DNA band patterns analysis revealed considerable genetic diversity in interspecies. Thus, the 450 and 550 bp sizes of the common bands in all E. coli isolates are highly diversified. Genotype I appeared as the most frequent with 77.3% (116 isolates), genotype II with 12% (18 isolates); genotype III with 9.7% (14 isolates), and the IV rarely occurred with 4% (2 isolates). Distributing the E. coli phylogroups showed 84 isolates (56%) of group A, 35 isolates (23.3%) of group B1, 28 isolates (18.7%) of group B2 and only three isolates (2%) of group D.

Bovine Milk Microbiota: Comparison among Three Different DNA Extraction Protocols To Identify a Better Approach for Bacterial Analysis.

The bovine udder is colonized by a huge quantity of microorganisms that constitute the intramammary ecosystem, with a specific role in modulating not only udder homeostasis and mastitis susceptibility, but also the quality of the dairy products. However, generating high-quality bacterial DNA can be critical, especially starting from a complex biological matrix like milk, characterized by high fat, protein, and calcium contents. Here, bacterial DNA was recovered from a commercial ultra-high-temperature (UHT) milk sample artificially spiked with a predetermined mock community composition and from three bulk tank milk (raw milk) samples. The DNA was isolated using three different protocols to evaluate the effect of the extraction procedures on the milk microbiota composition. In the mock community experiment, the bacterial profiles generated by the three DNA extraction protocols were profoundly different, with the genera Staphylococcus, Lactobacillus, Listeria, and Salmonella underestimated by all the protocols. Only one protocol revealed values close to the expected abundances for Escherichia/Shigella spp., Bacillus spp., Enterococcus spp., and Pseudomonas spp. On the other hand, the nonspiked UHT milk sample exhibited a similar microbiota composition, revealing the prevalence of Acinetobacter spp., for all the DNA extraction protocols. For the raw milk samples, the three DNA extraction kits performed differently, revealing significant separations in both the microbial richness (alpha diversity) and composition (beta diversity). Our study highlights the presence of significant differences among these procedures, probably due to the different DNA extracting capacities and to the different properties of the milk samples, revealing that the selection of DNA extraction protocol is a critical point. IMPORTANCE The advance of high-throughput technologies has increased our knowledge of the world of microorganisms, especially of microbial populations inhabiting living animals. This study provides evidence that milk, as other complex sources, could be critical for generating high-quality DNA for microbiota analysis. In addition, it demonstrates that the microbial population highlighted by metagenomic studies changes in relation to different DNA extraction procedures, revealing that attention should be paid especially when comparing different studies.

Bloody Diarrhea and Shiga Toxin-Producing Escherichia coli Hemolytic Uremic Syndrome in Children: Data from the ItalKid-HUS Network.

OBJECTIVE: To analyze the results of an enhanced laboratory-surveillance protocol for bloody diarrhea aimed at identifying children with Shiga toxin-producing Escherichia coli (STEC) infection early in the course of the disease toward the early identification and management of patients with hemolytic uremic syndrome (HUS). STUDY DESIGN: The study (2010-2019) involved a referral population of 2.3 million children. Stool samples of patients with bloody diarrhea were screened for Shiga toxin (Stx) genes. Positive patients were rehydrated and monitored for hemoglobinuria until diarrhea resolved or STEC-HUS was diagnosed. RESULTS: A total of 4767 children were screened; 214 (4.5%) were positive for either Stx1 (29.0%) or Stx2 (45.3%) or both Stx1+2 (25.7%); 34 patients (15.9%) developed STEC-HUS (0.71% of bloody diarrheas). Hemoglobinuria was present in all patients with HUS. Patients with Stx2 alone showed a greater risk of STEC-HUS (23.7% vs 12.7%) and none of the patients with Stx1 alone developed HUS. During the same period of time, 95 other patients were diagnosed STEC-HUS but were not captured by the screening program (26 had nonbloody diarrhea, 11 came from areas not covered by the screening program, and 58 had not been referred to the screening program, although they did meet the inclusion criteria). At HUS presentation, serum creatinine of patients identified by screening was significantly lower compared with that of the remaining patients (median 0.9 vs 1.51 mg/dL). CONCLUSIONS: Nearly 1% of children with bloody diarrhea developed STEC-HUS, and its diagnosis was anticipated by the screening program for Stx. The screening of bloody diarrhea for Stx is recommended, and monitoring patients carrying Stx2 with urine dipstick for hemoglobinuria is suggested to identify the renal complication as early as possible.

