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Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is one of the main food-, water- and soil-borne zoonotic disease worldwide. Over the past 20 years many papers were published on the transmission of T. gondii by marine animals, including mollusks, which can concentrate the oocysts and release them. Sporulated oocysts may remain viable and infective for 18 months in seawater. Therefore, raw or undercooked bivalve mollusks pose a risk to humans. This study aimed to apply and validate for the first time a very sensitive digital droplet polymerase chain reaction (ddPCR) protocol to detect and quantify T. gondii DNA in mussels. Four concentration levels: 8000 genomic copies (gc)/μL, 800 gc/μL, 80 gc/μL, 8 gc/μL of a T. gondii reference DNA were tested. DNA was extracted from 80 pools of mussels (Mytilus galloprovincialis). Forty pools were contaminated with T. gondii reference DNA and used as positive controls, while 40 pools were used as negative controls. DdPCR reaction was prepared using a protocol, previously developed by the authors, for detection of T. gondii in meat. Amplification was obtained up 8 gc/μL. All infected replicates resulted positive, as well as no droplets were detected in negative controls. The droplets produced in the reaction ranged from 8,828 to 14,075 (average 12,627 droplets). The sensitivity and specificity of ddPCR were 100% (95%CI = 94.3-99.9). In addition, 100 pools of mussels collected in the Gulf of Naples were used to validate the protocol. Of these 16% were positive (95% CI = 9.7-25.0) for T. gondii. Samples were also tested by real-time PCR and no positive samples were found. Data obtained from ddPCR showed good identification of negative and positive samples with higher specificity and efficiency than real-time PCR. This tool could be very useful for a rapid sensitive detection of low DNA concentrations of T. gondii in mussels, reducing the risk of toxoplasmosis in humans. [ABSTRACT FROM AUTHOR]

Background: Fasciolosis (Fasciola hepatica) and paramphistomosis (Calicophoron daubneyi) are two important infections of livestock. Calicophoron daubneyi is the predominant Paramphistomidae species in Europe, and its prevalence has increased in the last 10–15 years. In Italy, evidence suggests that the prevalence of F. hepatica in ruminants is low in the southern part, but C. daubneyi has been recently reported at high prevalence in the same area. Given the importance of reliable tools for liver and rumen fluke diagnosis in ruminants, this study evaluated the diagnostic performance of the Mini-FLOTAC (MF), Flukefinder® (FF) and sedimentation (SED) techniques to detect and quantify F. hepatica and C. daubneyi eggs using spiked and naturally infected cattle faecal samples. Methods: Briefly, negative bovine faecal samples were artificially spiked with either F. hepatica or C. daubneyi eggs to achieve different egg count levels: 10, 50 and 100 eggs per gram (EPG) of faeces. Moreover, ten naturally infected cattle farms from southern Italy with either F. hepatica and/or C. daubneyi were selected. For each farm, the samples were analysed individually only with MF technique and as pools using MF, FF and SED techniques. Bayesian latent class analysis (LCA) was used to estimate sensitivity and accuracy of the predicted intensity of infection as well as the infection rate in the naturally infected farms. Results: The outcome of this study showed that the highest number of eggs (F. hepatica and C. daubneyi) recovered was obtained with MF, followed by FF and SED in spiked infected samples at 50 and 100 EPG, while at lower infection levels of 10 EPG, FF gave the best results. Moreover, the sensitivity for all the techniques included in the study was estimated at > 90% at infection levels > 20 EPG for both F. hepatica and C. daubneyi eggs. However, MF was the most accurate of the three techniques evaluated to estimate fluke infection intensity. Nevertheless, all three techniques can potentially estimate infection rate at farm level accurately. Conclusions: Optimization and standardization of techniques are needed to improve the FEC of fluke eggs. [ABSTRACT FROM AUTHOR]

