The effects of a high-flavonoid corn cultivar on the gastrointestinal tract microbiota in chickens undergoing necrotic enteritis.

The search for alternative therapies to antimicrobial growth promoters (AGP) in poultry production has gained momentum in the past years because of consumer preference and government restrictions on the use of AGP in animal production. Flavonoids are plant-derived metabolites that have been studied for their health-promoting properties that could potentially be used as an alternative to AGP in poultry. In a previous study, we showed that the inclusion of a flavonoid-rich corn cultivar (PennHFD1) in the diet improved the health of broilers undergoing necrotic enteritis. However, the mechanisms of action by which the PennHFD1-based diet ameliorated necrotic enteritis are unknown. This study describes the microbial diversity and composition of the jejunum and ileum of chickens co-infected with Eimeria maxima and Clostridium perfringens and treated with a high-flavonoid corn-based diet. Luminal content and mucosal samples from the jejunum and ileum were collected for DNA extraction, 16S rRNA amplicon sequencing and data analyses. The infection model and the dietary treatments significantly changed the alfa diversity indices (Mucosal samples: ASVs, P = 0.04; Luminal content samples: ASVs, P = 0.03), and beta diversities (Mucosal samples: P < 0.01, Luminal content: P < 0.01) of the ileal samples but not those of the jejunal samples. The microbial composition revealed that birds fed the high-flavonoid corn diet had a lower relative abundance of C. perfringens compared to birds fed the commercial corn diet. The treatments also changed the relative abundance of other bacteria that are related to gut health, such as Lactobacillus. We concluded that both the infection model and the dietary high-flavonoid corn changed the broilers' gut microbial diversity and composition. In addition, the decrease in the relative abundance of C. perfringens corroborates with a decrease in mortality and intestinal lesions due to necrotic enteritis. Collecting different segments and sample types provided a broader understanding of the changes in the gut microbiota among treatments.

EtROP38 suppresses apoptosis of host cells infected with Eimeria tenella by inhibition of the p38MAPK pathway.

Coccidiosis is an important parasitic disease that has serious adverse effects on the global poultry industry. The mechanism by which the pathogenic factors of Eimeria tenella damage host cells is unknown. Some kinases from the rhoptry compartment can regulate apoptosis of host cells. This study focused on revealing the role and critical nodes of E. tenella rhoptry protein (EtROP) 38 in controlling the apoptosis of host cells via the P38 mitogen-activated protein kinase (MAPK) signaling pathway. The cells were treated with EtROP38 protein, siRNA p38MAPK, or both. The rate of infection, apoptosis, and the dynamic changes in the expression and activation of key factor genes of the P38MAPK signaling pathway in host cells infected with E. tenella were measured. The results showed that the addition of EtROP38 and/or knockdown of the host cells p38 gene reduced the apoptosis rate of cecal epithelial cells (CECS), decreased the mRNA expressions of p38, p53, c-myc, c-fos, and c-jun and increased the expression of p65, decreased the protein expressions of c-myc, c-fos, and c-jun, decreased the p38 protein phosphorylation level, and increased the p65 protein phosphorylation level in CECS. When E. tenella was inoculated for 4-96 h, the addition of Et ROP38 and/or host cell p38 knockdown both increased the infection rate of host cells, and this effect was more pronounced with the addition of EtROP38 with the host cell p38 knockdown. These observations indicate that E. tenella can inhibits the activation of the p38MAPK signaling pathway in host cells via EtROP38, which suppresses apoptosis in host cells.

Feasibility of implementing RPA coupled with CRISPR-Cas12a (RPA-Cas12a) for Hepatozoon canis detection in dogs.