Family Clusters of Shiga Toxin-producing Escherichia coli Infection: An Overlooked Source of Transmission. Data From the ItalKid-Hus Network.

BACKGROUND: The aim of the present work was to investigate family clusters of Shiga toxin-producing Escherichia coli (STEC) infection among the household members of STEC positive patients, identified within a screening program of bloody diarrhea (BD) for STEC in Northern Italy. METHODS: Stool samples from patients with BD or BD-associated-hemolytic uremic syndrome (HUS) and related households were investigated by molecular and bacteriologic methods to detect and characterize the virulence profile of STEC and Pulsed Field Gel Electrophoresis analysis were done on isolates. RESULTS: Thirty-nine cases of STEC infection (isolated BD in 16, BD-associated-HUS in 23) were considered, and a total of 130 stool samples from 1 to 8 households of the index patient were analyzed. The prevalence of positivity was higher in siblings (34.8%, 8/23) than in mothers (20%, 7/35), grandparents (9.5%, 2/21), fathers (8.8%, 3/34) or other households. In 14 clusters (36%), one or more household shared a STEC with the same virulence profile (stx, eae, serogroup) as the index case. In 7 clusters, STEC strains isolated from at least 2 subjects also shared identical Pulsed Field Gel Electrophoresis profile. The frequency of household infection does not appear to be associated to the index case's illness (HUS or BD), nor with the serotype or with the virulence profile of the involved STEC (stx2 or stx1-stx2). CONCLUSIONS: Our study shows that STEC infections, most likely related to human-to-human transmission, are common among households of patients with STEC BD or HUS and underlines the importance of extending the epidemiologic investigations to all family members, as the index case may not always be the primary infection in the family.

Heritability of Teat Condition in Italian Holstein Friesian and Its Relationship with Milk Production and Somatic Cell Score.

In spite of the impressive advancements observed on both management and genetic factors, udder health still represents one of most demanding objectives to be attained in the dairy cattle industry. Udder morphology and especially teat condition might represent the first physical barrier to pathogens' access. The objectives of this study were to investigate the genetic component of teat condition and to elucidate its relationship with both milk yield and somatic cell scores in dairy cattle. Moreover, the effect of selection for both milk yield and somatic cell scores on teat condition was also investigated. A multivariate analysis was conducted on 10,776 teat score records and 30,160 production records from 2469 Italian Holstein cows. Three teat scoring traits were defined and included in the analysis. Heritability estimates for the teat score traits were moderate to low, ranging from 0.084 to 0.238. When teat score was based on a four-classes ordinal scoring, its genetic correlation with milk yields and somatic cell score were 0.862 and 0.439, respectively. The scale used to classify teat-end score has an impact on the magnitude of the estimates. Genetic correlations suggest that selection for milk yield could deteriorate teat health, unless more emphasis is given to somatic cell scores. Considering that both at national and international level, the current selection objectives are giving more emphasis to health traits, a further genetic deterioration in teat condition is not expected.

Ventilation With Argon Improves Survival With Good Neurological Recovery After Prolonged Untreated Cardiac Arrest in Pigs.

