Parasites and zoonotic bacteria in the feces of cats and dogs from animal shelters in Carinthia, Austria.

Due to their close associations with humans, dogs and cats can be important reservoirs for zoonotic pathogens. In the current study 200 fecal samples of dogs (n = 70 samples) and cats (n = 130 samples) from animal shelters in Carinthia, southern Austria, were examined for the presence of parasites (fecal flotation and larval migration assay) and selected bacteria. Overall, 17.1% of the canine and 38.5% of the feline samples were positive for parasites (p < 0.001), most commonly Giardia duodenalis (dogs and cats), including potentially zoonotic genotypes revealed by multilocus genotyping, and Toxocara cati (cats). Cryptosporidium (C. felis), Cystoisospora spp. (dogs and cats), hookworms (dog), Trichuris (dog) Capillaria hepatica (cats), taeniids (cat), and Aelurostrongylus abstrusus (cat) were also found. Zoonotic bacteria were detected in 10.5% of the samples, Salmonella enterica (dogs), Campylobacter jejuni (dogs and cats) and Yersinia enterocolitica (cat) and were significantly associated with parasite infections in cats but not in dogs. Samples that were positive for several pathogens were common; especially G. duodenalis and T. cati were frequently found in association with each other, other parasites or bacteria. The spectrum of detected pathogens is comparable to that of other dog and cat populations in central Europe. However, since animals from shelters are frequently rehomed, diagnostic measures, appropriate hygiene and therapy as well as training of shelter staff are recommended to prevent zoonotic transmission of enteropathogens to staff or new owners. The presence of heteroxenic parasites, i.e. Aelurostrongylus abstrusus and Taenia taeniaeformis, and spurious excretion of Ca. hepatica in cats, indicates that these animals preyed on intermediate hosts, and that biosafety measures in pet shelters need to be evaluated for their efficacy in the prevention of pathogen transmission.

Absorption and Distribution of Toltrazuril and Toltrazuril Sulfone in Plasma, Intestinal Tissues and Content of Piglets after Oral or Intramuscular Administration.

Piglet coccidiosis due to Cystoisospora suis is a major cause of diarrhea and poor growth worldwide. It can effectively be controlled by application of toltrazuril (TZ), and oral formulations have been licensed for many years. Recently, the first parenteral formulation containing TZ in combination with iron (gleptoferron) was registered in the EU for the prevention of coccidiosis and iron deficiency anemia, conditions in suckling piglets requiring routine preventive measures. This study evaluated the absorption and distribution of TZ and its main metabolite, toltrazuril sulfone (TZ-SO2), in blood and intestinal tissues after single oral (20 mg/kg) or single intramuscular (45 mg/piglet) application of TZ. Fifty-six piglets were randomly allocated to the two treatment groups. Animals were sacrificed 1-, 5-, 13-, and 24-days post-treatment and TZ and TZ-SO2 levels were determined in blood, jejunal tissue, ileal tissue, and mixed jejunal and ileal content (IC) by high performance liquid chromatography (HPLC). Intramuscular application resulted in significantly higher and more sustained concentrations of both compounds in plasma, intestinal tissue, and IC. Higher concentrations after oral dosing were only observed one day after application of TZ in jejunum and IC. Toltrazuril was quickly metabolized to TZ-SO2 with maximum concentrations on day 13 for both applications. Remarkably, TZ and TZ-SO2 accumulated in the jejunum, the primary predilection site of C. suis, independently of the administration route, which is key to their antiparasitic effect.

Piglet coccidiosis in Belgium and the Netherlands: Prevalence, management and potential risk factors.

