First report on natural infection by Dirofilaria repens in a cat in Spain: case report and literature review of feline subcutaneous dirofilariosis in Europe.

Dirofilaria repens is an endemic mosquito-borne pathogen widely spread throughout Europe and other non-Europen regions. Infection by D. repens has been reported in dogs, although little is known about the occurrence and epidemiological features of this nematode in cats. During the surgical procedure within the framework of a trap, neuter and release sterilization program, two female filarial nematodes with spontaneous movement were removed from the internal part of the spermatic cord from asymptomatic stray cat in Spain (Grao de Castellón, Castellon de la Plana, province of Spain). Moreover, the presence of microfilariae was detected by using three different methods, including direct blood smear technique, the microhematocrit tube test, and Knott´s modified test. PCR performed from the nematodes extracted from the spermatic cord and from microfilariae in EDTA-blood sample tested both positive for D. repens. To the authors' knowledge, this is the first report that demonstrates the presence of D. repens in cats in Spain. In this sense, a review of the current scientific status of feline subcutaneous dirofilariosis in Europe was also performed. Future investigations should analyze the epidemiological role of cats in D. repens infection including the prevalence of infection. Veterinarians working in endemic areas should be aware of this infection in cats and their susceptibility.

Is there any change in the prevalence of intestinal or cardiopulmonary parasite infections in companion animals (dogs and cats) in Germany between 2004-2006 and 2015-2017? An assessment of the impact of the first ESCCAP guidelines.

Main objective of the present nationwide study was to assess the impact of the ESCCAP guideline for the control of worm infections in dogs and cats 8-10 years after its first publication in Germany. A secondary aim was to determine the prevalence of canine and feline cardiopulmonary nematodes and intestinal protozoa. Faecal samples of 53,693 dogs and 26,491 cats in 2004-2006 as well as of 129,578 dogs and 45,709 cats in 2015-2017 routinely submitted by veterinarians to a private veterinary laboratory were examined using appropriate parasitological methods. In dogs, the prevalence of Toxocara and taeniid egg shedding was significantly lower in 2015-2017 (3.8 % and 0.16 %, respectively) than in 2004-2006 (4.6 % and 0.27 %, respectively). The prevalence of hookworm and Capillaria eggs was higher in the second study period (2.3 % and 0.77 %, respectively) than in the first (1.3 % and 0.6 %, respectively). For Toxascaris leonina (0.55-0.6 %) and Trichuris (0.8-0.9 %), the difference was not significant between the study periods. Dogs shed more often Angiostrongylus vasorum larvae in the second study (3.1 %) than in the first (1.0 %), whereas the prevalence of Crenosoma vulpis did not change significantly (2.2-2.6 %). Cystoisospora canis and C. ohioensis-like infections were less detected in the second study period (1.0 % and 2.1 %, respectively) than in the first (1.8 % and 2.7 %, respectively). Neospora-like oocysts and Sarcocystis sporocysts were more prevalent in the second study period (0.19 % and 0.13 %, respectively) than in the first (0.13 % and 0.06 %, respectively). The percentage of Giardia or Cryptosporidium coproantigen-positive samples was lower in the second study period (18.9 % and 6.7 %, respectively) than in the first (22.8 % and 10.0 %, respectively). In cats, the prevalence of egg shedding of T. cati, Capillaria and taeniids was significantly lower in 2015-2017 (3.5 %, 0.25 % and 0.1 %, respectively) than in 2004-2006 (4.8 %, 0.54 % and 0.22 %, respectively). No difference was recorded for hookworms (0.12-0.13 %) and Ts. leonina (0.04-0.05 %). Aelurostrongylus-like larvae were detected more often in the second study period (6.5 %) than in the first (2.6 %). Infections with Cystoisospora felis, C. rivolta, Toxoplasma-like coccids and Sarcocystis were less prevalent in the second study period (1.9 %, 0.7 %, 0.24 % and 0.02 %, respectively) than in the first (2.7 %, 1.1 %, 0.36 % and 0.1 %, respectively). The percentage of Giardia or Cryptosporidium coproantigen-positive samples was significantly lower in the second study period (10.6 % and 4.8 %, respectively) than in the first (15.4 % and 8.3 %, respectively). Although these results indicate a decline of the occurrence of most canine and feline intestinal parasites in Germany over the years, a transmission risk of zoonotic parasites remains. Therefore, the control of helminth infections in domestic dogs and cats continues to be a challenge for veterinarians and pet owners.

In vitro activity of selected natural products against Eimeria tenella sporozoites using reproduction inhibition assay.

