Detection of zoonotic enteropathogens in captive large felids in Italy.

AIMS: Within the One Health paradigm, infectious disease surveillance have been developed for domestic and wild animals, leaving the role of captive non-domestic populations, especially felids in zoos and circuses, less explored. This study addresses the proximity of these captive animals to urban areas, necessitating focused monitoring for potential zoonotic enteropathogens. The present work aimed to investigate the presence of such zoonotic enteropathogens in faecal samples from captive large felid populations. METHODS AND RESULTS: A total of 108 faecal samples were collected in three circuses, five zoos and one rescue centre across Italy. Salmonella spp. isolation, serotyping and antimicrobial susceptibility testing were conducted on all samples. Additionally, 60 samples were also examined for gastrointestinal parasites using standard coprological techniques. Giardia spp. detection employed direct immunofluorescent staining and specific PCR, while Toxoplasma gondii was detected using PCR targeting B1 gene. A total of 51 Salmonella enterica subsp. enterica were isolated, with predominant serovariants including Infantis (43.1%), Coeln (11.8%) and Newport (11.8%). The captive felids likely act as asymptomatic carriers of foodborne Salmonella, with notable resistance ampicillin and trimethoprim-sulfamethoxazole, no resistance to enrofloxacin was noted. Microscopic analysis revealed Toxascaris leonina eggs in 11 faecal samples (18.3%) and Giardia duodenalis cysts in one animal (1.7%). CONCLUSIONS: Captive animals in public settings may act as sources of Salmonella infection and enteroparasitosis for both occupational and general exposure. The study emphasizes the role of captive animals in antimicrobial resistance dynamics, highlighting the need for routine pathogen screening in the management practices of zoological structures.

High Prevalence of Hymenolepis (Rodentolepis) nana in Amateur Breeding Facilities of Chinchillas (Chinchilla lanigera) and Sugar Gliders (Petaurus breviceps) from Italy.

PURPOSE: Hymenolepis (Rodentolepis) nana is an enteric tapeworm globally widespread in wild and captive rodents. The survey was carried out in three chinchilla breeding facilities and in one sugar glider breeding facility in Central and Southern Italy. METHODS: One hundred and four chinchilla fecal pools and 40 sugar glider fecal pools were collected from cages housing breeding pairs or females with their offspring. Fecal samples were examined with a qualitative and quantitative copromicroscopical approach. Hymenolepis eggs were identified based on morphological and morphometrical features, and the average number of eggs per gram (EPG) of feces was estimated. RESULTS: Hymenolepis nana eggs were detected in 39.42% of chinchilla samples with an average of 16.33 EPG; whereas, all of the sugar glider samples tested positive with an average of 454.18 EPG. Neither helminth eggs nor protozoan cysts/oocysts were detected in any of the breeding facilities. CONCLUSION: The results clearly show a diffuse parasitism in both chinchillas and sugar gliders, and should be taken into consideration given the rising popularity of these two pet species combined with the zoonotic complications presented by H. nana.

HSP70 upregulation in nasal mucosa of symptomatic children with allergic rhinitis and potential risk of asthma development.

Allergic rhinitis and asthma are the most common causes of chronic inflammation of the upper and lower airways in childhood. However, a nasal biomarker that can link to pulmonary inflammation is yet to be found. The present paper aims to investigate the possible role in inflammation of two inducible 70-kDa Heat Shock Proteins (HSP70) members, HSPA1A/B and HSPA6, in nasal mucosa cells of allergic children through their mRNA expression analysis, and their correlation to both spirometric and FeNO values. The relationship between FeNO in lower airways and ∆Cts of HSPA1A/B in nasal mucosa seems to be influenced by clinical symptoms regardless of age, sex, and sensitization patterns. Therefore, HSP70 expression, as well as FeNO levels, could have a predictive capability to identify lower airways inflammation and thus to recognize rhinitic children having a potential risk of asthma development.

Mismatches between Morphology and DNA in Italian Partridges May Not Be Explained Only by Recent Artificial Release of Farm-Reared Birds.