Piglet coccidiosis caused by Cystoisospora suis is one of the most important causes of diarrhea in suckling piglets. The parasite has a fast development and multiplies quickly and effectively under the conditions of a farrowing unit. Control measures include cleaning and disinfection and anticoccidial treatment. In Europe, toltrazuril-based products are authorized for this purpose and are applied to piglets on affected farms in the first week of life. To observe the effect of treatment and disinfection on the control of piglet coccidiosis in the field, 23 farms (11 from Belgium, 12 from the Netherlands, mean number of sows = 1413) were sampled twice by litter in the second and third week of life and fecal scores and the presence of C. suis oocysts were determined. A questionnaire was used to collect data on farm sizes, management and hygiene measures as well as treatment (product, dose and piglet age). Thirteen farms regularly treated with toltrazuril (treatment age: 1-6 days, mean 4.3 days) and 19 applied disinfection. Parasite excretion was documented on 60.9% and diarrhea on 78.3% of farms and in 34.3%/15.7% of the litters. Only 2.4% of the litters showed both, so subclinical infection appeared to be common. No significant differences between farms that did not treat against coccidiosis and farms that applied toltrazuril was observed with regard to C. suis oocysts shedding and/or diarrhea. However, in litters that were treated within the first three days of life, oocyst excretion was significantly less often observed than in litters with later treatment (p = 0.033). No significant effect of disinfection could be shown, but most farms applied disinfectants that have no proven effect against coccidia (oxygen-releasing agents or glutaraldehyde + ammonia) while the only farm that used chlorocresols (which are effective against coccidia) did not show oocyst shedding. Current control measures thus appear to be insufficient on most of the examined farms. It is therefore recommended to treat piglets timely and to apply effective disinfectants to reduce C. suis infections. Furthermore, regular evaluation of sustained efficacy of all implemented measures are necessary.

Cystoisospora suis Control in Europe Is Not Always Effective.

After introduction of the anticoccidial toltrazuril for the metaphylactic treatment of suckling piglet coccidiosis, only few field evaluations on the effect of treatment against the causative agent, Cystoisospora suis, were performed. In 2018, a field study was conducted to detect the presence of the parasite on pig farms in four different European countries, and to evaluate management parameters possibly associated with infection and disease. A total of 49 farms from Austria, the Czech Republic, Germany and Spain were included. Repeated pooled fecal samples from 603 litters were taken in the 2nd and 3rd week of life. Samples were examined by autofluorescence for the presence of C. suis, and fecal consistency was scored. For each farm a questionnaire was provided to document management and treatment history. Feces scored as diarrhoeic were not significantly more often positive for C. suis than non-diarrhoeic feces but samples from litters with previously reported occurrence of diarrhea were significantly more often positive (p = 0.000). Pasty feces were significantly more often positive than those of other consistency (p = 0.005). Overall, 71.4% of the farms and 50.1% of the litters were positive for C. suis at least once. The prevalence on the farms reached up to 100%. Diarrhea was seen in samples from 53.1% of the farms (9.6% of the litters). Cystoisospora suis was diagnosed on 80.8% of the farms with vs. 60.8% of those without diarrhea. Toltrazuril was applied on 30 farms, and of these 53.3% had diarrhoeic samples and 66.7% were positive for C. suis vs. 19 farms that did not use toltrazuril with 52.6% diarrhoeic and 79.0% C. suis positive samples (p > 0.05). Only on two farms a disinfectant with activity against coccidia was used, and C. suis was not detected there. Current control of C. suis appears to be insufficient on the majority of the examined farms. These findings highlight the importance of correct application of medication, and an effective hygiene management. To maintain effective parasite control, efficacy monitoring of the control measures should be implemented.

Molecular characterization of Giardia intestinalis and Cryptosporidium parvum from calves with diarrhoea in Austria and evaluation of point-of-care tests.

To obtain information about the occurrence and genotype distribution of G. intestinalis and C. parvum in Austrian cattle, faecal samples from diarrhoeic calves younger than 180 days of age originating from 70 farms were examined. Of the 177 faecal samples, 27.1% were positive for Giardia cysts (immunofluorescence microscopy) and 55.4% for Cryptosporidium oocysts (phase-contrast microscopy). Positive samples were characterized by nested PCR for Giardia, 83.3% (triosephosphate isomerase; tpi) and 89.6% (ß-giardin; bg) were positive, while the Cryptosporidium nested PCR returned 92.5% (60-kDa glycoprotein) positive results. Sequence analysis revealed one assemblage A-positive sample and 30 (bg) respectively 29 (tpi) assemblage E-positive samples for G. intestinalis. For C. parvum four subtypes within the IIa family (IIaA15G2R1, n = 29; IIaA19G2R2, n = 3; IIaA21G2R1, n = 2; IIaA14G1R1, n = 1) could be differentiated. Validation of two immunochromatographic point-of-care tests resulted in a sensitivity of 29.2% and 77.6%; a specificity of 98.4% and 91.1% for the detection of Giardia intestinalis and Cryptosporidium parvum, respectively. Results confirm the widespread occurrence of both protozoa in diarrhoeic calves in Austria.