Background Ventilation with the noble gas argon (Ar) has shown neuroprotective and cardioprotective properties in different in vitro and in vivo models. Hence, the neuroprotective effects of Ar were investigated in a severe, preclinically relevant porcine model of cardiac arrest. Methods and Results Cardiac arrest was ischemically induced in 36 pigs and left untreated for 12 minutes before starting cardiopulmonary resuscitation. Animals were randomized to 4-hour post-resuscitation ventilation with: 70% nitrogen-30% oxygen (control); 50% Ar-20% nitrogen-30% oxygen (Ar 50%); and 70% Ar-30% oxygen (Ar 70%). Hemodynamic parameters and myocardial function were monitored and serial blood samples taken. Pigs were observed up to 96 hours for survival and neurological recovery. Heart and brain were harvested for histopathology. Ten animals in each group were successfully resuscitated. Ninety-six-hour survival was 60%, 70%, and 90%, for the control, Ar 50%, and Ar 70% groups, respectively. In the Ar 50% and Ar 70% groups, 60% and 80%, respectively, achieved good neurological recovery, in contrast to only 30% in the control group (P<0.0001). Histology showed less neuronal degeneration in the cortex (P<0.05) but not in the hippocampus, and less reactive microglia activation in the hippocampus (P=0.007), after Ar compared with control treatment. A lower increase in circulating biomarkers of brain injury, together with less kynurenine pathway activation (P<0.05), were present in Ar-treated animals compared with controls. Ar 70% pigs also had complete left ventricular function recovery and smaller infarct and cardiac troponin release (P<0.01). Conclusions Post-resuscitation ventilation with Ar significantly improves neurologic recovery and ameliorates brain injury after cardiac arrest with long no-flow duration. Benefits are greater after Ar 70% than Ar 50%.

Staphylococcus aureus From Goats Are Genetically Heterogeneous and Distinct to Bovine Ones.

Staphylococcus aureus is one of the major pathogens responsible for intramammary infections in small ruminants, causing severe economic losses in dairy farms. In addition, S. aureus can contaminate milk and dairy products and produce staphylococcal enterotoxins, being responsible for staphylococcal food poisoning. Currently, data on the population structure and the virulence gene patterns of S. aureus strains isolated from goat milk is limited. Therefore, this study aimed at defining Ribosomal Spacer PCR (RS-PCR) genotypes, clonal complexes (CC), spa types, and virulence gene profiles of S. aureus isolated from goat milk samples from Lombardy region of Italy. A total of 295 S. aureus isolates from 65 goat bulk tank milk samples were genotyped by RS-PCR. spa typing and virulence gene patterns of a subgroup of 88 isolates were determined, and MLST was performed on a further subgroup of 39 isolates, representing all the spa types identified during the analysis. This study revealed 7 major genotypic clusters (CLR, CLAA, CLZ, CLAW, CLBW, CLS, and CLI), of which S. aureus CLR (19.8%) was the most common. A total of 26 different spa types were detected, the most prevalent types were t1773 (24%), t5428 (22.7%), and t2678 (12.5%). Overall, 44.3% of all isolates harbored at least one enterotoxin gene. The most prevalent was the combination of sec-sel genes (35.2%). Based on their MLST, isolates were assigned to 14 different CC, with majority grouped as CC133 (24%), CC130 (19.6%), and CC522 (19.6%). The caprine S. aureus population was depicted with a minimum spanning tree and an evolutionary analysis based on spa typing and MLST, respectively. Then, the variability of such strains was compared to that of bovine strains isolated in the same space-time span. Our results confirmed that S. aureus isolates from goats have wide genetic variability and differ from the bovine strains, supporting the idea that S. aureus from small ruminants may constitute a distinct population.

Toxoplasma gondii seroprevalence in beef cattle raised in Italy: a multicenter study.

