image/svg+xmlISSN Versión impresa 2218-6425ISSN Versión Electrónica 1995-1043Neotropical Helminthology, 2021, 15(2), jul-dic:199-209.ORIGINAL ARTICLE / ARTÍCULO ORIGINAL12 Facultad de Agronomía, UNCPBA, Azul, Buenos Aires, Argentina; Departamento de Zoonosis Rurales, Azul, Buenos Aires, 3 4 Argentina; Escuela de Educación Secundaria Agraria de Azul, Buenos Aires, Argentina; Facultad de Ciencias Veterinarias, 56UBA, Buenos Aires, Argentina; Instituto de Análisis Fares Taie, Mar del Plata, Argentina; Facultad de Ciencias Veterinarias, UNCPBA, Tandil, Buenos Aires, Argentina.*Corresponding author: escialfa@yahoo.com.arScialfa Exequiel: https://orcid.org 0000-0003-2871-5975Luz Allende: https://orcid.org/0000-0002-9765-2747 Fernando Fariña: https://orcid.org/0000-0003-4425-9973 Silvina Quintana: https://orcid.org/0000-0003-1845-7677Mariana Rivero: https://orcid.org/0000-0002-4792-2745Marcelo Rodriguez: https://orcid.org/0000-0003-3549-7582Soledad Pane: https://orcid.org/0000-0002-6778-40951,2,32456Exequiel Scialfa*; Luz Allende; Fernando Fariña; Silvina Quintana; Mariana Rivero; 63Marcelo Rodriguez & Soledad PaneABSTRACTCoccidiosis is cause of economic losses due to weight loss and growth retardation, decreased feeding efficiency, diarrhea and death of weaning rabbits. The main objective of the work was to evaluate the dynamic change of Eimeriaoocyst shedding in the reproductive stage and in weaned litters during the fattening period, in a farm for meat production, with a history of coccidiosis infection. A faecal sample of the following physiological condition was obtained weekly: gestation (G1, G2, G3 and G4) and lactation (L1, L2, L3 and L4), not pregnant or not lactating were also included (V). In weaned rabbits, faecal sample / litters were obtained weekly, until rabbits reached the slaughter weight (2.5 kg). All samples were examined by flotation and the McMaster method. Co-infection with six species of the genus Eimeriacould be observed in all positive animals. The infection rate in rabbits varied according to physiological state, being 22.7% (G3-L3), 13.4% (G4), 9.1% (G1-L4), 20% (GL), 4.6% (G2-L2). During the first week of lactation (L1), no positivity was observed. Parasitic loads ranged from 40 to 13820 OPG; the highest counts were observed in L2 and G3. In the second half of lactation, females have seven times the risk of excreting oocysts in the feces compared to the first half. Litter rabbits excreted oocyst during all fattening period; however, during weeks 3 to 7 the oocyst excretion was higher. This is the first report in which it is described the oocysts mixture of six Eimeriaspecies (E. vejdovskyi, E. coecicola, E. magna,E. exigua, E. media andE. stiedae) from the rabbit for meat production in the region. These findings show that breeding rabbits are asymptomatic carriers of coccidian infections and can shed oocysts at any time during the reproductive cycle. Neotropical Helminthology199doi:10.24039/rnh20211521276EIMERIAOOCYST EXCRETION IN NATURALLY INFECTED RABBIT IN A FARM FOR MEAT PRODUCTIONEXCRECIÓN DE OOQUISTES DE EIMERIAEN CONEJOS INFECTADOS NATURALMENTE EN UNA GRANJA DE PRODUCCIÓN DE CARNEKeywords: Eimeria– rabbits – coccidiosis – reproductive does – litters – OPGDDDDDDD
image/svg+xmlScialfa et al.200Coccidiosis, epizootic rabbit enteropathy (ERE) and colibacillosis are the main cause of digestive disorders as death in fattening rabbits (Licois, 2004). Rabbit's endoparasitosis are common, mainly in domestic breeding. The genera Eimeria spp. is the most frequent parasites found, however, other parasites as Passarulus ambiguus (Rudolphi, 1819), Giardia spp., Hymenolepis diminuta (Rudolphi, 1819), Trichostrongylus spp., Trichuris spp. and Cryptosporidium spp. are detected in feces samples of rabbits (Schafer da Silva et al.,2006; Scialfa, 2020). Mixed infection with nematodes species and Eimeria spp. is frequent in farm (Elshahawy & Elgoniemy, 2018), and the prevalence of coccidial oocyst in young rabbits is higher than in adult rabbits. Eimeriainfection is initiated by oral