Francisella novicida infection in a patient with pulmonary infection and pancreatitis in Italy.

Tularemia is a rare but potentially life threatening zoonotic disease caused by Francisella tularensis. F. novicida, previously considered a subspecies of F. tularensis, is currently considered a separate species. Human infections related to F. novicida are exceedingly rare but can cause morbidity and mortality in debilitated or immunocompromised individuals.A 42-year-old male presented at the hospital with vomiting, dehydration, constipation and pain in the right iliac fossa. He was first diagnosed with pancreatitis and admitted for further analysis. Chest computerized tomography scan showed the presence of parenchymal consolidation in the left upper and lower lobes of the lung with pleural effusion. Blood cultures isolated a Gram-negative coccobacillus, that was at first identified by MALDI-TOF as Francisella tularensis. Serological analysis for the detection of total antibodies against F. tularensis and Real-Time PCR targeting the gene coding for 23 kDa, resulting negative. Subsequently, PCR targeting helicases and tul4 genes, and the Regions of Difference RD1 and RD6 were performed allowing the identification of F. novicida. The isolate was further genetically characterized by whole genome sequencing (WGS).This is the first reported case of human infection caused by F. novicida in Italy.Given the rarity of human cases and the lack of specific symptoms, this pathogen is difficult to identify and the diagnosis can be extremely challenging. In this case report, despite the lack of amplification of the gene encoding for 23 kDa protein, the identification of Francisella species was achieved with the amplification of different genes and characterized by WGS.

Comparative analysis of two genomes of Chlamydia pecorum isolates from an Alpine chamois and a water buffalo.

BACKGROUND: To date, whole genome sequencing has been performed mainly for isolates of Chlamydia trachomatis, C. pneumoniae, C. psittaci and C. abortus, but only a few isolates of C. pecorum have been entirely sequenced and this makes it difficult to understand its diversity and population structure. In this study the genome of two C. pecorum strains isolated from the lung of an Alpine chamois affected with pneumonia (isolate PV7855) and the brain of a water buffalo affected with meningoencephalomyelitis (isolate PV6959), were completely sequenced with MiSeq system (Illumina) and analyzed in their most polymorphic regions. RESULTS: The genome length and GC content of the two isolates were found to be consistent with other C. pecorum isolates and the gene content of polymorphic membrane proteins and plasticity zone was found to be very similar. Some differences were observed in the phospholipase genes for both isolates and in the number of genes in the plasticity zone, such as the presence of some hypothetical proteins in PV6959, not present in any other genomes analyzed in this study. Interestingly, PV6959 possesses an extra pmp and has an incomplete tryptophan biosynthesis operon. Plasmids were detected in both isolates. CONCLUSIONS: Genome sequencing of the two C. pecorum strains did not reveal differences in length and GC content despite the origin from different animal species with different clinical disease. In the plasticity zone, the differences in the genes pattern might be related to the onset of specific symptoms or infection of specific hosts. The absence of a tryptophan biosynthesis pathway in PV6959 may suggest a strict relationship between C. pecorum and its host.

OCCURRENCE OF EUSTRONGYLIDES EXCISUS (NEMATODA:DIOCTOPHYMATIDAE) IN EUROPEAN PERCH (PERCA FLUVIATILIS) AND GREAT CORMORANT (PHALACROCORAX CARBO) IN LAKE ANNONE, NORTHERN ITALY.

The genus Eustrongylides includes zoonotic nematodes that infect fish species and fish-eating birds of freshwater ecosystems. This study aimed to evaluate the occurrence of Eustrongylides in the paratenic host Perca fluviatilis (European perch) and in the definitive host, Phalacrocorax carbo sinensis (great cormorant), in Lake Annone, a shallow eutrophic lake located in the pre-mountainous area of the Alps in northwest Italy where wintering cormorants coexist with new breeding colonies. A total of 114 European perch and 48 cormorants were examined for the occurrence of Eustrongylides. All parasites collected were identified with microscopic examination and molecular analysis. Overall, 11 specimens of European perch (9.6%) and 13 individuals of cormorants (27%) harbored nematodes identified as fourth-stage larvae and adults of Eustrongylides excisus. The observed prevalence of Eustrongylides spp. appears to be intermediate between the higher values in cormorant breeding areas in northern Europe and the lower prevalence observed in their wintering sites in southernmost Europe. Considering the eutrophication status of freshwater ecosystems and the increasing population of the cormorants, Eustrongylides has an increasing potential range of dispersion in Europe, including Italy; thus an extensive surveillance should be carried out, especially given the zoonotic potential of this nematode.

Detection of Chlamydial DNA from Mediterranean Loggerhead Sea Turtles in Southern Italy.

