RESUMEN

The aim of the present study was to assess the short-term behavioral and physiological responses of piglets to different treatment protocols for the control of iron-deficiency anemia (IDA) and cystoisosporosis. Piglets were treated with either (1) an injection of iron combined with an oral application of toltrazuril (TLZ) by drenching or (2) a combination injection of TZL + gleptoferron; the behavior of the piglets was then evaluated. For this study, 288 piglets were divided into three experimental groups: 96 piglets were kept untreated (control group); 96 piglets received an oral administration of a generic TZL-based anticoccidial agent (20 mg/kg BW) along with intramuscular administration of iron dextran (200 mg/mL; 1 mL/piglet) at the same handling (oral + parenteral group, O + P); and 96 piglets received an intramuscular application of the combination product (parenteral group, P). For each treated piglet, the total handling time, flight reaction, and the intensity and frequency of vocalizations were determined using the methodology described by Scollo et al. (2020). Piglets in the O + P group were found to emit more screams during treatment administration than animals in the P group (21.05% vs. 8.42% of animals; p < 0.05). Piglets in the O + P group reacted worse to manipulation and oral administration because a higher percentage of animals continued to fidget even after handling (32.63% vs. 12.63%; p < 0.05). Differences in growth performance between the groups were not observed in our study (p > 0.05). In conclusion, the administration of a combination product reduced stress during administration, as indicated by reduced vocalizations and reactions to manipulation.

RESUMEN

This study investigated the presence and abundance of Cystoisospora suis oocysts in faecal samples from 131 one- to three-week-old pig litters belonging to eight intensively raised, indoor herds in Spain. Seven herds used preventive anticoccidial toltrazuril treatments administered orally or by intramuscular injection, and one did not use preventive anticoccidial treatments. The diagnosis was performed using two oocyst flotation-concentration methods, Bailenger's method in every herd and the more recent Joachim's method in four herds. Oocysts were detected in every farm, the proportion of oocyst-positive samples was higher with Bailenger's technique, and the estimated overall prevalence (95% confidence interval) was 40 (32-49)%, including 47 (29-65)% in non-medicated litters, 52 (38-67)% in orally medicated litters and 28 (16-40)% in intramuscularly medicated litters (p < 0.05). However, mixed logistic regression models indicated that the risk of infection was not significantly associated with preventive anticoccidial treatments (p > 0.05), and increased with age, was higher in herds with partially compared to fully slatted dung floors in farrowing pens and in litters with pigs with diarrhoea (p < 0.05). The median (range) oocysts per gram of faeces (OpG) in infected litters by Bailenger's method was 623 (35-49048) and mixed negative binomial models revealed no significant association between infection intensity in positive litters and pen's floor type and piglets age, faecal consistency and treatment status (p > 0.05). The apparent low efficacy of Toltrazuril suggests treatment administration failures, reduced residual efficacy or low susceptibility of C. suis strains in study farms and needs further investigation.

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Cystoisospora belli causes chronic diarrhoea, acalculous cholecystitis, cholangiopathy and disseminated cystoisosporosis in patients with AIDS. Clinical manifestations and histological stages during C. belli infection in a patient with AIDS and liver disease were described. It was possible to identify sporozoite-like structures in the villus epithelium of the duodenum, close to the vascularization that underlies the basal membrane and unizoite tissue cysts near to the vascularization in the lamina propria. Unizoite tissue cysts were found inside of sinusoids in the liver communicating with the central vein and with a bile canaliculus and portal spaces. Based on these findings a hypothesis on C. belli life cycle could consider that the route of migration of unizoite tissue cysts up the liver is via the portal blood. The unizoite tissue cysts located in hepatic portal vein could migrated via sinusoid to central vein and general circulation through the venous system. The unizoite tissue cysts could also return via bile canaliculus to bile duct to portal triad. This hypothesis allows to understand the presence of unizoite stages in blood, the pathway by which the bile ducts become infected and unizoites in the liver being able to behave like hypnozoites that favour relapses and treatment failures.

