In vitro regulation of gene expression of pregnancy-associated proteins and cytokines in bovine endometrial epithelial cells and bovine trophoblastic cells by infection with Neospora caninum.

Abortion caused by the parasite Neospora caninum is an important threat to the livestock industry worldwide. Trophoblasts and caruncular cells play major roles in initiating innate immune responses and controlling parasite infection at the fetal-maternal interface. In the present study, bovine uterine epithelial cells (BUECs) and bovine trophoblastic (BT) cells treated with bovine interferon-gamma (IFN-γ), IFN-alpha (IFN-α) and IFN-tau (IFN-τ) followed by infection with N. caninum were examined by measuring the mRNA expression levels of numerous pregnancy-associated proteins and observing parasite growth to elucidate the host-parasite interaction at the uteroplacental region. N. caninum infection increased the expression of prolactin-related protein 1 (PRP1), pregnancy-associated glycoprotein 1 (PAG1), and cytokines (TNF-α, IL-8 and IL-10) in BUECs and of IL-8 in BT cells. Bovine IFN-γ inhibited IL-8 and TNF-α expression in BUECs and IL-8 in BT cells. In contrast, the expression of the interferon-stimulated gene OAS1 was significantly increased by treatment of the infected BT cells with IFN-γ. However, treatment with bovine IFNs did not inhibit N. caninum growth in either cell line. In conclusion, our results suggest that bovine IFN-γ plays a crucial role in control of pathogenesis in uterus and induction of inflammatory response in the placental region following N. caninum infection, rather than growth inhibition of the parasites.

Effects of dietary water-soluble extract of rosemary supplementation on growth performance and intestinal health of broilers infected with Eimeria tenella.

This study was conducted to explore the effect of dietary supplementation of water-soluble extract of rosemary (WER) on growth performance and intestinal health of broilers infected with Eimeria tenella (E. tenella), and evaluate the anticoccidial activity of WER. 360 1-d-old Chinese indigenous male yellow-feathered broiler chickens were randomly allocated to six groups: blank control (BC) group and infected control (IC) group received a basal diet; positive control (PC) group, received a basal diet supplemented with 200 mg/kg diclazuril; WER100, WER200, and WER300 groups received a basal diet containing 100, 200, and 300 mg/kg WER, respectively. On day 21, all birds in the infected groups (IC, PC, WER100, WER200, and WER300) were orally gavaged with 1 mL phosphate-buffered saline (PBS) of 8 × 104 sporulated oocysts of E. tenella, and birds in the BC group were administrated an aliquot of PBS dilution. The results showed that dietary supplementation of 200 mg/kg WER increased the average daily gain of broilers compared to the IC group from days 22 to 29 (P < 0.001). The anticoccidial index values of 100, 200, and 300 mg/kg WER were 137.49, 157.41, and 144.22, respectively, which indicated that WER exhibited moderate anticoccidial activity. Compared to the IC group, the groups supplemented with WER (100, 200, and 300 mg/kg) significantly lowered fecal oocyst output (P < 0.001) and cecal coccidia oocysts, alleviated intestinal damage and maintained the integrity of intestinal epithelium. Dietary supplementation with WER significantly improved antioxidant capacity, elevated the levels of secretory immunoglobulin A, and diminished inflammation within the cecum, particularly at a dosage of 200 mg/kg. The results of this study indicated that dietary supplementation with 200 mg/kg WER could improve broiler growth performance and alleviate intestinal damage caused by coccidiosis.

Avian coccidiosis, a prevalent parasitic disease caused by Eimeria protozoa, leads to significant economic losses in the global poultry industry. Currently, the control of coccidiosis in chickens primarily relies on chemical and ionophore anticoccidials. However, the long-term use of these compounds has resulted in the development of drug-resistant strains, presenting a critical challenge. Additionally, the toxic and side effects of ionophore anticoccidials have become increasingly apparent. Thus, there is an urgent need to find economical and environmentally friendly measures to control coccidiosis in chickens. In this study, we established a model of Eimeria tenella infection in broilers to explore whether the water-soluble extract of rosemary (WER) could serve as an alternative method for controlling avian coccidiosis. Our results showed that dietary supplementation with WER (100, 200, and 300 mg/kg) had a beneficial anticoccidial effect, alleviating intestinal damage caused by coccidiosis by enhancing the intestinal antioxidant defense and activating the immune function of the infected broilers. Specifically, dietary supplementation with 200 mg/kg WER emerged as a promising strategy for controlling avian coccidiosis in the poultry industry.