ingestion of sporulated oocysts by the susceptible rabbit. Concurrent infections with more than one Eimeria species were commonly recorded in rabbits infected, and were identified from naturally infected rabbits (Elshahawy & Elgoniemy, 2018): Eimeria media (Kessel, 1929), E. intestinalis (Cheissin, 1948), E. coecicola INTRODUCTION(Cheissin, 1947), E. magna (Pérard, 1925), E. exigua (Yakimoff, 1934), E. perforans (Leuckart, 1879) Sluiter & Swellengrebel, 1912, E. irresidua (Kessel & Jankiewicz, 1931), E. flavescens (Marotel & Guilhon, , E. piriformis Kotlán 1941)(& Pospesch, 1934), E. stiedae (Lindemann, 1865) Kisskalt Hartmann,1907& and E. vejdovskyi(Pakandl, 1988).Coccidiosis is cause of economic losses due to weight loss and growth retardation, decreased food efficiency, diarrhea and death of weaning rabbits (Pakandl, 2009). Intestinal coccidiosis cause more or less severe disease in rabbits, depending mainly on the infective dose, Eimeria spp.,rabbit immune system and age of animals (Coudert et al.,1995; Pakandl, 2009). Coccidia can be classified according to their pathogenicity: nonpathogenic (E. coecicola), slightly pathogenic (E. perforans, E. exigua and E. vejdovskyi), mildly pathogenic or pathogenic (E. media, E. magna, E. piriformis and E. irresidua), highly pathogenic (E. intestinalis and E. flavescens) (Coudert et al.,1995). Eimeria stiedae is associated with liver lesion of rabbits, causing hepatomegaly with numerous and scattered yellowish white nodules on the liver RESUMENPalabras clave: Eimeria– conejos – coccidiosis - conejas reproductoras – camadas - OPGLa coccidiosis es causa de pérdidas económicas debido a pérdida de peso, retraso del crecimiento, disminución de la eficiencia alimentaria, diarrea y muerte de conejos. El objetivo del trabajo fue evaluar la excreción de ooquistes de Eimeriaen hembras reproductoras y sus camadas destetadas durante el período de engorde, en una granja para la producción de carne, con antecedentes de infección por coccidiosis. Se recolectó semanalmente una muestra fecal de las conejas para las siguientes condiciones fisiológicas: gestación (G1, G2, G3 y G4) y lactancia (L1, L2, L3 y L4), también se incluyeron aquellas hembras vacías, es decir que no estaban gestando o lactando (V). En conejos destetados, se recolectaron muestras fecales / camadas / semana, hasta alcanzar el peso de faena. Las muestras se examinaron mediante el método de flotación la cámara McMaster. Se observó co-infección con seis especies del género Eimeriaen todos los animales positivos. La tasa de infección varió según la condición fisiológica de la coneja, siendo 22,7% (G3-L3), 13,4% (G4), 9,1% (G1-L4), 20% (GL), 4,6% (G2-L2). Las cargas parasitarias variaron de 40 a 13820 OPG; los recuentos más altos se observaron en L2 y G3. En la segunda mitad de la lactancia, las hembras tienen siete veces más riesgo de excretar ooquistes en las heces en comparación con la primera mitad. Los conejos excretaron ooquistes durante todo el período de engorde; sin embargo, durante las semanas 3 a 7 la excreción fue mayor. Este es el primer informe en el que se describe la presencia de ooquistes de seis especies de Eimeriaspecies (E. vejdovskyi, E. coecicola, E. magna,E. exigua, E. media andE. stiedae) en conejos de la región. Estos hallazgos muestran que los conejos reproductores son portadores asintomáticos de infecciones por coccidias y pueden arrojar ooquistes en cualquier momento durante el ciclo reproductivo.Neotropical Helminthology, 2021, 15(2), jul-dic