Eimeria tenella is the causative agent of cecal coccidiosis in poultry characterized by weight loss, hemorrhagic diarrhea, and high mortality rates. Research into herbal candidates with possible anticoccidial activity has increased lately. As an alternative to animal experiments, an in vitro reproduction inhibition assay (RIA) was previously designed to determine the sensitivity of E. tenella isolates against ionophores. In this study, the RIA was used to test the anticoccidial activity of nutmeg oil, cinnamon oil, and glabridin. The concentration of nutmeg oil used in this study ranged between 1.1 and 139.1 µg/ml. Nutmeg oil exhibited a moderate in vitro inhibitory activity ranging from 35.5 to 49.5%. In contrast, no inhibitory effect was detected when incubating E. tenella sporozoites for 24 h with cinnamon oil at concentrations of 0.3 to 80.5 µg/ml. Glabridin (0.08-41.7 µg/ml) prevented the replication of sporozoites at a rate of 14.1 to 81.7% of inhibition. The calculated minimum concentrations of glabridin needed to inhibit parasite replication by 75%, 50%, and 30% (MIC75, MIC50, and MIC30) were 21.43 µg/ml, 5.28 µg/ml, and 0.96 µg/ml, respectively. Further studies to assess the in vitro efficacy of glabridin were performed by studying mRNA gene expression of stress-induced protein genes (HSP-70, NADPH, and EtPP5) after exposure of E. tenella sporozoites to glabridin at MIC75 for 0.5 h, 1 h, 2 h, and 4 h (a time-dependent experiment). Moreover, a dose-dependent experiment was performed using glabridin at a concentration matching MIC75, MIC50, and MIC30 for 24 h. In the time-dependent experiment, a significant (p < 0.05) increase of expression in NADPH and EtPP5 were detected after 4 h of incubation with glabridin at a concentration of 21.43 µg/ml. The dose-dependent experiment exhibited a gradual increase of expression in all studied genes, which indicates stress imposed on E. tenella sporozoites by glabridin. In our hands, RIA was suitable to assess the anticoccidial activity exhibited by the tested natural products as a precursor to in vivo studies which will help in the identification of novel anticoccidial candidates.

Canine Dracunculus Nematode Infection, Toledo, Spain.

A fragment of a Dracunculus-like worm was extracted from the hind limb of a 2-year-old dog from Toledo, Spain. Cytochrome oxidase I and rRNA sequences confirmed an autochthonous mammalian Dracunculus worm infection in Europe. Sequence analyses suggest close relation to a parasite obtained from a North American opossum.

Passive immunization with Eimeria tenella gametocyte antigen 56 (EtGAM56) specific antibodies and active immunization trial with the epitope containing peptide.

Eimeria species cause avian coccidiosis leading to substantial economic losses in the poultry industry. Hence, anticoccidial drugs and vaccines have been used to combat this devitalizing disease. An effective vaccine based on gametocyte recombinant proteins would be very useful in terms of cost, labor and ethics (no animal experimentation). A mouse derived monoclonal antibody against Eimeria tenella gametocyte antigen 56 (EtGAM56) was used to immunize peritoneally E. tenella infected chicken a reduction of oocyst shedding by up to 78% was observed. The epitope recognized by the monoclonal antibody was mapped, recombinant expressed and used to immunize chicken (rEtGAM56N). After rEtGAM56N intramuscular immunization and parasite challenge, clinical parameters like faecal oocyst output, body weight gain, lesion score, feed conversion rate and serum antibody response were assessed to test the efficacy of vaccination against experimental infection with E. tenella. Chicken immunized with rEtGAM56N and challenged with E. tenella oocysts showed a robust antibody response against the rEtGAM56N peptide but no considerable effects on oocyst output and clinical parameters (weight gain, lesion score, feed conversion rate) compared to the mock control group. This study demonstrates the complexity of an effective vaccination. The immunoprotective epitope might be a conformational epitope that was recognized by the monoclonal mouse antibody but only weakly by the antibodies produced against the linear peptide leading to a divergent outcome between the passive and active immunization.

Anticoccidial efficacy testing: In vitro Eimeria tenella assays as replacement for animal experiments.

