In Vitro Evaluations and Comparison of the Efficacy of Two Commercial Products Containing Condensed Tannins and of Saifoin (Onobrychis viciifolia Scop.) Hay against Gastrointestinal Nematodes of Goats.

Gastrointestinal nematodes (GINs) is a limiting health factor for dairy goats, and the integration of the diet with fodder containing condensed tannins (CT) is becoming increasingly important to control GINs. To preliminary evaluate their potential role as part of GIN control in goat breeding, the in vitro anthelmintic efficacy of the CTs of Silvafeed BYPRO (SBP), Silvafeed Q powder (SQ), and sainfoin hay (SH) was evaluated, and the untargeted metabolomics profiling of the selected formulations was performed. CTs were extracted in water and in ethanol, their concentration was determined, and their chemical characterization was conducted using High-Performance Liquid Chromatography (HPLC) coupled to High-Resolution Mass Spectrometry (HRMS) platform. The in vitro anthelmintic activity of the extracts was evaluated using the Eggs Hatch Test (EHT) and the Larval Migration Inhibition Test (LMIT) using different extract concentrations (150, 300, 600, and 1200 µg/mL). The metabolomic profile of the ethanol extract showed a high number of flavonoids, while the water extract showed higher levels of hydrolysable tannins. The ethanol extracts were effective on both eggs hatching and larvae migration at low concentrations (150 µg/mL) for the three analyzed samples, while the water extracts showed more varied results: SH showed the greatest ovicidal efficacy (concentration 150 µg/mL, %IH = 40.9), while SBP and SQ were more effective against the larvae migration (concentration 600 µg/mL, %LMI = 69.7% and 88%), respectively. The integration of CT-rich fodder into the diet may be considered for the control of GIN infection in goats.

The Prophylactic Effect of Ivermectin Treatments on Nematode Infections of Mammals in a Faunistic Park (Northern Italy).

Nematode infections of mammals can spread in zoos and faunistic parks and lead to disease in humans and animals. Group treatment strategies with anthelminthic drugs are common. Still, their effectiveness should be verified by sensitive and specific copromicroscopic analyses. This study assessed longitudinal parasitological monitoring, by FLOTAC® dual technique, in mammals housed in an Italian faunistic park, in order to verify the effectiveness of the two adopted ivermectin prophylactic treatments. Twenty-one species of herbivorous mammals from ten families were treated twice per year with ivermectin in an in-feed formulation (medicated feed containing 1.7 g/ton ivermectin daily, for 30 days in March and November), while 13 species of carnivores and primates from five families were treated once a month with oral or subcutaneous administrations of ivermectin (200 µg/kg body weight (b.w.), from March to November). Fecal samples were collected in June-July and October 2019 (late spring-early summer and autumn sampling groups, respectively). All nematode infections, sustained by Nematodirus spp., Capillaria spp., Trichuris spp., Parascaris spp. and Strongylida, were detected in samples collected from herbivores, presenting prevalence rates of infection of 17.3% (9/52), 15.4% (8/52), 15.4% (8/52), 5.8% (3/52), and 3.8% (2/52), respectively. All carnivores and primates tested negative. The general linear mixed model showed that nematode eggs' excretion in herbivores were influenced by sampling and sampling-host family interaction. Results showed that frequency and dose of prophylactic treatments in herbivores should be improved according to host and parasite taxonomic groups. The treatment adopted in carnivores and primates, together with hygienic management, was effective in nematode control.

Maternal and neonatal canine cortisol measurement in multiple matrices during the perinatal period: A pilot study.