image/svg+xml201surface (Singla et al.,2000; Scialfa, 2020; Rampin et al.,2008); these nodules generally are detected in slaughtered rabbits or necropsy findings. The prevalence Eimeria spp.infection in rabbit farms is variable (33.9 to 85.1%), dependent of breeding system and risk factors (Li et al.,2010; Okumu et al.,2014; Elshahawy & Elgoniemy, 2018; Tanjung & Rangkuti, 2019).Local immune response plays more important role in immunity to coccidiosis than the systemic response (Pakandl, 2009). Apparently, the response immune it would associate with the specie of Eimeria, being E. intestinalis one of the most immunogenic, in contrast with E. flavescens and E. piriformis (Coudert et al.,1993).Coccidiosis is mainly controlled by prophylaxis with different anticoccidial drugs, usually mixed in feeding pellets, in drinking water and injected via subcutaneous (Pakandl, 2009; Qamar et al.,2013). Sulphonamides drugs (sulphadimethoxine, sulphadimidine, trimethoprim-sulphamethoxazole and sulphaquinoxaline) are currently used primarily for treatment of coccidiosis outbreak (Pakandl, 2009). The use of these drugs has some disadvantages, such as negative impact on the environment (excretion in faeces), resistance, toxicity for host and residues in meat. Vaccination with live attenuated lines of coccidia is another method for the control of coccidiosis (Pakandl, 2009).According to the accessible literature, the study of rabbit coccidiosisin Argentina is very limited, and there are few studies that describe the real impact of intestinal Eimeria spp. infection in rabbit farms, and even less about Eimeriaspecies that affect rabbits in the region. There are few reports about hepatic coccidiosis in domestic as wild rabbit of Argentina (Robles & Bonino, 1985; Scialfa, 2020).The main objective of the work was to evaluate the dynamic change of Eimeriaoocyst shedding in reproductive does and their weaned litters during fattening period, in a farm for meat production, with a history of coccidiosis infection. The specific objective was to determine the association between the parasite load (number of Eimeriaoocysts excreted by g of faeces) and the physiological condition of reproductive does. Rabbit farmThis study was carried out at Secondary School of Agricultural Education, Azul, Buenos Aires province, Argentina. Rabbit breeding was 2developed in a 100 m shed, with a cement floor and no artificial light; temperature, humidity and ventilation were controlled using curtains. Rabbit houses consist of rows of suspended wire mesh cages with shallow pits in the floor for litter. Male (6) and female (14) breeding rabbits were kept in individual cages, while the weaning fattening 2animals were kept in collective cages (0.25m) with the capacity to house 4 rabbits of 2.5-2.6 Kg of live weight. According to the available facilities, it was not possible to make an all-in-all-out system; according to the cleaning system at the end of each cycle, the system was considered continuous. Rabbits were fed a commercial laboratory pelleted feed free of anticoccidial drugs. On the farm, the presence of Eimeriainfection has been evidenced by flotation methods, and diarrhea and death have eventually been observed in weaned rabbits; chemoprophylaxis had not been applied at the time of the study.Faecal sample processingIndividual faecal samples (50 g) of 14 reproductive does and their respective weaning litters were collected weekly. Faecal samples were collected by means of plastic sheets placed under the wire mesh of the cage. In reproductive does, a faecal sample of the following physiological condition was obtained weekly: gestation (G1, G2, G3 and G4) and lactation (L1, L2, L3 and L4), those empty females were also included, that is, at some point in the reproductive cycle were not pregnant or not lactating (V). In weaned rabbits, faecal sample/litters were obtained weekly, until reach the slaughter weight (2.5 kg). All faecal samples were conserved with formalin solution (5%). In the Parasitology Laboratory of Rural Zoonotic Department, the samples were examined by flotation method using saturated solution of NaCl (Figure 1). Parasitological objects were observed microscopically under 400x (Figure 1). The invasion intensity of the Eimeria species was assessed using McMaster chamber and expressed as the number of oocysts per gram of faeces (OPG index). MATERIALS AND METHODSEimeriaoocyst excretion in naturally infected rabbitNeotropical Helminthology, 2021, 15(2), jul-dic