SCOPE: Availability of an accurate in vitro assay is a crucial demand to determine sensitivity of Eimeria spp. field strains toward anticoccidials routinely. In this study we tested in vitro models of Eimeria tenella using various polyether ionophores (monensin, salinomycin, maduramicin, and lasalocid) and toltrazuril. Minimum inhibitory concentrations (MIC95, MIC50/95) for the tested anticoccidials were defined based on a susceptible reference (Houghton strain), Ref-1. In vitro sporozoite invasion inhibition assay (SIA) and reproduction inhibition assay (RIA) were applied on sensitive laboratory (Ref-1 and Ref-2) and field (FS-1, FS-2, and FS-3) strains to calculate percent of inhibition under exposure of these strains to the various anticoccidials (%ISIA and%IRIA, respectively). The in vitro data were related to oocyst excretion, lesion scores, performance, and global resistance indices (GI) assessed in experimentally infected chickens. RESULTS: Polyether ionophores applied in the RIA were highly effective at MIC95 against Ref-1 and Ref-2 (%IRIA≥95%). In contrast, all tested field strains displayed reduced to low efficacy (%IRIA<95%).%IRIA values significantly correlated with oocyst excretion determined in the animal model (p<0.01) for polyether ionophores. However, this relationship could not be demonstrated for toltrazuril due to unexpected lack of in vitro sensitivity in Ref-2 (%IRIA=56.1%). In infected chickens, toltrazuril was generally effective (GI>89%) against all strains used in this study. However, adjusted GI (GIadj) for toltrazuril-treated groups exhibited differences between reference and field strains which might indicate varying sensitivity. CONCLUSION: RIA is a suitable in vitro tool to detect sensitivity of E. tenella towards polyether ionophores, and may thus help to reduce, replace, or refine use of animal experimentation for in vivo sensitivity assays.

In vitro efficacy of allicin on chicken Eimeria tenella sporozoites.

Chicken coccidiosis is a major parasitic disease caused by Eimeria spp. It is controlled and treated using chemical anticoccidial agents. Development of partial or complete resistance toward these anticoccidials is considered a major problem in poultry industry. Allicin is an organosulfur compound produced as a result of the reaction between alliin and alliinase after hacking of garlic. In this study, tenfold dilution from 180 mg/ml to 1.8 ng/ml of allicin in distilled water was tested against E. tenella in vitro. The percent of inhibition in allicin was from 99.9 to 71.53% using 180 mg/ml and 180 ng/ml, respectively. The percent of inhibition was 56.24% using 1.8 ng/ml. We used allicin as a treatment from plants against chicken coccidiosis; however, in vivo study should be performed to confirm these results.

Combination of cell culture and qPCR to assess the efficacy of different anticoccidials on Eimeria tenella sporozoites.

Three in vitro studies were designed to develop an assay for anticoccidial efficacy by use of laboratory (Houghton) and field (T-376) Eimeria tenella strains. In study (1), minimum inhibitory concentrations (MICs) of monensin (Mon), maduramicin (Mad), salinomycin (Sal), and lasalocid (Las) were determined that are able to inhibit more than 50% of sporozoites in host cell (Madin-Darby bovine kidney (MDBK)) penetration and more than 95% of Houghton sporozoites development to mature merozoites (treatment time 24 h) using quantitative real-time PCR (qPCR). MICs were 0.5, 2.5, 1, and 0.5 µg/ml for Mon, Mad, Sal, and Las, respectively. Applying the previous MIC on T-376 strain revealed a different sensitivity profile. Mad reduced T-376 gene copies by only 89.3% after 96 h of infection. In study (2), Houghton strain sporozoites were incubated with MIC of the different tested ionophores for 2 and 4 h, respectively; afterwards, their ability to invade MDBK cells was determined using phase-contrast microscopy and qPCR. Treatment of sporozoites with ionophores for 4 h resulted in significant inhibition of invasion compared with non-treated parasites as assessed both by microscopy as well as qPCR. Inhibition rates for Mon, Mad, Sal, and Las were 90.2, 75.0, 88.3, and 82.6% using phase-contrast microscopy and 83.9, 81.4, 85.8, and 75.4% using qPCR, respectively. T-376 sporozoite invasion into MDBK cells was reduced to 48.9% by Mad. Study (3) was conducted to determine inhibition exerted by toltrazuril (Tol). Tol at 5 µg/ml reduced reproduction of Houghton strain by 95%, whereas T-376 was only reduced by 86.5%. The presented experiments indicate that infectivity inhibition of sporozoites incubated for 4 h with anticoccidials and development inhibition after 96 h of infection by qPCR are suitable means to assess sensitivity of E. tenella strains to anticoccidials.

Efficacy of an anticoccidial live vaccine in prevention of necrotic enteritis in chickens.

