Gray Wolf (Canis lupus italicus) and Red Fox (Vulpes vulpes) Parasite Survey in Anthropized and Natural Areas of Central Italy.

Gastrointestinal nematodes and protozoa and other parasite occurrences were evaluated in free-ranging wolf (Canis lupus italicus) and red fox (Vulpes vulpes) populations from natural and anthropized areas of Central Italy. Analyzed fecal samples were collected from 60 foxes and 40 wolves in the anthropized areas, and 41 foxes and 39 wolves in the natural areas. In foxes, hookworm infections (p < 0.0001) were more frequently recorded in the anthropized environment, while coccidia (p < 0.05) and Cryptosporidium spp. (p < 0.0001) were more frequent in the natural area. In wolves, a higher frequency of hookworms (p < 0.0001) was observed in natural areas, while coccidia were more common in the anthropized area (p < 0.05). Moreover, in the natural environment, trichuroid nematodes (p < 0.0001) were significantly more frequent in wolves than in foxes, while Cryptosporidium (p < 0.001) and Giardia duodenalis (p < 0.001) were more common in foxes. In the anthropic area, the occurrence of hookworms was found to be significantly higher in foxes (p < 0.0001), while trichuroid nematodes were more common in wolves (p < 0.0001). The obtained data are indicative of a different diffusion of specific parasite taxa in wolves and foxes living in the natural and/or anthropized environments examined herein.

Zoonotic Giardia duodenalis Genotypes and Other Gastrointestinal Parasites in a Badger Population Living in an Anthropized Area of Central Italy.

The Eurasian badger (Meles meles) is widespread in Italy and occupies different habitats. The occurrence and species of gastrointestinal parasites were evaluated in a free-ranging badger population living in a highly anthropic area in central Italy. A total of 43 fecal samples were examined using the flotation test, the Mini-FLOTAC and Baermann techniques, and a rapid immunoassay for the detection of Giardia duodenalis and Cryptosporidium spp. fecal antigens. Molecular investigations were also performed that aimed at identifying Giardia genotypes. Overall, 37/43 samples (86%) were found positive. Specifically, 48.8% (21 samples) were positive for G.duodenalis, 23.2% (10/43) for Cryptosporidium spp., and 7% (3/43) for coccidian oocysts. Strongyloides sp. nematode larvae were detected in 3/43 samples (7%). Ascarid (1/43, 2.3%), capillariid (1/43, 2.3%), and strongyle-type eggs (76.7%, 33/43) were also identified. Among the 11 readable sequences of samples that were positive for G. duodenalis by end-point PCR (18/21), the zoonotic assemblage A sub-assemblage AII and mixed assemblage A and B were identified. This is the first report of zoonotic G. duodenalis genotypes in the Eurasian badger. Moreover, most of identified parasites have zoonotic potential and/or potential impact on the population health of wild badgers and other wild and domestic animals.

In Vitro Anthelmintic Activity of Sea Buckthorn (Hippophae rhamnoides) Berry Juice against Gastrointestinal Nematodes of Small Ruminants

Gastrointestinal nematodes are one of the major threats in small ruminant breeding. Their control is difficult due to the development of anthelmintic resistance, and the search for new molecules endowed with anthelmintic activity (AH) is considered a priority. In this context, we evaluated the in vitro AH activity of two commercial sea buckthorn (Hippophae rhamnoides) berry juices, namely SBT and SBF. The in vitro evaluation was based on the egg-hatch test and larval exsheathment assay at different concentrations. Data were statistically analysed, and the EC50 was calculated. Chemical analyses were performed to evaluate the total polyphenol content of the juices and chemical profile of the most represented compounds. The role of the polyphenolic fraction in the anthelmintic activity of the juices was also assessed. At the highest concentrations, the activity of SBT was high in both tests and comparable to that observed in the thiabendazole-treated positive controls, while SBF showed a lower efficacy. Glycosylated isorhamnetin and quercetin were the most represented polyphenolic compounds in both juices. In conclusion, both H. rhamnoides berry juices tested in this study showed interesting anthelmintic properties in vitro.