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Objetivou-se investigar a ocorrência de endoparasitos em veados-catingueiros (Mazama gouazoubira) pertencentes a uma propriedade conservacionista no município de Sousa, Paraíba. Foram realizadas coletas de fezes em sete animais que foram submetidas às técnicas de sedimentação espontânea, centrífugo-flutuação em solução de sacarose, contagem de ovos por grama de fezes (OPG), contagem de oocistos por grama de fezes (OoPG) e coproculturas. Foram avaliados os locais de criação dos animais, para obtenção das informações acerca do manejo sanitário e alimentar. Em todas as amostras observaram-se ovos da Superfamília Trichostrongyloidea (OPG 200 ± 100) e coccídios (OoPG 1500 ± 300). Nas coproculturas, 100% das larvas infectantes pertenciam ao gênero Trichostrongylus. Foram constatados oocistos do gênero Cystoisospora em todas as amostras. Em uma fêmea, que apresentou OoPG 1800, observaram-se sinais clínicos como apatia, anorexia, desidratação e diarreia intermitente, condizentes com Coccidiose. Este animal recebeu tratamento com a associação de sulfametoxazol e trimetropim, por sete dias, havendo melhora clínica. Erros de manejo foram observados na propriedade, como o convívio de M. gouazoubira com bovinos, ovinos e caprinos, alimentação inadequada à base de ração industrializada para equinos, bebedouros improvisados e não higienizados, e acúmulo de sujeira dos ambientes frequentados pelos animais. Concluiu-se que os veados-catingueiros estavam parasitados por helmintos (Trichostrongylus sp.) e protozoários (Cystoisospora spp.), podendo estas infecções estarem associadas ao convívio destes animais com ruminantes domésticos. Este estudo apresenta o primeiro relato da infecção por Cystoisospora spp. em M. gouazoubira.

The objective of this study was to investigate the occurrence of endoparasites in brown brocket deer (Mazama gouazoubira) belonging to a conservationist property in the municipality of Sousa, Paraíba. Fecal samples were collected in seven animals that were submitted to the techniques of spontaneous sedimentation, centrifugation-flotation in sucrose solution, egg count per gram of feces (EPG), oocyst count per gram of feces (OoPG) and coproculture. The breeding sites were evaluated to obtain information on Sanitary and food management. In all samples, eggs of the Trichostrongyloidea Superfamily (EPG 200 ± 100) and coccidia (OoPG 1500 ± 300) were observed. In the coprocultures, 100% of the infecting larvae belonged to the genus Trichostrongylus. Oocysts of the genus Cystoisospora spp. were found in all samples. In one female, who presented OoPG 1800, clinical signs such as apathy, anorexia, dehydration and intermittent diarrhea, consistent with Coccidiosis. This animal received treatment with the combination of sulfamethoxazole and trimetropim for seven days, with clinical improvement of the animal. Management errors were observed in the property, such as M. gouazoubira’s conviviality with as cattle, sheep and goats, inadequate feed based on industrialized ration for horses, improvised and unhygienic drinking fountains, accumulation of dirt in the environments frequented by animals. They concluded that veados-catingueiros were parasitized by helminths (Trichostrongylus sp.) and protozoa (Cystoisospora sp.) infections may be associated with the coexistence of animals with domestic ruminants. This study presents the first report of Cystoisospora spp. in M. gouazoubira.