Efficacy of injectable toltrazuril-iron combination product and oral toltrazuril against early experimental infection of suckling piglets with Cystoisospora suis.

BACKGROUND: Toltrazuril is frequently administered for the metaphylactic control of piglet cystoisosporosis. In a previous study, the efficacy of parenteral toltrazuril (45 mg/piglet, Group Forceris®) applied on the 2nd day of life (dol), and of oral toltrazuril (20 mg/kg of body weight, Group Baycox®) applied on the 4th dol was evaluated in an experimental model with Cystoisospora suis infection on the 3rd dol (late infection, LI). In a follow-up study, efficacy and safety were evaluated against infections with C. suis on the 1st dol (early infection, EI). Parameters included oocyst excretion and faecal consistency, body weight development, bacteriological examinations and animal health. RESULTS: All control piglets (n = 12) shed oocysts and had diarrhoea, while parasite excretion was completely suppressed in both treatment groups (n = 13 each) and diarrhoea was reduced to a single animal (Forceris® group), resulting in significant differences for these parameters between the treated groups and the controls without significant differences among the treatment groups. No treatment-related adverse events were noted. Body weight gain was reduced in the control group during the acute phase of infection, resulting in significantly lower body weight on the 15th dol. Sows and piglets shed high numbers of Escherichia coli. Clostridium perfringens type A was only detected in low amounts in pooled litter samples. In comparison to the LI study oocyst shedding was more intense in the control animals in EI, while diarrhea was more frequent in LI. In both infection models a high efficacy of toltrazuril in the control of parasitological and clinical outcomes of experimental C. suis infection could be demonstrated. Since in the LI study high numbers of Cl. perfringens type A were detected, it is hypothesized that colonization with these opportunistic pathogens has synergistic effects with C. suis and may explain variable clinical outcomes in untreated animals as well as the sporadic occurrence of diarrhea in toltrazuril-treated piglets. CONCLUSIONS: Parenteral and oral toltrazuril administered on the 2nd or 4th dol is safe and effective against experimental infections with C. suis on the 1st to 3rd dol. The clinical outcome of experimental infections seems influenced by bacterial coinfections.

W A A V P guideline for evaluating the efficacy of anticoccidials in mammals (pigs, dogs, cattle, sheep).

This guideline is intended as an aid in the design, implementation and interpretation of studies for the assessment of drug efficacy against Eimeria in cattle and sheep, Cystoisospora in pigs and dogs, and Cryptosporidium in cattle. It deals with the most important aspects of how to conduct both experimental and field studies for dose determination, dose confirmation and assessment of field effectiveness. Also, guidance on the selection of animals, diagnostic techniques, statistical evaluation and methods for the preparation, maintenance and use of parasites is provided. The specific management conditions that may influence the course of natural infections and consequently determine treatment schemes are mentioned and suggestions for best practice in sampling and evaluation of data prior to conducting of efficacy studies are given. The guideline is also intended to assist investigators in carrying out specific studies, provide relevant information for registration authorities involved in the decision-making process, assist in the approval of anticoccidial drugs in the target species, and facilitate the world-wide adoption of standard procedures. Although currently not implemented, issues of drug resistance testing and alternative methods for drug testing are also discussed as future issues in drug testing against mammalian coccidia.

Comparison of an injectable toltrazuril-gleptoferron (Forceris®) and an oral toltrazuril (Baycox®) + injectable iron dextran for the control of experimentally induced piglet cystoisosporosis.