In vitro anthelmintic activity of an aqueous extract of Glycyrrhiza glabra and of glycyrrhetinic acid against gastrointestinal nematodes of small ruminants.

This study evaluated the in vitro anthelmintic activity of a liquorice (Glycyrrhiza glabra) root aqueous extract and of glycyrrhetinic acid at 30, 10, 5, 1, and 0.5 mg/mL against sheep gastrointestinal nematodes (GINs), using the egg hatch test (EHT), the larval development test (LDT), and the larval migration inhibition test (LMIT). The compounds were applied on a mixture of GIN eggs and larvae, mainly Trichostrongylus spp. and Teladorsagia/Ostertagia spp. Cytotoxicity assays were also performed. In the EHT, both candidates showed significant concentration-dependent efficacy and were significantly more effective (p < 0.001) at the highest concentrations (30 and 10 mg/mL) than the lowest ones. In the LDT, only G. glabra showed a concentration-dependent effect (R2 = 0.924), but glycyrrhetinic acid (R2 = 0.910) had significantly higher efficacy than G. glabra root extract. Moreover, the efficacy of glycyrrhetinic acid at 30, 10, and 5 mg/mL was significantly higher (p < 0.001) than at lower concentrations. In the LMIT, G. glabra showed concentration-dependent efficacy (R2 = 0.971), while considerably reduced efficacy was observed for glycyrrhetinic acid (R2 = 0.855) at the lowest concentrations. These data suggest that the two compounds may have different mechanisms of action. In the LMIT, the 50% lethal concentration (LC50) of glycyrrhetinic acid (~5.12 mg/mL) was > 2.0-fold lower when compared to G. glabra (12.25 mg/mL). Analysis and previous findings indicated low toxicity for both compounds. The results obtained encourage in vivo studies aimed at evaluating the potential use of the tested compounds as natural de-wormers in ruminants.

TITLE: Activité anthelminthique in vitro d'un extrait aqueux de Glycyrrhiza glabra et de l'acide glycyrrhétinique contre les nématodes gastro-intestinaux des petits ruminants. ABSTRACT: Cette étude a évalué l'activité anthelminthique in vitro d'un extrait aqueux de racine de réglisse (Glycyrrhiza glabra) et de l'acide glycyrrhétinique à 30, 10, 5, 1 et 0,5 mg/mL contre les nématodes gastro-intestinaux (NGI) du mouton, en utilisant le test d'éclosion des œufs (TEO), le test de développement larvaire (TDL) et le test d'inhibition de la migration larvaire (TIML). Les composés ont été appliqués sur un mélange d'œufs et de larves de NGI, principalement Trichostrongylus spp. et Teladorsagia/Ostertagia spp. Des tests de cytotoxicité ont également été effectués. Dans le TEO, les deux candidats ont montré une efficacité concentration-dépendante significative et ont été significativement plus efficaces (p < 0,001) aux concentrations les plus élevées (30 et 10 mg/mL) qu'aux plus faibles. Dans le TDL, seul G. glabra a montré un effet concentration-dépendant (R2 = 0,924), mais l'acide glycyrrhétinique (R2 = 0,910) avait une efficacité significativement plus élevée que l'extrait de racine de G. glabra. De plus, l'efficacité de l'acide glycyrrhétinique à 30, 10 et 5 mg/mL était significativement plus élevée (P < 0,001) qu'à des concentrations plus faibles. Dans le TIML, G. glabra a montré une efficacité concentration-dépendante (R2 = 0,971), tandis qu'une forte réduction d'efficacité a été observée pour l'acide glycyrrhétinique (R2 = 0,855) aux concentrations les plus faibles. Ces données peuvent suggérer que les deux composés peuvent avoir des mécanismes d'action différents. Dans le TIML, la concentration létale à 50% (CL50) de l'acide glycyrrhétinique (~ 5,12 mg/mL) était > 2,0 fois inférieure à celle de G. glabra (12,25 mg/mL). L'analyse et les résultats précédents ont indiqué une faible toxicité pour les deux composés. Les résultats obtenus encouragent les études in vivo visant à évaluer l'utilisation potentielle des composés testés ici comme anthelminthique naturels chez les ruminants.

