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.

Parasitological Survey in European Brown Hare (Lepus europaeus Pallas, 1778) Breeding Facilities in Southern Italy.

Parasites are considered important regulating factors of hosts' population dynamics, not only in free-ranging wildlife, but also in captive bred animals. To date, only few studies have been carried out to assess the parasitic communities of the European brown hare in Southern Italy, and only one focused on animals in captivity. The aim of the present survey was to assess the composition of the endoparasite community in game hares bred for restocking purposes. For this purpose, 215 fecal pools collected in eight different breeding facilities were examined by qualitative and quantitative coprological techniques. Parasites characterized by a direct life cycle, including six species of coccidia from the genus Eimeria and the nematode Trichostrongylus retorataeformis, proved to be the most prevalent parasites. Further helminthic infestations by Passalurus ambiguous, Strongylosides papillosus, Cittotenia spp. and Dicrocoelium dendriticum were also detected, but with an overall prevalence lower than 20%. The present study contributes to increasing knowledge on the health status of a poorly investigated species, and is useful for optimizing breeding efforts in captivity.

The Use of Ascophyllum nodosum and Bacillus subtilis C-3102 in the Management of Canine Chronic Inflammatory Enteropathy: A Pilot Study.

The aim was to assess the effects of Ascophyllum nodosum (AN) with/without Bacillus subtilis C-3102 as alternative treatments for Chronic Inflammatory Enteropathy (CIE) of dogs. Fourteen CIE patients, which had received the same control (CTR) diet, were enrolled to serially receive three diets: (1) hydrolysed protein (HP) diet; (2) 4.0% AN supplemented HP (HPA) food, (3) HPA diet fortified with 125 billion B. subtilis C-3102 spores/10 kg body weight (HPAB diet). Clinical outcome was assessed by Canine Inflammatory Bowel Disease Activity Index (CIBDAI), whereas gut microbiota compositional variations were investigated via 16S rRNA gene analysis, and faecal fermentation end-products by liquid chromatography. Higher abundances of the Ruminococcaceae and Rikenellaceae families were shown in HPA relative to CTR treatment, with Bacillus genus being differentially abundant on HPAB diet. Concentrations of acetate were higher (p < 0.05) in dogs fed HPA compared to CTR diet, and amounts of isovalerate and isobutyrate were greater (p < 0.05) in HPA compared to HP food. A tendency for higher amounts of faecal butyrate was found for the HPAB treatment (p = 0.06). Comprehensively, while displaying potentially positive effects on faecal fermentations, the tested substances failed to improve CIBDAI scores and microbial richness in CIE dogs.

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.

Morishitium polonicum as a Cause of Severe Respiratory Disease in Eurasian Blackbirds (Turdus merula) in Central Italy.

Two Eurasian Blackbirds (Turdus merula) from central Italy were found with severe cyclocoelid trematodosis associated with airsacculitis. The birds were submitted with severe respiratory distress; one died shortly after hospitalization, while the second bird was euthanized. At necropsy, a massive presence of cyclocoelid flukes was observed in the coelomic cavity and air sacs of both birds. The air sacs were diffusely opaque, thickened, and covered by scant fibrinous exudate mixed with numerous parasites. Histologically, the air sacs showed diffuse and severe oedema with fibrinous exudate. Diffuse mononucleated and heterophilic infiltration mixed with multiple granulomas contained degenerated trematodes. Morishitium polonicum was identified using morphologic keys and molecular analysis of extracted DNA. Infections caused by M. polonicum are poorly documented in blackbirds and the findings in these birds support the pathogenic role of this trematode as a potential cause of death in blackbirds in Italy. Extended epidemiologic surveys are required to properly assess the potential importance of M. polonicum as a life-threatening pathogen in Blackbird populations.

Anthelmintic Efficacy and Pharmacokinetics of Ivermectin Paste after Oral Administration in Mules Infected by Cyathostomins.