Stress exposure during perinatal period may lead to maternal cortisol increase that negatively affects the offspring development. In recent years, the interest on non-invasive sampling methods to measure cortisol as a marker of stress is increasing in both humans and animals. Indeed, discomfort due to blood collection may compromise the diagnostic outcome, mainly in uncooperative patients. So far, some alternative matrices but not milk have been explored in adult dogs, while no data are available on the neonate and paediatric live pups. This study aimed to measure cortisol concentration in different biological substrates in both dams (blood, saliva, hair and milk) and pups (saliva and hair) at established times from proestrus up to two months after parturition. For this purpose, five female German shepherd bitches and their 22 pups were enrolled. Cortisol concentration was assessed using the enzyme immunoassay kit (Salivary Cortisol ELISA kit, Salimetrics) after matrices appropriate preparation if required. Cortisol was measurable in all the substrates, except some milk samples below the detection limit. Maternal cortisol concentrations differed among the matrices (P <0.0001) with the highest values recorded in plasma (median 0.596 µg/dL) compared to saliva (median 0.159 µg/dL), hair (median 0.083 µg/dL) and milk (median 0.045 µg/dL). Cortisol in dams did not vary within the same matrix over time. In pups, salivary (median 0.295 µg/dL) cortisol was always higher than hair (median 0.049 µg/dL; P <0.0001). At birth (P = 0.01) and two months later (P = 0.05), neonatal salivary cortisol was higher compared to other samplings. The present study demonstrates the suitability of these innovative substrates for cortisol measurement, suggesting them as potential diagnostic support in canine neonatology and welfare.

Spatial distance between sites of sampling associated with genetic variation among Neospora caninum in aborted bovine foetuses from northern Italy.

BACKGROUND: Neospora caninum, a coccidian protozoan, represents an important cause of bovine abortion. Available N. caninum strains show considerable variation in vitro and in vivo, including different virulence in cattle. To which extent sexual recombination, which is possible in the intestines of domestic dogs and closely related carnivores as definitive hosts, contributes to this variation is not clear yet. METHODS: Aborted bovine foetuses were collected between 2015 and early 2019 from Italian Holstein Friesian dairy herds suffering from reproductive problems. A total of 198 samples were collected from 165 intensive farms located in Lombardy, northern Italy. N. caninum samples were subjected to multilocus-microsatellite genotyping using ten previously established microsatellite markers. In addition to our own data, those from a recent study providing data on five markers from other northern Italian regions were included and analysed. RESULTS: Of the 55 samples finally subjected to genotyping, 35 were typed at all or 9 out of 10 loci and their individual multilocus-microsatellite genotype (MLMG) determined. Linear regression revealed a statistically significant association between the spatial distance of the sampling sites with the genetic distance of N. caninum MLMGs (P < 0.001). Including data from this and a previous North Italian study into eBURST analysis revealed that several of N. caninum MLMGs from northern Italy separate into four groups; most of the samples from Lombardy clustered in one of these groups. Principle component analysis revealed similar clusters and confirmed MLMG groups identified by eBURST. Variations observed between MLMGs were not equally distributed over all loci, but predominantly observed in MS7, MS6A, or MS10. CONCLUSIONS: Our findings confirm the concept of local N. caninum subpopulations. The geographic distance of sampling was associated with the genetic distance as determined by microsatellite typing. Results suggest that multi-parental recombination in N. caninum is a rare event, but does not exclude uniparental mating. More comprehensive studies on microsatellites in N. caninum and related species like Toxoplasma gondii should be undertaken, not only to improve genotyping capabilities, but also to understand possible functions of these regions in the genomes of these parasites.

Lyme borreliosis incidence in Lombardy, Italy (2000-2015): Spatiotemporal analysis and environmental risk factors.

Lyme borreliosis cases have been reported from Lombardy in northern Italy, where Ixodes ricinus is the main vector of Borrelia burgdorferi sensu lato. However, spatial and temporal variation in the incidence of Lyme borreliosis is not well understood. In the present study, based on new notified cases of Lyme borreliosis from 2000 to 2015, an average of 1.24 new cases per million residents per year was documented. New cases, georeferenced at the municipal level, were analyzed by retrospective space-time analysis (using SaTScan v. 9.3.1); and land cover, extrapolated from a Corine Land Cover dataset (using QGIS 2.8.1), was used to implement an environmental risk factor analysis. Firstly, a temporal high-risk cluster was detected in Lombardy: the relative risk of Lyme borreliosis was 3.73 times higher during 2008-2015 compared with the entire study period. Moreover, in a spatiotemporal high-risk cluster with a circular base, land cover consisting of wildland-urban interface, meadow, forest and meadow-forest transition were significantly higher compared to low-risk areas. Results of the present study demonstrate that the incidence of Lyme borreliosis is increasing in Lombardy and that environmental conditions are suitable for I. ricinus ticks infected with B. burgdorferi s.l.: citizens and health systems should be aware of Lyme borreliosis to reduce tick bites with personal protective behaviors and to avoid misdiagnosis, particularly within the area including the observed high-risk cluster. Economic resources should be invested to inform about methods to prevent tick bites, how to check people and pets after frequenting risk areas, and ways of removing the biting ticks when they are found.