The effect of an injectable toltrazuril - gleptoferron (Forceris®) on Cystoisospora suis oocyst excretion and growth of neonatal piglets pre- and post-weaning.

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.

The impact of different levels of functional oil supplementation in combination with salinomycin on growth performance and intestinal microbiota of broilers undergoing Eimeria challenge: An analysis of dynamics.

The effect of salinomycin sodium alone and in combination with functional oils on performance and microbiota of broiler infected Eimeria were evaluated. 512 broilers were randomly assigned to 4 treatments (8 replicates, 16 birds/pen): a Control group (any additives); Ionophore group: salinomycin supplementation at 66 ppm (SS66); Ionophore +0.075% Functional oil (FO) group (SS66 + FO supplementation at 750 ppm); and Ionophore +0.10% FO group (SS66 + FO supplementation at 1000 ppm). At 14 days of age, birds were gavaged with 1 mL of a saline solution containing sporulated oocysts of E. tenella, E. acervulina and E. maxima. Performance indices were measured weekly. At 28 days, intestinal content was collected for microbiota analysis. Broilers of Control group presented the worst performance indices. Broilers of Ionophore + FO (0.075% and 0.10%) groups exhibited a higher BW at 28 days of age. The supplementation of Ionophore +0.075% FO resulted in a higher relative proportion of Firmicutes and a lower proportion of Actinobacteria in the ileum-jejunum. Lactobacillaceae was the dominant family in the jejunal, and ileal microbiotas of broilers fed diets supplemented with Ionophore, Ionophore +0.075% FO and Ionophore +0.10% FO. The supplementation of ionophore yielded higher numbers of Lactobacillaceae, Enterobactereaceae and Cloritridiaceae in the cecal. Ionophore associated with FO controlled the Lactobacillaceae, Enterobactereaceae and Cloritridiaceae families present in the cecum. Therefore, the combination of salinomycin with functional oil showed synergistic effect on performance and modulation of intestinal microbiota of broilers challenged with Eimeria.

Examining the molecular epidemiology of Giardia and Eimeria species in Japan: a comprehensive review.

Globally, animals and humans suffer from diarrheal illnesses due to protozoan parasites such as Giardia and Eimeria species. The molecular epidemiology of these parasites in Japan is summarized in this review. In humans, researchers found only one main species of Giardia, which is most referred to as G. lamblia, but it's also known by different names like G. duodenalis or G. intestinalis. However, within this species, six assemblages (A, B, C, D, E, and F) were found in animals, and assemblage B was frequently recorded in human and monkey populations, whereas assemblages A and E were predominant in calves. Assemblage A was found in sika deer and assemblages A, C, D, and F were predominant in dogs, cats, and ferret. Eimeria bovis, E. zuernii, and other species found in animals made up the group of species known as Eimeria spp., with E. bovis and E. zuernii being the most common in cattle. Our review highlighted a notable lack of data investigations regarding these two pathogens in water and environmental sources. Giardia cysts were found in the few studies that have been done on water sources, suggesting that water may play a significant role in the transmission of Giardia species. Our review suggests that further research is necessary to fully comprehend the molecular diversity and dynamics of transmission of Giardia spp. and Eimeria spp. in humans, animals, and environmental sources in Japan.

Construction of luciferase-expressing Neospora caninum and drug screening.