Ivermectin (IVM) is an anthelmintic compound commonly used off-label in mules due to its broad-spectrum of activity. Despite the general use of IVM in mules with the same dose and route of administration licensed for horses, significant pharmacokinetic differences might exist between horses and mules, as already observed for donkeys. The aim of the present study was to evaluate the pharmacokinetic profile and anthelmintic efficacy of an oral paste of IVM in mules naturally infected with cyathostomins. Fifteen adult mules with fecal egg counts (FEC) ≥200 eggs per gram (EPG), with exclusive presence of cyathostomins, were included in the study. All mules were orally treated with IVM according to the manufacturer's recommended horse dosage (200 µg/kg body weight). FECs were performed before (day-10 and day-3) and after treatment at days 14 and 28 by using a modified McMaster method. The FEC reduction (FECR%) was also calculated. Blood samples were collected from five animals at various times between 0.5 h up to 30 days post treatment to determine pharmacokinetic parameters. The maximum IVM serum concentration (Cmax) was 42.31 ± 10.20 ng/mL and was achieved at 16.80 ± 9.96 hours post-treatment (Tmax), area under the curve (AUC) was 135.56 ± 43.71 ng × day/mL. FECR% remained high (>95%) until the 28th day.

Absence of Viable Toxoplasma gondii in Artisanal Raw-Milk Ewe Cheese Derived from Naturally Infected Animals.

The presence of viable Toxoplasma gondii was investigated in artisanal cheeses made from milk of naturally infected ewes. Ewe milk was analyzed beforehand for the presence and vitality of T. gondii by loop-mediated isothermal amplification (LAMP) and reverse-transcriptase PCR (RT-PCR), respectively. Cheeses were prepared from raw milk following a traditional cheesemaking process. The cheese obtained from T. gondii-positive milk was analyzed by LAMP to detect Toxoplasma DNA-positive samples. RT-PCR was then carried out to assess the viability of the parasites in T. gondii-positive milk samples and fresh cheese, after 5 and 15 days of ripening. Physical-chemical parameters of cheeses were also investigated. All cheese samples derived from T. gondii-positive milk were positive according to LAMP, at both 5 and 15 days of ripening, while none of the samples were positive according to RT-PCR. Thus, while the presence of the parasite was demonstrated by the detection of specific DNA, the absence of detectable T. gondii RNA supports the hypothesis that changes in the chemical and physical characteristics occurring during the cheesemaking process and ripening period, could be sufficient to inactivate viable T. gondii in milk, minimizing the risk of human infection through consumption of raw sheep milk cheese.

Emerging feline vector-borne pathogens in Italy.

BACKGROUND: The epidemiology of feline vector-borne pathogens (FeVBPs) has been less investigated in cats than in dogs. The present study assessed the prevalence of Rickettsia spp., Babesia spp., Cytauxzoon spp. and Leishmania infantum infections in cat populations living in central Italy, by molecular and serological tools. RESULTS: A total of 286 healthy cats were randomly selected from catteries and colonies in central Italy. Peripheral blood and conjunctival swab (CS) samples were collected during surgical procedures for regional neutering projects. Sera were analysed by IFAT to detect anti-Rickettsia felis, R. conorii, Babesia microti and Leishmania IgG antibodies using commercial and home-made antigens. DNA extracted from buffy coats (BCs) was tested for Rickettsia spp., and Piroplasmida species, including Cytauxzoon spp. and Babesia spp. by PCR. Buffy coats and CS samples were assayed by a nested (n)-PCR for Leishmania spp. Sixty-two cats (21.67%) were seropositive to at least one of the tested pathogens. The serological assay revealed 23 (8.04%) and 18 (6.29%) positive cats for R. felis and R. conorii, respectively, with low titers (1/64-1/128). No antibodies against B. microti were detected. Neither Rickettsia nor Piroplasmida DNA were amplified using the specific PCR assays. Thirty-one cats (10.83%) tested positive to anti-Leishmania IgG, with titers ranging from 1:40 to 1:160 and 45 animals (15.73%) tested positive to Leishmania CS n-PCR, whereas none of the animals tested positive to BC n-PCR. Considering the results obtained by IFAT and CS n-PCR, a moderate agreement between the two tests was detected (κ = 0.27). CONCLUSIONS: The results of the serological and molecular surveys showed a moderate exposure to Leishmania in the investigated cats and highlighted the limited molecular diagnostic value of BC versus CS samples for this pathogen. Conversely no evidence supported the circulation of Cytauxzoon spp. in domestic cats, in contrast with previous detections in European wild cats in the same areas monitored. The low positive titres for R. felis in association with no DNA BC amplification prevent speculation on the exposure of feline populations to this FeVBP due to the cross-reactivity existing within spotted fever group rickettsiosis (SFGR).

