BackgroundUnlike mouse models of congenital toxoplasmosis, pregnant sheep models provide the opportunity to evaluate treatment strategies that more closely resemble clinical practice in pregnant women, including chemotherapeutic interventions initiated after specific IgM seroconversion. BKI-1748, which targets Toxoplasma gondii CDPK1 and MAPKL-1 protein kinases, has demonstrated an excellent safety profile and efficacy when administered repeatedly to pregnant sheep, starting at 2 and 7 days after challenge.MethodsIn this study, treatment was initiated at day 14 post-infection (p.i.), following T. gondii IgM seroconversion. Twenty-three sheep were orally inoculated with 10 TgShSp1 oocysts at 90 days of gestation, while three sheep remained uninfected. On day 14 p.i., infected sheep carrying live fetuses (n = 10) received 10 doses of BKI-1748 orally at 15 mg/kg every 2 days, whereas 10 infected sheep were left untreated.ResultsAll infected sheep, both treated and untreated, seroconverted to serum IgG by day 21 p.i., with treated sheep showing a marked reduction in IgG levels from day 28 p.i. onward. Administration of the compound significantly enhanced lamb viability in infected sheep, resulting in 91% viable lambs in treated animals compared to 52% in untreated sheep. Whereas all lambs born to untreated sheep were congenitally infected, only 17% of lambs in the treated group were infected. Nevertheless, congenitally infected lambs in the treated group had lower birth weights than T. gondii-free lambs.ConclusionThis study highlights the potential utility of BKI-1748 for prenatal treatment of human congenital toxoplasmosis, in which IgM seroconversion prompts the need for intervention.

Abstract Toxoplasma gondii is an apicomplexan parasite causing reproductive failure in small ruminants. In this study, we evaluated the capacity of an inactivated vaccine to prevent reproductive failure caused by congenital toxoplasmosis in sheep. The vaccine is based on an antigen extract obtained from a low passage T. gondii Type III isolate (TgPigSp1) preserving the ability to spontaneously produce cysts in vitro and following a procedure involving parasite inactivation via hyperosmotic shock and membrane proteins solubilisation. The vaccine prototype, consisting of 40 µg of parasite extract adjuvanted with QuilA®, was evaluated in two different trials using a pregnant ovine model of T. gondii infection based on orally challenging sheep at 90 days of gestation with 10 sporulated oocysts of the heterologous Type II isolate TgShSp1. Two subcutaneous immunizations at days 55 and 76 of pregnancy caused mild and transient local reactions and had no discernible impact on gestation. Vaccination triggered both specific cellular and humoral immune responses. The proportion of viable gestations resulted in 100% (vaccine trial 1) and 78% (vaccine trial 2) in vaccinated/challenged ewes versus 50% in unvaccinated/challenged sheep. This increase in viable gestations was associated with a significant increase of lambs born viable for vaccine trial 1 (62.5%) and a decrease of early foetal losses (i.e. abortion) for vaccine trial 2 (70%), associated with control of T. gondii multiplication in the cotyledons. This inactivated vaccine could be a suitable and practical tool to mitigate economic losses in sheep caused by T. gondii outbreaks.