BACKGROUND: Neospora caninum is an apicomplexan parasite that is particularly responsible for abortions in cattle and neuromuscular disease in dogs. Due to the limited effectiveness of currently available drugs, there is an urgent need for new therapeutic approaches to control neosporosis. Luciferase-based assays are potentially powerful tools in the search for antiprotozoal compounds, permitting the development of faster and more automated assays. The aim of this study was to construct a luciferase-expressing N. caninum and evaluate anti-N. caninum drugs. METHODS: Luciferase-expressing N. caninum (Nc1-Luc) was constructed using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9). After testing the luciferase expression and phenotype of the Nc1-Luc strains, the drug sensitivity of Nc1-Luc strains was determined by treating them with known positive or negative drugs and calculating the half-maximal inhibitory concentration (IC50). The selective pan-rapidly accelerated fibrosarcoma (pan-RAF) inhibitor TAK-632 was then evaluated for anti-N. caninum effects using Nc1-Luc by luciferase activity reduction assay and other in vitro and in vivo studies. RESULTS: The phenotypes and drug sensitivity of Nc1-Luc strains were consistent with those of the parental strains Nc1, and Nc1-Luc strains can be used to determine the IC50 for anti-N. caninum drugs. Using the Nc1-Luc strains, TAK-632 showed promising activity against N. caninum, with an IC50 of 0.6131 µM and a selectivity index (SI) of 62.53. In vitro studies demonstrated that TAK-632 inhibited the invasion, proliferation, and division of N. caninum tachyzoites. In vivo studies showed that TAK-632 attenuated the virulence of N. caninum in mice and significantly reduced the parasite burden in the brain. CONCLUSIONS: In conclusion, a luciferase-expressing N. caninum strain was successfully constructed, which provides an effective tool for drug screening and related research on N. caninum. In addition, TAK-632 was found to inhibit the growth of N. caninum, which could be considered as a candidate lead compound for new therapeutics for neosporosis.

Different methionine to cysteine supplementation ratios altered bone quality of broilers with or without Eimeria challenge assessed by dual energy X-ray absorptiometry and microtomography.

Despite the acknowledged significance of nutrition in bone development, effects of methionine (Met) and cysteine (Cys) on bone quality remain under-researched, particularly during Eimeria challenge. We investigated the effects of different supplemental Met to Cys ratios (MCR) on bone quality of broilers under Eimeria challenge. A total of 720 fourteen-day old Cobb500 broilers were allocated into a 5 × 2 factorial arrangement. Five diets with Met and Cys supplemented at MCR of 100:0, 75:25, 50:50, 25:75, and 0:100 were fed to the birds with or without Eimeria challenge. Body composition was measured by dual energy x-ray absorptiometry, and the femur bone characteristics were assessed by microtomography. Data were analyzed by two-way ANOVA and orthogonal polynomial contrast. The results reaffirmed the detrimental effects of Eimeria challenge on bone quality. On 9 d post inoculation (DPI), significant interaction effects were found for whole body bone mineral content (BMC), lean tissue weight, and body weight (P < 0.05); in the nonchallenged group (NCG), these parameters linearly decreased as MCR decreased (P < 0.05). In the challenged group (CG), body weight and lean tissue weight were unaffected by MCR, and BMC linearly increased as MCR decreased (P < 0.05). For the cortical bone of femoral metaphysis on 6 DPI, bone mineral density (BMD) linearly increased as MCR decreased (P < 0.05). Bone volume to tissue volume ratio (BV/TV) in the CG linearly increased as MCR decreased (P < 0.05). On 9 DPI, BMC and TV linearly increased as MCR decreased (P < 0.05) in the NCG. BMD and BV/TV changed quadratically as MCR decreased (P < 0.05). For the trabecular bone of femoral metaphysis on 9 DPI, BV/TV, and trabecular number linearly increased as MCR decreased (P < 0.05) in the NCG. For the femoral diaphysis, BV, TV, BMC on 6 DPI, and BMD on 9 DPI linearly increased as MCR decreased (P < 0.05). In conclusion, this study showed that both Eimeria challenge and varying supplemental MCR could influence bone quality of broilers.

Identification and characterization of the receptors of a microneme adhesive repeat domain of Eimeria maxima microneme protein 3 in chicken intestine epithelial cells.