Simple Summary: Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by the tapeworm Echinococcus granulosus. Dogs and other carnivores represent the definitive hosts in the life cycle of this parasite, whereas herbivores/omnivores, including humans, are the intermediate hosts, where the larval stage (metacestode) develops in the organs (mainly liver and lungs). Among the intermediate hosts, sheep have recently gained more attention as reservoirs of infection by E. granulosus, but there is still poor information about the local inflammatory response associated with liver cystic echinococcosis. With our study, we aimed to contribute to "finding the gaps" and further define the immunological mechanisms involved during the different stages of the development of ovine hydatidosis. In order to characterize the inflammatory phenotype of livers of sheep naturally infected by cystic echinococcosis, 100 sheep livers have been macroscopically assessed for the presence of hydatid cysts and sampled for histopathological and molecular analysis. According to gross and microscopic examination, livers were subsequently classified into three groups: normal liver (Group A), liver with the presence of fertile hydatid cysts (Group B), and liver with the presence of sterile hydatid cysts (Group C). Immunohistochemical analyses were accomplished using primary antibodies anti-Iba1, anti-CD3, anti-CD20, anti-TGF-β, and anti-MMP9. Finally, real-time PCR was performed in order to estimate the concentration levels of tumor necrosis factor-α (TNF-α), interferon-γ (INF-γ), interleukin (IL)-12, IL-10, and TGF-β. Immunohistochemical analysis showed a diffuse immunolabelling of mononuclear cells for Iba-1 and TGF-β and a higher amount of CD20+ B cells compared to CD3+ T cells in both Groups B and C. The expression levels of Th-1-like immune cytokines TNF-α, INF-γ, and IL-12 did not show significant statistical differences. However, we found a significant increase in expression levels of Th-2 immune cytokines TGF-β and IL-10 in Groups B and C compared to Group A. Taken together, our findings suggest that macrophages have a predominant role in the local immune response to cystic echinococcosis. Moreover, we can speculate that Th2 immunity may be dominant, corroborating the idea that B cells are decisively essential in the control of the immune response during parasitic infection and that the immunomodulatory role of IL-10 and TGF-β may ensure the persistence of the parasite within the host. [ABSTRACT FROM AUTHOR]

Simple Summary: Inappropriate use of regular anthelmintic drugs has led to the development of anthelmintic resistance in sheep gastrointestinal nematodes (GINs) and consequently caused huge economic losses. Therefore, researchers worldwide are making efforts in finding novel strategies to control these parasites, which are mostly based on the rational use of synthetic drugs and the use of various alternatives, such as botanical anthelmintics. The aim of this study was to evaluate the anthelmintic efficacy of the essential oil (EO) of oregano (Origanum vulgare (L.)) against sheep GINs and to demonstrate the possibility of its use in veterinary practice. For obtaining clear results, both in vitro (egg hatch test) and in vivo efficacy tests (fecal egg count reduction test), as well as toxicity studies, on the hosts (clinical examination, blood count, and liver or kidney function test) were performed. The egg hatch test demonstrated the anthelmintic potential of the tested EO with an efficacy of 71.3%–93.7%, which was also demonstrated in the fecal egg count reduction test with an efficacy of 60.1% in total (on the second farm, 78.26%) shown on day 14 after treatment. Moreover, no negative or side effects of the applied EO formulation to the sheep were noticed in toxicity studies. The obtained results indicate the high potential of the O. vulgare EO for use in veterinary practice to control sheep GINs, as a part of an integrated strategy aimed to reduce the use of conventional anthelmintics. Therefore, these results may be significant for the future management of these infections. The development of anthelmintic resistance in sheep gastrointestinal nematodes (GINs) requires novel strategies for the sustainable control of these parasites. This study aimed to evaluate the anthelmintic efficacy of the Origanum vulgare (L.) essential oil (EO) against sheep GINs and to evaluate the possibility of its use in control practice. The in vitro egg hatch test was conducted at eight different concentrations (50, 12.5, 3.125, 0.781, 0.195, 0.049, 0.025, and 0.0125 mg/mL) of the tested EO. For the in vivo fecal egg count reduction test, the EO of O. vulgare was administrated orally at a mean single dose of 150 mg/kg to sheep from two farms in Southern Italy, whereby potential toxic effects to the hosts were also evaluated. In the egg hatch test, the inhibition of egg hatchability varied from 71.3% to 93.7%, depending on the concentration used. The high anthelmintic potential was confirmed in the fecal egg count reduction test with an average reduction of nematode eggs in feces of 43.2% and 60.1% on days 7 and 14 after treatment, respectively. In addition, no toxic effects were noticed during the clinical examination of sheep or by observing blood count and liver or kidney function test results. The obtained results suggest the strong activity of the O. vulgare EO against sheep GINs, probably due to a high percentage of carvacrol (76.21%), whereby it can be considered safe for sheep at the dose tested in vivo. Therefore, it is suitable for use in veterinary practice as a part of an integrated strategy for the control of sheep GINs. [ABSTRACT FROM AUTHOR]