Toxoplasmosis represents an important public health issue, with the consumption of raw or undercooked meat being a major way of human infection. The role of beef in the transmission of the parasite to humans is questioned due to lower quantity of tissue cysts compared with other meat-producing species. However, the habit of consuming raw beef is regionally diffused, and the risk posed by Toxoplasma gondii infection in cattle should not be overlooked. Therefore, to update information on T. gondii in cattle reared in Italy, a multicentric seroepidemiological survey was designed and implemented in four Northern regions (Liguria, Lombardy, Piedmont, and Trentino Alto Adige) and Sardinia. Overall, a convenience sampling was performed, collecting 1444 serum samples from 57 beef cattle herds. Thirteen beef breeds were sampled, besides cross-breed; bovines age varied from 3 months to over 12 years. Sera were tested with a commercial ELISA for the detection of anti-T. gondii antibodies. Individual and herd data were analyzed by binary logistic regression analysis. A T. gondii seroprevalence of 10.2% was recorded, with differences among regions and values ranging from 5.3% in Liguria to 18.6% in the Piedmont region (p value = 0.0001). Both young and adult animals and males and females tested positive, without any significant difference (age and gender: p value > 0.05). Lower seroprevalence values were recorded in cattle born in Italy (8.7%) if compared with animals imported from abroad (13.4%) (p value = 0.046). The spread of T. gondii in beef cattle destined to Italian consumers is confirmed, suggesting the need of continuous monitoring of the infection.

Is Shigatoxin 1 protective for the development of Shigatoxin 2-related hemolytic uremic syndrome in children? Data from the ItalKid-HUS Network.

BACKGROUND: Shigatoxin (Stx)-producing Escherichia coli (STEC) are the most common causes of hemolytic uremic syndrome (STEC-HUS). The aim of our study is to compare the risk of developing STEC-HUS in relation to the type of Stx genes (Stx1, Stx2, or both). METHODS: This is a prospective, observational, multicenter study involving 63 pediatric units in Northern Italy (ItalKid-HUS Network). STEC-infected children were identified within a screening program for bloody diarrhea during a 10-year period (2010-2019). Stx genes were detected by reverse dot blot or real-time PCR. After the identification of STEC infection, children were followed until diarrhea complete recovery for the possible development of STEC-HUS. RESULTS: Of the 214 Stx-positive patients, 34 (15.9%) developed STEC-HUS. The risk of HUS in STEC-infected children with Stx1 (n: 62; 29.0%) and Stx2 (n: 97; 45.3%) was respectively 0% and 23.7%, while in patients carrying both Stx1 and Stx2 (n: 55; 25.7%), the risk was 12.7% (p: 0.001). CONCLUSIONS: Our data confirm that Stx1 is a very rare cause of STEC-HUS and demonstrate that the risk of STEC-HUS halves in the case of Stx1+2-producing Escherichia coli infection compared with infections where Stx2 is present alone. This observation is helpful in assessing the risk of individual STEC-infected patients for the development of HUS and suggests that Stx1, in the presence of Stx2, might exert a protective role possibly by receptor competition.

Technical note: Development of multiplex PCR assays for the molecular characterization of Streptococcus uberis strains isolated from bovine mastitis.

Streptococcus uberis is an important causative agent for clinical and subclinical mastitis in dairy cattle. The aim of this study was to develop 2 multiplex PCR assays (mPCR) for the simultaneous detection of virulence factors and housekeeping genes for use when investigating the genetic variability and distribution of Strep. uberis virulence factors. The tuf, cpn60, pauA, sodA, sua, oppF, and gapC genes were grouped in assay 1 (mPCR1) and the hasA, hasB, and hasC genes were included in assay 2 (mPCR2). The detection limits were 11.8 pg and 5.9 pg of DNA for mPCR1 and mPCR2, respectively. The 2 mPCR assays were validated with 56 Strep. uberis strains isolated from mastitis milk samples collected from different bovine herds in northern Italy. Results revealed that gapC and oppF were detected in 98.2% of the strains, whereas sua and hasC genes were detected in 94.6 and 89.2% of the strains, respectively. The most common pattern was gapC+, oppF+, cpn60+, sua+, sodA+, pauA+, tuf+, hasA+, hasB+, and hasC+, which appeared in 59% of the strains analyzed. The molecular assays developed in the present study represent a powerful tool for the evaluation of virulence pattern distribution in Strep. uberis strains associated with intramammary infections.