image/svg+xml202Identification of EimeriaspeciesEimeria species were identified based on the clinical signs, prepatent period, oocyst morphology and molecular methods (Baker, 2007; Taylor et al., 2007). During the study period, litters rabbits were checked daily to observe clinical signs, such as diarrhea, indicating a clinical form of coccidiosis and their weight was recorded at the weaning and at the end of fattening period. The morphological features of the sporulated oocyst, including the size of the oocysts, the presence or absence of residual bodies of the oocyst, the size of the sporocysts, and the clinical manifestation were also used to identify the Eimeria species. To determine the size of the oocysts, photomicrographs were taken through the ICC-50HD camera incorporated into the LEICA DM-500 microscope (Figure 1). The interactive measurement module of the LAS V4.5 software was used to carry out the measurements. For molecular Eimeria species identification, total genomic DNA was extracted from stool samples using ADN Puriprep K1210 kit Inbio Highway®). Obtained DNA was quantified using DeNovix Spectrophotometer. Samples were subjected to real-time polymerase chain reaction (qPCR) with generic primers (ITS1-F- 3´-GGGAAGTTGCGTAAATAGA-5´ and ITS1-R CTGCGTCCTTCATCGAT)that amplify all Eimeriaspecies of domestic rabbit (Oliveira et al.,2011) generating amplification products from 400bp to 600 bp depending on the specie. The thermal cycling conditions were as follows: an initial denaturation of 3 min at 95° C, and 45 cycles of 94°C 25", 50°C 30", 72°C 40". After amplification, a melting curve analysis to confirm specific amplification of the PCR product was performed. All qPCR reactions were carried out in 3a thermocycler QTower (Analytic Jena, Germany) in a final volume of 20 µl using EvaGreen® Fluorescent DNA intercalating dye (KAPA Fast, Biosystems, Woburn, E.E.U.U. To corroborate size and amplification specificity, PCR products were resolved on agarose gel. Amplified DNA fragments were purified using the AccuPrep® PCR/Gel Purification Kit (Bioneer, South Korea) and directly sequenced (ABI 3500 Genetic Analyzer, Applied Biosystems, Foster City, CA, USA). The sequences similarities were determined by the Basic Local Alignment Search Tool (BLAST, NCBI).Statistical analysisIn order to compare the oocyst excretion (OPG) in reproductive does, throughout of the different physiological condition (G1, G2, G3, G4, L1, L2, L3, L4 and V), a Chi squared and Fisher Test was performed. However, to compare the oocyst excretion (OPG) by litter's rabbits, throughout the weeks of fattening period, an ANOVA test was performed for repeated measures. The response variable was the log of OPG, and the classification variable was the week with its 12 levels. The repeated measurement of the litters rabbits was contemplated throughout the time that the test lasted (slaughter weight: 2.5 kg). The statistical language R was used for the analysis. The data with negative (OPG value= 0) result or without data were excluded from the analysis, since these values cannot be transformed using logarithm. Spearman coefficient correlation was used for to determine the association between fattening days and OPG, GMD and OPG, and weaning weight and OPG. Ethic aspects: The authors point out that they fulfilled all national and international ethical aspects.A total of 119 faecal samples of 14 reproductive does were studied, and the presence of Eimeriainfection was observed in 10 does (71.4%), which oocyst shedding at certain moments of the reproductive cycle (Table 1). The 16.8% of samples (20/119) were positive, and co-infection with at least three species of the genus Eimeriacould be observed in all positive animals. The infection rate varied according to the physiological status, being of 25% (G3-L3), 15% (G4), 10% (G1-L4), and 5% (G2-L2). During the first week of lactation (L1), no positivity was observed. Parasitic loads ranged from 40 to 13820 OPG; the highest counts were observed in L2 and G3 (Table 1). In the second half of gestation period there was a greater probability of finding positive animals, however, the differences were not significant (p= 0.09). During the second half of lactation period the reproductive does are seven times more likely to be positive (p= 0.02). Seven does are found lactating RESULTSScialfa et al.Neotropical Helminthology, 2021, 15(2), jul-dic