Eimeria maxima microneme protein 3 (EmMIC3) is pivotal in the initial recognition and attachment of E. maxima sporozoites to host cells. EmMIC3 comprises 5 tandem Type I microneme adhesive repeat (MAR) domains, among which MAR2 of EmMIC3 (EmMAR2) has been identified as the primary determinant of EmMIC3-mediated tissue tropism. Nonetheless, the mechanisms through which EmMAR2 guides the parasite to its invasion site through interactions with host receptors remained largely uncharted. In this study, we employed yeast two-hybrid (YTH) screening assays and shotgun LC-MS/MS analysis to identify EmMAR2 receptors in chicken intestine epithelial cells. ATPase H+ transporting V1 subunit G1 (ATP6V1G1), receptor accessory protein 5 (REEP5), transmembrane p24 trafficking protein (TMED2), and delta 4-desaturase sphingolipid 1 (DEGS1) were characterized as the 4 receptors of EmMAR2 by both assays. By blocking the interaction of EmMAR2 with each receptor using specific antibodies, we observed varying levels of inhibition on the invasion of E. maxima sporozoites, and the combined usage of all 4 antibodies resulted in the most pronounced inhibitory effect. Additionally, the spatio-temporal expression profiles of ATP6V1G1, REEP5, TMED2, and DEGS1 were assessed. The tissue-specific expression patterns of EmMAR2 receptors throughout E. maxima infection suggested that ATP6V1G1 and DEGS1 might play a role in early-stage invasion, whereas TMED2 could be involved in middle and late-stage invasion and REEP5 and DEGS1 may participate primarily in late-stage invasion. Consequently, E. maxima may employ a multitude of ligand-receptor interactions to drive invasion during different stages of infection. This study marks the first report of EmMAR2 receptors at the interface between E. maxima and the host, providing insights into the invasion mechanisms of E. maxima and the pathogenesis of coccidiosis.

Integrated application of transcriptomics and metabolomics provides insight into the mechanism of Eimeria tenella resistance to maduramycin.

Avian coccidiosis, caused by Eimeria parasites, continues to devastate the poultry industry and results in significant economic losses. Ionophore coccidiostats, such as maduramycin and monensin, are widely used for prophylaxis of coccidiosis in poultry. Nevertheless, their efficacy has been challenged by widespread drug resistance. However, the underlying mechanisms have not been revealed. Understanding the targets and resistance mechanisms to anticoccidials is critical to combat this major parasitic disease. In the present study, maduramycin-resistant (MRR) and drug-sensitive (DS) sporozoites of Eimeria tenella were purified for transcriptomic and metabolomic analysis. The transcriptome analysis revealed 5016 differentially expressed genes (DEGs) in MRR compared to DS, and KEGG pathway enrichment analysis indicated that DEGs were involved in spliceosome, carbon metabolism, glycolysis, and biosynthesis of amino acids. In the untargeted metabolomics assay, 297 differentially expressed metabolites (DEMs) were identified in MRR compared to DS, and KEGG pathway enrichment analysis indicated that these DEMs were involved in 10 pathways, including fructose and mannose metabolism, cysteine and methionine metabolism, arginine and proline metabolism, and glutathione metabolism. Targeted metabolomic analysis revealed 14 DEMs in MRR compared to DS, and KEGG pathway analysis indicated that these DEMs were involved in 20 pathways, including fructose and mannose metabolism, glycolysis/gluconeogenesis, and carbon metabolism. Compared to DS, energy homeostasis and amino acid metabolism were differentially regulated in MRR. Our results provide gene and metabolite expression landscapes of E. tenella following maduramycin induction. This study is the first work involving integrated transcriptomic and metabolomic analyses to identify the key pathways to understand the molecular and metabolic mechanisms underlying drug resistance to polyether ionophores in coccidia.

Host protein EPCAM interacting with EtMIC8-EGF is essential for attachment and invasion of Eimeria tenella in chickens.

