Veterinary Parasitology: Regional Studies and Reports 48 (2024) 100971 Available online 13 December 2023 2405-9390/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). Original Article Cystoisospora spp. infection at a dog breeding facility in the Madrid region: Infection rate and clinical management based on toltrazuril metaphylaxis Juan P. Barrera, Ana Montoya *, Valentina Marino, Juliana Sarquis, Rocío Checa, Guadalupe Miró * Department of Animal Health, Veterinary Faculty, Universidad Complutense de Madrid, Spain A R T I C L E I N F O Keywords: Cystoisospora spp. Dogs Toltrazuril Control Metaphylaxis Treatment A B S T R A C T Canine coccidiosis caused by Cystoisospora canis and Cystoisospora ohioensis-complex is common in kennels. While often underestimated, coccidiosis may cause severe clinical signs in puppies and sometimes even lead to death, so preventative measures are important. This study examines Cystoisospora spp. infection at a Labrador retriever breeding facility in Madrid, Spain. To identify environmental factors associated with infection, dams were examined throughout a reproductive cycle (from oestrus to 60 days postpartum) and their puppies during their first 60 days of life. Also assessed was the efficacy of combined treatment with emodepside (0.9 mg/ml) and toltrazuril (18 mg/ml) at a dose of 0.5 ml/kg of weight, equivalent to 0.45 mg/kg and 9 mg/kg, respectively, in puppies on day 35 of life. Oocyst shedding was detected in 4.6–18.6% of 45 dams examined and in 2.2–9.1% of their litters (315 puppies). In both cases, peak opg elimination was recorded on day 30 postpartum/of life. The species of Cystoisospora detected were C. canis (91.3%) and C. ohioensis-complex (8.7%). While in both dams and puppies opg counts were higher in autumn when rainfall was at its highest, correlation between opg and rainfall emerged as significant only in puppies (p = 0.031). The treatment of 35 day-old puppies with toltrazuril was 100% effective in controlling this infection in the kennel. Our findings therefore suggest the need for a strict hygiene regime and the use of toltrazuril as blanket treatment to reduce Cystoisospora transmission in dog breeding facilities. 1. Introduction Infectious diseases are a main health concern in kennels where dogs live in close contact. Examples are infections caused by intestinal par asites such as Toxocara canis, Giardia duodenalis and Cystoisospora spp., which mainly affect animals under one year of age (Barutzki and Schaper, 2013) and are more prevalent in breeding kennels than among household dogs (Gothe and Reichler, 1990). The phylum Apicomplexa comprises numerous genera of protozoan parasites including the coccidia Cystoisospora canis and Cystoisospora ohioensis-complex (C. ohioensis, C. burrowsi and C. neorivolta), which cause coccidiosis sensu stricto in dogs (Fig. 1). These species are distributed worldwide and have a monoxenous life cycle, meaning that they only need one host. They are also strictly host-specific (Dubey and Lindsay, 2019). Cystoisospora spp. are transmitted through the faecal-oral route and complete their life cycle in the small intestine of the host. Asexual multiplication is followed by sexual multiplication (gametogony) to produce thick-walled non-sporulated oocysts, which are excreted by the host into the environment. Then, within 12 h or a few days, depending on the species (Lappin, 2010) and environmental conditions like tem perature and humidity (Lindsay et al., 1982), the oocysts sporulate becoming infectious for other hosts when ingested by feeding on the soil, and the cycle ends. The prepatent period is 9–11 days for C. canis (Lepp and Todd, 1974) and about 6–7 days for C. ohioensis-complex (Buehl et al., 2006). Sporulated oocysts can survive in the environment for months and are resistant to general disinfectants, but cresols can inac tivate coccidian oocysts (Deplazes et al., 2016). In addition, it is important that animal handlers adopt strict hygiene measures as * Corresponding authors. E-mail addresses: jbarrera@ucm.es (J.P. Barrera), amontoya@ucm.es (A. Montoya), vamarino@ucm.es (V. Marino), juguimar@ucm.es (J. Sarquis), rocichec@ ucm.es (R. Checa), gmiro@ucm.es (G. Miró). Contents lists available at ScienceDirect Veterinary Parasitology: Regional Studies and Reports journal homepage: www.elsevier.com/locate/vprsr https://doi.org/10.1016/j.vprsr.2023.100971 Received 30 May 2023; Received in revised form 11 October 2023; Accepted 5 December 2023 mailto:jbarrera@ucm.es mailto:amontoya@ucm.es mailto:vamarino@ucm.es mailto:juguimar@ucm.es mailto:rocichec@ucm.es mailto:rocichec@ucm.es mailto:gmiro@ucm.es www.sciencedirect.com/science/journal/24059390 https://www.elsevier.com/locate/vprsr https://doi.org/10.1016/j.vprsr.2023.100971 https://doi.org/10.1016/j.vprsr.2023.100971 https://doi.org/10.1016/j.vprsr.2023.100971 http://crossmark.crossref.org/dialog/?doi=10.1016/j.vprsr.2023.100971&domain=pdf http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ Veterinary Parasitology: Regional Studies and Reports 48 (2024) 100971 2 oocysts, like other parasite stages, attach to the coat of the animal and remain infective over hours or even a few days (Wolfe and Wright, 2003). Sanitation measures are also needed to minimize the spread of sporulated oocysts in the environment. These include daily removal of faeces from kennels and metaphylactic treatment targeted at decreasing oocyst excretion and thus reducing oocyst pressure in the field. It is also important to implement control measures against paratenic hosts, such as rodents, to reduce the transmission of these parasites (Daugschies et al., 2000; ESCCAP, 2018). Although Cystoisospora spp. in dogs have no zoonotic potential (Lappin, 2010), a feature of coccidiosis in all animal species is that only a fraction of those individuals that are infected and shed oocysts develop disease. Of these individuals, some are only transiently affected while others suffer from serious enteral disturbances followed by weight loss and other long-lasting health problems, such as poor appetite, diarrhoea, dehydration, vomiting, and even death (Daugschies et al., 2000; Junker and Houwers, 2000). Microscopic lesions include villous atrophy, dila tion of lacteals and hyperplasia of lymph nodes in Peyer's patches (Mitchell et al., 2007). It cannot be predicted which individuals will suffer the most, so breeders usually opt for “blanket treatment” to prevent oocyst shedding and therefore reduce transmission. It should also be considered that if tested animals belong to a com mercial dog breeder's in which many animals coexist and the purpose is their sale, once a disease occurs, treatment is usually more expensive than prevention as in other types of animal farms such as those of small ruminants and pigs (Daugschies and Najdrowski, 2005; Kreiner et al., 2011). Some treatments for cystoisosporosis in dogs have been reported but the Spanish Agency for Medical and Health Products (AEMPS) only authorizes toltrazuril for this purpose, which is commercialized in combination with emodepside at concentrations of 0.9 mg of emodep side/kg and 18 mg of toltrazuril/kg (Procox®, Vetoquinol S. A., Lure, France). Emodepside is an anthelmintic agent with efficacy against several gastrointestinal nematodes. This treatment is safe for puppies from two weeks of age at 0.4 kg of weight and is given as a single oral 0.5 ml/kg dose, equivalent to 0.45 mg/kg (emodepside) and 9 mg/kg (toltrazuril) (Agencia Española de Medicamentos y Productos Sanitarios (AEMPS), n.d). The objectives of this study were: a) to determine the dynamics of Cystoisospora spp. oocyst excretion in puppies faeces in a breeding kennel; b) to explore the impacts of seasonality and the correlation be tween shedding in dams and their litters throughout a reproductive/life cycle; c) to assess the efficacy of toltrazuril treatment in puppies testing positive for coccidiosis at the age of 35 days; and d) to assess the met aphylactic efficacy of this active substance within a coccidiosis control programme. 