The family Sarcocystidae includes several intracellular coccidial parasites such as Toxoplasma gondii, Neospora caninum, Sarcocystis spp. and Hammondia spp. with heteroxenous life cycles involving different parasitic stages (oocysts/sporocysts, tachyzoites and bradyzoites in tissue cysts). The aim of this work was to evaluate monoclonal antibodies (MAb) (anti NcSAG1, anti NcSAG4 and anti TgCC2) and/or polyclonal antibodies (PAb) (anti NcSAG4 and anti TgBAG1) to label specific immunodominant antigens in different parasitic stages of N. caninum (oocyst, bradyzoite and tachyzoite), T. gondii (oocyst, cyst and tachyzoite), H. heydorni (oocyst), S. cruzi (cyst and bradyzoite) and S. falcatula (sporocyst). It was observed that the MAb directed against NcSAG1 reacted exclusively with N. caninum tachyzoites. In contrast, the MAb directed against NcSAG4 did not react with any of the parasites tested at any stage. The MAb directed against NcSAG4 reacted with both N. caninum and T. gondii tachyzoites, T. gondii tissue cysts and S. cruzi tissue cysts and bradyzoites. As expected, the MAb directed against the T. gondii tissue cyst wall antigen TgCC2 reacted with T. gondii tissue cysts, N. caninum bradyzoites, but also with T. gondii and H. heydorni oocysts and S. falcatula sporocysts. Finally, the PAb directed against the T. gondii bradyzoite proteinTgBAG1 reacted with T. gondii tissue cysts, N. caninum bradyzoites, and also with S. cruzi tissue cysts and bradyzoites. These data reveal a wide range of cross-reactions between different species of protozoa and between different developmental stages, which should be taken into account in the design and evaluation of diagnostic tests, as well as in the assessment of vaccination and challenge studies.

BACKGROUND: Bovine besnoitiosis (elephant skin disease) caused by Besnoitia besnoiti is a costly endemic disease in the Middle East, Asia, and tropical and subtropical Africa and is also emerging as a significant problem in Europe. This study is aimed at determining the prevalence of B. besnoiti in blood and skin biopsies of cattle as well as evaluating the risk factors associated with the infection among cattle in Mosul, Iraq.
RESULTS: To achieve this aim, four hundred and sixty apparently healthy cattle of different breeds, ages, and sexes were sampled from seven different locations in Mosul, Iraq. Blood and skin biopsies were carefully collected from each cattle, and these samples were subjected to molecular analysis. The detection of B. besnoiti was molecularly confirmed by the presence of 231 bp of ITS-1 in the rDNA gene of the protozoan. Besnoitia besnoiti DNA was present in 74 (16.09%; 95% CI = 13.01-19.72) and 49 (10.65%; 95% CI = 8.15-13.80) of the blood and skin biopsies, respectively, that were analyzed. Age, breed, and sex were significantly (p < 0.05) associated with the occurrence of B. besnoiti among cattle in the study area.
CONCLUSIONS: Findings from this study will serve as baseline data in the epidemiology, prevention, and control of the protozoan among cattle in Iraq.

In this study the efficacy of an intramuscular formulation of toltrazuril combined with gleptoferron for the control of porcine cystoisosporosis caused by Cystoisospora suis was investigated. The study was carried out on three Belgian farms with a confirmed history of C. suis infections. As none of the farms implemented a standardized toltrazuril treatment regimen for their piglets, the presence of resistant C. suis strains seems improbable. In total 90 litters, representing 1249 piglets, were included in the study and randomly allocated to either the treatment or control group. Piglets in the treatment group received a single intramuscular injection, containing 45 mg toltrazuril and 200 mg gleptoferron, between 1 and 3 days of age. Piglets in the control group received a single injection with only 200 mg gleptoferron. The effect of treatment on oocyst excretion, expressed in oocysts per gram of feces (OPG), average daily weight gain (ADG) and mortality was determined both pre- and post-weaning. A significant decrease in OPG as well as a decrease in the number of litters (pre-weaning) and pens (post-weaning) that tested positive for cystoisosporosis, was observed in the treated animals compared to the controls. Furthermore, treatment resulted in an increased ADG during the period from day 1 to day 21 (p-value: 0.03881). There was no significant difference in mortality observed between the treatment group to the control group (p-value: 0.2167). To our knowledge, this is the first report on the effect of toltrazuril on oocyst excretion after weaning. This finding highlights the potential long-term benefits of the treatment beyond the initial administration.