The southernmost foci of Dermacentor reticulatus in Italy and associated Babesia canis infection in dogs.

BACKGROUND: Two clustered clinical cases of canine babesiosis were diagnosed by veterinary practitioners in two areas of northeastern Italy close to natural parks. This study aimed to determine the seroprevalence of babesial infection in dogs, the etiological agents that cause canine babesiosis and the potential tick vector for the involved Babesia spp. METHODS: The study area was represented by two parks in northeastern Italy: Groane Regional Park (Site A) and the Ticino Valley Lombard Park (Site B). From March to May 2015 ticks were collected from the vegetation in three transects in each site. In the same period, blood samples were collected from 80 dogs randomly chosen from veterinary clinics and kennel located in the two areas. Morphological identification of the ticks was performed and six specimens were molecularly characterised by the amplification and sequencing of partial mitochondrial 12S rRNA, 16S rRNA and cox1 genes. For phylogenetic analyses, sequences herein obtained for all genes and those available from GenBank for other Dermacentor spp. were included. Dog serum samples were analysed with a commercial indirect fluorescent antibody test to detect the presence of IgG antibodies against Babesia canis. Ticks and blood samples were tested by PCR amplification using primers targeting 18S rRNA gene of Babesia spp. RESULTS: Ticks collected (n = 34) were morphologically identified as adults of D. reticulatus. Twenty-eight ticks were found in all transects from Site A and the remaining six were collected in Site B. Blast analysis of mitochondrial sequences confirmed the morphological identification of processed tick specimens by revealing a highest nucleotide similarity (99-100%) with those of D. reticulatus available in the GenBank database. The phylogenetic trees were concordant in clustering D. reticulatus in a monophyletic clade. Seven dogs (8.8%) had antibodies against B. canis, most of which (n = 6) came from Site A. Analysis of nucleotide sequences obtained from one tick and from one dog identified B. canis displayed a 100% similarity to those available in GenBank. CONCLUSIONS: This study morphologically and molecularly confirms the presence of D. reticulatus in Italy and links it, for the first time, with the occurrence of B. canis infection in dogs in this country.

Parasitic and Bacterial Infections of Myocastor coypus in a Metropolitan Area of Northwestern Italy.

Coypus (Myocastor coypus) are widespread throughout Europe. In northern Italy, they are abundant in the flatland areas, and their high population densities can cause economic loss and ecosystem damage. We examined 153 coypus for selected parasitic and bacterial infections. We found Strongyloides myopotami (63.4% prevalence), Trichostrongylus duretteae (28.1%), Eimeria coypi (86.3%), and Eimeria seideli (6.8%), but did not find Giardia duodenalis or Cryptosporidium spp. We also isolated Staphylococcus aureus (10.1%), Escherichia coli (4.5%), and Streptococcus spp. (3.4%) from lung samples; no Salmonella spp. were isolated from fecal samples. Coypus had antibodies to Toxoplasma gondii (28.9%) and to four serovars of Leptospira interrogans (44.9%); Australis/Bratislava was the serovar most frequently detected. It is clear that coypu can be infected with pathogens of human and veterinary importance.

Coinfection with Tritrichomonas foetus and Giardia duodenalis in Two Cats with Chronic Diarrhea.

A Tritrichomonas foetus and Giardia duodenalis mixed infection was diagnosed in two Maine Coon cats aged six months. One of them presented a history of chronic liquid diarrhea and of several unsuccessful treatments. In both cats, G. duodenalis and trichomonads were detected in fecal smears from freshly voided feces; the presence of T. foetus was confirmed by a real-time PCR assay. The cats completely recovered after treatment with ronidazole. In a refrigerated fecal sample collected from the cat with chronic diarrhea, drop-shaped trichomonad pseudocysts smaller than G. duodenalis cysts were detected. They appeared brownish or light-bluish when stained with Lugol's solution or with Giemsa stain, respectively, and their morphological features were similar to those expressed by bovine T. foetus pseudocysts in vitro. Existence of pseudocysts even in feline trichomonads is noteworthy as they could represent a form of protozoan resistance due to unfavorable conditions whose detection in refrigerated feces can be a useful clue for clinicians.