The five epidermal growth factor-like domains (EGF) of Eimeria tenella microneme protein 8 (EtMIC8) (EtMIC8-EGF) plays a vital role in host cell attachment and invasion. These processes require interactions between parasite proteins and receptors on the surface of host cells. In this study, five chicken membrane proteins potentially interacting with EtMIC8-EGF were identified using the GST pull-down assay and mass spectrometry analysis, and only chicken (Gallus gallus) epithelial cell adhesion molecule (EPCAM) could bind to EtMIC8-EGF. EPCAM-specific antibody and recombinant EPCAM protein (rEPCAM) inhibited the EtMIC8-EGF binding to host cells in a concentration-dependent manner. Furthermore, the rEPCAM protein showed a binding activity to sporozoites in vitro, and a significant reduction of E. tenella invasion in DF-1 cells was further observed after pre-incubation of sporozoites with rEPCAM. The specific anti-EPCAM antibody further significantly decreased weight loss, lesion score and oocyst output during E. tenella infection, displaying partial inhibition of E. tenella infection. These results indicate that chicken EPCAM is an important EtMIC8-interacting host protein involved in E. tenella-host cell adhesion and invasion. The findings will contribute to a better understanding of the role of adhesion-associated microneme proteins in E. tenella.

Supplementing broiler diets with bacterial selenium nanoparticles enhancing performance, carcass traits, blood indices, antioxidant status, and caecal microbiota of Eimeria tenella-infected broiler chickens.

Nanomedicine is a critical therapeutic approach for treating most poultry illnesses, particularly parasitic infections. Coccidiosis is a severe protozoan infection affecting poultry; the emergence of drug-resistant Eimeria strains demands the development of new, safe therapies. Consequently, the objective of this work was to investigate the efficacy of the biosynthesized selenium nanoparticles (SeNPs) by Paenibacillus polymyxa (P. polymyxa) against Eimeria tenella (E. tenella) experimental infection in broiler chickens. The prepared SeNPs absorbed the UV at 270 nm were spherical with a size of 26 nm, and had a surface negative charge of -25 mV. One hundred and fifty, 1-day-old male broiler chicks were randomly allocated into 5 groups (30 birds/group with triplicates each) as follows: T1: negative control (noninfected and nontreated with SeNPs); T2: delivered SeNPs (500 µg/kg diet) for 35 successive days, T3: E. tenella-infected (positive control birds), T4: E. tenella-infected and treated with SeNPs (500 µg/kg diet) and T5: E. tenella-infected chicks and treated with anticoccidial agent (sulfadimidine, 16% solution 8 mL/L of drinking water) for 5 successive days. At 14 d of age, each bird in infected groups was orally treated with 3 × 103 sporulated oocyst of E. tenella. SeNPs considerably decreased the number of oocysts in broiler feces compared to positive control and anticoccidial drug, followed by a substantial reduction of parasite phase count in the cecum (15, 10, and 8 for meronts, gamonts, and developing oocysts) when compared with positive control birds. The Eimeria experimental infection lowered the activity of antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx), and reduced glutathione (GSH) while increasing the stress parameters nitric oxide (NO) and malonaldehyde (MDA). Moreover, the production of proinflammatory (TNF-α and IL-6) and apoptotic genes (BcL2 and Cas-3) were significantly elevated. Administrating SeNPs to chicks significantly decreased oxidative stress, inflammation, and apoptotic markers in the cecum tissue. Therefore, growth performance, carcass weights, antioxidant enzymes, and blood properties of infected chicks were enhanced. The findings compared the protecting role of Se-nanoparticles against cecum damages in E. tenella-infected broilers.

Hepatozoon spp. infection in wild canids in the eastern United States.

BACKGROUND: Hepatozoon spp. are apicomplexan parasites known to cause musculoskeletal disease in a variety of animals. Two species are known to infect wild and domestic canids in the US: Hepatozoon canis and H. americanum. METHODS: In this study, blood, heart, and/or spleen samples were collected from 278 wild canids (180 coyotes, 93 red foxes, and 5 gray foxes) in the eastern US and tested via PCR for Hepatozoon. Histology slides of heart and skeletal muscle were assessed for Hepatozoon cysts and associated inflammation when fresh tissue was available (n = 96). RESULTS: Hepatozoon spp. were found in 24.2% (59/278) of individuals, with Hepatozoon canis in 14.0% (34/278) and H. americanum in 10.7% (26/278). One coyote was positive for both H. canis and H. americanum. Foxes were more likely to be positive for H. canis than coyotes (23% and 7% respectively, P = 0.0008), while only coyotes were positive for H. americanum. Of the eight sampled states, H. canis was present in six (Louisiana, North Carolina, Pennsylvania, South Carolina, Tennessee, and Virginia) while H. americanum was found in two southern states (South Carolina and Louisiana). Infection status was positively correlated with myositis and myocarditis, and heart or muscle cysts were found in 83% (5/6) of H. americanum-positive coyotes. CONCLUSION: This survey showed a moderate prevalence of H. canis and H. americanum in states where the parasite was previously unrecorded including South Carolina and Pennsylvania.