The Kubic FLOTAC microscope (KFM) is a compact, low-cost, versatile and portable digital microscope designed to analyse fecal specimens prepared with Mini-FLOTAC or FLOTAC, in both field and laboratory settings. In this paper, we present the characteristics of the KFM along with its first validation for fecal egg count (FEC) of gastrointestinal nematodes (GINs) in cattle. For this latter purpose, a study was performed on 30 fecal samples from cattle experimentally infected by GINs to compare the performance of Mini-FLOTAC either using a traditional optical microscope (OM) or the KFM. The results of the comparison showed a substantial agreement (concordance correlation coefficient = 0.999), with a very low discrepancy (−0.425 ± 7.370) between the two microscopes. Moreover, the KFM captured images comparable with the view provided by the traditional OM. Therefore, the combination of sensitive, accurate, precise and standardized FEC techniques, as the Mini-FLOTAC, with a reliable automated system, will permit the real-time observation and quantification of parasitic structures, thanks also to artificial intelligence software, that is under development. For these reasons, the KFM is a promising tool for an accurate and efficient FEC to improve parasite diagnosis and to assist new generations of operators in veterinary and public health. [ABSTRACT FROM AUTHOR]

Canine leishmaniosis (CanL) is caused by protozoans of the genus Leishmania and characterized by a broad spectrum of clinical signs in dogs. Early diagnosis is of great importance in order to perform an appropriate therapy and to prevent progression towards severe disease. The aim of this study was to compare a point-of-care molecular technique, i.e., the loop-mediated isothermal amplification (LAMP), with a real-time polymerase chain reaction (Rt-PCR), and three serological techniques, i.e., immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), and a rapid SNAP Leishmania test, to develop an integrated approach for the diagnosis of CanL. Sixty dogs were chosen after physical examination and collection of blood and sera samples, fine-needle aspiration of lymph nodes, and conjunctival swabs were performed. Lymphadenopathy (82.3%), as well as clinicopathological alterations of total proteins (70.6%), were the most frequent signs. Forty-one (68.3%) samples resulted positive at least to one technique. IFAT resulted in the best serological diagnostic method (specificity = 100%, sensitivity = 97.2%), detecting a higher number of positive samples than those revealed by other techniques. Among the samples used for molecular analysis, fine-needle aspiration of lymph nodes was revealed as the best sample source. LAMP showed a substantial agreement (κ = 0.80; p <0.0001) with Rt-PCR; therefore, it could be promising for the rapid diagnosis of CanL. Nevertheless, further studies should be performed to confirm these findings. [ABSTRACT FROM AUTHOR]

