Dataset Information

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

ABSTRACT: 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.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.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.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.

SUBMITTER: Shrestha A

PROVIDER: S-EPMC5492287 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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

Shrestha Aruna A Freudenschuss Barbara B Jansen Rutger R Hinney Barbara B Ruttkowski Bärbel B Joachim Anja A

Parasites & vectors 20170629 1

<h4>Background</h4>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 oo ...[more]

PMID: 28662668

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Project description:The protozoan parasite Cystoisospora suis causes diarrhea and reduced weight gain in suckling piglets. Infections occur in the first days of life; it is transient but can lead to dysbiosis, exacerbating disease and increasing mortality. Cystoisosporosis is effectively controlled by toltrazuril treatment; however, alterations of the gut microbial composition upon infection and treatment have not been investigated. This study evaluated the development of fecal microbiota of C. suis infected piglets in response to treatment with toltrazuril. Thirty-eight conventional piglets were infected with C. suis on the first day of life (dol 1). Twenty-six of them received either parenteral or oral toltrazuril 2 days later. Fecal samples were collected pre- and post-weaning (dol 1-15 and 31-38) for microbiota analysis using 16S rRNA amplicon sequencing and during dol 5-18 to determine fecal consistency and parasite excretion. All control animals shed parasites at least once and the majority developed diarrhea, while toltrazuril-treated piglets did not excrete parasites and only had low levels of diarrhea. Age-related shifts in the fecal microbiota composition and increase in diversity and species richness were seen until after weaning. Parasite infection disrupted bacterial maturation 2 weeks after infection. Irrespective of the route of administration, fecal communities of piglets in the treated groups clustered separately and were more diverse compared to that of control piglets during the acute phase of infection on dol 11. Control piglet feces showed higher levels of Fusobacteriaceae and Veillonellaceae, while Ruminococcaceae, Lachnospiraceae, S24-7, Clostridiaceae, and Erysipelotrichaceae were more abundant in feces of treated piglets on dol 11. Thereafter, treatment-related effects on the microbial communities were small and mainly detectable on dol 34 (5 days post-weaning), potentially indicating that the oral toltrazuril treatment might have had long-term effects on host physiological responses post-weaning. Irrespective of the administration route, toltrazuril prevented C. suis-related dysbiosis and maintained species richness and diversity on dol 11. In addition to cystoisosporosis prevention, toltrazuril seems to contribute to the stabilization of the gut microbial development during the suckling phase and thus may reduce the need for antibiotics to control infections with secondary bacterial enteropathogens in C. suis-infected suckling piglets.

Project description:BackgroundCoccidiosis due to Eimeria spp. infections in lambs causes increased mortality and substantial production losses, and anticoccidials are important for control of the infection. Anticoccidial resistance has been reported in poultry and swine, and we recently described reduced toltrazuril efficacy in ovine Eimeria spp. in some Norwegian sheep farms using a newly developed faecal oocyst count reduction test (FOCRT). The aim of the present study was to use a controlled efficacy trial to assess the efficacy of toltrazuril against a field isolate suspected of being resistant.MethodsTwenty lambs, 17-22 days old and raised protected against exposure to coccidia, were infected with a field isolate of 100,000 Eimeria spp. oocysts. This isolate was obtained from a farm with a previously calculated drug efficacy of 56% (95% confidence interval: -433.9 to 96.6%). At day 7 post-infection, 10 of the lambs were orally treated with 20 mg/kg toltrazuril (Baycox Sheep vet., Bayer Animal Health), while the other 10 lambs (controls) were given physiological saline. Clinical examinations were conducted, and weight gains recorded. Daily faecal samples were scored for diarrhoea on a scale from 1 to 5, and oocyst excretion was determined using a modified McMaster technique. Oocysts were morphologically identified to species level. At 17-24 days post-infection, the lambs were euthanized and necropsied.ResultsThe tested Eimeria isolate was resistant against toltrazuril, and resistance was seen in both pathogenic and non-pathogenic species. In addition, no significant differences in faecal score, growth, gross pathology or histological changes were identified between the two groups. The pathogenic E. ovinoidalis was the dominant species, and no significant difference in the individual prevalence of E. ovinoidalis post-treatment was found between treated (66.9%) and control lambs (61.9%). Other species identified included E. crandallis/weybridgensis, E. parva, E. marsica, E. faurei, E. pallida, E. ahsata and E. bakuensis.ConclusionsThis study confirms toltrazuril resistance in ovine Eimeria spp.; in addition, the data support the use of FOCRT as an appropriate tool for field evaluation of anticoccidial efficacy. Due to limited anticoccidial treatment alternatives, these findings may have important implications for the sheep industry, particularly in northern Europe.