2. Materials and methods 2.1. Site selection, animal husbandry and hygiene conditions The site selected was a Labrador retriever breeding centre in Gua darrama, located in the NW Madrid Community (40.629,-4.107; 981 m a.s.l) (Spain). The climate is described as Continental Mediterranean, with dry warm summers and very cold winters. Average annual rainfall is 1223 mm, mainly falling in autumn (González-Flórez et al., 2022). The breeding facility was located in a peri-urban area close to a forest, and the adults were housed in kennels with cement floors and hosted individually or in small groups, depending on sex, age, repro ductive cycle and health status. According to the kennel's protocol, pregnant females were housed individually one week before parturition and their health status checked postpartum by a veterinarian. Dams and their litters were housed together in stalls with straw bedding, which was changed each repro ductive cycle enforcing a strict all-in-all-out regime in each stall. A plan for disinfection and insect and rodent control was applied to ensure the health and safety of the dogs in the breeding facility. Faeces in the stalls was removed daily every 12 h, and then, each stall was disinfected with 10% hypochlorite solution and wait for desiccation before house again the animals. All puppies from the 7th week of age according to the vaccine manufacturer's instructions were vaccinated and revaccinated against canine distemper virus, canine parvovirus, canine adenovirus and canine parainfluenza virus. Dams and puppies were fed with commercial food (Advance Mother Dog & Initial® (Advance-Affinity®, Barcelona, Spain)) twice daily, following the manufacturer's recommendations. Puppies of the same litter were collectively fed with the same food, but this time moistened, from the age of 3 weeks, and individually from 6 weeks three times daily. Water was always provided ad libitum. 2.2. Study design 2.2.1. Faecal analysis and epidemiological variables Forty-five Labrador retriever dams aged 2 to 5 years along with their litters comprising 315 puppies were followed until 8 weeks of age. The group of dams served as a control group. Among the inclusion re quirements were that participants were healthy animals without alter ations in their biochemical profile subjected to the correct vaccination and deworming schedule without including active agents effective against coccidia. Faecal samples of dams were collected individually from the cubicle substrate at the following time points: oestrus-service (nearly 60 days before parturition), one-day prepartum, and 1, 15, 30, 45 and 60 days postpartum. Puppies in the litters were administered toltrazuril both as a treatment and metaphylaxis. The unit of this group of animals was Fig. 1. Sporulated oocyst of C. canis (36 × 30 μm) (a) and C. ohioensis-complex (24 × 21 μm) (b) under the light microscope (40×). J.P. Barrera et al. Veterinary Parasitology: Regional Studies and Reports 48 (2024) 100971 3 considered the complete litter of each dam and the available samples from each puppy that made up the litter were collected from the cubicle substrate and pooled on days 30, 35, 36, 45 and 60 of life. Faecal samples from dams and litters were tested from September 2015 to August 2018. Samples were stored at 4 ◦C until processing within 48 h by the Mini-FLOTAC® technique (Cringoli et al., 2013), and the number of excreted oocysts per gram of faeces (opg) was determined by light microscopy. Oocysts were obtained by a concentration method using a 33% saturated sodium chloride solution, and sporulation was induced by storing samples in potassium dichromate solution (2.5%) for 48 h (Ryley et al., 1976). Cystoisospora species were then identified ac cording to oocyst morphological characteristics and measurements (Dubey, 1978; Lepp and Todd, 1974). Daily climate records (maximum, mean and minimum temperatures, as well as mean and total rainfall) were compiled from the website of the Spanish Meteorology Agency - AEMET (Accessed 27 December 2020), from weather stations close to the study area NW of Madrid. The months of the year were clustered into seasons: autumn (October–November- December), winter (January–February-March), spring (April–May- June) and summer (July–August-September). 