Characterization of Neospora caninum virulence factors NcGRA7 and NcROP40 in bovine target cells.

Bovine neosporosis is one of the major causes of reproductive failure in cattle worldwide, and differences in virulence between isolates have been widely shown. However, the molecular basis and mechanisms underlying virulence in Neospora caninum are mostly unknown. Recently, we demonstrated the involvement of NcGRA7 and NcROP40 in the virulence of N. caninum in a pregnant murine model using single knockout mutants in these genes generated by CRISR/Cas9 technology. In this study, the role of these proteins was investigated in two in vitro models using bovine target cells: trophoblast (F3 cell line) and monocyte-derived macrophages (BoMØ). The proliferation capacity of the single knockout mutant parasites was compared to the wild-type strain, the Nc-Spain7 isolate, using both cell populations. For the bovine trophoblast, no differences were observed in the growth of the defective parasites compared to the wild-type strain, neither in the proliferation kinetics nor in the competition assay. However, in naïve BoMØ, a significant decrease in the proliferation capacity of the mutant parasites was observed from 48 h pi onwards. Stimulation of BoMØ with IFN-γ showed a similar inhibition of tachyzoite growth in defective and wild-type strains in a dose-dependent manner. Finally, BoMØ infected with knockout parasites showed higher expression levels of TLR3, which is involved in pathogen recognition. These results suggest that NcGRA7 and NcROP40 may be involved in the manipulation of innate immune defense mechanisms against neosporosis and confirm the usefulness of the BoMØ model for the evaluation of N. caninum virulence mechanisms. However, the specific functions of these proteins remain unknown, opening the way for future research.

Transcriptional changes associated with apoptosis and type I IFN underlie the early interaction between Besnoitia besnoiti tachyzoites and monocyte-derived macrophages.

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.

In vivo anticoccidial, antioxidant, and anti-inflammatory activities of avocado fruit, Persea americana (Lauraceae), against Eimeria papillata infection.

Apicomplexan parasites, especially Eimeria sp., are the main intestinal murine pathogens, that lead to severe injuries to farm and domestic animals. Many anticoccidial drugs are available for coccidiosis, which, leads to the development of drug-resistant parasites. Recently, natural products are considered as an alternative agent to control coccidiosis. This study was designed to evaluate the anticoccidial activity of the Persea americana fruit extract (PAFE) in male C57BL/6 mice. A total of 35 male mice were divided into seven equal groups (1, 2, 3, 4, 5, 6, and 7). At day 0, all groups except the first group which served as uninfected-untreated control were infected orally with 1 × 103E. papillata sporulated oocysts. Group 2 served as uninfected-treated control. Group 3 was considered an infected-untreated group. After 60 min of infection, groups 4, 5, and 6 were treated with oral doses of PAFE aqueous methanolic extract (100, 300, and 500 mg/kg of body weight, respectively). Group 7 was treated with amprolium (a reference drug for coccidiosis). PAFE with 500 mg/kg, was the most effective dose, inducing a significant reduction in the output of oocysts in mice feces (by about 85.41%), accompanied by a significant decrease in the number of the developmental parasite stages and a significant elevation of the goblet cells in the jejunal tissues. Upon treatment, a significant change in the oxidative status due to E. papillata infection was observed, where the levels of glutathione (GSH) increased, while, levels of malondialdehyde (MDA) and nitric oxide (NO) were decreased. In addition, the infection significantly upregulated the inflammatory cytokines of interleukin-1ß (IL-1ß), tumor necrosis factor-alpha (TNF-α), and interferon-γ (IFN-γ). This increase in mRNA expression of IL-1ß, TNF-α, and IFN-γ was about 8.3, 10.6, and 4.5-fold, respectively, which significantly downregulated upon treatment. Collectively, P. americana is a promising medicinal plant with anticoccidial, antioxidant, and anti-inflammatory activities and could be used for the treatment of coccidiosis.

