Automated Analysis of Intracellular Phenotypes of <em>Salmonella</em> Using ImageJ

Salmonella is an enteric pathogen able to invade the intestinal epithelium and replicate in enterocytes, both inside Salmonella-specific vacuoles and free ...

Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia-Romagna (IZSLER)

An aborted fetus of 7 months gestation, the associated placenta, and a single blood sample from the dam were submitted for diagnostic investigation to the diagnostic laboratory of the Lombardy and Emilia-Romagna Experimental Zooprophylactic Institute in Parma, Italy. The serum was negative for Neospora caninum, Coxiella burnetii, Chlamydophila abortus, Bovine herpesvirus 1 (BHV-1), Bovine viral diarrhea virus (BVDV), Brucella abortus, and Brucella melitensis. Fetal tissues and placental cotyledons were pooled and tested by polymerase chain reaction (PCR) for the presence of BHV-1, Bovine herpesvirus 4, BVDV, N. caninum, C. burnetii, Chlamydophila spp., Schmallemberg virus, and Leptospira interrogans. All PCR assays were negative. Bacteriological examinations performed on the fetal organs revealed a pure growth of Staphylococcus lugdunensis in all organs cultured. In human beings, S. lugdunensis is responsible for community-acquired and nosocomial infections, in both immunocompetent and immunocompromised patients. In veterinary medicine, the pathogenic potential of S. lugdunensis has not been fully investigated. The incidence of S. lugdunensis is regarded as being underreported because it could be easily misidentified as Staphylococcus aureus. The current report documents the ability of S. lugdunensis to cause abortion in cattle, indicating the need for accurate diagnostic procedures to identify this emerging and zoonotic pathogen whose incidence is likely underestimated in both human and veterinary medicine. 

Abortion in cattle due to infection with Staphylococcus lugdunensis.

We retrospectively analyzed a rare Salmonella enterica serovar Manhattan outbreak that occurred in Italy in 2009 to evaluate the potential of new genomic tools based on differential single nucleotide polymorphism (SNP) analysis in comparison with the gold standard genotyping method, pulsed-field gel electrophoresis. A total of 39 isolates were analyzed from patients (n=15) and food, feed, animal, and environmental sources (n=24), resulting in five different pulsed-field gel electrophoresis (PFGE) profiles. Isolates epidemiologically related to the outbreak clustered within the same pulsotype, SXB_BS.0003, without any further differentiation. Thirty-three isolates were considered for genomic analysis based on different sets of SNPs, core, synonymous, nonsynonymous, as well as SNPs in different codon positions, by Bayesian and maximum likelihood algorithms. Trees generated from core and nonsynonymous SNPs, as well as SNPs at the second and first plus second codon positions detailed four distinct groups of isolates within the outbreak pulsotype, discriminating outbreak-related isolates of human and food origins. Conversely, the trees derived from synonymous and third-codon-position SNPs clustered food and human isolates together, indicating that all outbreak-related isolates constituted a single clone, which was in line with the epidemiological evidence. Further experiments are in place to extend this approach within our regional enteropathogen surveillance system.

Differential single nucleotide polymorphism-based analysis of an outbreak caused by Salmonella enterica serovar Manhattan reveals epidemiological details missed by standard pulsed-field gel electrophoresis.

The quantitative and qualitative patterns of environmental contamination by Listeria monocytogenes were investigated in the production chain of dry-cured Parma ham. Standard arrays of surfaces were sampled in processing facilities during a single visit per plant in the three compartments of the food chain, i.e., ham production (19 plants) and postproduction, which was divided into deboning (43 plants) and slicing (25 plants) steps. The numbers of sampled surfaces were 384 in ham production, with 25 positive for L. monocytogenes, and 1,084 in postproduction, with 83 positives. Statistical analysis of the prevalence of contaminated surfaces showed that in ham production, contamination was higher at the beginning of processing and declined significantly toward the end, while in postproduction, prevalence rose toward the end of processing. Prevalence was higher in the deboning facilities than in slicing facilities and was dependent on the type of surface (floor/drainage > clothing > equipment). The qualitative pattern of contamination was investigated through an analysis of the survey isolates and a set of isolates derived from routine monitoring, including longitudinal isolations. Pulsed-field gel electrophoresis (PFGE) and whole-genome single-nucleotide polymorphism (SNP) analysis revealed a remarkable clonality of L. monocytogenes within plants, with the detection of 16 plant-specific clones out of 17 establishments with multiple isolates. Repeated detections of clonal isolates >6 months apart were also observed. Six was the maximum number of between-isolate differences in core SNPs observed within these clones. Based on the same six-SNP threshold, three clusters of clonal isolates, shared by six establishments, were also identified. The spread of L. monocytogenes within and between plants, as indicated by its clonal behavior, is a matter of concern for the hygienic management of establishments. 