2.2.2. Treatment efficacy trial The efficacy of toltrazuril treatment was tested in the six litters (57 puppies) in which oocysts were detected on day 35 or earlier and follow- up exams were performed on days 36, 45 and 60 of life. The decision to use the combined treatment of emodepside/toltrazuril at a dose of 0.45 mg/kg and 9 mg/kg respectively (according to the instruction labels and after written owner consent) on day 35 of life of the puppies was based on our previous experience and considered the feasibility of adminis tration of the medication to the puppies, prepatent periods of Cys toisospora species, and management practices in the breeding centre. The health status and weight of the puppies was first evaluated and all other treatments avoided. Treatment efficacy (E) was calculated according to the guidelines issued by the World Association for the Advancement of Veterinary Parasitology (Joachim et al., 2018) and European Medicines Agency (EMA, 2021) using the equation: E = ( mean opg before treatment − mean opg after treatment mean opg before treatment ) x 100 2.2.3. Metaphylaxis trial Based on knowledge that Cystoisopora spp. were the most prevalent enteric parasites in the kennel, another 39 litters, totalling 262 puppies that had tested negative for coccidia from birth to 35 days, were also treated with emodepside/toltrazuril as metaphylaxis. Faecal samples were collected at the same time points and doses were similar to those described for the treatment efficacy trial. 2.3. Statistical analysis To identify risk factors for infection (relative humidity and temper ature), correlations were assessed between epidemiological variables and the excretion of oocysts by dams and litters through Pearson's cor relation analysis using the SPSS software package (version 21). Signifi cance was set at p ≤ 0.05. 3. Results 3.1. Faecal analysis and epidemiological variables A total of 524 faecal samples were obtained. Of these samples, 46 (8.8%) tested positive for Cystoisospora spp. In the case of dams, the percentage of positive samples was 10.6% (32/300) and in litters was 6.2% (14/224). The species of Cystoisospora identified were C. canis in 91.3% (42/46) of the samples and C. ohioensis-complex in 8.7% (4/46). No co-infections by both species of Cystoisospora were observed. For results in the litters, only samples obtained on days 30 and 35 of life were considered because no litter had been treated to avoid affecting the parasite's life cycle. The percentage of litters excreting oocysts was 6.6% (day 35) to 8.8% (day 30) and the average of oocysts shed per gram of faeces (opg) was 780 (day 35) to 1135 (day 30). The maximum number of excreted oocysts in litters was recorded on day 30 (mean 1135 opg) and the minimum on day 35 (mean 780 opg) (Fig. 2). The percentage of dams which excreted oocysts throughout the reproductive cycle was between 4.6% (day 15) and 18.6% (day − 1). Peak oocyst excretion took place on day 30 postpartum (mean 844 opg) and minimum excretion (mean 52 opg) was recorded at oestrus (Fig. 3). Throughout the study, all puppies were apparently healthy and returned a good body condition score. Diarrhoea was not considered as diets were very wet and faecal samples were mixed with absorbent straw so, while stool consistency could not be easily graded, it was considered normal for the type of feed given to the puppies. A reduction in stool consistency was neither detected in mothers. We also examined correlations between excreted opg and the epidemiological variables: maximum, mean, and minimum temperature and total rainfall. Significant correlation was detected between total rainfall and the number of oocysts eliminated in the faeces of litters (p = 0.031) (Table 1). In addition, highest numbers of excreted oocysts were observed in untreated puppies in autumn 2016, averaging 1630 opg, which is when total rainfall was highest (281.4 mm) (Fig. 4). In contrast, in the months of spring 2017 and summer 2018, when temperatures were higher and total rainfall lower, the excretion of oocysts was almost null in both dams and their offspring. 3.2. Treatment study Four litters tested positive for Cystoisospora spp. oocysts in the samples analysed on day 30 and two litters on day 35. Therefore, six litters were included in this treatment study and administered combined treatment with emodepside and toltrazuril. By the end of follow up, these litters showed a 100% reduction in opg counts (Table 2). 3.3. Metaphylaxis study Thirty-nine litters testing negative for coccidiosis up until 35 days of life were included in this trial. After the administration of combined treatment, the data collected revealed that 33 of the litters (84.6%) remained negative until the end of follow up, and the remaining 6 litters (15.4%) tested positive sometime after treatment onset: two litters on day 45 and four litters on day 60 (Table 3). 