Orally delivered Bacillus subtilis expressing chicken NK-2 peptide stabilizes gut microbiota and enhances intestinal health and local immunity in coccidiosis-infected broiler chickens.

We recently reported a stable Bacillus subtilis-carrying chicken NK-lysin peptide (B. subtilis-cNK-2) as an effective oral delivery system of an antimicrobial peptide to the gut with therapeutic effect against Eimeria parasites in broiler chickens. To further investigate the effects of a higher dose of an oral B. subtilis-cNK-2 treatment on coccidiosis, intestinal health, and gut microbiota composition, 100 (14-day-old) broiler chickens were allocated into 4 treatment groups in a randomized design: 1) uninfected control (CON), 2) infected control without B. subtilis (NC), 3) B. subtilis with empty vector (EV), and 4) B. subtilis with cNK-2 (NK). All chickens, except the CON group, were infected with 5,000 sporulated Eimeria acervulina (E. acervulina) oocysts on d 15. Chickens given B. subtilis (EV and NK) were orally gavaged (1 × 1012 cfu/mL) daily from d 14 to 18. Growth performances were measured on d 6, 9, and 13 postinfection (dpi). Spleen and duodenal samples were collected on 6 dpi to assess the gut microbiota, and gene expressions of gut integrity and local inflammation makers. Fecal samples were collected from 6 to 9 dpi to enumerate oocyst shedding. Blood samples were collected on 13 dpi to measure the serum 3-1E antibody levels. Chickens in the NK group showed significantly improved (P < 0.05) growth performance, gut integrity, reduced fecal oocyst shedding and mucosal immunity compared to NC. Interestingly, there was a distinct shift in the gut microbiota profile in the NK group compared to that of NC and EV chickens. Upon challenge with E. acervulina, the percentage of Firmicutes was reduced and that of Cyanobacteria increased. In NK chickens, however, the ratio between Firmicutes and Cyanobacteria was not affected and was similar to that of CON chickens. Taken together, NK treatment restored dysbiosis incurred by E. acervulina infection and showed the general protective effects of orally delivered B. subtilis-cNK-2 on coccidiosis infection. This includes reduction of fecal oocyst shedding, enhancement of local protective immunity, and maintenance of gut microbiota homeostasis in broiler chickens.

Analytical sensitivity of a multiplex quantitative PCR for Toxoplasma gondii and Neospora caninum.

Cyst-forming coccidia, Toxoplasma gondii and Neospora caninum, are recognised as important causes of animal disease. Molecular diagnostics based on the presence of DNA in animal tissue are required to specifically detect T. gondii and N. caninum while achieving high levels of analytical sensitivity. We optimised available single-plex probe base qPCR assays into a multiplexed qPCR panel to detect cyst-forming coccidia, i.e. T. gondii and N. caninum. The T. gondii assay is based on a 529-bp repetitive (REP) element and the N. caninum assay on the NC5 repetitive region. Using target sequence synthetic DNA, the limit of detection (LOD) was determined to be 100 copies, that is less than a single tachyzoite of either T. gondii or N. caninum. The T. gondii and N. caninum multiplexed qPCR assay optimised in this study can be used to effectively detect parasite DNA for diagnostic purposes in animal tissue.

Neospora caninum and Toxoplasma gondii infections in one-humped camels (Camelus dromedarius) in central desert of Iran.