image/svg+xml203and pregnant simultaneously, and five were positives; however, the 60% (3/5) of oocyst shedding in feces during this reproductive period (Figure 2). During the study, the age of the reproductive doe, was on average 13 and 15.7 months, for the negative and positive does respectively; respect the number of births, it was 6 and 6.7 for the negative and positive does respectively. The lactation period was of 31.3 days (range: 27-36 days), and the weaned rabbits weighed of 644 g (range: 486-1000 g). The fattening period was an average of 63.5 days (range: 49-80 days), reaching slaughter weight (2540 g on average) at age of 92.4 days (range: 83-116 days). The 71.4 % of the suckling rabbits reaching slaughter weight at week 8 and 9, with an average weight gain per day of 33.4 g. A total of 130 faecal samples of 14 weaned litters were analyzed, and the 100% were positive by flotation method. The highs values reached by the different litters during the fattening period were of 12000, 12880, 13460, 15240, 19800, 23240, 25000, 27360, 29500, 30120, 46500, 68000 and 75680 respectively. The 28.6% presented values higher than 30000 OPG.During all fattening period the weaned litters excreted oocyst; however, at weeks 3 to 7 the oocyst excretion was higher (Figure 3), and the differences observed were statistically significant (p= 0.01). The means of oocyst shedding per week of fattening varied from 160 to 16474 OPG, the lowest loads being observed at the beginning and at the end of the fattening period (Figure 3). A high variability of oocyst excretion by litter's rabbits in the different weeks was observed. In 50% of the weaned litters expressed a negative result to the flotation method (Value=0) before or after a positive result with oocyst count (OPG), even after high counts (Figure 4). Table 1.Eimeriainfection in reproductive does according to the physiological condition: Negative = flotation methods negative; (-) = without sample; 0 = flotation method positive and counting negative.GestationLactationDoeAge(months)N° birthEmptyG1G2G3G4L1L2L3L4612512-NegativeNegative7880NegativeNegativeNegative480Negative11471-NegativeNegative70080Negative13820NegativeNegative412814-Negative88040NegativeNegativeNegative880Negative492614NegativeNegativeNegative4860120----802111-NegativeNegative420NegativeNegativeNegativeNegativeNegative87197-NegativeNegativeNegativeNegativeNegativeNegative2200Negative1137123000NegativeNegativeNegativeNegativeNegative03920101135-NegativeNegativeNegativeNegativeNegativeNegativeNegativeNegative104112NegativeNegativeNegativeNegative120Negative NegativeNegativeNegative91135NegativeNegativeNegativeNegativeNegativeNegativeNegative20Negative10793NegativeNegativeNegativeNegativeNegativeNegativeNegativeNegativeNegative11092-NegativeNegativeNegativeNegativeNegativeNegativeNegativeNegative109102-160NegativeNegativeNegativeNegativeNegativeNegative160522414-NegativeNegativeNegativeNegativeNegativeNegativeNegativeNegativeEimeriaoocyst excretion in naturally infected rabbitNeotropical Helminthology, 2021, 15(2), jul-dic
image/svg+xml204Figure 1.A) Laboratory and faecal sample processing. B) Eimeria species observed microscopically under 100x. C) Mixed Eimeriaoocysts observed by photomicrographs under 400x.Figure 2. Eimeriainfection according to the physiological condition of reproductive does. In grey color are observe the moment of oocyst excretion and OPG.Oocyst shedding by weaned litters during fattening period was very variable (Table 3), regardless of whether or not they came from positive mothers. No association between fattening days and OPG (p= 0.51), GMD and OPG (p= 0.31) and weaning weight and OPG (p= 0.72) was observed. A great variability was observed in oocyst excretion, both between litters and between fattening weeks (Table 3 and 4), however, the differences found were not significant (p= 0.05).In all analysed stool samples, we detected the presence of Eimeria spp. DNA. Three different size PCR products (400 bp ,450 bp and 600 bp) were amplified. BLAST analysis as E. mediaand the 400 bp product as E. vejdovskyiidentified the 450 bp Scialfa et al.Neotropical Helminthology, 2021, 15(2), jul-dic