Sarcocystosis is a protozoal disease affecting a wide range of animals. The aims of this study were to characterize the following in sheep: (1) the muscle pathology in Sarcocystis infection, (2) the inflammatory infiltrate and its relationship to severity of infection, and (3) immune markers expressed by parasitized muscle fibers and parasitic cysts. Skeletal muscle samples from 78 sheep slaughtered in southern Italy were snap frozen and analyzed by histopathology, immunohistochemistry, and immunofluorescence. Polymerase chain reaction (PCR) and sequencing were used for Sarcocystis species identification. All 40 muscle samples tested were PCR-positive for Sarcocystis tenella. Histologically, cysts were identified in 76/78 cases (97%), associated with an endomysial infiltrate of lymphocytes and plasma cells. The T cells were predominantly CD8+, with fewer CD4+ or CD79α+ cells. Eosinophils were absent. Notably, sarcolemmal immunopositivity for major histocompatibility complex (MHC) I and II was found in 76/78 cases (97%) and 75/78 cases (96%), respectively, both in samples with and in those without evident inflammatory infiltrate. The number of cysts was positively correlated with inflammation. In addition, MHC I was detected in 55/78 cyst walls (72%), and occasionally co-localized with the membrane-associated protein dystrophin. The findings suggest that muscle fibers respond to the presence of cysts by expression of MHC I and II. The possible role of MHC I and II in the inflammatory response and on the cyst wall is also discussed. [ABSTRACT FROM AUTHOR]

Animal Assisted Interventions (AAIs) programmes have been considered useful in different settings, such as hospital, therapeutic, educational and assisted living environments. In these contexts, all animals, and particularly dogs, should be subjected to appropriate health controls to prevent a potential risk of transmission of zoonotic agents. Domestic dogs are reservoirs of many zoonotic pathogens including several gastrointestinal parasites (protozoa and helminths). Therefore, the aim of the present study was to investigate the presence of the protozoanGiardia duodenalisand zoonotic gastrointestinal nematodes (geohelminths) in dogs hosted in a dog educational centre in the city of Naples (southern Italy) where the animals were trained to AAI. Between April and June 2016, 74 dog faecal samples were analysed using the FLOTAC dual technique to detectG. duodenaliscysts and other parasitic elements. Out of the 74 faecal samples examined, 18 (24.3%; 95% CI = 15.4–35.9) were positive for parasitic elements. Specifically, 8 were positive forG. duodenalis(44.4%; 95% CI = 22.4–68.7). In addition, some co-infections were also found: one sample (5.6%; 95% CI = 0.3–29.4) resulted positive to bothToxocara canisandTrichuris vulpisand two samples (11.1%; 95% CI = 1.9–36.1) were positive to bothG. duodenalisand Ancylostomidae. Given that children, young adults and immunocompromised individuals are among the main users of the AAIs, specific guidelines targetingG. duodenalisand other gastrointestinal zoonotic parasites should be formulated in order to develop effective control and prevention strategies and reduce the zoonotic risk favoured by the human-dog interaction. [ABSTRACT FROM PUBLISHER]

Background: Nematode infections in horses are widespread across the world. Increasing levels of anthelmintic resistance, reported worldwide in equine parasites, have led to the creation of programs for the control of nematodes based on faecal egg counts (FEC). To improve nematode egg counting in equine faecal samples and establish whether the matrix of equine faeces or the eggs affect the counts, the analytical sensitivity, accuracy and precision of Mini-FLOTAC (combined with Fill-FLOTAC), McMaster and Cornell-Wisconsin techniques were compared. Known numbers of eggs extracted from equine or ovine faeces were added to egg free ovine and equine faeces to give counts of 10, 50, 200 and 500 eggs per gram (EPG) of faeces. Results: The Cornell-Wisconsin significantly underestimated egg counts and McMaster showed a low analytical sensitivity, revealing 100% of sensitivity only for concentrations greater than 200 EPG. EPG values detected by Mini-FLOTAC did not differ significantly from expected counts at any level of egg density. Conclusions: Mini-FLOTAC combined to Fill-FLOTAC which provides an accurate method of weighing without need for a balance and filtering out debris, could be used for FEC on the farm as well as in the laboratory. [ABSTRACT FROM AUTHOR]