Drug development for congenital toxoplasmosis is challenging since first-line therapy has a high rate of adverse effects and exhibits suboptimal efficacy. Bumped kinase inhibitors (BKIs), targeting protein kinases with small gatekeeper residues, have been found to be effective against Toxoplasma gondii. The efficacy of BKI-1748 administered later than 2 days post-infection (p.i.), a scenario that may better reflect its real-world use as a therapeutic candidate, has not been investigated in T. gondii-infected pregnant sheep. For this purpose, 19 pregnant sheep were assigned to three experimental groups. Group 1 (G1, n = 8) and group 2 (G2, n = 8) were dosed orally with 10 TgShSp1 sporulated oocysts at 90 days of gestation (dg). Animals from group 3 (G3, n = 3) were simultaneously mock dosed with phosphate-buffered solution (PBS). In G1, BKI-1748 was administered orally from day 7 p.i. (fever and increased serum IFN gamma levels) onward, maintaining drug exposure for 20 days (10 doses at 15 mg/kg every 2 days). Treated animals (G1) exhibited significantly lower rectal temperatures (on days 8 and 9 p.i.), serum IFN gamma levels (on day 10 p.i.), and specific IgG levels when compared with non-treated animals (G2). At delivery, significantly higher percentages of healthy lambs were found in infected/treated sheep in G1 (73.3%) and in uninfected sheep in G3 (80%) compared with infected/untreated sheep in G2 (31.3%). Concerning congenital transmission, parasite DNA was neither detected in placenta nor target tissues (brain and lungs) from the fetuses/lambs in G1(infected/treated) and G3 (uninfected). By contrast, parasite DNA was detected in all placentas and lambs from G2 (infected/untreated), except for one sheep that aborted on day 13 p.i.

Toxoplasma gondii, a zoonotic protozoan parasite, represents a major threat to public health and food safety worldwide, particularly through the consumption of undercooked or raw pork containing tissue cysts. Controlling T. gondii infection in pigs is therefore critical to reduce zoonotic transmission. This study evaluated a novel inactivated vaccine formulated with antigens from the Type III TgPigSp1 isolate, comprising both tachyzoite and bradyzoite stages, and adjuvanted with Quil-A. Safety, immunogenicity, and efficacy were assessed first in a murine model as proof of concept and subsequently in piglets challenged with 1000 oocysts of the heterologous Type II-PRU TgShSp1 isolate. The vaccine showed no systemic adverse effects in either model, and an absence of local reactions was observed in piglets. In mice, vaccination elicited strong parasite-specific IgG responses and significantly reduced the parasite burden in brain and muscle tissues by more than 80

Type II reference isolates of Toxoplasma gondii are widely used in animal toxoplasmosis models, but studies with Type III isolates remain scarce. In addition, these methods often rely on laboratory-adapted parasite stages that may not reflect natural infection. This study presents a new murine model based on an oral infection with oocysts from a recently obtained Type III isolate, TgShSp24, which exhibited remarkable morbidity and a distinct tissue distribution during chronic infection, differing from the recently obtained Type II isolate TgShSp1. This novel model aims to better mimic natural infection and provides a valuable tool for testing drugs and vaccines.

Congenital toxoplasmosis in humans and in other mammalian species, such as small ruminants, is a well-known cause of abortion and fetal malformations. The calcium-dependent protein kinase 1 (CDPK1) inhibitor BKI-1748 has shown a promising safety profile for its use in humans and a good efficacy against Toxoplasma gondii infection in vitro and in mouse models. Ten doses of BKI-1748 given every other day orally in sheep at 15 mg/kg did not show systemic or pregnancy-related toxicity. In sheep experimentally infected at 90 days of pregnancy with 1000 TgShSp1 oocysts, the BKI-1748 treatment administered from 48 hours after infection led to complete protection against abortion and congenital infection. In addition, compared to infected/untreated sheep, treated sheep showed a drastically lower rectal temperature increase and none showed IgG seroconversion throughout the study. In conclusion, BKI-1748 treatment in pregnant sheep starting at 48 hours after infection was fully effective against congenital toxoplasmosis. Toxoplasmosis is a zoonotic disease and the first-line treatments for congenital Toxoplasma gondii infections in humans lack sufficient safety and efficacy. BKI-1748 is a lead compound and here we demonstrate its favorable safety and efficacy in sheep.