image/svg+xml205Figure 3.Oocyst excretion (average of oocyst per gram: OPG) by weaned litters during fattening weeks.PCR product, although other species that amplify the same molecular weight could be present in the samples. The 600 bp PCR product did not contain enough material for sequencing analysis, although according to the molecular size it could correspond to E. coecicola orE. stiedae. According to the distribution of the Eimeriaspecies in the rabbit farm, E. vejdovskyiwas found in all breeding sector, E. mediain weaning sector, fattening and reproductive does sector, while E. coecicolaand E. stiedae, only in fattening sector.Figure 4. Oocyst excretion (oocyst per gram: OPG) in weaning litters during fattening weeks. Litters that expressed a negative result to the flotation method (Value= 0) before or after a positive result with oocysts count (OPG), can be observed.Eimeriaoocyst excretion in naturally infected rabbitNeotropical Helminthology, 2021, 15(2), jul-dic
image/svg+xml206Table 2.Oocyst excretion (average of oocyst per gram: OPG) in weaning litters during fattening weeks. In addition, the time of the fattening period as the daily weight gain (GMD) can be observed.Litter doeLitteraverage OPGFatteningweeksGMDOPGrange805131833.940 a 12880875796834.2280 a 2500010960561224.60 a 1524011366621125.60 a 13460527057928.60 a 273606188091028.30 a 23240919722932.3820 a 1980010410349832.70 a 3012010110931932.10 a 4650011412435836.020 a 68000110125261028.280 a 295004117598932.60 a 756804921564938.50 a 7680010737564933.10 a 12000Table 3.Variability in the oocyst excretion (oocyst per gram; OPG) by weaning litters during fattening weeks.FatteningweeksOPG averageOPGrange138850 a 9000247950 a 1200031367820 a 756804164740 a 680005120610 a 47680613986680 a 7680079380280 a 30120855350 a 21600981130 a 4650010251080 a 4200117195240 a 1415012160160The overall prevalence of Eimeriaspp. of this study (97.2%) is higher than the 78.6%, 70.3%, 64.7% observed in Nigeria, Indonesia and Malaysia respectively (Ola-Fadunsin et al., 2019; Hamid et al., 2019; Mohamad-Radzi et al.,2021). The prevalence of coccidia oocysts in does and weaned litters were 71.4% and 100% respectively. Eimeriainfestation in weaned rabbits was similar to that the described in a study of three meat rabbit farms in Malaysia, with a prevalence of 76.5% in young animals; however, in a large-scale farm the DISCUSSIONprevalence was 100%. Possibly have a stocking density of 5–6 animals per cage is an important factor of the occurrence of coccidia (Mohamad-Radzi et al.,2021). The variation in the prevalence of this study with other studies may be due to study location, environmental conditions and system breeding. Difference between four seasons and infection rate was observed in Egypt, were the highest rates of infection (53.5% and 39%) were recorded in autumn and winter respectively (El-Sayed et al., 2020).Although the morphological characteristics of coccidia oocyst helped to identify the EimeriaScialfa et al.Neotropical Helminthology, 2021, 15(2), jul-dic
image/svg+xml207weight (83- 116 d age). According previous studies, intestinal Eimeriainfections occur in kits just after weaning (Papeschi et al., 2013). In open-air breeding systems, weaned rabbits reach the maximum oocyst excretion earlier compared to semi-intensive indoor systems, being during the 45 to 60 days of age (Papeschi et al., 2003).Findings from this study suggest that mothers, mainly in the last half of gestation and lactation, may play an important role in Eimeria transmission to the litters. Weaning involves changes that can affect your immune response capacity, due to a stressful situation for the animal due