Background: Analyses of environmental correlates of the composition of gastrointestinal parasite communities in black howler monkeys (Alouatta pigra) have been hindered by inadequate calibration techniques of detection and quantification methods of the parasites. Here we calibrate samples and compare the likelihood of parasite detection using two flotation techniques, FLOTAC and Mini-FLOTAC, and compare flotation solution, preservation method and dilution ratio for egg detection and counts of the most common parasites (Controrchis spp. and Trypanoxyuris spp.) in howler monkeys. Results: For samples preserved in 5% formalin, the Mini-FLOTAC technique was the best option for qualitative and quantitative copro-microscopic analysis. This technique displays an 83.3% and 100% detection of Controrchis spp. and Trypanoxyuris spp. infections, respectively. For the trematode Controrchis spp., more eggs per gram of feces (EPG) were recorded with the flotation solution (FS) #7 (zinc sulfate; specific gravity SG = 1.35) at 1:20 and 1:25 dilution than other methods. By contrast, for the nematode Trypanoxyuris spp., the best results were recorded with FS1 (sucrose and formaldehyde; SG = 1.20) at 1:10 dilution. Conclusions: We recommend the Mini-FLOTAC technique for general use with parasite analysis on frugivore/ folivores like the howler monkey, especially if many samples are analyzed. The technique has a high detection rate and the best EPG counts, allowing the qualitative and quantitative analysis of parasite load among the species or populations without the need for specialized equipment. [ABSTRACT FROM AUTHOR]

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Copyright of Summa, Animali da Compagnia is the property of Point Veterinaire Italie s.r.l. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background: The cardiopulmonary nematodes Dirofilaria immitis and Angiostrongylus vasorum are increasingly reported in dogs and are responsible for two diseases with overlapping endemic areas, especially in Europe: dirofilariosis and angiostrongylosis. The reasons for their apparent emergence are unknown, but several factors (e.g. increased disease awareness, better diagnostic tools, climatic changes, seasonal population dynamics and movements of animals) may play a role in the recent rise in reports of infection in the various countries of Europe. The aim of this study was to investigate the seroprevalence of D. immitis (by DiroCHECK® ELISA) and the fecal presence of first stage larvae (L1) of A. vasorum (by FLOTAC) in dogs from 68 kennels of the Campania region (southern Italy). The fecal samples were collected from pooled samples using the box as epidemiological unit. To the authors's knowledge, this is the first cross-sectional survey conducted at regional-scale in Italy and in Europe on the contemporaneous detection of D. immitis antigens and A. vasorum L1 in kennels. Results: Antigens of D. immitis were detected in 24/537 (4.4 %; 95 % Confidence Interval = 3.0-6.7) dogs in 6 out of the 68 kennels (8.8 %; 95 % CI = 3.6-18.9). The 24 positive samples for D. immitis antigen were tested also with AngioDetect® and only 1 sample was seropositive for A. vasorum with a prevalence of 4.2 %. A. vasorum L1 were detected in dogs from 9 out of the 68 kennels (13.2 %; 95 % CI = 21.8-44.9). Pooled fecal samples from 25 boxes out of the 1360 analyzed resulted positive to A. vasorum L1 (1.8 %; 95 % CI = 1.2-2.7). Conclusions: The present study indicates that cardiopulmonary nematodes are present in Campania region in symptomatic dogs as well as in asymptomatic ones. Therefore, regular parasitological surveillance, appropriate treatment strategies and high quality standard of hygiene are required to guarantee the health and welfare of kennel dogs. [ABSTRACT FROM AUTHOR]