Serological Survey on the Occurrence of Rickettsia spp., Neospora caninum, Bartonella henselae and Toxoplasma gondii in Cats from Tuscany (Central Italy).

Asymptomatic cats often harbor pathogens, some of which have not been largely investigated in feline populations. The aim of this study was to evaluate the occurrence of antibodies against Rickettsia conorii, Rickettsia felis, Rickettsia typhi, Neospora caninum, Bartonella henselae and Toxoplasma gondii in cats from Tuscany. Ninety-five blood serum samples, previously collected, were analyzed by indirect immunofluorescence assay. Fifty-six (58.94%) cats had antibodies to at least one investigated pathogen: 28 (29.47%) cats were positive for B. henselae, 17 (17.89%) for R. felis, 14 (14.73%) for R. conorii, 14 (14.73%) for T. gondii, 2 (2.1%) for N. caninum. No cats were positive for R. typhi. Positive reactions to two or more pathogens were detected in 18 (18.94%) cats. The occurrence of antibodies against these microorganisms suggests that cats, even though asymptomatic, may be infected by pathogens, often zoonotic, and thus may be a source of infections for other animals and humans.

Cyathostomin fecal egg count and milk quality in dairy donkeys.

This study was performed to assess the possible relationship between gastrointestinal strongylid infections and milk quantity and quality in donkeys used for milk production. Individual fecal and milk samples were collected from 16 multiparous lactating jennies of Amiatina breed at their 6th month of lactation. Statistical analysis was carried out between strongylid eggs per gram of faeces (EPG) and milk parameters. All animals were found to shed cyathostomin eggs, with EPG ranging from 150 to 1900. A higher milk production was observed in animals showing an EPG< 250, but this result was statistically not significant. Conversely, significant higher concentrations of milk urea and pH values, and lower concentrations of milk lactose, were observed in jennies with an EPG >1000. In conclusion, results from this study suggest that cyathostomin EPG values > 1000 may affect milk quality in dairy donkeys. Considering the novelty of these findings, more data needs to be collected to determine EPG thresholds to be used as a good marker for parasite infections influencing milk performance and quality in donkeys.

Prevalence, Risk Factors and Genotypes of Giardia duodenalis in Sheltered Dogs in Tuscany (Central Italy).

In sheltered dogs, the prevalence of Giardia duodenalis is frequently high and may include potential zoonotic genotypes. The prevalence, genotypes and potential risk factors of G. duodenalis were assessed in 168 dogs from four kennels (Pistoia, Prato, Florence, Valdarno) in Tuscany, central Italy and compared with data from previous Italian studies. The prevalence of other intestinal parasites was also investigated. Individual dog faecal samples collected from each kennel were examined by parasitological techniques and a rapid immunoassay for the detection of G. duodenalis and Cryptosporidium faecal antigens. On Giardia-positive samples, molecular analysis was performed for genotype identification. Overall, 69 dogs scored positive for G. duodenalis (41%), but significant differences (p ≤ 0.05) were found among the four kennels and sampling seasons. The potentially zoonotic assemblages A and B and the canine-specific assemblage C (Pistoia: A-AII, B, C; Prato: A-AII, B; Florence: A-AII; Valdarno: A and C) were identified. Toxocara canis (8.9%), Trichuris vulpis (3.6%), hookworms (1.19%) and Cryptosporidium sp. (0.6%) were also identified. The high prevalence of G. duodenalis and the identification of potentially zoonotic genotypes in all examined kennels underline the need to improve routine parasite monitoring and control measures and to provide insights into the zoonotic potential of G. duodenalis.