Hepatozoonosis, caused by the protozoan Hepatozoon canis, is a prevalent blood disease affecting owned and stray dogs and cats. The prevalence of these parasites among companion animals in Thailand remains poorly understood. Diagnosing the old-world form of the disease is challenging due to the wide range of nonspecific clinical signs and the reliance on finding low levels of Hepatozoon gamonts in blood smears for conventional diagnosis. PCR demonstrates high specificity and sensitivity but it requires sophisticated instrumentation. Therefore, we established recombinase polymerase amplification (RPA) coupled with Cas12a for H. canis detection based on 18S rRNA. Our findings showed that RPA-Cas12a using gRNA_H was highly specific to H. canis, without yielding positives for other pathogen species including Babesia species. Even in cases of co-infection, RPA-Cas12a only detected positives in samples containing H. canis. This approach detected minimal amounts of H. canis18S rRNA-harboring plasmid at 10 copies per reaction, whereas plasmid-spiked canine blood enabled detection at a minimal amount of 100 copies per reaction. The performance of RPA-Cas12a was validated by comparing it with quantitative PCR-high resolution melting analysis (qPCR-HRM) and sequencing based on 35 canine blood samples. RPA-Cas12a demonstrated precision and accuracy values of 94 % and 90 %, respectively comparable to qPCR-HRM. Overall, these results indicate that RPA-Cas12a serves as a promising tool for H. canis detection as indicated by comparable performance to qPCR-HRM and is suitable for implementation in small animal hospitals or clinics due to its minimal resource requirements, thereby contributing to effective diagnosis and treatment for infected dogs.

Molecular diagnosis and phylogenetic insights of Eimeria species infecting buffaloes (Bubalus bubalis) in Meghalaya's subtropical hilly region, India.

Coccidiosis is an intestinal infection caused by Eimeria spp. that results in economic losses owing to morbidity and mortality in young buffalo calves. This study aimed for molecular diagnosis and phylogenetic analysis of Eimeria spp. in buffaloes of Meghalaya's sub-tropical mountainous terrain. Fresh buffaloes' fecal samples were collected from buffalo farms of Umling, Umsning and Bhoirymbong blocks, Ri Bhoi, Meghalaya and screened for Eimeria oocysts using flotation and modified McMaster methods. Fecal sample examination revealed 27.44 % (87/317) infection in buffaloes. Age wise, 64.44 % (29/45), 25.35 % (36/142) and 16.92 % (22/130) infections were recorded in <6 months, 6 months to 1 year and 1-2 year old buffaloes, respectively. Morphological characterization of Eimeria spp. revealed E. bovis (21.83 %), E. bareillyi (18.39 %), E. zuernii (11.49 %), E. ellipsoidalis (3.44 %) and mixed infection (44.82 %). Amplification of ITS-1 gene confirmed Eimeria spp. (410 bp), E. bovis (238 bp) and E. zuernii (344 bp). Phylogenetic analysis of E. bovis Umiam isolate revealed that these were closely related to the E. bovis isolate from South Korea (MH245198.1), and Turkey (KU351711.1) and distantly related to the isolates from Jammu and Kashmir (OQ103422.1) and Uttar Pradesh, Mathura (OK486542.1). E. zuernii isolate from Umiam, Meghalaya was observed to be phylogenetically close to the isolates from South Korea (MH245202.1), Japan (LC171339.1) and Turkey (KU351715.1), whereas phylogenetic divergence was observed between, E. zuernii isolate from Umiam, Meghalaya with isolates of Andhra Pradesh, Tirupati (MN601278.1) and Jammu and Kashmir (OQ103424.1). Therefore, treatment and effective control strategies should be implemented immediately to prevent spread of infection in the buffaloes.

Metabolomic analysis reveals altered amino acid metabolism and mechanisms underlying Eimeria infection in laying hens.