Endothelial injury, inflammatory infiltrate and fibrosis are the predominant lesions in the testis of bulls with besnoitiosis that may result in sterility. Moreover, fibroblasts, which are key players in fibrosis, are parasite target cells in a Besnoitia besnoiti chronic infection. This study aimed to decipher the molecular basis that underlies a drift toward fibrosis during the disease progression. Transcriptomic analysis was developed at two times post-infection (p.i.), representative of invasion (12 h p.i.) and intracellular proliferation (32 h p.i.), in primary bovine aorta fibroblasts infected with B. besnoiti tachyzoites. Once the enriched host pathways were identified, we studied the expression of selected differentially expressed genes (DEGs) in the scrotal skin of sterile infected bulls. Functional enrichment analyses of DEGs revealed shared hallmarks of cancer and early fibrosis. Biomarkers of inflammation, angiogenesis, cancer, and MAPK signaling stood out at 12 h p.i. At 32 h p.i., again MAPK and cancer pathways were enriched together with the PI3K–AKT pathway related to cell proliferation. Some DEGs were also regulated in the skin samples of naturally infected bulls (PLAUR, TGFβ1, FOSB). We have identified potential biomarkers and host pathways regulated during fibrosis that may hold prognostic significance and could emerge as potential therapeutic targets.

Ovine coccidiosis is a widespread intestinal parasitic disease caused by Eimeria spp. Lambs are infected by the ingestion of sporulated oocysts, experiencing diarrhea and low growth rates. Control should be based on measures to reduce infection pressure and stress on the animals as well as on appropriate diagnosis and strategic treatment. To obtain information on how control measures are implemented in the ovine sector in Spain, a questionnaire-based survey was completed in 2022 by 154 veterinarians and 173 farmers working in this sector. Coccidiosis was highlighted as a relevant disease by 34% of the respondents. The period of greatest risk seemed to differ between production systems, being mainly early after weaning (7-15 days after weaning) in meat flocks and feedlots and later (1-2 months after weaning) in dairy flocks. The absence of cleaning and disinfection measures was identified as a risk factor by 51% of the veterinarians, with 22% mentioning overcrowding of animals and 22% indicating that coccidiosis has more incidence in flocks with large number of animals. The use of laboratory diagnosis methods (fecal oocyst count) was unusual in 70 and 84% of the veterinarians and farmers, respectively. Regarding control, dairy flocks usually housed a larger number of animals under intensive conditions, and they implemented more frequently control measures for coccidiosis than meat flocks. Anticoccidial drugs were used in 79% of the flocks, and in 74-82% of them, they were applied based on clinical criteria. Comparing protocols for anticoccidial treatment among different production systems, in meat flocks, anticoccidial drugs were applied more frequently when clinical signs were observed, and coccidiostats were used for less than 28 days compared to dairy flocks. These results highlight the need for improvement in the use of anticoccidial treatments adjusted to the new regulatory framework in the EU, which in turn will rationalize the use of antimicrobial compounds and may help to mitigate the impact of coccidiosis in flocks.

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 highthroughput 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 (IFNa) 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.& COPY; 2023 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Livestock animals, such as swine, are an important source of Toxoplasma gondii in the human population. Currently, there is limited knowledge regarding the potential influence that the T. gondii genotype might exert on establishing infection in swine. Herein, we investigated the role of 2 T. gondii isolates, type II and III, representative of the genotypes circulating in Europe, in the immune responses and infection dynamics in piglets. Recently obtained oocysts (103) from the T. gondii field isolates TgShSp1 (type II, ToxoDB genotype #3) and TgShSp24 (type III, #2) were used for oral infection. Thirteen 50-day-old female piglets of the Landrace-Large White crossbreed were randomly allocated into three different groups: Group 1 (G1, n=5), inoculated with TgShSp1; Group 2 (G2, n=5), inoculated with TgShSp24; and Group 3 (G3, n=3), a non-infected control group. Clinical signs were monitored daily until 42 days post-infection (dpi) when piglets were euthanized. Blood samples were collected weekly to test the cellular immune response in parasite-stimulated peripheral blood and specific IgG, IgG1 and IgG2, responses in sera. Parasite distribution and burden were evaluated in target tissues using a mouse bioassay and quantitative RT−PCR (qPCR). Apathy and a moderate decrease in feed consumption were observed in G1 and G2 piglets between 5 and 8 dpi, coinciding with fever (>40°C). G2 piglets had higher temperatures for a longer duration. Using mouse bioassay and qPCR, the detection frequency was higher in G2 vs. G1, and the highest parasite burdens in target tissues were also found in G2. Seroconversion was detected at 14 dpi in both infected groups, but higher antibody levels were observed in G2 piglets. Cytokine analyses revealed the production of IL-8, IL-1β and IFN-ɤ from 7 dpi in both infected groups. Moreover, IL-12 was produced from 7 dpi in G1 and from 14 dpi in G2. Levels of IL-8 were higher in G2, but IL-1β, IL-12 and IFN-ɤ were higher in G1 at 14 dpi. This cytokine profile reveals a predominant proinflammatory response that could be involved in limiting T. gondii infection in piglets, although it is more efficient against TgShSp1 type II-driven infection.