Cyathostomin fecal egg count and milk quality in dairy donkeys / Contagem de ovos de Cyathostominae nas fezes e a qualidade do leite em jumentas lactantes

Abstract This study was performed to assess the possible relationship between gastrointestinal strongylid infections and milk quantity and quality in donkeys used for milk production. Individual fecal and milk samples were collected from 16 multiparous lactating jennies of Amiatina breed at their 6th month of lactation. Statistical analysis was carried out between strongylid eggs per gram of faeces (EPG) and milk parameters. All animals were found to shed cyathostomin eggs, with EPG ranging from 150 to 1900. A higher milk production was observed in animals showing an EPG< 250, but this result was statistically not significant. Conversely, significant higher concentrations of milk urea and pH values, and lower concentrations of milk lactose, were observed in jennies with an EPG >1000. In conclusion, results from this study suggest that cyathostomin EPG values > 1000 may affect milk quality in dairy donkeys. Considering the novelty of these findings, more data needs to be collected to determine EPG thresholds to be used as a good marker for parasite infections influencing milk performance and quality in donkeys.

Resumo Este estudo foi realizado com o objetivo de determinar a possível relação entre a infecção por estrongilídeos gastrintestinais e a quantidade e qualidade do leite de jumentas em lactação. Amostras individuais de fezes e leite foram coletados em 16 jumentas multíparas e lactantes da raça Amiatina no seu sexto mês de lactação. A análise estatística foi realizada entre a contagem de ovos de Cyathostominae por grama de fezes (OPG) e parâmetros do leite. Todos os animais estavam eliminando Cyathostominae nos ovos das fezes amostradas, variando entre 150 a 1.900 OPG. Embora não estatisticamente significativo, houve uma maior produção de leite em animais com OPG < 250. Inversamente, houve um aumento significativo na concentração de ureia e pH no leite, e baixa concentração de lactose foi observada nas jumentas com OPG > 1.000. Em conclusão, os resultados deste estudo sugerem que valores de OPG > 1.000 para ovos de Cyathostominae, podem comprometer a qualidade do leite em jumentas lactantes. Essa informação é original e mais dados necessitam ser obtidos para determinar os limites de OPG que poderão ser usados como marcadores para infecção parasitária, bem como na qualidade e "performance" de jumentas.

Fatal Pulmonary Hypertension and Right-Sided Congestive Heart Failure in a Kitten Infected with Aelurostrongylus abstrusus.

Aelurostrongylus abstrusus is considered the most important respiratory nematode of domestic cats worldwide. This parasite inhabits the alveoli, alveolar ducts, and bronchioles and causes a subacute to chronic respiratory clinical disease. Clinical signs may occur in domestic cats of any age, though they are more often described in young animals. Physical examination, echocardiography, thoracic radiography, pulmonary and cardiac pathological findings, classical, and molecular parasitological analysis of a six-month-old kitten referred at the Veterinary Teaching Hospital of the University of Pisa (Italy) led to a diagnosis of parasitic bronchopneumonia caused by A. abstrusus, which was complicated by severe pulmonary hypertension (PH) and right-sided congestive heart failure (R-CHF) that caused the death of the animal. Cases of reversible PH associated with A. abstrusus infection have been seldom reported in cats. This is the first report of fatal PH and R-CHF in a kitten with clinical aelurostrongylosis.

Intestinal Strongyle Genera in Different Typology of Donkey Farms in Tuscany, Central Italy.