Translocations and releases of farm-reared birds are considered among the major drivers of genetic pollution with consequent loss of genetic diversity in wild populations. In this study, we aimed to assess the extent of hybridization and introgression in the Italian partridges as a consequence of translocation. We surveyed two mitochondrial markers and one nuclear marker of Alectoris and Perdix from collections (museums and private collections), extant wild populations and farms. Consistent with previous studies, we found haplotypes of allochthonous species within the same genus, likely due to introductions for hunting activities. In addition, we found hybrids between Perdix and Alectoris species with genetic markers from both genera in single individuals. Such introgression was bidirectional and in both mitochondrial and nuclear markers. Counterintuitively, most of the hybrid samples came from collections before the 1950s, when large-scale translocations started, from wild populations where Grey Partridge (Perdix perdix) and Rock Partridge (Alectoris graeca) overlap in their distribution, whereas only one hybrid occurred among the farmed birds. Our results suggest that Perdix and Alectoris species can hybridize in nature and that artificial translocations and releases of farm-reared birds for restocking or reintroduction purposes may be only partially responsible for the genomic mismatches of Italian partridges.

One in a Million: Genetic Diversity and Conservation of the Reference Crassostrea angulata Population in Europe from the Sado Estuary (Portugal).

The production of cupped oysters is an important component of European aquaculture. Most of the production relies on the cultivation of the Pacific oyster Crassostrea gigas, although the Portuguese oyster Crassostrea angulata represents a valuable product with both cultural and economic relevance, especially in Portugal. The authors of the present study investigated the genetic diversity of Portuguese oyster populations of the Sado estuary, both from natural oyster beds and aquaculture facilities, through cox1 gene fragment sequencing. Then, a comparison with a wide dataset of cupped oyster sequences obtained from GenBank (up to now the widest available dataset in literature for the Portuguese oyster) was performed. Genetic data obtained from this work confirmed that the Pacific oyster does not occur in the natural oyster beds of the Sado estuary but showed that the species occasionally occurs in the oyster hatcheries. Moreover, the results showed that despite the founder effect and the bottleneck events that the Sado populations have experienced, they still exhibit high haplotype diversity. Risks are arising for the conservation of the Portuguese oyster reference populations of the Sado estuary due to the occurrence of the Pacific oyster in the local hatcheries. Therefore, researchers, local authorities, and oyster producers should work together to avoid the loss of this valuable resource.

Developmental stages of Amphibiocystidium sp., a parasite from the Italian stream frog (Rana italica).

Amphibian parasites of the genus Amphibiocystidium are members of the class Ichthyosporea (=Mesomycetozoea), within the order Dermocystida. Most of the species in the Dermocystida fail to grow in ordinary culture media, so their life cycle has only been partially constructed by studies in host tissues. However, to date, there have been few reports on the life cycle of Amphibiocystidium parasites with respect to the developmental life stages of both Dermocystidium and Rhinosporidium parasites. In this study, we provide light and electron microscopic findings of developmental phenotypes of Amphibiocystidium sp., a parasite previously characterized in the Italian stream frog (Rana italica), which has caused an ongoing infection in a natural population of Central Italy. These phenotypes exhibited distinct morphological characteristics that were similar to A. ranae from the skin of R. temporaria, but showed histochemical properties particularly comparable with those of maturing phenotypes of Rhinosporidium seeberi, and compatible with fungal-like parasites. Therefore, for Amphibiocystidium sp. phenotypes, we suggest adopting the terminology used for maturing stages of R. seeberi, such as juvenile sporangia, early mature sporangia and mature sporangia. The characterization of these developmental stages will be useful to increase the understanding of the life cycle of parasites of the genus Amphibiocystidium and of the interactions with their amphibian hosts.

Small heat shock proteins in the amphibian Pelophylax bergeri: Cloning and characterization of Hsp27 and Hsp30 cDNAs and their expression analysis in ex vivo skin exposed to abiotic stresses.