4. Discussion Natural canine Cystoisospora spp. infection is usually a cause of concern only when dogs are kept in large groups such as in breeding facilities or shelters (Gothe and Reichler, 1990), or when they are young or immunocompromised. It is important that the impacts of outbreaks of coccidiosis are not underestimated as, while infection does not usually produce patent clinical signs, it can worsen the health state of its hosts (Dubey and Lindsay, 2019). In addition, correlations between cys toisosporosis and other intestinal infections such as canine parvovirus have been reported in collective canine facilities likely as the conse quence of common risk factors for these infections in dogs which often harbour more than one pathogen (Duijvestijn et al., 2016). In our study, dams excreted fewer oocysts than their litters at all time points in accordance with the results of Becker et al. (1981) in which it was observed that acquired immunity was boosted after successive re infections, reducing the number of oocysts eliminated and leading to a lack of clinical signs. However, perhaps the ingestion by the dams of the faeces of their pups was the cause of them shedding oocysts in smaller numbers than the puppies infected in a higher percentage in general, J.P. Barrera et al. Veterinary Parasitology: Regional Studies and Reports 48 (2024) 100971 4 Fig. 2. Mean opg excreted in litters not treated throughout the study period. Fig. 3. Mean opg excreted in dams throughout the study period. Table 1 Pearson correlation between climate variables and oocysts excreted by dams and their litters. % Positive opg Total Litters Dams Total Litters Dams − 0.265 0.124 − 0.275 − 0.095 − 0.170 − 0.092 Pearson correlation 0.118 0.471 0.104 0.581 0.322 0.595 p-value Minimum temp. (C◦) − 0.231 0.176 − 0.250 − 0.033 − 0.042 − 0.074 Pearson correlation 0.175 0.304 0.141 0.850 0.807 0.667 p-value Average temp. (C◦) − 0.280 0.185 − 0.308 − 0.010 − 0.136 − 0.008 Pearson correlation 0.099 0.279 0.067 0.956 0.428 0.963 p-value Total rainfall (mm) 0.044 0.165 0.019 0.103 0.360a − 0.021 Pearson correlation 0.801 0.335 0.911 0.551 0.031 0.902 p-value Average rainfall (mm) 0.072 0.118 0.063 0.131 0.317 0.033 Pearson correlation 0.676 0.494 0.714 0.445 0.059 0.848 p-value n = 36. a Correlation significant at the 0.05 level. J.P. Barrera et al. Veterinary Parasitology: Regional Studies and Reports 48 (2024) 100971 5 thus maintaining Cystoisospora infection in the dog breeding site at basal levels. In this study, the time points when opg of Cystoisospora were greatest in the dams were near oestrus and prepartum, and a peak of oocyst excretion was detected on day 30 postpartum. Considering the prepatent periods of both C. canis (about 9–11 days) and C. ohioensis-complex (about 6–7 days) and according to the findings of Penzhorn et al. (1992), these time points could coincide with the slight immunocompromise that occurs physiologically near postpartum day in dams, allowing for greater multiplication of the parasite and thus an increase in opg, sug gesting dams are potential reservoirs (Daugschies et al., 2000; Yun et al., 2000). Moreover, such physiological immunocompromise could in crease the susceptibility of mothers to infection through alternative routes such as the ingestion of paratenic hosts (Markus, 1983; Hilali et al., 1992, 1995; Zayed and El-Ghaysh, 1998; Dubey and Lindsay, 2019), which although numerous include mice as the most likely can didates in dogs. Although the animals at the breeding centre examined were selected for their characteristics as working dogs, they could ingest rodents while playing outdoors. Despite adequate control measures such as the removal of faeces within 12 h to avoid oocyst sporulation in the environment, dams played an important role in the transmission of these parasites to their litters, as they had access to the outside meaning they had contact with a possibly contaminated environment or could feed on infected paratenic hosts. In 13 out of 45, dams but not their litters, shed oocysts. Conversely, in 7 out of 45 dams were negative and their litters positive, indicating that puppies were not infected by their mothers or that