Avian coccidiosis, caused by Eimeria spp, is a devastating disease in laying hens. Previous studies have suggested that amino acids may be involved in Eimeria infection of broiler chickens. However, their metabolic features in laying hens, as well as the effect of multiple Eimeria species challenges on poultry hosts have not been elucidated yet. Here, a targeted metabolomics approach was employed to identify altered amino acid metabolism and mechanisms in laying hens with multiple Eimeria species challenges. Laying hens, Hy-Line W-36 aged 25 wk, were randomly assigned to a control group and groups inoculated with varying levels of mixed Eimeria species (E. maxima, E. tenella, and E. acervulina). Serum samples from each group were collected at 6 d and 14 d of postinoculation (6 and 14 DPI) for metabolite profiling. Metabolomic analysis revealed notable metabolic variations between control and infected groups, especially at 6 DPI stage. Varying levels of Eimeria dosages did not show a significant metabolic difference, and metabolites were sensitive to low-level infection. With statistical analysis, differentially expressed compounds (3-methylhistidine, alanine, aspartate, lysine, asparagine, methionine, ornithine, and tryptophan) were selected, and their metabolic network was identified by pathway enrichment analysis. In the network, the lysine biosynthesis pathway was upregulated, while the arginine and proline metabolic pathway was downregulated under infection. Other pathways showed complex patterns of metabolic relationships. Based on the results, biological implications of metabolic changes were elucidated and discussed. Last, the results were further confirmed with our previous study (phenotype and gene expression results) using the same set of samples. Our finding provides in-depth information on altered amino acid metabolism and mechanisms in laying hens upon multiple Eimeria species infection.

Molecular phylogenetic analysis and seasonal dynamics of Eimeria species infecting broilers of Kashmir, India.

Globally, the poultry industry is seriously threatened by coccidiosis caused by various species of Eimeria. This protozoan parasite inhabits the epithelial lining of the gastrointestinal tract of poultry globally and can cause serious clinical disease. The present study was carried out on poultry farms located in various regions of Kashmir, India, to investigate the prevalence and phylogenetic relationships of Eimeria species affecting broiler chickens. Over a period of one year, fecal samples were collected from 60 poultry farms in Kashmir and morphological and molecular techniques were employed for Eimeria species identification. Results revealed a high prevalence of coccidiosis, with 58.3% (35/60) of farms positive for Eimeria. The most prevalent species were E. tenella (31/35, 88.6%) followed by E. acervulina (25/35, 71.4%), E. maxima (19/35, 54.3%), E. mitis (18/35, 51.4%), and E. necatrix (9/35, 25.7%). Seasonal variation in prevalence was also observed, with the highest rates in autumn (86.7%) and summer (66.7%). Additionally, younger birds (3-4 weeks) exhibited higher infection rates (85.7%) compared to older birds (57.9%) (5-6 weeks). Mixed infection was found in 94.2% (33/35) of positive farms. Phylogenetic analysis using ITS1 sequences confirmed species clustering and revealed evolutionary relationships among Eimeria species. E. tenella and E. necatrix formed a distinct clade, while E. acervulina formed another. The study underscores the importance of molecular techniques in accurate species identification and provides valuable insights into the epidemiology of coccidiosis in poultry in Kashmir. Effective control strategies, including vaccination and improved management practices, are necessary to mitigate the economic losses associated with this widespread poultry disease.

Prevalence of Neospora caninum and associated risk factors in semi-intensive female water buffaloes reared in Greece, using an ELISA kit on milk samples.

Neospora caninum is an apicomplexan (family: Sarcocystidae) protozoan parasite with a global distribution. In the N. caninum life cycle, dogs and other related canids are the definitive hosts, while other animals such as water buffaloes (Bubalus bubalis) constitute the intermediate host for this parasite. In many countries, the water buffalo is of high economic importance, providing valuable high-quality products for human needs. Although knowledge concerning the prevalence of this parasite in intermediate animal host populations is crucial, data from water buffalo are scarce. Keeping this in mind, the aim of this study was to examine the presence and assess the prevalence rates of N. caninum infection in water buffaloes in Northern Greece, where this animal husbandry sector started to raise, as well as to determine associated risk factors, with the application of a commercially available Neospora ISCOM ELISA test kit, developed for the detection of antibodies against N. caninum in milk samples The study was conducted during January-June 2023 in a total of 172 individual raw milk samples, collected from dairy buffaloes, reared under a semi extensive system, in three different farms. Information on the status of N. caninum infection in buffaloes from Greece was so far unknown with a lack of epidemiological or risk factors associated. For the detection of N. caninum, the commercially available Neospora ISCOM ELISA test kit (SANOVIR ®, Svanova Biotech AB, Uppsala, Sweden) was utilized. Results demonstrated the presence of N. caninum in water buffaloes from Greece for the first time. All positive N. caninum animal were asymptomatic, with absence of any disease signs. The overall prevalence of infection was 22.10%, whereas the main risk factors include the presence of dogs as well as the low biosecurity measures.

