Diagnostic challenges for Aelurostrongylus abstrusus infection in cats from endemic areas in Italy

Background: The lungworm Aelurostrongylus abstrusus infects wild and domestic feline species worldwide and is considered a primary respiratory parasite of cats. Definitive diagnosis is based on the identification of first-stage larvae (L1s) released in faeces approximately 5 to 6 weeks after infection. More recently, serology has been shown to be a diagnostic alternative for A. abstrusus infection in cats. The present study aimed at evaluating the diagnostic performance of serological antibody detection compared to faecal examination for A. abstrusus infection in a population of cats with known infection status from endemic areas in Italy and to identify factors (larval scores, age, co-infections with other helminths) that may influence test sensitivity and specificity of serology. Methods: All cats resulting positive using the Baermann technique (n = 78) were tested with the A. abstrusus ELISA. An additional 90 serum samples from cats living in three geographical areas with infection prevalence > 10%, but that resulted negative on Baermann, were also tested. Results: Among 78 cats copromicroscopically positive for L1s of A. abstrusus (Group 1), 29 (37.2%) were seropositive in ELISA. Of the 90 cats from Group 2 (cats living in three geographical areas in Italy with A. abstrusus prevalence > than 10%, but negative on Baermann examination), 11 (12.2%) were positive on ELISA. The overall seroprevalence was 23.8%. There was no statistical difference either between average optical density (OD) values of cats excreting > 100 L1s vs. cats excreting < 100 L1s (0.84 vs. 0.66; P value = 0.3247) or comparing the OD values with age of infected cats. Few Baermann-negative cats positive for Toxocara cati or hookworms were seropositive, supporting lack of cross-reactivity to these nematodes. Conclusions: Results from the present study suggest that relying solely on faecal examination may underestimate prevalence of A. abstrusus infection in cats and that field surveys based on antibody detection are useful for establishing true prevalence of infected and/or exposed animals. Graphical Abstrac

Shiga toxin-producing Escherichia coli O157, O26 and O111 in cattle faeces and hides in Italy

Introduction: Ruminants are regarded as the natural reservoir for Shiga toxin-producing Escherichia coli (STEC), especially of serogroup O157. Materials and methods: During 2011 and 2012, 320 samples (160 faecal samples from the rectum and 160 hide samples from the brisket area) were collected from 160 cattle at slaughter in Northern Italy during warm months (May to October). Cattle were reared in different farms and their age at slaughter ranged between nine months and 15 years, most of them being culled cattle (median age: six years; average age: 4.6 years). Samples were tested by immunomagneticseparation technique for E coli O157 and O26 and by a screening PCR for stx genes followed by cultural detection of STEC. The virulence genes stx1, stx2, eae, and e-hlyA were detected and among stx2-positive isolates the presence of the stx2a and stx2c variants was investigated. Results: Twenty-one of 160 cattle (13.1 per cent; 95 per cent CI 8.3 to 19.4 per cent) were found to be faecal carriers of STEC. STEC O157 was found in 10 (6.3 per cent) samples, STEC O26 in six (3.8 per cent) and STEC O111 in one (0.6 per cent). Four isolates (2.5 per cent) were O not determined (OND). Six out of 160 (3.8 per cent; 95 per cent CI 1.4 to 8.0 per cent) hide samples were positive for STEC; four hides (2.5 per cent) were contaminated by STEC O157 and two (1.3 per cent) by STEC O26. In three cattle (1.9 per cent) STEC from both faeces and hides were detected. Among STEC O157, 87.5 per cent of them carried the stx2c gene and 12.5 per cent carried both stx1 and stx2c genes. No O157 isolate harboured stx2a variant. STEC O26 and O111 carried the stx1 gene only. One OND strain carried both the stx2a and stx2c genes. Conclusions: This study shows that STEC O157 from cattle can harbour the stx2c variant, which is associated with haemolytic uraemic syndrome in humans, and that cattle hides may be a source of human pathogenic STEC O157 and O26 in the slaughterhouse environment

Arbovirus Screening in Mosquitoes in Emilia-Romagna (Italy, 2021) and Isolation of Tahyna Virus

Several viruses can be transmitted by mosquitoes. We searched some of these viruses in 20,778 mosquitoes, collected in 95 traps on the plains of Emilia-Romagna (North of Italy) in 2021. We detected West Nile virus (WNV) and Usutu virus (USUV) in pools of Culex (Cx.) pipiens. In addition, we detected two insect-specific flaviviruses in three pools of Aedes (Ae.) caspius and in two of Ae. vexans. Tahyna virus (TAHV) was detected in six pools, three of Ae. caspius and three of Cx. pipiens, and one isolated strain was obtained from one of the Ae. caspius pools. Moreover, we detected TAHV in pools of several mosquito species (Ae. caspius, Ae. vexans, Ae. albopictus, Anopheles maculipennis s.l.) collected in the previous year of surveillance. Our data indicate Ae. caspius as the species most infected with TAHV in the surveyed area. Together with the likely plasticity of the cycle, we reported strong genome stability of the TAHV, probably linked to a successful adaptation of the virus to its ecological niche. Interestingly, in six pools of Cx. pipiens we detected two associated viruses among USUV, WNV, TAHV and all the three viruses in two pools. This result allows us to assume the presence of particular conditions that prompt the circulation of arboviruses, creating the conditions for viral hot spots. While no human diseases related to Tahyna virus were reported in Italy, its detection over the years suggests that it is worth investigating this virus as a potential cause of disease in humans in order to assess its health burden. IMPORTANCE We reported in this work the detection of three Arboviruses (Arthropod-borne viruses) in mosquitoes collected in Emilia-Romagna in 2021. In addition to West Nile and Usutu viruses, which were reported from more than 10 years in the study area, we detected and isolated Tahyna virus (TAHV). We also reported detections of TAHV obtained in previous years of surveillance in different species of mosquitoes. TAHV is the potential causative agent of summer influenza-like diseases and also of meningitis. Even if human cases of disease referable to this virus are not reported in Italy, its relevant presence in mosquitoes suggests investigating the possibility they could