Molecular Survey on Rickettsia spp., Anaplasma phagocytophilum, Borrelia burgdorferi Sensu Lato, and Babesia spp. in Ixodes ricinus Ticks Infesting Dogs in Central Italy.

Dogs are a common feeding hosts for Ixodes ricinus and may act as reservoir hosts for zoonotic tick-borne pathogens (TBPs) and as carriers of infected ticks into human settings. The aim of this work was to evaluate the presence of several selected TBPs of significant public health concern by molecular methods in I. ricinus recovered from dogs living in urban and suburban settings in central Italy. A total of 212 I. ricinus specimens were collected from the coat of domestic dogs. DNA was extracted from each specimen individually and tested for Rickettsia spp., Borrelia burgdorferi sensu lato, Babesia spp., and Anaplasma phagocytophilum, using real-time and conventional PCR protocols, followed by sequencing. Sixty-one ticks (28.8%) tested positive for TBPs; 57 samples were infected by one pathogen, while four showed coinfections. Rickettsia spp. was detected in 39 specimens (18.4%), of which 32 were identified as Rickettsia monacensis and seven as Rickettsia helvetica. Twenty-two samples (10.4%) tested positive for A. phagocytophilum; Borrelia lusitaniae and Borrelia afzelii were detected in two specimens and one specimen, respectively. One tick (0.5%) was found to be positive for Babesia venatorum (EU1). Our findings reveal the significant exposure of dogs to TBPs of public health concern and provide data on the role of dogs in the circulation of I. ricinus-borne pathogens in central Italy.

Sequence variation in the B1 gene among Toxoplasma gondii isolates from swine and cats in Italy.

The evaluation of the genetic variations of Toxoplasma gondii among isolates of a wide variety of animal hosts can provide significant information for better understanding the epidemiology and population structure of the parasite in different geographical areas. The aim of this study was to provide information on T. gondii genetic diversity in host species living in central Italy, which could act as a potential source of human infection. Seventy-seven feline faecal samples, and 36 and 20 diaphragm pillar tissue samples from pigs and wild boars were collected in Umbria (central Italy). The samples were tested by a nested-PCR protocol amplifying an informative region within the B1 gene, a multi-copy genetic target, showing a good rate of variability. Thirty-six specimens (27.07%) belonging to 10 pigs, 13 wild boars and 13 cats, tested positive to the B1 nested-PCR screening. Of these, 23 good quality sequences (8 from wild boars, 5 from pigs, and 10 from cats) were analyzed. A comparison of the B1 DNA sequences showed that a single homogeneous nucleotide substitution (C/T) was present at position 31 in the isolates from pigs and wild boars compared with the sampled cats and other hosts (including humans) available in GenBank™. The present results suggest the existence of a T. gondii genetic diversity for swine host species, based on a SNP (C/T) of the B1 gene. Further studies are needed to draw more solid conclusions on the discriminatory power of the B1 target by collecting more swine samples from much broader geographical areas.

Comparison of PCR assays targeting the multi-copy targets B1 gene and 529 bp repetitive element for detection of Toxoplasma gondii in swine muscle.

The comparison of the sensitivities of two molecular assays designed to target the multi-copy sequences of the Toxoplasma gondii genomic B1 region and 529 bp-RE respectively, in detecting T. gondii in swine muscle was assessed. Diaphragm pillars were obtained from 498 slaughtered pigs managed in intensive farms in Central Italy. Genomic DNA was extracted from the tissues and T. gondii-B1 and 529 bp-RE sequences were amplified by specific PCR protocols. Toxoplasma gondii DNA was detected in 165 samples (33.13%). There was a good correlation (κ = 0.77) between the results obtained targeting the two different genetic markers, however the 529 bp RE-PCR assay overall detected a significantly higher (P < 0.05) number of T. gondii-positive samples (150 samples) than the B1-PCR protocol (134). Our results show that: i) standardized B1 and 529 bp-RE PCRs applied to muscle tissues can detect a high rate of T. gondii-infection; ii) a multi-target PCR approach is recommended for the accurate diagnosis of infection in swine and can also be used in food testing.