BACKGROUND: Cystoisospora suis causes diarrhoeal disease and reduced weight gain in suckling piglets, and a toltrazuril-based oral suspension is available for treatment. Recently a combinatorial product with toltrazuril plus iron has been developed for parenteral application. In this study we compared the efficacy of the injectable product with the oral suspension against experimentally induced piglet cystoisosporosis. METHODS: In a randomised controlled study, three groups of piglets (n = 10-13) were treated either with a fixed dose of 45 mg toltrazuril + 200 mg gleptoferron i.m. per piglet (Forceris®) on the second day of life (study day 2; SD 2) or with 20 mg toltrazuril/kg body weight as an oral suspension (Baycox® 5%) on SD 4 or left untreated (Control group). The Baycox® and the Control group received 200 mg of iron dextran/piglet on SD 2. All piglets were infected with 1000 sporulated C. suis oocysts on SD 3. Faecal samples were taken daily from SD 7 to SD 20 to determine faecal consistency, oocyst shedding and other diarrhoeal pathogens. Body weight was recorded on SD 1 and then weekly until SD 29. Animals were observed daily for general health and after treatment for possible adverse events. RESULTS: In the Control group all animals shed oocysts for 3.1 days on average and all animals showed diarrhoea for an average of five days. Excretion peaked on SD 9 (max. 48,618 oocysts per gram of faeces). Treatment with Forceris® completely suppressed oocyst excretion. In the Baycox® group, low levels of excretion could be detected. Diarrhoea was reduced to single piglets in the treated groups. Body weight development was reduced in the Control group compared to the treated groups. Enteropathogenic bacteria (Escherichia coli, Clostridium perfringens) could be detected. All parameters related to oocyst excretion, faecal consistency and weight gain were significantly improved in the treated groups compared to the Control group without significant differences between the treated groups. Both products were safe to use. CONCLUSIONS: Treatment with both the injectable (Forceris®) and the oral (Baycox®) formulation of toltrazuril in the prepatent period were safe and highly effective against experimental infection with C. suis in newborn piglets.

Comparative efficacy of two parenteral iron-containing preparations, iron gleptoferron and iron dextran, for the prevention of anaemia in suckling piglets.

Iron-deficiency anaemia (IDA) is a serious health problem in neonatal piglets and is controlled by routine application of iron in various formulations. The efficacy and safety of two iron-containing products for the prevention of IDA in suckling piglets were compared in a randomised, parallel study. Newborn piglets were treated with 200 mg iron supplied by intramuscular injection in the neck as either Forceris (gleptoferron; n=13) or Uniferon 200 (iron dextran; n=12) 24-48 hours after birth. Blood samples were collected before and after treatment (2nd, 18th and 31st day of life) for complete haematology. The treatments were well tolerated with only mild transient swelling observed in two piglets (Forceris group). Piglets treated with Forceris had significantly higher haemoglobin, haematocrit, mean corpuscular volume and haemoglobin concentration values, as well as significantly higher plasma iron and transferritin saturation and a lower total iron binding capacity than those treated with Uniferon. No animals in the Forceris group but 17 per cent of piglets in the Uniferon group had haemoglobin levels <9 g/dl after treatment, indicating anaemia. These results suggest that both products were safe and effective in the prophylaxis of IDA in piglets, and that Forceris was superior to Uniferon in preventing IDA in piglets.

Examination of anonymous canine faecal samples provides data on endoparasite prevalence rates in dogs for comparative studies.