Oestrus ovis L. (Diptera: Oestridae) Induced Nasal Myiasis in a Dog from Northern Italy.

A companion dog from Milan province (northern Italy), presenting with frequent and violent sneezing, underwent rhinoscopy, laryngoscopy, and tracheoscopy procedures. During rhinoscopy, a dipteran larva was isolated from the dog and identified as first instar larval stage of O. ovis by morphological features. Reports of O. ovis in domestic carnivores are sporadic and nevertheless this infestion should be considered as a possible differential diagnosis of rhinitis in domestic carnivores living in contaminated areas by the fly as consequence of the presence of sheep and goats. This report described a case of autochthonous infestion in a dog from an area where O. ovis was not historically present but it could be affected by a possible expansion of the fly as a consequence of climate change. This is the first record of Oestrus ovis infestion in a dog in Italy and, at the same time, the most northerly finding of larvae of sheep bot fly in the country.

Detection of Cyclospora in captive chimpanzees and macaques by a quantitative PCR-based mutation scanning approach.

BACKGROUND: Cyclospora is a protistan parasite that causes enteritis in several species of animals including humans. The aim of this study was to investigate the presence of Cyclospora in captive non-human primates. METHODS: A total of 119 faecal samples from Pan troglodytes, Macaca sylvanus, Cercopithecus cephus, Erythrocebus patas, Chlorocebus aethiops and Macaca fascicularis from a wildlife animal rescue center as well as from Macaca fascicularis from an experimental primate research center were tested for the presence of Cyclospora by quantitative real-time PCR (qPCR) and single-strand conformation polymorphism (SSCP) analysis. RESULTS: Cyclospora was detected in three Pan troglodytes (13.6%) and nine (9.3%) Macaca fascicularis. CONCLUSIONS: The present study represents the first record of Cyclospora in captive primates in Europe, suggesting the presence of Cyclospora cayetanensis, which is transmissible to humans.

Besnoitia besnoiti among cattle in insular and northwestern Italy: endemic infection or isolated outbreaks?

BACKGROUND: Bovine besnoitiosis, caused by the apicomplexan Besnoitia besnoiti, is a chronic and debilitating disease considered as emerging in Europe. In Spain, Portugal and France it is endemic and foci of infection were recorded in Germany, Switzerland, Hungary, Greece and Italy. In Italy, cases of bovine besnoitiosis were registered both in imported and autochthonous cattle, and mostly in central regions; high seroprevalence was also revealed by an epidemiological survey performed in the southern part of the country. Aiming to update information on the disease in northwestern and insular areas of Italy, where data on bovine besnoitiosis were missing, a serosurvey was designed for the present study. METHODS: Three thousand one hundred and forty bovine blood samples from both dairy and beef farms (n = 126) were collected in northwestern regions (Lombardy, Piedmont and Liguria) and in the island of Sardinia. Samples were analyzed by a standardized in-house ELISA and those resulted positive were re-tested by Western Blot (WB) for confirmation. On results obtained by both ELISA and WB, apparent (AP) and true prevalence (TP) were calculated at individual and herd levels. Further, a panel of sera resulted positive to ELISA was analyzed by IFAT. RESULTS: A total of 712 animals (AP = 22.7%; TP = 18.8%) and 109 farms (AP = 86.5%; TP = 88.2%) showed a positive reaction in ELISA. Only ten (AP = 0.3%; TP = 0%) specimens proceeding from five farms (AP = 3.9%; TP = 1.7%) from Lombardy were confirmed positive to the WB, corresponding to two Holstein Friesian cows and eight beef cattle. IFAT showed a low sensitivity (44.4%) scoring positive in only four samples out of 9 positive to WB. CONCLUSIONS: The survey demonstrated that bovine besnoitiosis cannot still be considered endemic in whole Italy. In fact, independent foci of infection were registered only in Lombardy region. Therefore, a sanitary strategy aimed to increase control measures and to organize monitoring plans, by adequate diagnostic tools is necessary to avoid overestimation of B. besnoiti in Italy.