Detection of Toxoplasma gondii in faeces of privately owned cats using two PCR assays targeting the B1 gene and the 529-bp repetitive element.

Toxoplasma gondii infection is a worldwide parasitic zoonosis with a high-health risk for humans. The key epidemiological role played by felids is related to oocyst shedding. The present study compared two amplification protocols for the molecular diagnosis of Toxoplasma infection in owned cats. A total of 78 owned cats referred to an Italian university-teaching hospital and exposed to various T. gondii-associated risk factors were sampled for blood and faeces. Faecal specimens were processed by flotation and tested using 2 copro-PCRs targeting the widely used B1 gene and the 529-bp repetitive element (RE). The sera were tested by the indirect immunofluorescent antibody test (IFAT) for the detection of immunoglobulins against T. gondii. Sixteen faeces (20.52%) tested positive for T. gondii DNA; 12 samples were positive only at B1-PCR, two at 529-bp RE-PCR and two at both genetic targets (overall agreement = 82.11%). The amplicons obtained were sequenced, and the Basic Local Alignment Search Tool analysis showed a high homology with the T. gondii strains available in reference databases. Two stool samples were microscopically positive for T. gondii-like oocysts and also tested positive by both B1 and 529-bp RE-PCRs. Thirty-three (42.3%) sera tested positive for antibodies; of which, seven were found to have T. gondii DNA-positive results using the B1 genetic target (overall agreement = 57.77%). The amplification sets targeting B1 and 529-bp RE showed substantially different yields. Further research is needed to better understand the significance and the sensitivities of using these multi-copy-targeted molecular methods from cat faeces before being used for routine diagnosis.

Metastrongyloid infection by Aelurostrongylus abstrusus, Troglostrongylus brevior and Angiostrongylus chabaudi in a domestic cat.

The "cat lungworm", Aelurostrongylus abstrusus, affects cats worldwide. Recently, other cardio-pulmonary parasites, e.g. Troglostrongylus brevior and Angiostrongylus chabaudi, have been isolated from cats either for the first time or a long time after they were first described. This paper describes the first known mixed infection by A. abstrusus, T. brevior and A. chabaudi in a domestic cat. Biological and epidemiological implications of old and "new" metastrongyloid-caused infections in cats are discussed.

Molecular and serological detection of tick-borne pathogens in donkeys (Equus asinus) in Italy.

Donkeys, owing to the frequent outdoor activity, are exposed to a high risk of infection with tick-borne pathogens. This work aimed to detect exposure to Theileria equi, Babesia caballi, Anaplasma phagocytophilum and Borrelia burgdorferi s.l. of donkeys reared in Central Italy. For this purpose 122 adult donkeys were selected within 11 herds and submitted to blood collection. IgG antibodies to T. equi, B. caballi, A. phagocytophilum and B. burgdorferi s.l. were detected by IFAT. Conventional PCRs targeting the genes MSP2 and the flagellin were used for the detection of A. phagocytophilum and B. burgdorferi s.l. respectively and a Real Time PCR Sybr Green was used to detect Babesia/Theileria spp…. The species identity was determined by amplicons sequencing. Forty eight (39.3%) and 58 (47.5%) animals tested positive for T. equi and B. caballi antibodies, respectively; nine animals (7.4%) were found positive for antibodies against A. phagocytophilum whereas negative results were obtained for B. burgdorferi s.l. Twenty-six (21.3%) animals showed antibodies for both T. equi and B. caballi. Twenty-three (18.8%) donkeys were positive to Babesia/Theileria spp. PCR assay. Out of 21 sequenced amplicons, 20 were identified as T. equi, belonging to three main groups designated A, B and D and one as B. caballi group A. Neither A. phagocytophilum nor B. burgdorferi PCR results were positive. The study showed a high exposure of donkeys to tick-borne pathogens and provides information on the genetic identity of the T. equi strains circulating in Central Italy.