Expression of IL-1ß in transgenic Eimeria necatrix enhances the immunogenicity of parasites and promotes mucosal immunity against coccidiosis.

Anticoccidial vaccines comprising living oocysts of Eimeria tenella, Eimeria necatrix, Eimeria maxima, and Eimeria acervulina are used to control coccidiosis. This study explored the potential of IL-1ß to act as a molecular adjuvant for enhancing the immunogenicity of Eimeria necatrix and mucosal immunity. We engineered E. necatrix to express a functional chIL-1ß (EnIL-1ß) and immunized chickens with oocysts of the wild type (EnWT) and tranegenic (EnIL-1ß) strains, respectively. The chickens were then challenged with EnWT oocysts to examine the immunogenicity-enhancing potential of chIL-1ß. As expected, the oocyst output of EnIL-1ß-immunized chickens was significantly reduced compared to those immunized using EnWT. No difference in body weight gain and lesion scores of EnIL-1ß and EnWT groups was observed. The parasite load in the small intestine and caeca showed that the invasion and replication of EnIL-1ß was not affected. However, the markers of immunogenicity and mucosal barrier, Claudin-1 and avian ß-defensin-1, were elevated in EnIL-1ß-infected chickens. Ectopic expression of chIL-1ß in E. necatrix thus appears to improve its immunogenicity and mucosal immunity, without increasing pathogenicity. Our findings support chIL-1ß as a candidate for development of effective live-oocyst-based anticoccidial vaccines.

Molecular evidence of hepatozoonosis in tigers of Vidarbha region of Maharashtra State of India.

BACKGROUND: Hepatozoonosis has been reported in many species around the world. Few incidences have been reported in various species of wild felids. Tigers are endangered large cats and are protected under the Wildlife Protection Act, 1972 under Schedule I. The study was carried out to estimate the positivity rate of hepatozoonosis in tigers of the Vidarbha region of Maharashtra, India. METHODS: Blood (n = 21) or tissue samples (n = 5) were collected from 26 wild captured / zoo-born or dead tigers during the quarantine period/post-mortem examination. Blood smear examination along with Polymerase Chain Reaction (PCR) studies were conducted for the detection of hepatozoonosis. All the amplicons from the positive samples were purified and sequenced, and the sequences were subjected to nBLAST analysis to detect the species of Hepatozoon. The sequences were deposited into public domain database of National Center for Biotechnology Information (NCBI) and accession numbers were allotted. A phylogenetic study was undertaken to understand the evolutionary lineage of the pathogen. Tissue distribution studies were carried out on tissue samples received during post mortem. A clinical case in a tiger cub was managed and sub-clinical cases were monitored for relapse. Age-wise, sex-wise, region-wise and captive time-wise positivity rate was estimated. The data was analyzed using statistical tools. RESULTS: A total of 12 tigers were found positive for H. felis during the screening. A clinical case was diagnosed and successfully treated. The age group of 0-3 years reported a positivity rate of 66.66%, and all the cases found positive were reported between the age group of 0-7 years. Males reported a positivity rate of 58.33 per cent, while females reported 35.71%. Taboba and Andhari Tiger Reserve of the state had a positivity rate of 52.94 per cent. However, the statistical analysis for blood parameters and positivity rate by 't' test and Chi-squared test were found to be non-significant. CONCLUSIONS: An overall positivity rate of 46.15% indicates the wide distribution of hepatozoonosis among wild tigers of the Vidarbha region of Maharashtra, India, which is strategically important considering the gene flow and migration of tigers. Hepatozoonosis can progress to clinical outcomes in young animals and require veterinary intervention. Molecular tools and phylogenetic studies can supplement important data on circulating species of Hepatozoon in the field. Further studies on the clinical management and epidemiology of the infection in wild felids will comprehend the cause of wildlife conservation.