Antimicrobial resistance, biofilm synthesis and virulence genes in Salmonella isolated from pigs bred on intensive farms

Salmonella is the second cause of foodborne infection in humans in the USA and Europe. Pigs represent the second most important reservoir for the pathogen and the consumption of pork meat is a major risk factor for human salmonellosis. Here, we evaluated the virulence patterns of eleven Salmonella isolated from pigs (carcasses and faces) bred in intensive farms in the north of Italy. The two serotypes identified were S. Typhimurium and its monophasic variant 1,4,5,12:i:-. None of the isolates was an ESBL producer, as confirmed also by PCR. However, the presence of a multidrug resistant pattern was evident, with all the isolates being resistant to at least to five antimicrobial agents belonging to various classes. Moreover, six out of eleven isolates showed important resistance profiles, such as resistance against colistin and ciprofloxacin, with nine to twelve recorded resistances. The isolates were negative for the biofilm synthesis test, while four different virulotypes were characterized. All the isolates showed the presence of invA, hilA, stn, ssrA, sipC. One sample also harbored ssaR and spvC genes. One strain was positive for all the virulence genes tested and was resistant to 12 antimicrobial agents. The present study contributes new data to the surveillance program for antibiotic resistance. Furthermore, the presence of eleven highly virulent isolates poses concern for human health in relation to their diffusion in the environment

Exposure to amitraz, fipronil and permethrin affects cell viability and ABC transporter gene expression in an Ixodes ricinus cell line

Background: Over-expression of ATP-binding cassette (ABC) transporter proteins has been implicated in resistance of ticks to acaricides. Tick cell lines are useful for investigating resistance mechanisms, as development of an in vitro model for the study of acaricide resistance would contribute to improving knowledge of the molecular basis behind drug processing and exclusion in ticks. In the present study, cultures of the Ixodes ricinus-derived cell line IRE/CTVM19 were treated with the acaricides amitraz, permethrin or fipronil to determine modulation of ABC transporter gene expression. Cells were treated with different drug concentrations (25, 50, 100, 150 \u3bcM) and incubated for ten days. Cell morphology, viability, metabolic activity and relative expression of ABC (B1, B6, B8 and B10) genes were determined at day 10 post-treatment. Results: Cell morphology determined by light microscopy was altered following treatment with all drugs, but only at high concentrations, while total cell numbers decreased with increasing drug dose. Cell viability determined by trypan blue exclusion was not significantly different from untreated controls (P > 0.1) following treatment with amitraz and permethrin, but high concentrations of fipronil caused decrease (up to 37%, P < 0.01) in viability. At all drug concentrations, fipronil and permethrin induced dose-dependent reduction in cell metabolic activity measured by MTT assay (P < 0.01). Quantitative RT-PCR showed that the drugs significantly affected expression of ABC genes. In particular, fipronil treatment downregulated ABCB1 (P < 0.001) and upregulated ABCB6, ABCB8 and ABCB10 (P < 0.01); amitraz treatment down regulated ABCB1 (significant difference between 25 and 150 \u3bcM, P < 0.001) and upregulated ABCB8 and ABCB10 at lower concentrations (25 and 50 \u3bcM, P < 0.05); and permethrin upregulated ABCB6, ABCB8 and ABCB10 only at 150 \u3bcM (P < 0.01). Conclusions: The adverse effects on cell viability and metabolic activity, and changes in expression of different ABC transporter genes, detected in IRE/CTVM19 cells following treatment with amitraz, permethrin and fipronil, support the proposed application of tick cell lines as in vitro models for the study of resistance to these acaricides in ticks

Prevalence and risk factors associated with cat parasites in Italy: a multicenter study

Background: Parasites that infect cats include protozoa, helminths and arthropods, many of which are transmissible to humans. Effective control relies on a good knowledge of parasite distribution and the risk factors for infection. The present study was aimed at evaluating the prevalence of major feline parasites in Italy and the risk factors associated with their occurrence. Methods: Over a 12-month study period, feces, hair and ectoparasites from naturally infected cats from feral colonies, shelters and private households were analyzed at 13 study centers across Italy. Samples from these cats (n = 987) were analyzed at all centers using the same diagnostic methods. Prevalence values and risk factors were evaluated statistically for the identification of predictors of risk. Results: The overall prevalence of gastro-intestinal and broncho-pulmonary (BP) nematodes was 35.9% (354/987). Toxocara cati was the most prevalent species (253/987; 25.6%), followed by Ancylostomatidae (98/987; 9.9%). Among BP nematodes, Aelurostrongylus abstrusus was the most common (76/987; 7.7%). Approximately 35.7% (352/987) of the study population was infested by ectoparasites, of which the most common were fleas (29.4%, 290/987), followed by ear mites Otodectes cynotis (9.8%, 97/987). Predictors of risk for parasite infection included age, a predominantly or exclusively outdoor lifestyle, geographic area and lack of antiparasitic treatment. Conclusions: Both ecto- and endoparasites are still common in cats throughout Italy, many of them being of zoonotic concern and vectors of pathogens to humans. Given the presence of parasites throughout the entire study period, year-round treatment should be considered. Furthermore, data confirm the need to protect the human–animal bond using proper endo- and ectoparasiticides to reduce the risk of human infection, in application of the One-Health concept