the parasite load was too small and/or patent time was short, and the oocysts were unde tectable in the faeces samples. In our study, the only similarities in the oocyst excretion rate be tween dams and their litters were increased opg in autumn 2016 and on day 30 postpartum/postnatal, when peak opg values were recorded in both dams and litters. This association between oocyst excretion in mothers and their puppies has not been seen in similar studies (Penzhorn et al., 1992). One explanation could be that the physiological decrease in immunity produced in the dams in the peripartum coincides with the first weeks of life of the puppies. Thus, periods of patency would be synchronized in both mothers and puppies and the maximum peak of elimination of oocysts on the 30th day would coincide with recovery of the mothers' immunity therefore eliminating fewer oocysts in the faeces. In agreement with reports by other authors (Bode, 1999), the spread of oocysts may be affected by seasonal variables as observed in this study in the autumn of 2016. During this period, weather conditions in the area were favourable for these obligate parasites. Cystoisospora spp. require mild temperatures (Dubey et al., 2009) and specifically the sporulation of C. canis takes place from 30 to 35 ◦C in 16 h (Lepp and Todd, 1976). Regarding the correlation observed in this study between total rainfall and increased oocyst excretion, it has been described that rainfall effectively plays a role in coccidian oocyst dissemination in the environment, both in the case of Toxoplasma gondii (Cotey et al., 2022) and Cryptosporidium spp. oocysts (Ramirez et al., 2009). This may be one of the reasons for our observation of increased oocyst excretion in wetter periods, as environmental oocysts are not eliminated. Indeed, the autumn of 2016 was when the highest percentage of litters tested pos itive for Cystoisospora oocysts at 28.5% (Supplementary material). This figure coincides with those described by others who have examined rainfall as a variable correlated with the prevalence of the Eimeria spp. infection in small ruminants (Alcala-Canto et al., 2020). To effectively control coccidiosis in dog litters, a single dose of tol trazuril is sufficient (Daugschies et al., 2000) and this was observed in our metaphylaxis trial. Most of the litters remained negative (84.6%) until day 60 of life, and no clinical signs were observed. However, 6 of the 39 litters subjected to metaphylaxis with this drug obtained a Fig. 4. Mean opg excreted in untreated litters and dams across the seasons of the year and total rainfall data. Table 2 Cystoisospora oocyst counts and other data obtained in the treatment efficacy trial. Number of litter Day 30 Day 35 Day 36a Day 45 Day 60 1 1,260 80 0 0 0 16 2,320 0 0 0 0 19 0 2,200 0 0 0 20 920 0 ND 0 0 22 40 0 0 0 0 24 0 60 40 0 0 Positives/total (%) 4/6 (66,6) 3/6 (50.0) 1/6 (16.6) 0 (0.0) 0 (0.0) Mean opg 1,135 780.00 40 0 0 Standart deviation 942.53 1,499.07 0 0 0 ND - No data. a After toltrazuril administration. J.P. Barrera et al. Veterinary Parasitology: Regional Studies and Reports 48 (2024) 100971 6 positive result on days 45–60. According to the results of other trials, this could be attributed to decreased bioavailability of toltrazuril in tissues, such that when animals are exposed to parasite infections over long periods of time, additional treatment approximately two weeks after the first dose is needed (Altreuther et al., 2011b). In this dog breeding facility, litters started sharing a large feeder at 3 weeks of age and at 6 weeks they were given individual feeders, which were washed and disinfected after each use. However, cases of cys toisosporosis reinfections are not uncommon (Litster et al., 2014) and this could be the consequence of insufficient cleanliness of bowls or even of the dogs' perianal hair after treatment, as with other intestinal para site infections such as Giardia duodenalis (ESCCAP, 2018) and Toxocara canis (Roddie et al., 2008; Rojas et al., 2017; Wolfe and Wright, 2003). In effect, these factors could mask the effectiveness of treatment. In agreement with the results of other studies, the oocyst reduction percentage by toltrazuril was 100% in treated animals (Altreuther et al., 2011a, 2011b). Probably, the