The protozoan parasite Neospora caninum infects carnivores as definitive and a wide range of mammals as intermediate hosts. This parasite is regarded as an important cause of abortion in cattle worldwide, causing significant economic losses. Although there is serological evidence of infection in Old World camelids, the significance of N. caninum in these animal species is still poorly understood. The aim of this study was to use molecular and histological methods to detect N. caninum in the blood and tissues of 100 slaughtered one-humped camels (Camelus dromedarius) in Iran. For this, genomic DNA was extracted from blood, brain, portal lymph node and liver of the camels, and nested-PCR assay followed by sequencing were performed. Besides, paraffin-embedded tissue sections were stained with hematoxylin and eosin (H&E) and studied microscopically. In addition, immunohistochemical staining for N. caninum was attempted on brain samples with positive PCR results. All animals were tested for antibodies against N. caninum and Toxoplasma gondii by whole tachyzoite-agglutination tests. N. caninum DNA was detected in blood, brain, and portal lymph node, but not in the liver of two (2%) camels. Histopathological examination revealed cysts resembling N. caninum in brain samples of one of these camels; however, immunohistochemical staining for N. caninum and T. gondii did not allow a morphological identification. IgG antibodies to N. caninum and T. gondii were detected in 36% and 35% of the camels, respectively. This study provides the first insight into direct detection of N. caninum in C. dromedarius in Iran. Further molecular studies on aborted fetuses, stillborn animals and cases of perinatal mortality are needed to understand the possible involvement of N. caninum in cases of reproductive failure. As the definitive hosts of N. caninum are domestic and wild canids, producers should be advised to monitor and limit exposure of their camelids to these species and their feces.

Cystoisospora suis infection in suckling piglets in Brazil: Prevalence and associated factors.

Piglet coccidiosis is a parasitic disease caused by the protozoan Cystoisospora suis, which is regarded as the most prevalent gastrointestinal parasite in intensive pig farms. Despite the availability of highly effective chemo-metaphylaxis (toltrazuril), coccidiosis is still prevalent in European and other countries. We conducted a cross-sectional study on swine farms in Brazil, to assess the prevalence of Cystoisospora suis in fecal samples and determined the associated factors. In total, 666 litters from 50 farms were sampled twice within one week between samplings (mean age at sampling: 10.75 and 17.7 days). Of 666 litters, 225 (33.8%) were positive at least once, and the expected within farm prevalence of C. suis oocysts was 32.9% (25.4-41.3%; 95% confidence interval). Oocysts were more prevalent in fecal samples collected from farms with diarrhea (odds ratio = 6.75). The room temperature was also positively associated with oocyst detection; a one-degree increase in room temperature increased the chance of a litter being positive by 23.2%. Up-to-date, this is the most comprehensive technical evidence of factors associated with C. suis infection in Brazilian industrial piglet farms.

Absence of anti-Besnoitia spp. specific antibodies in European wild lagomorphs from the Iberian Peninsula.

Besnoitia besnoiti is an apicomplexan parasite whose life cycle is not completely understood. It is assumed that this parasite might have an indirect life cycle with a carnivore as a definitive host able to shed oocysts after the ingestion of mature cysts in tissues of an infected intermediate host. Cattle and wild cervids on the Iberian Peninsula can act as intermediate hosts of B. besnoiti, and exposure to the parasite has been demonstrated in equids. In this study, we aimed to assess the presence of members of the genera Besnoitia in wild lagomorphs from the Iberian Peninsula and the potential role of these host species in the life cycle of B. besnoiti, as all the animals were sampled from 19 regions of the Iberian Peninsula where cases of bovine besnoitiosis have been previously detected. Serum samples (Oryctolagus cuniculus: n = 552; Lepus europaeus: n = 122) were first analysed by ELISA and subsequently confirmed by Western blot (WB). Specific antibodies against B. besnoiti were not found in any sampled animal by WB. In addition, lung samples from a subset of wild rabbits (n = 16) were tested by PCR and Besnoitia spp. DNA was not detected. These results suggest that Besnoitia spp. are unlikely to circulate in wild lagomorphs in the Iberian Peninsula. Thus, lagomorphs are not expected to play a key role in the biological cycle of B. besnoiti. Further studies are necessary to assess whether different micromammal species, such as rodents, can serve as natural reservoirs of Besnoitia spp. in other European regions.