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.

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.

BACKGROUND: In September 2014, there was sudden upsurge in the number of Eurasian red squirrels (Sciurus vulgaris) found dead in the Netherlands. High infection levels with the parasite Toxoplasma gondii were demonstrated, but it was unclear what had caused this increase in cases of fatal toxoplasmosis. In the present study, we aimed to gain more knowledge on the pathology and prevalence of T. gondii infections in Eurasian red squirrels in the Netherlands, on the T. gondii genotypes present, and on the determinants of the spatiotemporal variability in these T. gondii infections. The presence of the closely related parasite Hammondia hammondi was also determined.
METHODS: Eurasian red squirrels that were found dead in the wild or that had died in wildlife rescue centres in the Netherlands over a period of seven years (2014-2020) were examined. Quantitative real-time polymerase chain reaction was conducted to analyse tissue samples for the presence of T. gondii and H. hammondi DNA. Toxoplasma gondii-positive samples were subjected to microsatellite typing and cluster analysis. A mixed logistic regression was used to identify climatic and other environmental predictors of T. gondii infection in the squirrels.
RESULTS: A total of 178 squirrels were examined (49/178 T. gondii positive, 5/178 H. hammondi positive). Inflammation of multiple organs was the cause of death in 29 squirrels, of which 24 were also T. gondii polymerase chain reaction positive. Toxoplasma gondii infection was positively associated with pneumonia and hepatitis. Microsatellite typing revealed only T. gondii type II alleles. Toxoplasma gondii infection rates showed a positive correlation with the number of days of heavy rainfall in the previous 12 months. Conversely, they showed a negative association with the number of hot days within the 2-week period preceding the sampling date, as well as with the percentage of deciduous forest cover at the sampling site.
CONCLUSIONS: Toxoplasma gondii infection in the squirrels appeared to pose a significant risk of acute mortality. The T. gondii genotype detected in this study is commonly found across Europe. The reasons for the unusually high infection rates and severe symptoms of these squirrels from the Netherlands remain unclear. The prevalence of T. gondii in the deceased squirrels was linked to specific environmental factors. However, whether the increase in the number of dead squirrels indicated a higher environmental contamination with T. gondii oocysts has yet to be established.

Rectal contents of 56 adult bobcats (Lynx rufus) in 2014 and 2017 from remote areas of Mississippi were examined microscopically for parasite stages after the sugar flotation method. Among the helminths, eggs/larvae found were: Paragonimus sp. in 12, Toxocara cati-like in 16, trichurid-capillarid-like in 3, hookworms in 27, and lungworms in 28. Among the protozoa, oocysts/cysts found were: Cystoisospora felis-like in 2, Cystoisospora rivolta-like in 4, Cryptosporidium sp. in 1, and Giardia sp. in 1. Additionally, numerous Sarcocystis sporocysts were detected in the feces of 12 bobcats; sporocysts were described morphologically. The status of C. felis derived from the bobcat and other wild felids is reviewed and compared with C. felis from the domestic cat. It is the first record of C. rivolta from the bobcat. The presence of eggs of Paragonimus sp. and T. cati in feces of 21.4% and 28.5%, respectively, suggests a role for the bobcat in the dissemination of these zoonotic helminths in the environment in the wild. Taxonomy of coccidia of wild Felidae is discussed and Isospora lyncisLevine and Ivens, 1981 from the Lynx is now regarded as a species inquirenda.

To investigate the prevalence and molecular characteristics of Cystoisospora sp. in blue fox (Alopex lagopus), Sheather\'s sugar floatation method was conducted to detect coccidia in 423 fresh fecal samples randomly collected from blue fox farms from three cities in China. The overall prevalence of coccidia was 1.4% (6/423), and three Cystoisospora sp. (Cystoisospora fennechi, Cystoisospora sp. I and Cystoisospora vulpina) were identified by their morphological characteristics. The 18S ribosomal RNA (rRNA) and cytochrome c oxidase subunit I (COI) locus sequences were sequenced for molecular biological identification, homology comparison, and phylogenetic analysis of Cystoisospora sp. by single-oocyst selection technology and multi-locus-nested PCR amplification. At the 18S rRNA and COI loci, C. vulpina had 99.48% and 99.59% homology, respectively, with Cystoisospora canis and Cystoisospora ohioensis from canines. Phylogenetic analysis indicated that C. vulpina was clustered in a clade with Cystoisospora sp. from Canidae, which the relatives are consistent with the hosts. To our knowledge, this is the first report on molecular identification and evolutionary analysis of C. vulpina at two different loci.