Chlamydiae are obligate intracellular bacteria that include pathogens of human and veterinary importance. Several reptiles were reported to host chlamydial agents, but pathogenicity in these animals still needs clarification. Given that only one report of chlamydiosis was described in sea turtles, and that chlamydiae might also be detected in hosts without clinical signs, the current study examined asymptomatic Mediterranean loggerhead sea turtles for the presence of chlamydial DNA. Twenty loggerhead sea turtles, rehabilitated at the Marine Turtle Research Centre (Portici, Italy), were examined collecting ocular-conjunctival, oropharyngeal and nasal swabs. Samples were processed through quantitative and conventional PCR analyses to identify Chlamydiales and Chlamydiaceae, with particular attention to C. pecorum, C. pneumoniae, C. psittaci, and C. trachomatis. Although it was not possible to determine the species of chlamydiae involved, the detection of chlamydial DNA from the collected samples suggests that these microorganisms might act as opportunistic pathogens, and underlines the role of sea turtles as potential carriers. This study highlights the presence of chlamydial agents in sea turtles, and encourages further research to fully characterize these microorganisms, in order to improve the management of the health and conservation of these endangered species, and prevent potential zoonotic implications.

Chlamydia Species and Related Risk Factors in Poultry in North-Western Italy: Possible Bird-to-Human Transmission for C. gallinacea.

Chlamydiaceae are obligatory intracellular bacteria causing acute and chronic diseases in animals and humans worldwide, with recently discovered species with a still unclear pathogenic potential (i.e., C. gallinacea). In Italy, Chlamydiaceae infections are underestimated both in animals and humans. To estimate the prevalence of Chlamydiaceae species in poultry and occupationally exposed workers on farm, a cross-sectional study was carried out in north-western Italy. A total of 2063 samples from 83 commercial and 31 backyard poultry farms were analysed using real-time PCRs for Chlamydiaceae screening and species typing. Chlamydiaceae were detected in 23 farms, with a herd prevalence of 20.2% (95%CI: 13.2-28.7), higher in backyard farms (38.7%; 95%CI: 21.8-57.8) compared to commercial ones (13.3%; 95%CI: 6.8-22.5). C. gallinacea was found in 18 chicken farms, both commercial and backyard, and C. psittaci only in 3 backyard farms. Exposure to wild birds and factors related to biosecurity resulted the main risk factors associated with Chlamydia positivity. Out of the 113 sputum samples collected from farmers, 16 tested positive to Chlamydiaceae, with a prevalence of 14.2% (95%CI: 8, 3-22). To the best of our knowledge, for the first time at international level, C. gallinacea was detected in humans with farmer positivity associated with farm infectious status, suggesting a bird-to-human transmission.

Complete Genome Sequence of Chlamydia avium PV 4360/2, Isolated from a Feral Pigeon in Italy.

We report here the whole-genome sequence of a Chlamydia avium isolate recovered from a feral pigeon in 1999 in Italy. Only one complete genome of a C. avium strain has been published so far. Future comparative analyses could provide valuable insights on the genomic evolution of the pathogen.

The influence of centrifugation and incubation temperatures on various veterinary and human chlamydial species.

The Chlamydiaceae are Gram-negative bacteria causing diseases in humans and in both, endothermic (mammals and birds) and poikilothermic (e.g. reptiles, amphibians) animals. As most chlamydial species described today were isolated from humans and endothermic animals, the commonly used culturing temperature in vitro is 37 °C, although the centrifugation temperature during experimental infection, a technique necessary to improve the infection rate, may vary from 25 to 37 °C. The aim of this study was to investigate the influence of different centrifugation (28° or 33 °C) and incubation temperatures (28 °C or 37 °C) on the average inclusion size, infectivity and ultrastructural morphology of human and animal chlamydial strains, as well as two recently described species originating from snakes, C. poikilothermis and C. serpentis, in LLC-MK2 cells at 48 h post infection. Infectivity and average inclusion size was reduced at an incubation temperature of 28 °C compared to 37 °C for all strains including C. poikilothermis, although the latter formed larger, fully matured inclusions at 28 °C in comparison to the other investigated Chlamydia species. C.psittaci displayed a shorter developmental cycle than the other species confirming previous studies. Higher centrifugation temperature increased the subsequent inclusion size of C. trachomatis, C. abortus and C. suis but not their infectivity, while the incubation temperature had no discernable effect on the morphology, inclusion size and infectivity of the other chlamydial strains. In conclusion, we found that all Chlamydia species are viable and can grow at low incubation temperatures, although all strains grew better and more rapidly at 37 °C compared to 28 °C.

Lack of viable parasites in cured 'Parma Ham' (PDO), following experimental Toxoplasma gondii infection of pigs.

Twelve Large White pigs were experimentally infected with 1000 Toxoplasma gondii oocysts/each. Serology was carried out at different time points post infection (p.i.) and animals were slaughtered at four months p.i. One of two thighs was examined for T. gondii infection status by PCR and bioassay in mice. The other thigh was processed for Parma ham production. Four thighs were examined after twelve months of curing, four after fourteen months and four were examined after sixteen months. Cured hams were analyzed by PCR, bioassay and in-vitro cultivation on Vero cells followed by real-time PCR. Pigs seroconverted from day 21 p.i. Bioassays were positive for all fresh thighs, but negative for cured hams. PCR was positive for parasite DNA from most thighs both at slaughter and post curing, but parasite growth was not observed following in vitro cultivation and real-time PCR. Results indicate that the curing process of Parma Ham (PDO), when carried out according to the Parma Ham consortium regulations, can inactivate T. gondii tissue cysts. Results would suggest that food-borne transmission of T. gondii to consumers from Parma ham can be excluded.