Background Acute and chronic besnoitiosis in extensive natural-service herds can have relevant effects in the health of bulls and negative consequences in their productive performance. Recent progress has been made in order to elucidate the pathogenesis of this disease. In this context, the study of biomarkers of inflammation in serum would contribute to gaining knowledge about the physiopathology of bovine besnoitiosis. Serological biomarkers could help in early diagnosis and prognosis, as seropositive bulls may have mild or severe testicular lesions. Methods Herein , we have investigated the diagnostic and/or prognostic value of a panel of serum (serological) biomarkers related to inflammation, including total protein, globulin and albumin, haptoglobin (Hp), adenosine deaminase (ADA) paraoxonase-1 (PON-1) and acetylcholinesterase (AChE) in naturally and experimentally B. besnoiti -infected males classified according to different clinical phases of the disease (acute, chronic and subclinical besnoitiosis). Results Results showed a similar response pattern in these biomarkers for naturally and experimentally infected cattle, with a few relevant variations. Most significant changes occurred during the acute phase of infection, although significant changes in a few biomarkers were also observed during the chronic infection. Haptoglobin, albumin, PON-1 and ADA were identified as the biomarkers that showed changes of higher magnitude in the acute phase of the infection, whereas high total protein and globulin values were found in chronically infected cattle. We have described the changes of a panel of inflammatory biomarkers of acute and chronic bovine besnoitiosis. Conclusions In summary, several biomarkers with promising diagnostic value have been identified. The biomarkers associated with acute infection are related to previously reported molecular biomarkers in testicular parenchyma of infected bulls and could help in the diagnosis of early infections and complement results from specific immunoglobulin M (IgM) detection. Graphical abstract

The Neospora caninum Calcium-dependent protein kinase 1 (NcCDPK1) inhibitor BKI-1294 had demonstrated excellent efficacy in a pregnant mouse model of neosporosis, and was also highly efficacious in a pregnant sheep model of toxoplasmosis. In this work, we present the efficacy of BKI-1294 treatment (dosed 5 times orally every 48 h) starting 48 h after intravenous infection of sheep with 105 Nc-Spain7 tachyzoites at mid-pregnancy. In the dams, BKI-1294 plasma concentrations were above the IC50 for N. caninum for 12-15 days. In treated sheep, when they were compared to untreated ones, we observed a minor increase in rectal temperature, higher IFNγ levels after blood stimulation in vitro, and a minor increase of IgG levels against N. caninum soluble antigens through day 28 post-infection. Additionally, the anti-NcSAG1 and anti-NcSAG4 IgGs were lower in treated dams on days 21 and 42 post-infection. However, BKI-1294 did not protect against abortion (87% foetal mortality in both infected groups, treated and untreated) and did not reduce transplacental transmission, parasite load or lesions in placentomes and foetal brain. The lack of foetal protection was likely caused by short systemic exposure in the dams and suboptimal foetal exposure to this parasitostatic drug, which was unable to reduce replication of the likely established N. caninum tachyzoites in the foetus at the moment of treatment. New BKIs with a very low plasma clearance and good ability to cross the blood-brain and placental barriers need to be developed.