Project description:BACKGROUND:The porcine coccidium Cystoisospora suis is characterized by a complex life-cycle during which asexual multiplication is followed by sexual development with two morphologically distinct cell types, the micro- and macrogametes. Genes related to the sexual stages and cell cycle progression were previously identified in related Apicomplexa. Dynein light chain type 1 and male gamete fusion factor HAP2 are restricted to microgametes. Tyrosine-rich proteins and oocyst wall proteins are a part of the oocyst wall. The Rad51/Dmc1-like protein and Nima-related protein kinases are associated with the cell cycle and fertilization process. Here, the sexual stages of C. suis were characterized in vitro morphologically and for temporal expression changes of the mentioned genes to gain insight into this poorly known phase of coccidian development. METHODS:Sexual stages of C. suis developing in vitro in porcine intestinal epithelial cells were examined by light and electron microscopy. The transcriptional levels of genes related to merozoite multiplication and sexual development were evaluated by quantitative real-time PCR at different time points of cultivation. Transcription levels were compared for parasites in culture supernatants at 6-9 days of cultivation (doc) and intracellular parasites at 6-15 doc. RESULTS:Sexual stage of C. suis was detected during 8-11 doc in vitro. Microgamonts (16.8 ± 0.9 µm) and macrogamonts (16.6 ± 1.1 µm) are very similar in shape and size. Microgametes had a round body (3.5 ± 0.5 µm) and two flagella (11.2 ± 0.5 µm). Macrogametes were spherical with a diameter of 12.1 ± 0.5 µm. Merozoite gene transcription peaked on 10 doc and then declined. Genes related to the sexual stages and cell cycle showed an upregulation with a peak on 13 doc, after which they declined. CONCLUSIONS:The present study linked gene expression changes to the detailed morphological description of C. suis sexual development in vitro, including fertilization, meiosis and oocyst formation in this unique model for coccidian parasites. Following this process at the cellular and molecular level will elucidate details on potential bottlenecks of C. suis development (applicable for coccidian parasites in general) which could be exploited as a novel target for control.

Project description: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.

Project description:BackgroundThe genome of the apicomplexan parasite Cystoisospora suis (syn. Isospora suis) has recently been sequenced and annotated, opening the possibility for the identification of novel therapeutic targets against cystoisosporosis. It was previously proposed that a 42 kDa uncharacterized merozoite protein, encoded by gene CSUI_005805, might be a relevant vaccine candidate due to its high immunogenic score, high expression level and species-specificity as determined in silico.MethodsThe 1170 bp coding sequence of the CSUI_005805 gene was PCR amplified and cloned into the bacterial expression vector pQE-31. The specificity of the expressed recombinant protein was evaluated in an immunoblot, and relative levels of expression in different developmental stages and subcellular localization were determined by quantitative real-time PCR and indirect immunofluorescence assay, respectively.ResultsThe CSUI_005805 gene encoded for a 389 amino acid protein containing a histidine-rich region. Quantitative RT-PCR showed that CSUI_005805 was differentially expressed during the early development of C. suis in vitro, with higher transcript levels in merozoites compared to sporozoites. The recombinant protein was specifically recognized by sera from chicken immunized with recombinant CSUI_005805 protein and sera from piglets experimentally infected with C. suis, all of which suggested that despite prokaryotic expression, the recombinant CSUI_005805 protein maintained antigenic determinants and could elicit an immune response in the host. Immunofluorescence labelling and confocal microscopy revealed localization primarily at the surface of the parasite.ConclusionsThe results suggest that CSUI_005805 is highly expressed in merozoites and might thus be critical for their survival and establishment inside host cells. Owing to its specificity, localization and expression pattern, CSUI_005805 could be exploited as an attractive candidate for alternative control strategies against C. suis such as vaccines.