to separation from the mother, to the change of accommodation and social group. The fact that all rabbit litters were positive post-weaning, regardless of whether they came from positive does and the negative results between counts of oocysts with high values, this could be due to the sensitivity of the flotation method and McMaster method (sensitivity of 20 oocysts / gram of feces sample). That is, negative results can probably be interpreted as false positives where oocyst counts are less than 20 OPG. The time of the fattening period as well as the observed daily weight gain (GMD) are the usual ones for the farm, where the litters rabbits reaching slaughter weight (2.5 kg) at week 8 and 9 the litters rabbits reaching slaughter weight with an average of 33.4 gram/day. The growth rate and daily weight gain of current hybrid lines of domestic rabbits, reach the highest level (45-50 g / day) at 8 weeks of age (Gidenne et al., 2010). Probably, the daily weight gain in the farm was influenced by feed (formulation of balanced diet, the quality of raw materials and the manufacturing process); however, could be attributed to non-feed factors as genetic, environment and housing. No evident clinical manifestations in weaning rabbits, even in litters with high counts (mayors of 30000 OPG) could be attributed to moderately and low pathogenic Eimeriaspecies observed, being E. vejdovskyithe most frequent in the analyzed feces samples. Despite the presence of E. stiedaeon the farm, no liver lesions were observed in the slaughtered animals, possibly this is due to a low load of these coccidian in relation to E. vejdovskyiand E. media, and the age of the slaughtered animals.species, it did not allow a good differentiation of the oocysts, mainly in samples with a high load. The unsporulated morphology characteristics between E. vejdovskyiand E. coecicola, were similar, so it was impossible to differentiate them only on the basis of unsporulated morphology features. In this study, the E. magnaand E. exiguawere identified based on the morphological characteristics, but could not be detected by the PCR methods. The complementation of the morphological features of the oocysts with the application of molecular methods allowed to identify E. vejdovskyi, E. media, E. stiedae andE. coecicola. The molecular technique applied in the present work has the limitation that the number of Eimeriaspecies present in each sample can be underestimated. As various species show the same amplicon size, the obtained sequence corresponds to the most prevalent specie in the sample, PCR amplification with specific primers as described by Oliviera et al. (2011) could be a more suitable diagnostic assay.Female rabbits examined sowed two periods de oocyst excretion: in the second half of lactation (preceding weaning of the litter) and after parturition (second half); as being lactating and pregnant simultaneously, the most vulnerable periods. The high nutritional requirements during gestation and lactation may lead to lowered resistance to Eimeria infections, increasing the growth of the pathogen at the intestinal level, which is reflected by the presence of oocysts in feces. The period preceding weaning of the litter born was a frequent period of oocyst shedding, and this finding was previously reported (Papeschi et al.,2013). The 50% of the females eliminated oocysts in a single moment or physiological condition, 30% in two and 20% in three (Figure 1). Although the range of oocyst clearance was wide (20-13820), the 66.7% of the does had counts of 1000 OPG (Median: 480).Oocyst shedding by litters weaned during fattening period was very variable, with values of 0 to 76800 OPG. A great variability was observed in oocyst excretion, both between litters and between fattening weeks. From weaning moment, the excretion of oocyst increases to week seven (74 d of age) and then it decreases to reaching slaughter Eimeriaoocyst excretion in naturally infected rabbitNeotropical Helminthology, 2021, 15(2), jul-dic