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Objetivou-se investigar a ocorrência de endoparasitos em veados-catingueiros (Mazama gouazoubira) pertencentes a uma propriedade conservacionista no município de Sousa, Paraíba. Foram realizadas coletas de fezes em sete animais que foram submetidas às técnicas de sedimentação espontânea, centrífugo-flutuação em solução de sacarose, contagem de ovos por grama de fezes (OPG), contagem de oocistos por grama de fezes (OoPG) e coproculturas. Foram avaliados os locais de criação dos animais, para obtenção das informações acerca do manejo sanitário e alimentar. Em todas as amostras observaram-se ovos da Superfamília Trichostrongyloidea(OPG 200 ± 100) ecoccídios(OoPG 1500 ± 300). Nas coproculturas, 100% das larvas infectantes pertenciam ao gênero Trichostrongylus. Foram constatados oocistos do gênero Cystoisospora em todas as amostras. Em uma fêmea, que apresentou OoPG 1800, observaram-se sinais clínicos como apatia, anorexia, desidratação e diarreia intermitente, condizentes com Coccidiose. Este animal recebeu tratamento com a associação de sulfametoxazol e trimetropim, por sete dias, havendo melhora clínica. Erros de manejo foram observados na propriedade, como o convívio de M. gouazoubira com bovinos, ovinos e caprinos, alimentação inadequada à base de ração industrializada para equinos, bebedouros improvisados e não higienizados, e acúmulo de sujeira dos ambientes frequentados pelos animais. Concluiu-se que os veados-catingueiros estavam parasitados por helmintos (Trichostrongylus sp.) e protozoários (Cystoisospora spp.), podendo estas infecções estarem associadas ao convívio destes animais com ruminantes domésticos. Este estudo apresenta o primeiro relato da infecção por Cystoisospora spp. em M. gouazoubira.

The objective of this study was to investigate the occurrence of endoparasites in brown brocket deer (Mazama gouazoubira) belonging to a conservationist property in the municipality of Sousa, Paraíba. Fecal samples were collected in seven animals that were submitted to the techniques of spontaneous sedimentation, centrifugation-flotation in sucrose solution, egg count per gram of feces (EPG), oocyst count per gram of feces (OoPG) and coproculture. The breeding sites were evaluated to obtain information on Sanitary and food management. In all samples, eggs of the Trichostrongyloidea Superfamily (EPG 200 ± 100) and coccidia (OoPG 1500 ± 300) were observed. In the coprocultures, 100% of the infecting larvae belonged to the genus Trichostrongylus. Oocysts of the genus Cystoisospora spp. were found in all samples. In one female, who presented OoPG 1800, clinical signs such as apathy, anorexia, dehydration and intermittent diarrhea, consistent with Coccidiosis. This animal received treatment with the combination of sulfamethoxazole and trimetropim for seven days, with clinical improvement of the animal. Management errors were observed in the property, such as M. gouazoubira's conviviality with as cattle, sheep and goats, inadequate feed based on industrialized ration for horses, improvised and unhygienic drinking fountains, accumulation of dirt in the environments frequented by animals. They concluded that veados-catingueiros were parasitized by helminths (Trichostrongylus sp.) and protozoa (Cystoisospora sp.) infections may be associated with the coexistence of animals with domestic ruminants. This study presents the first report of Cystoisospora spp. in M. gouazoubira.

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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.

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BACKGROUND: The 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. METHODS: The 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. RESULTS: The 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. CONCLUSIONS: The 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.

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Cystoisospora belli is an opportunistic protozoan that causes human cystoisosporiasis, an infection characterized by diarrhea, steatorrhea, abdominal pain, fever, and weight loss. The lack of animal models susceptible to C. belli, and the difficulty in obtaining clinical samples with fair amounts of oocysts have limited the research pertaining to the basic biology of this parasite. This study aimed to describe the ultrastructure of endogenous stages of C. belli in Monkey Rhesus Kidney Cells (MK2) and Human Ileocecal Adenocarcinoma cells (HCT-8). Zoites of C. belli exhibited typical morphological features of coccidia, which included a trilaminar pellicle, an apical complex formed by a conoid, polar rings, rhoptries, and micronemes, in addition to dense granules and the endoplasmic reticulum. No crystalloid body was observed but various lipid and amylopectin granules were usually present in the cytoplasm of zoites. We observed a tendency of the endoplasmic reticulum of the host cell to be located near the parasitophorous vacuole membrane. Merozoites were formed by endodyogeny and during replication, the apical complex of the mother cell remained intact. The formation of gametes or oocysts was not observed. The ultrastructural findings of C. belli are further evidence of its proximity to Sarcocystidae family members and corroborate their reclassification as Cystoisospora spp.

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