Project description:BackgroundTo date, investigations on the immune response to Cystoisospora suis infections focused on suckling piglets, the age group clinically most affected. Actively immunizing piglets is unfeasible due to their immature immune system and the typically early infection in the first days after birth. Therefore, understanding and possibly enhancing the immune response of immune-competent animals is the prerequisite to develop a passive immunization strategy for piglets which currently rely on very limited treatment options.MethodsTo investigate antibody and cytokine responses of immune-competent animals and the impact of the oral immunization protocol on their immune response, growers with unknown previous exposure to C. suis (10-11 weeks-old) were infected one or three times with different doses (600 and 6000 or 200 and 2000, respectively) of C. suis oocysts, and compared to uninfected controls. Oocyst excretion was evaluated, and blood and intestinal mucus antibody titers were determined by IFAT. Systemic production of Th1, Th2, inflammatory and regulatory cytokines was determined in different immune compartments at mRNA and (after stimulation with a recombinant merozoite-protein) at protein level by PCR and multiplex fluorescent immunoassay, respectively.ResultsInfection generated significantly increased serum IgA and IgG levels against C. suis sporozoites and merozoites, irrespective of infection mode, with IgG against merozoites showing the strongest increase. No clinical signs and only occasional excretion were observed. The systemic cytokine response to C. suis was only weak. Nonetheless, in white blood cells, IL-4, IL-6 and IL-10 mRNA-levels significantly increased after infection, whereas IFN-ɣ, IL-2 and TGF-β expression tended to decrease. In mesenteric lymph nodes (MLN), IL-10 and TNF-α levels were elevated while splenic cytokine expression was unaltered upon infection. Stimulated MLN-derived lymphocytes from infected pigs produced slightly more IL-12 and less IFN-α than controls.ConclusionsAn infection and a subsequent systemic immune response can be induced in immune-competent animals by all evaluated infection models and growers can be used as models to mimic sow immunizations. The immune response to C. suis, although mild and with considerable variation in cytokine expression, was characterized by a Th2-associated and regulatory cytokine profile and antibody production. However, none of the parameters clearly stood out as a potential marker associated with protection. Antibody titers were significantly positively related with oocyst excretion and might thus serve as correlates for parasite replication or severity of infection.

Project description:Cystoisosporosis is a leading diarrheal disease in suckling piglets. With the confirmation of resistance against the only available drug toltrazuril, there is a substantial need for novel therapeutics to combat the infection and its negative effects on animal health. In closely related apicomplexan species, bumped kinase inhibitors (BKIs) targeting calcium-dependent protein kinase 1 (CDPK1) were shown to be effective in inhibiting host-cell invasion and parasite growth. Therefore, the gene coding for Cystoisospora suis CDPK1 (CsCDPK1) was identified and cloned to investigate activity and thermal stabilization of the recombinant CsCDPK1 enzyme by BKI 1369. In this comprehensive study, the efficacy, safety and pharmacokinetics of BKI 1369 in piglets experimentally infected with Cystoisospora suis (toltrazuril-sensitive, Wien-I and toltrazuril-resistant, Holland-I strains) were determined in vivo and in vitro using an established animal infection model and cell culture, respectively. BKI 1369 inhibited merozoite proliferation in intestinal porcine epithelial cells-1 (IPEC-1) by at least 50% at a concentration of 40 nM, and proliferation was almost completely inhibited (>95%) at 200 nM. Nonetheless, exposure of infected cultures to 200 nM BKI 1369 for five days did not induce structural alterations in surviving merozoites as confirmed by transmission electron microscopy. Five-day treatment with BKI 1369 (10 mg/kg BW twice a day) effectively suppressed oocyst excretion and diarrhea and improved body weight gains in treated piglets without obvious side effects for both toltrazuril-sensitive, Wien-I and resistant, Holland-I C. suis strains. The plasma concentration of BKI 1369 in piglets increased to 11.7 μM during treatment, suggesting constant drug accumulation and exposure of parasites to the drug. Therefore, oral applications of BKI 1369 could potentially be a therapeutic alternative against porcine cystoisosporosis. For use in pigs, future studies on BKI 1369 should be directed towards ease of drug handling and minimizing treatment frequencies.

Dataset Information

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

Publications

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

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