Intestinal strongyles are common helminths of donkeys, in which they may be responsible for disease and poor performance. This study aimed to identify intestinal strongyle genera in 55 naturally infected donkeys from three different farm typologies in Tuscany, central Italy, using morphological and metrical analysis of third stage larvae (L3) obtained from faecal cultures. Larvae were identified using two previous reported morphological identification keys. Moreover, eggs per gram (EPG) data were also evaluated to assess differences, if any, according to the farm typology, sex, and age of the examined donkeys. The results showed that small strongyles were prevalent in all donkey farms. In all examined farms, most (92-100%) of L3 were identified as cyathostomin species of the genera Cylicocyclus spp. and Cylicostephanus spp. Large strongyles of the genera Strongylus spp. and Triodontophorus spp., were identified at low percentage (8%), only in the single organic farm included in the study. A high agreement was observed between the two different morphometric keys used. No significant differences were found for EPG according to farm typology, and sex and age from the examined donkeys. This is the first report about genera identification of intestinal strongyles infecting donkeys in Tuscany, Italy.

Pearsonema spp. (Family Capillariidae, Order Enoplida) Infection in Domestic Carnivores in Central-Northern Italy and in a Red Fox Population from Central Italy.

Pearsonema spp. nematodes infect the urinary bladder of domestic and wild carnivores. The red fox is considered a reservoir of Pearsonema plica, while the prevalence of Pearsonema spp. in domestic carnivores is still poorly known. This study aimed to assess the occurrence of Pearsonema spp. infection in privately owned cats (26) and dogs (83) from central and northern Italy alongside occurrence in red foxes (42) from central Italy. In positive dogs and cats, associated clinical signs were also evaluated. Urine samples were first examined under a stereomicroscope; then, they were centrifuged and microscopically examined after a flotation test. As for foxes, the urinary bladders were opened and urine was collected and processed as above, while collected nematodes were identified at the species level. Among examined animals, 2/26 cats (7.7%), 1/83 dogs (1.2%), and 38/42 foxes (90.5%) scored positive. Recurrent cystitis, pollakiuria, and hematuria were the main clinical signs in symptomatic dogs and cats. This is the first study on Pearsonema spp. infection in domestic carnivores examining a large number of privately owned pet animals. Obtained results confirm the role of the red fox as a reservoir for P. plica and suggest a possible high occurrence of Pearsonema spp. infection in domestic cats of central Italy.

In Vitro Inhibiting Effects of Three Fungal Species on Eggs of Donkey Gastrointestinal Strongyles.

Recently, donkeys have gained popularity mainly due to the use of donkey milk by the cosmetic industry and for human consumption. Gastrointestinal strongyles (GIS) are considered a potential cause of disease and reduced production in infected donkeys. European laws limit the use of anthelmintic drugs for the control of GIS in dairy donkey farms, thus the need to develop alternative control methods. This study aimed to test the in vitro inhibiting effects of three chitin degrading fungi (Scopulariopsis brevicaulis, Metarhizium anisopliae, and Beauveria bassiana) on the hatch and viability of donkey GIS eggs by using the egg hatch test, and to compare their activity to that of Pochonia chlamydosporia. About 150 eggs were added to 0.5 mL of sterile saline solution containing about 1.4 × 108 spores of each fungal species or with 0.5 mL of sterile saline solution only (untreated controls). After incubation, the percentage of egg hatch reduction was calculated, and data were statistically analyzed. All fungi were able to significantly reduce (p < 0.05) the hatch of GIS eggs compared to the untreated controls. Further studies that aim to investigate the efficiency of these fungi in reducing donkey GIS eggs in contaminated environments are encouraged.

First report of Giardia duodenalis infection in the crested porcupine (Hystrix cristata L., 1758).