Diagnostic value of composite milk sample vs single quarter milk sample for the detection of Staphylococcus aureus intra-mammary infections in cattle.

Staphylococcus aureus (S. aureus) is one of the most important causes of mastitis in dairy cattle. Control and eradication programs of S. aureus intra-mammary infections (IMI) are based on different factors included the correct detection and management of the infected cows. The present study aimed at evaluating the efficacy of composite milk samples (CMS) analysis, compared to quarter milk samples (QMS) analysis, for the bacteriological detection of S. aureus intra-mammary infections. During 2016, 661 CMS (hygienically collected) and 2644 QMS (aseptically collected) were obtained from 661 cows in 5 herds. All the samples were submitted to S. aureus bacteriological culture and somatic cell count (SCC) analysis. QMS bacteriological analysis on blood agar plates was able to detect 236 cows excreting S. aureus, while the bacteriological analysis of CMS, using selective agar, identified 229 positive cows. The concordance was 95% with an excellent Cohen's κ (0.89). Relative sensitivity and specificity of CMS vs QMS, considered as the reference test, were 91.5% ± 2.1 and 96.9% ± 1.3 (CI 95%), respectively. In addition, the relative sensitivity of CMS improved as the number of infected quarters per cow and the number of colony forming units (cfu) per sample increased. The predictive value of CMS results was better when paired with SCC data, in particular CMS showed better negative predictive value when SCC was <200,000 cells/mL and better positive predictive value when SCC was>200,000 cells/mL. The probability for a cow to be S. aureus positive was 56.4% in case of SCC > 200,000 cells/mL, while it was 18.6% in case of SCC < 200,000 cells/mL. The average SCC in CMS was significantly higher in positive cows and the value rose as the number of infected quarters per cow increased. Given the intermittent excretion of S. aureus in milk from dairy cows, it could be more advantageous to carry out several serial CMS, rather than few QMS, being CMS an easier to collect and less expensive milk sampling method. Thus, bacteriological examination of CMS, combined with SCC data of the same sample, could be extremely useful for the success of S. aureus IMI control plans, because repeated CMS are easier to be performed and could be more easily proposed to the farmers.

Genome sequencing of Prototheca zopfii genotypes 1 and 2 provides evidence of a severe reduction in organellar genomes.

Prototheca zopfii (P. zopfii, class Trebouxiophyceae, order Chlorellales, family Chlorellaceae), a non-photosynthetic predominantly free-living unicellular alga, is one of the few pathogens belonging to the plant kingdom. This alga can affect many vertebrate hosts, sustaining systemic infections and diseases such as mastitis in cows. The aim of our work was to sequence and assemble the P. zopfii genotype 1 and genotype 2 mitochondrial and plastid genomes. Remarkably, the P. zopfii mitochondrial (38 Kb) and plastid (28 Kb) genomes are models of compaction and the smallest known in the Trebouxiophyceae. As expected, the P. zopfii genotype 1 and 2 plastid genomes lack all the genes involved in photosynthesis, but, surprisingly, they also lack those coding for RNA polymerases. Our results showed that plastid genes are actively transcribed in P. zopfii, which suggests that the missing RNA polymerases are substituted by nuclear-encoded paralogs. The simplified architecture and highly-reduced gene complement of the P. zopfii mitochondrial and plastid genomes are closer to those of P. stagnora and the achlorophyllous obligate parasite Helicosporidium than to those of P. wickerhamii or P. cutis. This similarity is also supported by maximum likelihood phylogenetic analyses inferences. Overall, the P. zopfii sequences reported here, which include nuclear genome drafts for both genotypes, will help provide both a deeper understanding of the evolution of Prototheca spp. and insights into the corresponding host/pathogen interactions.

In Vitro Activity of 30 Essential Oils against Bovine Clinical Isolates of Prototheca zopfii and Prototheca blaschkeae.