Efficacy of toltrazuril 5 % suspension (Baycox®, Bayer) and diclazuril (Vecoxan®, Janssen-Cilag) in the control of Eimeria spp. in lambs.

Sheep coccidiosis is a pathology caused by protozoan parasites of the genus Eimeria spp. with clinical and economic effect especially in young animals (Ambrosi 1995; Pellérdy 1974). A field study was conducted to evaluate the efficacy of a metaphylactic treatment with toltrazuril 5 % suspension (Baycox®, Bayer) in comparison with diclazuril (Vecoxan®, Janssen-Cilag) and untreated controls against naturally acquired Eimeria infections in housed lambs. A total of 170 animals, aged 24 to 34 days and randomly divided in three homogeneous groups, were included in the study. The assessment of treatment efficacy was based on total faecal oocyst excretion (opg) and count reduction (FOCR) in the two groups of animals treated with toltrazuril (TOLT) and diclazuril (DICL) compared with untreated control group (C). The animals treated with toltrazuril showed a considerably lower mean opg to that of group C (5.78 opg versus 144.62 opg) (p < 0.05) and a FOCR of 97.7 %. The higher efficacy (99.23 %) was observed at 15 days post treatment; however, the average efficacy of the drug remained extremely high (> 90 %) for all the study. The lambs treated with diclazuril showed an intense, persistent oocyst excretion with average levels of 97.54 opg, (p < 0.05). This study demonstrates the good efficacy of toltrazuril administered orally to lambs in the prepatent period in subclinical natural Eimeria infections in housed lambs.

Influence of a metaphylactic treatment with Baycox(®) Bovis on the reproductive performances of Fresian heifers: a preliminary study.

A blind, randomised, controlled, multicentric field trial was conducted to assess the influence of a metaphylactic treatment with an oral solution of toltrazuril on some reproductive parameters of Italian Fresian heifers during the first 18-20 months of life. For this goal 40 calves were selected from two dairy farms and randomly divided into two homogeneous groups: MTol, treated with toltrazuril and NegC, left untreated. The calves were clinically and coprologically examined over the entire study period. The body condition scores, the body weights and the age at the first service were recorded and compared between the two groups, in addition to some other reproductive parameters including number of pregnancies, average service per pregnancy, conception rate, conception rate at first service and post first service conception rate. The analysis of the results showed that the metaphylactic treatment with toltrazuril influenced positively the average age of the first service (MTol 461.4 days versus NegC 485.45 days), the overall conception rate (MTol 95 % versus NegC 85 %), the success at first (MTol 60 % versus NegC 45 %) and second (MTol 75 % versus NegC 45 %) services and, consequently, the mean number of services to be carried out for each animal (MTol 1.4 ± 0.6 versus NegC 1.6 ± 0.79). Furthermore, the results confirmed that toltrazuril treatment, applied in accordance with the epidemiological aspects of each farm, is highly efficacious in persistent reduction of oocyst excretions with particular reference to Eimeria zuernii, Eimeria bovis, considered to be mainly responsible for clinical coccidiosis.

Long-term effect of toltrazuril on growth performances of dairy heifers and beef calves exposed to natural Eimeria zuernii and Eimeria bovis infections.

The long-term effects of a toltrazuril treatment against natural Eimeria bovis and/or Eimeria zuernii infections were investigated in comparison with diclazuril and untreated controls on two dairy (Italian Friesian breed) and two beef (Chianina breed) farms. At each trial site, 30 calves were allocated into three groups of 10 calves each: T (treated with toltrazuril), D (treated with diclazuril) and C (left untreated). For 40 weeks post-treatment, the calves were weighed and examined clinically and parasitologically. The oocyst counts as well as the number of scour days were significantly lower in the T group than in the D and C groups. Final bodyweights and body condition scores of the T group exceeded those of groups C and D. The results confirmed that toltrazuril was highly efficacious, safe and provided productive benefits in dairy and beef calves.