Italy is the only European country where the crested porcupine (Hystrix cristata) lives. A parasitological investigation was performed on faecal samples, aimed to evaluate Giardia and other parasites in a free-ranging crested porcupine population in Central Italy. Samples were collected from captured and road-killed individuals as well as from feeding areas and pathways. Collected faecal samples were examined by the Mini-FLOTAC technique and a rapid immunoassay for the search of Giardia and Cryptosporidium spp. faecal antigens. For the identification of Giardia species and genotypes, molecular analysis was performed on Giardia-positive samples, by using PCR protocols able to amplify glutamate dehydrogenase, triosephosphate isomerase and a fragment of the small subunit ribosomal RNA genes. A total of 52 crested porcupine faecal samples were collected and analysed. At microscopical examination, 39 out of 52 samples were found positive for at least a single parasite species and six different parasite taxa were identified. Forty-eight percent (25/52) of faecal samples were positive for Giardia spp. and 1.9% (1/52) for Cryptosporidium spp. at the immunoassay. Among 12 faecal samples belonging to different individuals, 33.3% (4/12) were positive for Giardia spp. By using the Mini-FLOTAC technique, positivity for Trichuris spp. (32.7%, 17/52), gastrointestinal strongyles (32.7%, 17/52), capillariid eggs (3.8%, 2/52) and coccidian oocysts (1.9%; 1/52) was also evidenced. Molecular analysis was performed on 17 out of 25 Giardia-positive isolates. At the SSU rDNA locus, expected bands were achieved for 12 out of 17 isolates and all samples were assigned to Giardia duodenalis assemblage B. Sequencing at tpi locus revealed potentially zoonotic G. duodenalis assemblage AII (two isolates) and assemblage BIV (one isolate). The present study provides the first report of G. duodenalis infection in H. cristata. More in depth studies are needed on the impact and epidemiology of G. duodenalis and other identified parasites in crested porcupines.

In Vitro Anthelmintic Activity of Saponins from Medicago spp. Against Sheep Gastrointestinal Nematodes.

Gastrointestinal strongyle nematodes (GIS) are included among the most important parasites of small ruminants. The widespread drug resistance and drug residues in products of animal origin have increased the interest in the search for natural compounds with anthelmintic activity as a valid alternative to current synthetic drugs. The aim of the present investigation was to test the 'in vitro' anthelmintic activity of saponins and prosapogenins from different Medicago species, selected for their importance as a forage crop worldwide for animal feeding. From these plants, saponin mixtures were extracted, purified and used at scalar concentrations to evaluate their anthelmintic activities against sheep gastrointestinal strongyles (GISs), by the egg hatch test. Treated and untreated controls were used as the comparison. Data were statistically analyzed, and EC50 and EC90 were also calculated. All saponins and prosapogenins showed inhibiting effects on GIS eggs in a concentration-dependent manner. At higher concentrations, most of them showed an efficacy comparable to the reference drug (Thiabendazole 3 µg/mL) (P < 0.001). With 1.72 mg/mL EC50 and 3.84 mg/mL EC90, saponin from M. polymorpha cultivars Anglona was the most active. Obtained results encourage further studies aimed at evaluating the efficacy 'in vivo' of saponins which resulted as most effective 'in vitro' in this study.

In Vitro Anthelmintic Activity of Saponins Derived from Medicago spp. Plants against Donkey Gastrointestinal Nematodes.

With the aim to find new effective natural compounds for the control of nematodes, the in vitro anthelminthic properties of purified 1% saponins showing different chemical compositions and derived from Medicago sativa (MS), Medicago arborea (MA), Medicago polymorpha cultivar 'Santiago' (MPS), M. polymorpha cultivar 'Anglona' (MPA), and 1% prosapogenins from M. sativa (MSp), were evaluated and compared. As a source of nematode eggs, pooled fresh fecal samples taken from dairy donkeys naturally infected by gastrointestinal nematodes were used. From fecal samples, eggs were recovered, suspended in deionized water, and used immediately in the bioassay (egg hatch test). The activity of the tested compounds was compared to positive (0.1% thiabendazole) and negative (deionized water and 1% DMSO) controls. All experiments were repeated in triplicate and the obtained data were statistically analyzed. All the tested plant compounds caused a significant (p < 0.05) inhibition of nematode egg hatching (>80%). Moreover, all saponins and prosapogenins showed in vitro anthelmintic properties statistically comparable to that of the reference drug (p < 0.05), except for MPS extract. Obtained results showed that the different Medicago saponins evaluated in this study possess high anthelmintic properties against gastrointestinal nematodes of dairy donkeys, although to a different extent depending on their composition.