Protothecal mastitis poses an emergent animal health problem in dairy herds, with a high impact on dairy industries, causing heavy economic losses. Current methods of treating protothecal infections are ineffective, and no drug is licensed for use in cattle. The aim of the present study was to check the antialgal activity of 30 chemically defined essential oils (EOs) against Prototheca zopfii and Prototheca blaschkeae isolated from the milk of dairy cows with mastitis. A microdilution test was carried out to estimate the antialgal effectiveness of the selected chemically defined EOs. The microdilution test showed different degrees of inhibition among the examined Prototheca species. The activity of some of the examined EOs seem interesting. In particular, Citrus paradisi yielded the lowest minimal inhibitory concentration values (0.75%) for both algal species. P. zopfii appeared to be more sensitive to EOs in comparison to P. blaschkeae. The present study investigated the in vitro susceptibility of P. zopfii and P. blaschkeae to a wide range of EOs, obtained from different botanical families. Further investigations are necessary to evaluate the efficacy of EO-based formulations intended for the disinfection of both udder and milking products.

Antibiotic resistance phenotypes and virulence-associated genes in Escherichia coli isolated from animals and animal food products in Tunisia.

Livestock and food products of animal origin constitute important reservoirs of intestinal and extraintestinal pathogenic Escherichia coli including antibiotic-resistant E. coli isolates. To assess potential risks to public health related to E. coli strains of animal origin in Tunisia, 65 E. coli isolates recovered from healthy animals and food products of animal origin were studied. Antimicrobial susceptibility was determined according to CLSI guidelines and genes encoding antibiotic resistance as well as virulence factors were investigated by PCR. High rates of antibiotic resistance were observed to kanamycin (78.4%), gentamicin (75.3%) and streptomycin (75.3%, encoded by strA-strB (7 isolates)), amoxicillin (64.6%), amoxicillin/clavulanic acid (60%), tetracycline (44.6%; tetA (8 isolates) and tetB (7 isolates)), nalidixic acid (27.6%, qnrS (3 isolates), qnrB (2 isolates) and qnrA (one isolate)) and sulfonamides (36.9%; sul1 (1 isolate), sul2 (4 isolates), and sul3 (1 isolate)). Virulotypes classified some isolates as STEC (3%), MNEC (1.5%) and atypical EPEC (1.5%). This study demonstrated high rates of antimicrobial resistance and the presence of some pathogenic pathovars from animal origins that are a cause of concern for public health.

Duration of Untreated Cardiac Arrest and Clinical Relevance of Animal Experiments: The Relationship Between the "No-Flow" Duration and the Severity of Post-Cardiac Arrest Syndrome in a Porcine Model.

INTRODUCTION: The study investigated the effect of untreated cardiac arrest (CA), that is, "no-flow" time, on postresuscitation myocardial and neurological injury, and survival in a pig model to identify an optimal duration that adequately reflects the most frequent clinical scenario. METHODS: An established model of myocardial infarction followed by CA and cardiopulmonary resuscitation was used. Twenty-two pigs were subjected to three no-flow durations: short (8-10 min), intermediate (12-13 min), and long (14-15 min). Left ventricular ejection fraction (LVEF) was assessed together with thermodilution cardiac output (CO) and high sensitivity cardiac troponin T (hs-cTnT). Neurological impairment was evaluated by neurological scores, serum neuron specific enolase (NSE), and histopathology. RESULTS: More than 60% of animals survived when the duration of CA was ≤13 min, compared to only 20% for a duration ≥14 min. Neuronal degeneration and neurological scores showed a trend toward a worse recovery for longer no-flow durations. No animals achieved a good neurological recovery for a no-flow ≥14 min, in comparison to a 56% for a duration ≤13 min (P = 0.043). Serum NSE levels significantly correlated with the no-flow duration (r = 0.892). Longer durations of CA were characterized by lower LVEF and CO compared to shorter durations (P < 0.05). The longer was the no-flow time, the higher was the number of defibrillations delivered (P = 0.043). The defibrillations delivered significantly correlated with LVEF and plasma hs-cTnT. CONCLUSIONS: Longer no-flow durations caused greater postresuscitation myocardial and neurological dysfunction and reduced survival. An untreated CA of 12-13 min may be an optimal choice for a clinically relevant model.