Small Heat Shock Proteins (sHSP) are molecular chaperones that play an essential role in maintaining protein homeostasis and promoting cell survival. In this work, for the first time, multiple cDNAs encoding for small Hsp27 and Hsp30, designed, respectively, as PbHsp27-(1-2) and PbHsp30-(1-5), were cloned and characterized in the amphibian Pelophylax bergeri, which is a suitable model for studying biological responses to environmental perturbations. Domain architecture analysis showed that PbHsp27 and PbHsp30 cDNAs displayed the typical signature motifs of the sHSP family such as the conserved α-crystallin domain flanked by variable N-terminal and C-terminal regions. Phylogenetic analysis revealed that PbHsp27 and PbHsp30 clustered, respectively, with Hsp27 and Hsp30 members of other vertebrates, but more closely with amphibians. Overall PbHsp27 and PbHsp30 transcriptional activity, analyzed by qRT-PCR, evidenced that, in ex vivo skin exposed to thermal shock and cadmium treatment, PbHsp27 and PbHsp30 mRNAs were inducible and regulated differently. This study provides the basis for future research on the potential use of PbHsp27 and PbHsp30 as biomarkers of proteotoxic stress in amphibians.

Longitudinal study of Amphibiocystidium sp. infection in a natural population of the Italian stream frog (Rana italica).

Mesomycetozoean-induced infections (order Dermocystida, genus Amphibiocystidium) in European and North American amphibians are causing alarm. To date, the pathogenicity of these parasites in field conditions has been poorly studied, and demographic consequences on amphibian populations have not been explored. In this study, an Amphibiocystidium sp. infection is reported in a natural population of the Italian stream frog (Rana italica) of Central Italy, over a 7-year period from 2008 to 2014. Light and electron microscope examinations, as well as partial 18S rDNA sequence analysis were used to characterize the parasite. Moreover, a capture-mark-recapture study was conducted to assess the frog demographics in response to infection. Negative effects of amphibiocystidiosis on individual survival and population fitness were absent throughout the sampling period, despite the high estimates of disease prevalence. This might have been due to resistance and/or tolerance strategies developed by the frogs in response to the persistence of Amphibiocystidium infection in this system. We hypothesized that in the examined R. italica population, amphibiocystidiosis is an ongoing endemic/epidemic infection. However, ecological and host-specific factors, interacting in a synergistic fashion, might be responsible for variations in the susceptibility to Amphibiocystidium infection of both conspecific populations and heterospecific individuals of R. italica.

High Levels of Variation Within Gene Sequences of Olea europaea L.

Gene sequence variation in cultivated olive (Olea europaea L. subsp. europaea var. europaea), the most important oil tree crop of the Mediterranean basin, has been poorly evaluated up to now. A deep sequence analysis of fragments of four genes, OeACP1, OeACP2, OeLUS and OeSUT1, in 90 cultivars, revealed a wide range of polymorphisms along all recognized allele forms and unexpected allele frequencies and genotype combinations. High linkage values among most polymorphisms were recorded within each gene fragment. The great sequence variability corresponded to a low number of alleles and, surprisingly, to a small fraction of genotype combinations. The distribution, frequency, and combination of the different alleles at each locus is possibly due to natural and human pressures, such as selection, ancestrality, or fitness. Phylogenetic analyses of allele sequences showed distant and complex patterns of relationships among cultivated olives, intermixed with other related forms, highlighting an evolutionary connection between olive cultivars and the O. europaea subspecies cuspidata and cerasiformis. This study demonstrates how a detailed and complete sequence analysis of a few gene portions and a thorough genotyping on a representative set of cultivars can clarify important issues related to sequence polymorphisms, reconstructing the phylogeny of alleles, as well as the genotype combinations. The identification of regions representing blocks of recombination could reveal polymorphisms that represent putatively functional markers. Indeed, specific mutations found on the analyzed OeACP1 and OeACP2 fragments seem to be correlated to the fruit weight.

New Evidences of Mitochondrial DNA Heteroplasmy by Putative Paternal Leakage between the Rock Partridge (Alectoris graeca) and the Chukar Partridge (Alectoris chukar).