Bovine besnoitiosis is a re-emerging cattle disease caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti. Neutrophil extracellular trap (NET) formation represents an efficient innate immune mechanism of polymorphonuclear neutrophils (PMN) against apicomplexan parasites, including B. besnoiti. PMN purinergic signaling was proposed as a critical factor for NET formation. One important purinergic ligand is ATP, which is recognized as a danger signal and released into the extracellular space acting as an autocrine/paracrine signaling molecule. ATP-driven effects on PMN via the nucleotide P2 receptor family include chemotaxis, reactive oxygen species (ROS) production, and NET formation. So far, data on both PMN ATP concentrations and the role of ATP as a key modulator of purinergic signaling in B. besnoiti tachyzoite-triggered bovine NETosis is scarce. Current data showed that B. besnoiti tachyzoite exposure to bovine PMN neither changed total PMN ATP nor extracellular ATP quantities even though it significantly triggered NET formation. Moreover, B. besnoiti tachyzoite-exposed PMN revealed enhanced oxygen consumption rates (OCR) as quantified by the Seahorse metabolic analyzer. Exogenous supplementation of ATP or non-hydrolizable ATP (ATPγS) led to increased extracellular acidification rates (ECAR) but failed to alter tachyzoite-induced oxidative responses (OCR) in exposed PMN. In addition, exogenous supplementation of ATPγS, but not of ATP, boosted B. besnoiti tachyzoite-induced anchored NET formation. Referring to purinergic signaling, B. besnoiti tachyzoite-triggered anchored NET formation revealed P2X1 purinergic as receptor-dependent since it was blocked by the P2X1 inhibitor NF449 at an IC50 of 1.27 µM. In contrast, antagonists of P2Y2, P2Y6, P2X4, and P2X7 purinergic receptors all failed to affect parasite-driven NETosis. As an interesting finding, we additionally observed that B. besnoiti tachyzoite exposure induced PMN clustering in a P2X1-dependent manner. Thus, we identified P2X1 purinergic receptor as a pivotal molecule for both B. besnoiti tachyzoite-induced PMN clustering and anchored NET formation.

Besnoitia besnoiti-infected bulls may develop severe systemic clinical signs and orchitis that may ultimately cause sterility during the acute infection. Macrophages might play a relevant role in pathogenesis of the disease and the immune response raised against B. besnoiti infection. This study aimed to dissect the early interaction between B. besnoiti tachyzoites and primary bovine monocyte-derived macrophages in vitro. First, the B. besnoiti tachyzoite lytic cycle was characterized. Next, dual transcriptomic profiling of B. besnoiti tachyzoites and macrophages was conducted at early infection (4 and 8 h p.i.) by high-throughput RNA sequencing. Macrophages inoculated with heat-killed tachyzoites (MO-hkBb) and non-infected macrophages (MO) were used as controls. Besnoitia besnoiti was able to invade and proliferate in macrophages. Upon infection, macrophage activation was demonstrated by morphological and transcriptomic changes. Infected macrophages were smaller, round and lacked filopodial structures, which might be associated with a migratory phenotype demonstrated in other apicomplexan parasites. The number of differentially expressed genes (DEGs) increased substantially during infection. In B. besnoiti-infected macrophages (MO-Bb), apoptosis and mitogen-activated protein kinase (MAPK) pathways were regulated at 4 h p.i., and apoptosis was confirmed by TUNEL assay. The Herpes simplex virus 1 infection pathway was the only significantly enriched pathway in MO-Bb at 8 h p.i. Relevant DEGs of the Herpes simplex virus 1 infection (IFNα) and the apoptosis pathways (CHOP-2) were also significantly regulated in the testicular parenchyma of naturally infected bulls. Furthermore, the parasite transcriptomic analysis revealed DEGs mainly related to host cell invasion and metabolism. These results provide a deep overview of the earliest macrophage modulation by B. besnoiti that may favour parasite survival and proliferation in a specialized phagocytic immune cell. Putative parasite effectors were also identified.