Several endoparasites of dogs cannot only be detrimental to their primary host but might also represent a threat to human health because of their zoonotic potential. Due to their high dog population densities, metropolitan areas can be highly endemic for such parasites. We aimed to estimate the prevalence of endoparasites in dogs in the Austrian capital of Vienna by examining a representative number of canine faecal samples and to compare the prevalences with two neighbouring peri-urban and rural regions. In addition we analysed whether the density of dog populations and cleanliness of dog zones correlated with parasite occurrence. We collected 1001 anonymous faecal samples from 55 dog zones from all 23 districts of the federal state of Vienna, as well as 480 faecal samples from the Mödling district and Wolkersdorf with a peri-urban and rural character, respectively. Faeces were examined by flotation and by Baermann technique. Additionally we evaluated 292 Viennese, 102 peri-urban and 50 rural samples for Giardia and Cryptosporidium by GiardiaFASTest® and CryptoFASTest®. Samples from "clean" dog zones were compared to samples from "dirty" zones. The infection rate of Toxocara was surprisingly low, ranging from 0.6% to 1.9%. Trichuris was the most frequent helminth (1.8-7.5%) and Giardia the most frequent protozoan (4.0-10.8%). Ancylostomatidae, Crenosoma, Capillaria, Taeniidae, Cystoisospora and Sarcocystis were found in 1.8-2.2%, 0-0.9%, 0-0.9%, 0-0.6%, 0.3-3.1% and 0-0.6% of the samples, respectively. Samples from "dirty" dog zones in Vienna showed a significantly higher rate of parasites overall (p=0.003) and of Trichuris (p=0.048) compared to samples from "clean" dog zones. There were no statistically significant differences in densely vs. less densely populated areas of Vienna. Samples from the rural region of Wolkersdorf had significantly higher overall parasite, Trichuris and Cystoisospora prevalences than the peri-urban Mödling district and Vienna (p=0.000-0.039), while samples from the Mödling district had a significantly higher Giardia, Crenosoma and Capillaria prevalence than those from Vienna (p=0.002-0.047). Parasite excretion is dynamic and representative sampling and monitoring are necessary for parasite surveillance. Dog owners should be informed about the zoonotic risk and encouraged to remove dog faeces and dispose of them properly to reduce the infection risk for both other dogs and humans.

Experimentally confirmed toltrazuril resistance in a field isolate of Cystoisospora suis.

BACKGROUND: Constant treatment regimens with toltrazuril against Cystoisospora suis infection in piglets are being applied in the intensive production systems for the last two decades, but the possibility of resistance development has not been addressed so far despite limited availability of treatment alternatives. Recently, a pig producer in The Netherlands who routinely used toltrazuril complained about diarrhea in suckling piglets in the absence of bacterial and viral pathogens, and oocysts of C. suis could be isolated from feces of affected litters. METHODS: Piglets from two litters were infected with a field isolate of C. suis, Holland-I, and treated with 0 (Holl-Ctrl), 20 (Holl-20) or 30 (Holl-30) mg/kg of body weight (BW) of toltrazuril (Baycox®). The efficacy of toltrazuril was measured by assessment of oocyst excretion, fecal consistency and BW gain. A separate litter was infected with a toltrazuril-susceptible strain of C. suis, Wien-I, and treated with 0 (Wien-Ctrl) or 20 (Wien-20) mg/kg BW of toltrazuril for comparison. RESULTS: Treatment with the recommended (20 mg/kg) dose of toltrazuril completely suppressed oocyst shedding and diarrhea in group Wien-20. The prevalence of oocyst excretion was 100% in the groups infected with Holland-I and 80% in the group Wien-Ctrl. Most days with diarrhea were observed in group Holl-20 with an average of 6.40%, followed by 5.71% in Wien-Ctrl, while in Holl-Ctrl and Holl-30 diarrhea was only seen in 1.79% of the samples (n = 14/piglet). Oocyst excretion, fecal consistency and BW gain did not differ significantly among groups infected with Holland-I, indicating loss of efficacy to toltrazuril. CONCLUSION: Experimental infections and treatment confirmed toltrazuril resistance against the field isolate even at increased dosage. Such isolates are a potential threat to pig production as no other effective and economically sustainable alternative treatment is currently available. In the absence of a standardized protocol for resistance testing in C. suis, regular parasitological examination and, if possible, experimental confirmation should be considered to evaluate the extent and consequences of toltrazuril resistance.

Cystoisospora suis - A Model of Mammalian Cystoisosporosis.