image/svg+xml208Although in our study the presence of six Eimeriaspecies was observed, similar studies in other countries (Indonesia, Malaysia, Nigeria and Poland) have shown mixed infection with seven and ten Eimeria species (Hamid et al., 2019; Ola-Fadunsin et al., 2019; Balicka-Ramisz et al., 2020; Mohamad-Radzi et al.,2021).This is the first report in which it is described the oocysts mixture of six Eimeriaspecies species (E. vejdovskyi, E. coecicola, E. magna,E. exigua, E. media andE. stiedae)from the rabbit for meat production in the region. These findings show that breeding rabbits are asymptomatic carriers of coccidian infections and can shed oocysts at any time during the reproductive cycle. However, at the time of further development of the fetuses during gestation and at the peak of milk production there is a decrease in resistance to infection by Eimeria species, expressed in a greater excretion of oocysts, being a potential source of infection for kits. PCR is an important method for diagnosing rabbit Eimeriadisease in rabbits and should be complemented by identification based on morphological characteristics. These molecular tests will make it possible to carry out epidemiological surveillance in other rabbit farms of the region, and to know the distribution of Eimeriaspecies that affect rabbits. Further studies will aim to evaluate the impact of infection by Eimeriaon the efficiency of feed conversion in litter rabbits of farm.ence, Research and Development Environment Research Programme, pp. 52-73.Coudert, P, Licois, D, Provôt, F & Drouet-Viard, F. 1993. Eimeria sp. from rabbit (Oryctolagus cuniculus): pathogenicity and immunogenicity of Eimeria intestinalis. Parasitology Research,vol. 79, pp. 186-190.Elshahawy, S & Elgoniemy, A. 2018. An epidemiological study on endoparasites of domestic rabbits(Oryctolagus cuniculus) in Egypt with special reference to their health impact.Sains Malaysiana, vol. 47, pp. 9-18.El-Sayed, N, Metwally, MMM & Ras, R. 2020.Prevalence and morphological identification of Eimeriaspp. in domestic rabbit (Oryctolagus cuniculus) in Sharkia province, Egypt.Egyptian Veterinary Medical Society of Parasitology Journal, vol. 16, pp. 114-130. Gidenne, TL, Lebas, F & Fortun-Lamothe, L (eds). 2010. Feeding behavior of rabbits. De Blas, C., Wiseman, J., "Nutrition of the rabbit", Cab International, pp. 233-252.Hamid, PH, Prastowo, S & Kristianingrum, YP. 2019. Intestinal and hepatic coccidiosis among rabbits in Yogyakarta, Indonesia. Veterinary World, vol.12, pp. 1256-1260.Li, MH, Huang, HI & Ooi, HK. 2010. Prevalence, infectivity and oocyst sporulation time of rabbit-coccidia in Taiwan.Tropical Biomedicine, vol. 27, pp. 424-429.Licois, D. 2004.thDomestic rabbit enteropathies. 8 World Rabbit Congress, 7-10 September, 2004. Puebla, Mexico, pp. 385-403.Mohamad-Radzi, NN, Che-Amat, A, Aziz, NAA, Amin Babjee, SM, Mazlan, M, Saudol Amid, NF & Lekko, YM. 2021. Preliminary detection of mites and coccidia with their zoonotic potential in meat-farmed rabbits in three districts in Selangor, Malaysia. Journal of Parasitic Diseases, vol. 45, pp. 169-175.Okumu, PO, Gathumbi, PK, Karanja, DN, Mande, JD, Wanyoike, MM, Gachuiri, CK, Kiarie, N, Mwanza, RN & Borter DK. 2014. Prevalence, pathology and risk factors for coccidiosis in domestic rabbits (Oryctolagus cuniculus) in selected regions in Kenya, Veterinary Quarterly, vol. 34, pp. 205-210.Baker, DG (eds). 2007. Flynn's Parasitology of Laboratory Animals. Blackwell Publishing Company.Balicka-Ramisz, A, Laurans, Ł, Pohorecki, K, Batko, M & Ramisz, A. 2020. Short communication: prevalence of Eimeriaspp. infection in domestic rabbits of Polish farms.World Rabbit Science, vol. 28, pp. 181-185.Coudert, P, Licois, D & Drouet-Viard, F. 1995. Eimeriaspecies and strains of the rabbits. In: J. Eckert, R. Braun, M.W. Shirley & P. Coudert (eds), Guidelines on techniques in coccidiosis research. European Commission, Directorate-General XII, Sci-BIBLIOGRAPHIC REFERENCESScialfa et al.Neotropical Helminthology, 2021, 15(2), jul-dic
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