Necrotic enteritis (NE) is an important disease in poultry caused by Clostridium perfringens combined with predisposing factors, mainly eimeriosis. In the present study, we investigated the protective effect of a commercial attenuated anticoccidial live vaccine against NE in a clinical infection model using 60 day-old chicks. Vaccination was performed on study day (SD) 1 with natural booster-infections for 4 weeks from Eimeria spp. oocysts present in litter. On SD 28, five groups were formed (n=12): group V+/C-E- (vaccinated, uninfected), group V+/C-E+ (vaccinated, infected with Eimeria spp.), group V+/C+E+ (vaccinated, infected with clostridia and Eimeria spp.), group V-/C+E+ (unvaccinated, infected with clostridia and Eimeria spp.), and group NC (negative control). Efficacy was measured by clinical parameters, pathogen multiplication, and pathological parameters assessed during two necropsies on SD 34 and SD 40, respectively. Additionally, cytokine expression was measured in gut and spleen tissues at necropsy. Clinical signs of NE were observed only in the coinfected groups, mainly in group V-/C+E+. Accordingly, lowest body weight gain was observed in group V-/C+E+ (301.8 g from SD 28 to SD 40; group NC: 626.2 g). Oocyst excretion varied significantly (P<0.01) between all Eimeria spp. infected groups and was highest in group V-/C+E+, followed by V+/C+E+, and lowest in group V+/C-E+. NE typical intestinal lesions showed only in groups V+/C+E+ and V-/C+E+. The intestinal mucosa featured partly severe lesions in the jejunum, C. perfringens colonization was histologically visible. Upregulation of IFN-γ, was observed in the jejunal tissue of group V-/C+E+ (P<0.01 (SD 34) or P<0.05 (SD 40) compared to all other groups). IL-10 and IL-12 were upregulated in group V-/C+E+, IL-10 also in group V+/C+E+ (SD 40) while IL-2 expression remained unaltered. In conclusion, vaccination against coccidiosis was effective in preventing NE in a mixed infection comparable to field situations.

Efficacy of early treatment with toltrazuril in prevention of coccidiosis and necrotic enteritis in chickens.

In the present study, efficacy of the toltrazuril treatment for prevention of coccidiosis and necrotic enteritis was tested. Ninety-six 14-day-old commercial broiler chickens were caged and divided into eight groups (n=12), designated groups 1 to 8. Chickens of groups 1 to 6 were inoculated orally at 18 days of age with 25,000 oocysts of Eimeria tenella and 75,000 oocysts of Eimeria brunetti. At 22 days of age, chickens of groups 1 to 6 were infected with 10(9) colony-forming unit Clostridium perfringens. Chickens of group 1 were treated with 75 parts/10(6) toltrazuril in drinking water for 8 h on two consecutive days up to 12 h before Eimeria infection, while chickens of groups 2 to 5 were treated with the same dose of toltrazuril at 12 h, 36 h, 60 h and 84 h after Eimeria infection, respectively. The non-treated group 6 served as a positive control. Chickens in group 7 were treated with toltrazuril at 17 and 18 days of age, and those of group 8 remained uninfected and non-treated as a negative control. The feed conversion ratio was higher in the positive control compared with other groups. The mortality rates were 16.8% and 41.7% in the late toltrazuril-treated (at 84 h) and infected non-treated chickens, respectively. Lesions scores of necrotic enteritis or coccidiosis in infected, non-treated chickens were significantly more severe compared with negative controls (P<0.01) and late toltrazuril-treated (at 84 h) chickens (P<0.05). In conclusion, application of toltrazuril before Eimeria challenge protected chickens from coccidiosis and indirectly from successive necrotic enteritis caused by C. perfringens infection.

Embryonated chicken eggs as an alternative model for mixed Clostridium perfringens and Eimeria tenella infection in chickens.

The chorioallantoic membrane (CAM) of chicken embryo eggs is a suitable model for viral and bacterial infections. In the present study, a new approach for testing the pathogenesis and virulence of Clostridium perfringens and Eimeria tenella dual infections as a model using the CAM of embryonated chicken eggs was developed. For this purpose, 24 specific pathogen-free (SPF) embryonated chicken eggs were divided into four groups (n = 6) and designated group E, group CP, group CPE, and NC. Sporozoites of E. tenella (20,000 sporozoites) were inoculated into 10-day-old embryonated SPF chicken eggs (groups E and CPE) via allantoic sac route. At 15-day-old, eggs of groups CP and CPE were infected with 10 (4)  cfu C. perfringens via the same route. Assessment of pathogenicity was assessed using gross and histopathological lesions. Embryo mortality reached 17 % after mono-infection with C. perfringens and/or E. tenella and 50 % in the mixed-infected group. Lesions in the CAMs were most numerous and most severe in co-infected eggs (group CPE), reaching the maximum score of 3 in 50 % of the inoculated eggs (P < 0.01). In Eimeria spp.-infected eggs (group E), lesions of score were between 1 and 2. Mono-infection with C. perfringens did not lead to a significant occurrence of lesions. Histopathological investigations of the CAM revealed clusters of Gram-positive bacteria, infiltration with leukocytes, lymphocytes, and developmental stages of E. tenella in the co-infected group. These data suggest that embryonated eggs could be an in ovo model for studying the pathogenesis of mixed infection with Eimeria and C. perfringens.