The rock partridge, Alectoris graeca, is a polytypic species declining in Italy mostly due to anthropogenic causes, including the massive releases of the closely related allochthonous chukar partridge Alectoris chukar which produced the formation of hybrids. Molecular approaches are fundamental for the identification of evolutionary units in the perspective of conservation and management, and to correctly select individuals to be used in restocking campaigns. We analyzed a Cytochrome oxidase I (COI) fragment of contemporary and historical A. graeca and A. chukar samples, using duplicated analyses to confirm results and nuclear DNA microsatellites to exclude possible sample cross-contamination. In two contemporary specimens of A. graeca, collected from an anthropogenic hybrid zone, we found evidence of the presence of mtDNA heteroplasmy possibly associated to paternal leakage and suggesting hybridization with captive-bred exotic A. chukar. These results underline significant limitations in the reliability of mtDNA barcoding-based species identification and could have relevant evolutionary and ecological implications that should be accounted for when interpreting data aimed to support conservation actions.

Mitochondrial genome of Esox flaviae (Southern pike): announcement and comparison with other Esocidae.

Pikes are fish species belonging to order Esociformes, family Esocidae, genus Esox. Species of the genus Esox are common, large, and economically important for food and fishing activities. Recently, a new species, southern pike E. flaviae, was described for a well-studied area such as Italy, using also two mtDNA markers: cox1 and cytb. A scant number of remnant populations of the species persist in Italy, threatened by habitat loss and degradation and by competition and possible hybridization with E. lucius, massively and recurrently stocked to sustain angling pressure. The availability of new mtDNA markers will possibly contribute to the conservation of the species. Currently, whole mitogenome information for the genus is available only for E. lucius and for E. reichertii. The aim of the present paper is to report novel mitogenomic information for southern pike.

Short-term cadmium exposure induces stress responses in frog (Pelophylax bergeri) skin organ culture.

There have been a few studies on the negative effects of pollutants on amphibian skin, the first structural barrier that interacts with the environment and its potential contaminants. In this study an ex vivo skin organ culture from the amphibian Pelophylax bergeri was used to evaluate cell stress responses induced by short-term exposure to cadmium (Cd), a toxic heavy metal known to be an environmental hazard to both humans and wildlife. Histopathological studies were carried out on skin explants using light microscopy and changes in the expression of stress proteins, such as Metallothionein (MT) and Heat shock proteins (HSPs), were investigated by Real-time RT-PCR. Results revealed that amphibian skin reacts to Cd-induced stress by activating biological responses such as morphological alterations and dose- and time-dependent induction of Mt and Hsp70 mRNA expression, suggesting their potential role as biomarkers of exposure to Cd. This work provides a basis for a better understanding of the tissue-specific responses of amphibian skin as a target organ to Cd exposure and its in vitro use for testing potentially harmful substances present in the environment.

In planta expression of a mature Der p 1 allergen isolated from an Italian strain of Dermatophagoides pteronyssinus.

European (Dermatophagoides pteronyssinus) and American (Dermatophagoides farinae) house dust mite species are considered the most common causes of asthma and allergic symptoms worldwide. Der p 1 protein, one of the main allergens of D. pteronyssinus, is found in high concentration in mites faecal pellets, which can became easily airborne and, when inhaled, can cause perennial rhinitis and bronchial asthma. Here we report the isolation of the Der p 1 gene from an Italian strain of D. pteronyssinus and the PVX-mediated expression of its mature form (I-rDer p 1) in Nicotiana benthamiana plants. Human sera from characterized allergic patients were used for IgE binding inhibition assays to test the immunological reactivity of I-rDer p 1 produced in N. benthamiana plants. The binding properties of in planta produced I-rDer p 1 versus the IgE of patients sera were comparable to those obtained on Der p 1 preparation immobilized on a microarray. In this paper we provide a proof of concept for the production of an immunologically active form of Der p 1 using a plant viral vector. These results pave the way for the development of diagnostic allergy tests based on in planta produced allergens.