Cystoisospora suis is a coccidian species that typically affects suckling piglets. Infections occur by oral uptake of oocysts and are characterized by non-hemorrhagic transient diarrhea, resulting in poor weight gain. Apparently, primary immune responses to C. suis cannot readily be mounted by neonates, which contributes to the establishment and rapid development of the parasite, while in older pigs age-resistance prevents disease development. However, the presence of extraintestinal stages, although not unequivocally demonstrated, is suspected to enable parasite persistence together with the induction and maintenance of immune response in older pigs, which in turn may facilitate the transfer of C. suis-specific factors from sow to offspring. It is assumed that neonates are particularly prone to clinical disease because infections with C. suis interfere with the establishment of the gut microbiome. Clostridia have been especially inferred to profit from the altered intestinal environment during parasite infection. New tools, particularly in the area of genomics, might illustrate the interactions between C. suis and its host and pave the way for the development of new control methods not only for porcine cystoisosporosis but also for other mammalian Cystoisospora infections. The first reference genome for C. suis is under way and will be a fertile ground to discover new drugs and vaccines. At the same time, the establishment and refinement of an in vivo model and an in vitro culture system, supporting the complete life cycle of C. suis, will underpin the functional characterization of the parasite and shed light on its biology and control.

Enteric protozoa of cats and their zoonotic potential-a field study from Austria.

Domestic cats can be infected with a variety of enteric protozoa. Genotyping of protozoan species, especially Giardia as the most common, can improve assessment of their relevance as zoonotic agents. For an overview on the occurrence of feline enteric protozoa, 298 faecal samples of cats from private households, catteries and animal shelters in Austria were collected. All samples were examined by flotation and using a rapid test for Giardia (FASTest). For the detection of Tritrichomonas blagburni, freshly voided faeces (n = 40) were processed using a commercial culturing system (InPouch TF-Feline). Genotyping was done at the ß-giardin gene loci (each sample) and triosephosphate isomerase gene loci (positive samples) for Giardia and at the 18S rRNA gene (positive samples) for Cryptosporidium. Thirty-seven samples (12.4%) were positive for Giardia by flotation and/or using a rapid test. Cryptosporidium was present in 1.7%, Cystoisospora in 4.0%, Sarcocystis in 0.3% and T. blagburni in 2.5% of the samples. Genotyping revealed Giardia cati, the potentially zoonotic Giardia duodenalis and Cryptosporidium felis. Most of the infected cats had no diarrhoea. Cats from shelters were significantly more often infected than owned cats (p = 0.01). When comparing Giardia detection methods, the rapid test had a higher sensitivity than flotation. Polymerase chain reaction (PCR) results were mostly independent from the other two tests.

Which factors influence the outcome of experimental infection with Cystoisospora suis?

For reliable predictions of clinical and parasitological outcome of experimental infections with parasites, different models must be evaluated for possible influences of infection time point, infection dose and host-specific parameters such as breed or litter size. To address these issues for Cystoisospora (syn. Isospora) suis, the causative agent of porcine neonatal coccidiosis, 181 piglets from 90 litters (hybrid crosses of different breeds) were included in a retrospective study to evaluate differences in time point and dose of infection in four different experimental models ((1) 1,500 oocysts on the 4th day of life, d.o.l.; (2) 1,000 oocysts, 4th d.o.l.; (3) 1,000 oocysts, 1st d.o.l.; (4) 5,000 oocysts, 4th d.o.l.). The target variables body weight gain, faecal consistency and oocyst excretion were evaluated during the acute phase of infection (5-10 days post infection), and the influences of the dependent variables breed or litter size were estimated. Despite differences in the time course of excretion and faecal consistency, neither the average amount of excretion nor the average faecal consistency differed among models, breeds or litters of different size. High individual variability was seen in all four models as described earlier for higher infection doses. When infections on the 1st vs. 4th day of life were compared, no differences in averages could be found, in contrast to previous observations on the influence of age. Other, not yet defined, variables appear to have a greater impact on the outcome of infection than doses and time points in the tested range, despite the reliable outcome of infection with high excretion rates and signs of clinical disease.