main sources of infection of litters were the dams because they had access to the outside and likely maintained the parasite burden of the kennel. Approximately, three months after starting the puppies on combination treatment (at 35 days of age), the number of oocysts excreted by dams was significantly reduced and remained low thereafter (Fig. 4). Accordingly, the measures taken were the administration of this treatment to litters on day 35 of life and an additional dose on day 60. This second dose was given to avoid any reinfections in the kennel and ensure that dogs were free of coccidia infection and completely healthy when handed over to their future owners. Despite these measures, it is not possible to completely remove coccidia from this type of environ ment. In the absence of appropriate treatment, litters may shed more oocysts into the environment, and some of them could develop clinical signs. 5. Conclusions Dams were responsible for transmitting Cystoisospora spp. infection to their litters, and both showed peak oocyst elimination on postpartum or postnatal day 30. Relative humidity was found to be directly related to the number of oocysts detected, as peak oocyst excretion was observed in the wetter autumn months. In addition to being an excellent active agent for the treatment of cystoisosporosis in 35-day old puppies, toltrazuril emerged as a useful metaphylactic tool for its control in dog kennels, where the risk of oocyst shedding is particularly high, as most of the study animals remained negative for Cystoisospora infection. Declaration of Competing Interest The authors declare the following financial interests/personal re lationships which may be considered as potential competing interests: Guadalupe Miró reports equipment, drugs, or supplies and travel were provided by Complutense University of Madrid. Guadalupe Miró reports a relationship with Complutense University of Madrid that in cludes: board membership, employment, non-financial support, and travel reimbursement. Aknowledgements We want to thank Mr. Pedro García Riaño as the owner of the breeding site where this study has carried out for his valuable help. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.vprsr.2023.100971. References Agencia Estatal de Meteorología - AEMET. Gobierno de España. http://www.aemet.es/e s/portada (Accessed 27 December 2020). Agencia Española de Medicamentos y Productos Sanitarios (AEMPS). Medicamentos Veterinarios (CIMAvet). Anexo I Ficha Técnica o Resumen de las Características del Producto. https://cimavet.aemps.es/cimavet/pdfs/es/p/EU%402%4011%40123% 40001/P_EU-2-11-123-001.pdf. (Accessed 02 February 2023). Alcala-Canto, Y., Figueroa-Castillo, J.A., Ibarra-Velarde, F., Vera-Montenegro, Y., Cervantes-Valencia, M.E., Alberti-Navarro, A., 2020. First database of the spatial distribution of Eimeria species of cattle, sheep and goats in Mexico. Parasitol. Res. 119, 1057–1074. https://doi.org/10.1007/s00436-019-06548-8. Altreuther, G., Gasda, N., Adler, K., Hellmann, K., Thurieau, H., Schimmel, A., Hutchens, D., Krieger, K.J., 2011a. Field evaluations of the efficacy and safety of Emodepside plus Toltrazuril (Procox® Oral suspension for dogs) against naturally acquired nematode and Isospora spp. Infections in Dogs. Parasitol. Res. 109, 21–28. https://doi.org/10.1007/s00436-011-2399-z. Altreuther, G., Gasda, N., Schroeder, I., Joachim, A., Settje, T., Schimmel, A., Hutchens, D., Krieger, K.J., 2011b. 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Litter Day 30 Day 35 Day 36a Day 45 Day 60 2 0 0 0 0 0 3 0 0 0 0 0 4 0 0 0 0 0 5 0 0 0 0 0 6 0 0 0 0 0 7 0 0 0 0 0 8 0 0 0 0 0 9 0 0 0 0 0 10 0 0 0 0 0 11 0 0 0 0 0 12 0 0 0 0 0 13 0 0 0 0 0 14 0 0 0 0 0 15 0 0 0 40 0 17 0 0 0 0 0 18 0 0 0 0 20 21 0 0 0 0 0 23 0 0 0 0 0 25 0 0 0 0 0 26 0 0 0 0 0 27 0 0 0 0 0 28 0 0 0 0 2832 29 0 0 0 0 0 31 0 0 0 0 0 32 0 0 0 0 0 33 0 0 0 0 0 34 0 0 0 0 0 35 0 0 0 0 0 36 0 0 0 0 3700 37 0 0 0 0 0 38 0 0 0 0 60 39 0 0 0 0 0 40 0 0 0 0 0 41 0 0 0 0 0 42 0 0 0 0 0 43 0 0 0 20 0 44 0 0 0 0 0 45 0 0 0 0 0 46 0 0 0 0 0 Positives/total (%) 0/39 (0.0) 0/39 (0.0) 0/39 (0.0) 2/39 (5.1) 4/39 (10.2) Mean opg 0 0 0 30 1653 Standard deviation 0 0 0 14.14 1896.01 a After toltrazuril administration. J.P. 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