Molecular and phenotypic evidence of a new species of genus Esox (Esocidae, Esociformes, Actinopterygii): the southern pike, Esox flaviae.

We address the taxonomic position of the southern European individuals of pike, performing a series of tests and comparisons from morphology, DNA taxonomy and population genetics parameters, in order to support the hypothesis that two species of pike, and not only one, exist in Europe. A strong relationship emerged between a northern genotype supported by COI, Cytb, AFLP and specific fragments, and a phenotype with round spot skin colour pattern and a large number of scales in the lateral line, clearly separated from a southern genotype with other skin colour pattern and a low number of scales in the lateral line. DNA taxonomy, based on a coalescent approach (GMYC) from phylogenetic reconstructions on COI and Cytb together with AFLP admixture analysis, supported the existence of two independently evolving entities. Such differences are not simply due to geographic distances, as northern European samples are more similar to Canadian and Chinese samples than the southern Europe ones. Thus, given that the differences between the two groups of European pike are significant at the phenotypic, genotypic and geographical levels, we propose the identification of two pike species: the already known northern pike (Esox lucius) and the southern pike (E. flaviae n.sp.). The correct identification of these two lineages as independent species should give rise to a ban on the introduction of northern pikes in southern Europe for recreational fishing, due to potential problems of hybridisation.

Muscle actin isoforms are differentially expressed in human satellite cells isolated from donors of different ages.

Myogenesis is mainly sustained by a subpopulation of myogenic cells known as satellite cells (SC). In this paper we studied alpha-smooth muscle (alphaSMA) and alpha-sarcomeric muscle (alphaSRA) actin isoform expression in cultures of human satellite cells (HSC) isolated from skeletal muscle biopsies from a 5-day-old newborn, a 34-year-old young adult and a 71-year-old donor. Myogenicity of cultures was assessed using immunocytochemical detection of desmin and myosin heavy chain. Time-course expression of alphaSRA and alphaSMA were studied with both immunocytochemistry and western blotting procedures. Although alphaSMA was never detected in whole skeletal muscle, both alphaSMA and alphaSRA were detected in proliferating and differentiating HSC derived from donors of all examined ages. The expression level experiments showed that alphaSRA was gradually up-regulated during HSC differentiation, but no significant differences were observed between newborn, young, and elderly HSC cultures. Our data demonstrated that HSC, isolated from subjects of different ages, re-expressed alphaSMA, but its levels and expression pattern varied considerably in the newborn with respect to the young adult and elderly donors. These results are discussed in relation to the myogenic differentiation capability of HSC during human muscle senescence.

Low molecular weight phosphotyrosine protein phosphatase from PC12 cells. Purification, some properties and expression during neurogenesis in vitro and in vivo.

The purification and partial characterization of low molecular weight phosphotyrosine phosphatase (LMW-PTP) was reported for the first time in PC12 cells. In addition, the expression levels during neuronal phenotype induction by nerve growth factor (NGF) and during neurogenesis in chick embryos were investigated. LMW-PTP was purified to homogeneity and showed a single band of about 18 kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis. A native molecular mass of 20.1 kDa was determined by gel filtration on Sephadex G-75 column. The LMW-PTP from PC12 cells displays structural and biochemical characteristics similar to the enzyme isolated for normal tissues. It was specifically immunoprecipitated by an affinity purified antibody directed against the bovine liver enzyme. The enzyme is present in the cytosolic and cytoskeletal cell compartment where is tyrosine phosphorylated. Time course expression of LMW-PTP in PC12 cells was investigated after NGF treatment and showed an increase of about 30% in the basal level of LMW-PTP from 0 to 72 h. These changes were related to the appearance in PC12 cells of neuronal processes and to a decrease in cell proliferation. An increase of the LMW-PTP expression was also observed in vivo during chick embryo neurogenesis from 8-day-old embryos to adult chicks. The protein level, assayed by immunoblotting, increases from 14-day-old embryos to the hatched chicks reaching the adult levels within the first week after birth. These data indicate that the neurogenesis process is accompanied by a physiological increment of LMW-PTP expression in vitro and in vivo.