This is a review of the development of bumped-kinase inhibitors (BKIs) for the therapy of One Health parasitic apicomplexan diseases. Many apicomplexan infections are shared between humans and livestock, such as cryptosporidiosis and toxoplasmosis, as well as livestock only diseases such as neosporosis. We have demonstrated proof-of-concept for BKI therapy in livestock models of cryptosporidiosis (newborn calves infected with Cryptosporidium parvum), toxoplasmosis (pregnant sheep infected with Toxoplasma gondii), and neosporosis (pregnant sheep infected with Neospora caninum). We discuss the potential uses of BKIs for the treatment of diseases caused by apicomplexan parasites in animals and humans, and the improvements that need to be made to further develop BKIs.

Breeding bulls infected with Besnoitia besnoiti may develop sterility during either acute or chronic infection. The aim of this study was to investigate the molecular pathogenesis of B. besnoiti infection with prognosis value in bull sterility. Accordingly, five well-characterized groups of naturally and experimentally infected males were selected for the study based on clinical signs and lesions compatible with B. besnoiti infection, serological results and parasite detection. A broad panel of molecular markers representative of endothelial activation and fibrosis was investigated and complemented with a histopathological approach that included conventional histology and immunohistochemistry. The results indicated the predominance of an intense inflammatory infiltrate composed mainly of resident and recruited circulating macrophages and to a lesser extent of CD3+ cells in infected bulls. In addition, a few biomarkers were associated with acute, chronic or subclinical bovine besnoitiosis. The testicular parenchyma showed a higher number of differentially expressed genes in natural infections (acute and chronic infections) versus scrotal skin in experimental infections (subclinical infection). In subclinical infections, most genes were downregulated except for the CCL24 and CXCL2 genes, which were upregulated. In contrast, the acute phase was mainly characterized by the upregulation of IL-1α, IL-6 and TIMP1, whereas in the chronic phase, the upregulation of ICAM and the downregulation of MMP13, PLAT and IL-1α were the most relevant findings. Macrophages could be responsible for the highest level of gene regulation in the testicular parenchyma of severely affected and sterile bulls, and all these genes could be prognostic markers of sterility.

The quinolone decoquinate (DCQ) is widely used in veterinary practice for the treatment of bacterial and parasitic infections, most notably, coccidiosis in poultry and in ruminants. We have investigated the effects of treatment of Toxoplasma gondii in infected human foreskin fibroblasts (HFF) with DCQ. This induced distinct alterations in the parasite mitochondrion within 24 h, which persisted even after long-term (500 nM, 52 days) treatment, although there was no parasiticidal effect. Based on the low half-maximal effective concentration (IC50) of 1.1 nM and the high selectivity index of >5000, the efficacy of oral treatment of pregnant mice experimentally infected with T. gondii oocysts with DCQ at 10 mg/kg/day for 5 days was assessed. However, the treatment had detrimental effects, induced higher neonatal mortality than T. gondii infection alone, and did not prevent vertical transmission. Thus, three quinoline-O-carbamate derivatives of DCQ, anticipated to have better physicochemical properties than DCQ, were assessed in vitro. One such compound, RMB060, displayed an exceedingly low IC50 of 0.07 nM, when applied concomitantly with the infection of host cells and had no impact on HFF viability at 10 µM. As was the case for DCQ, RMB060 treatment resulted in the alteration of the mitochondrial matrix and loss of cristae, but the changes became apparent at just 6 h after the commencement of treatment. After 48 h, RMB060 induced the expression of the bradyzoite antigen BAG1, but TEM did not reveal any other features reminiscent of bradyzoites. The exposure of infected cultures to 300 nM RMB060 for 52 days did not result in the complete killing of all tachyzoites, although mitochondria remained ultrastructurally damaged and there was a slower proliferation rate. The treatment of mice infected with T. gondii oocysts with RMB060 did reduce parasite burden in non-pregnant mice and dams, but vertical transmission to pups could not be prevented.