Background Kennel dogs are at the high risk of infections with intestinal and extra-intestinal helminths. Therefore, regular parasitological surveillance, appropriate treatment strategies and high quality standard of hygiene are required to guarantee the health and welfare of kennel dogs. The aim of the present study was to evaluate the efficacy of helminth control in kennels using different broad-spectrum anthelmintics that are routinely used in canine veterinary practice. Particular attention was given to the field efficacy and ease-of-use of each product. Methods The study was conducted in 3 public kennels in the Campania region (southern Italy). Eighteen boxes from each of the three kennels were selected for treatment based on faecal egg counts (FECs) at Day -30. The treatments were conducted using tablets containing combinations of: i) milbemycin oxime and praziquantel (Milbemax®); ii) pyrantel embonate, febantel and praziquantel (Drontal Plus Flavour®), and; iii) pyrantel pamoate, oxantel pamoate and praziquantel (Nemex® POP). All dogs were treated on Day 0 and sampled on Days 0, 7, 14 and 21 for copromicroscopic analyses. The FLOTAC dual technique on pooled samples was used with efficacy determined by reduction in FECs. Results At Day -30 intestinal nematodes (hookworms, Toxocara, Trichuris) and cestodes (Dypilidium caninum) were found. Milbemax® showed 100% efficacy against all the helminths in all the kennels. Drontal Plus Flavour® was 100% effective against hookworms in all the kennels but gave lower efficacy against T. canis (range = 97.1-100%) and T. vulpis (range = 95.6-100%). Nemex® POP was also 100% effective against hookworms in all kennels but less effective against T. canis (range = 95.7-100%) and T. vulpis (range = 95.7- 100%). All three drugs were 100% effective against D.caninum. Conclusions Milbemax®was fully effective against all the helminthes and was palatable and ease-of-use for all the dogs. It is the optimum choice for the treatment and prevention of helminth infections in kennel dogs under field conditions. [ABSTRACT FROM AUTHOR]

Background: Capillaria plica (syn. Pearsonema plica) is a nematode that resides in the urinary bladder and rarely in ureters or in the kidney pelvis of various carnivores, especially foxes and dogs. Urine sedimentation technique is actually the only diagnostic tool that permits the identification of C. plica eggs, but its sensitivity is low and when an infection is suspected (or when it is necessary to confirm treatment efficacy) more than one examination of urine sediment should be performed. The present paper reports a clinical case of natural C. plica infection in a dog from southern Italy. In addition, two new techniques, FLOTAC and Mini-FLOTAC, were used for the diagnosis of C. plica in dog urine and compared with the technique of sedimentation. Results: Using FLOTAC with fresh urine and sodium chloride as flotation solution, were obtained the best results for the diagnosis of C. plica in dog urine in term of eggs counted (mean eggs per 10 ml of urine = 70.3 FLOTAC vs 40.3 Mini FLOTAC vs 32.8 sedimentation) and coefficient of variation (CV%) (6.2 FLOTAC vs 13.4 Mini-FLOTAC vs 32.9 sedimentation). Conclusions: The FLOTAC was the more sensitive method, but also the Mini-FLOTAC could be a valid alternative diagnostic method because gave better results than the classical sedimentation and can be used in place of the FLOTAC in laboratories where the centrifugation step cannot be performed. [ABSTRACT FROM AUTHOR]

Herbal products such as essential oils may play a promising role in the treatment of infections caused by gastrointestinal nematodes (GINs). The aim of this study was to evaluate the in vitro potential of 11 essential oils (EOs) and one binary combination of isolated EO compounds, as well as the in vivo anthelmintic efficacy of two EO formulations. Four GIN genera were identified in the coproculture examination: Haemonchus, Trichostrongylus, Teladorsagia and Chabertia. The in vitro egg hatch test (EHT) was performed at six different concentrations (50, 12.5, 3.125, 0.781, 0.195 and 0.049 mg/mL) for each EO, whereas in the in vivo faecal egg count reduction test (FECRT), each EO sample was diluted in sunflower oil and orally administrated at a dose of 100 mg/kg to the different group of animals. In the EHT, the EOs of Origanum vulgare, Foeniculum vulgare, Satureja montana, Satureja hortensis and two types of Thymus vulgaris were the most effective. The dominant compounds of these EOs were carvacrol, thymol, anethol, p-cymene and γ-terpinene, indicating their importance for the anthelmintic activity. In the FECRT, both T. vulgaris EO type 1 and linalool:estragole combination show an anthelmintic potential with a mean effect on FECR of approximately 25%. The results suggest the possible role of tested EOs as anthelmintic agents in sheep farms, although further in vivo tests are needed. [ABSTRACT FROM AUTHOR]