Prototheca blaschkeae subsp. brasiliensis subsp. nov., isolated from cow milk.

A strain of an achlorophyllic alga, named PR24T, was isolated from cow milk samples from the state of Minas Gerais, Brazil. Based on 18S rDNA, 28S rRNA, D1/D2 region of the LSU rDNA and SSU rRNA gene sequence similarities, this strain was found to be a member of the genus Prototheca and closely related to Protothecablaschkeae SAG2064T. However, the novel strain could easily be distinguished from recognized Prototheca species by internal transcribed spacer, species-specific PCR, single-strand conformation polymorphism-PCR analysis and phenotypic characteristics. The inability to grow in Sabouraud broth at pH 4.0 and the different cellular fatty acid composition clearly distinguished PR24T from the reference strain of P. blaschkeae. The combination of genotypic and phenotypic data indicates that strain PR24T represents a subspecies of P. blaschkeae, for which the name Prototheca blaschkeae subsp. brasiliensis subsp. nov. is proposed. The respective type strain is PR24T (=DSM 103592T=IHEM 26958T).

Influenza D in Italy: towards a better understanding of an emerging viral infection in swine.

Influenza D virus (IDV), a new member of the Orthomyxoviridae family, was first reported in 2011 in swine in Oklahoma, and consequently found in cattle across North America and Eurasia. To investigate the circulation of IDV among pigs in Italy, in the period between June 2015 and May 2016, biomolecular and virological tests were performed on 845 clinical samples collected from 448 pig farms affected by respiratory distress located in the Po Valley. Serological tests were conducted on 3698 swine sera, including archive sera collected in 2009, as well as samples collected in 2015 from the same region. Viral genome was detected in 21 (2.3%) samples from 9 herds (2%), while virus was successfully isolated from 3 samples. Genetic analysis highlighted that Italian swine IDVs are closely related to the D/swine/Oklahoma/1334/2011 cluster. Sera collected in 2015 showed a high prevalence of IDV antibody titers (11.7%), while archive sera from 2009 showed statistically significant lower positivity rates (0.6%). Our results indicate an increasing epidemiological relevance of the pathogen and the need for in-depth investigations towards understanding its pathogenesis, epidemiology and possible zoonotic potential of this emerging virus.

Identification of additional loci associated with antibody response to Mycobacterium avium ssp. Paratuberculosis in cattle by GSEA-SNP analysis.

Mycobacterium avium subsp. paratuberculosis: (MAP) causes a contagious chronic infection results in Johne's disease in a wide range of animal species, including cattle. Several genome-wide association studies (GWAS) have been carried out to identify loci putatively associated with MAP susceptibility by testing each marker separately and identifying SNPs that show a significant association with the phenotype, while SNP with modest effects are usually ignored. The objective of this study was to identify modest-effect genes associated with MAP susceptibility using a pathway-based approach. The Illumina BovineSNP50 BeadChip was used to genotype 966 Holstein cows, 483 positive and 483 negative for antibody response to MAP, data were then analyzed using novel SNP-based Gene Set Enrichment Analysis (GSEA-SNP) and validated with Adaptive Rank Truncated Product methodology. An allele-based test was carried out to estimate the statistical association for each marker with the phenotype, subsequently SNPs were mapped to the closest genes, considering for each gene the single variant with the highest value within a window of 50 kb, then pathway-statistics were tested using the GSEA-SNP method. The GO biological process "embryogenesis and morphogenesis" was most highly associated with antibody response to MAP. Within this pathway, five genes code for proteins which play a role in the immune defense relevant to response to bacterial infection. The immune response genes identified would not have been considered using a standard GWAS, thus demonstrating that the pathway approach can extend the interpretation of genome-wide association analyses and identify additional candidate genes for target traits.