Processing-Dependent and Clonal Contamination Patterns of Listeria monocytogenes in the Cured Ham Food Chain Revealed by Genetic Analysis.

Occurrence of mcr-1 in Colistin-Resistant Salmonella enterica Isolates Recovered from Humans and Animals in Italy, 2012 to 2015.

Usutu virus is an arbovirus closely related to West Nile virus (genus Flavivirus), which circulates between mosquitoes and wild birds. This virus has been increasingly reported in Europe, raising concerns for its possible pathogenic potential for wild birds and humans. This study reports the whole genome sequences of 15 strains of USUV, isolated between 2010 and 2014 from mosquitoes and wild birds in the course of West Nile virus surveillance in the Emilia-Romagna and Lombardy regions of Northern Italy. Both whole and 656 partial genome sequences, obtained from isolated viruses and field samples (mosquitoes and wild birds), were analyzed to describe the temporal and geographical spread of USUV in the surveyed area. The detected sequences belonged to two groups, with one circulating primarily in the northwestern part of the area and the other in the southeastern part. This pattern is likely the result of different routes of introduction from the North (over the Alps) and from the East, respectively. The phylogenetic analysis of obtained sequences and other European sequences demonstrated that the majority of European strains belonged to one main clade, while less common strains, mainly from Western Europe, fell in other two clades. This analysis strongly suggested an autochthonous evolution process of strains of the main clade from a common ancestor with an estimated time of arrival in Europe at the beginning of the 1990s. In addition to causing mass mortality in wild birds, Usutu virus can infect humans and can sporadically cause disease. These factors and the endemization of the Usutu virus in a large area of Europe, sustained by the obtained data, strongly support the need to adequately survey Usutu virus in areas in which its circulation is detected.

Co-circulation of two Usutu virus strains in Northern Italy between 2009 and 2014.

An expert opinion elicitation, based on a modified Delphi technique, was organized to collect the opinion of 16 Italian veterinarians with the aim of conducting a hazard and a welfare promoter characterization for defining and weighing a list of management and housing factors potentially associated with negative or positive welfare outcomes in dairy cows kept in loose housing systems. In addition, the 16 experts judged a set of animal-based measures in order to rate them by appropriateness and by the level of animal pain and suffering due to the welfare consequences they measure. Veterinary experts were asked to score 52 hazards, 47 welfare promoters and 18 animal-based measures. Management and housing hazards, that were determined to be associated with a very high impact on the welfare of dairy cows, were mainly referred to lactating cows (absence of bedding material, presence of inadequate or slippery floor in walking areas, wrong design of the lying area), but also the use of harmful tools for animal handling and the lack of scheduled foot inspection, trimming and foot bathing reached very high impact scores. Management and housing welfare promoters dealing with optimal cow comfort around resting obtained the highest ratings, together with the presence of experienced and trained stockpersons, the implementation of an attentive animal grouping strategy and the control of temperature, humidity and ventilation. Considering animal-based measures, the highest ranking of importance was given to observation of lameness, records of mortality of adult cows and calves, observation of integument alterations and body condition scoring. This study has been the starting point for the development of the first Italian national protocol for the assessment of the welfare of dairy cows farmed in loose housing systems.

Stefano Pongolini

Risk Analysis and Genomic Epidemiology Unit

Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia-Romagna (IZSLER)

Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

Stefano Pongolini

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Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia-Romagna (IZSLER)

Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

Risk Analysis and Genomic Epidemiology Unit