High serological and molecular prevalence of Ehrlichia canis and other vector-borne pathogens in dogs from Boa Vista Island, Cape Verde.

Despite the high global impacts of canine vector-borne diseases (CVBD) due to their wide distribution and zoonotic potential, the current epidemiological situation of CVBD in many tropical and subtropical regions remains unknown. This study examines the seroprevalence and molecular prevalence of Ehrlichia canis and other pathogens causing CVBDs (Leishmania infantum, Dirofilaria immitis, Babesia spp., Anaplasma spp. and Hepatozoon canis) in dogs living on the island of Boa Vista (Cape Verde Republic). Blood samples and infesting ticks were taken from 150 dogs across the island (stray, shelter, and pet dogs). Serum samples were tested using a rapid immunochromatographic test (Uranotest® Quattro) that detects antibodies against E. canis, L. infantum, Anaplasma spp. and D. immitis antigen. Levels of serum antibodies against E. canis were measured using the immunofluorescence antibody test (IFAT). In addition, tick-borne pathogens in blood samples (Anaplasma spp., Babesia spp., Hepatozoon spp., and Ehrlichia canis) were detected by microscopy observation and/or PCR plus sequencing. The seroprevalence of E. canis was extremely high at 82% (123/150), as revealed by both immunochromatography and IFAT. Most dogs returning a seropositive test result (82.92%; 102/123) had antibody titres > 1:1280 but showed no clinical signs or notable laboratory abnormalities. Of the 123 animals testing seropositive for E. canis, 67 (54.47%) also presented antibodies against Anaplasma spp., and 13 (10.56%) showed the presence of Hepatozoon spp. gamonts in the blood smear. Ehrlichia canis infection was detected in 17.1% (25/146) of dogs tested by direct sequencing of polymerase chain reaction (PCR) products. Co-infections were detected in seven of these dogs: four dogs tested PCR-positive for both E. canis and A. platys, two dogs tested positive for E. canis and Hepatozoon spp., and one dog tested positive for E. canis, A. platys and Hepatozoon spp. Rhipicephalus sanguineus sensu lato was the only tick species found infesting the canine study population. The high prevalence of tick-borne pathogens detected in dogs from Boa Vista Island highlights a need for improved control measures designed to prevent the transmission of these pathogens.

Identification and function characterization of NcAP2XII-4 in Neospora caninum.

BACKGROUND: Neospora caninum is a protozoan parasite in the Apicomplexa controlled by complex signaling pathways. Transcriptional control, an important way to regulate gene expression, has been almost absent in the N. caninum life process. However, to date, research on the transcriptional regulation of the AP2 family factors in N. caninum has been extremely limited. A prior study demonstrated that removing rhoptry protein 5 (ROP5), a significant virulence factor, resulted in abnormal expression levels of predicted NcAP2XII-4 in N. caninum, suggesting that the factor may regulate the function of ROP5. This study aimed to identify NcAP2XII-4 and its function in transcriptional regulation. METHODS: The NcAP2XII-4 gene was identified by analyzing the N. caninum genome. A polyclonal antibody against the protein was prepared and purified, and its expression and localization in the parasite were detected using western blot (WB) and immunofluorescence assay (IFA). The ΔNcAP2XII-4 strain was constructed from the Nc1 strain using CRISPR/Cas9 to study its effect on the growth and development of N. caninum, and DAP-Seq and electrophoretic mobility shift assay (EMSA) were used to verify the transcriptional regulatory functions of the gene. RESULTS: Bioinformatic analysis showed that NcAP2XII-4 consists of 11,976 bp and encodes 3991 amino acids, with a predicted molecular mass of 410 kDa. The protein has two AP2 domains, 1207aa-1251aa and 3453aa-3500aa, and is predicted to be located in the nucleus. The results of PCR, WB, and IFA were in accordance with the bioinformatics analysis. ΔNcAP2XII-4 was successfully constructed, but the strain could not be released and ultimately succumbed within parasitophorous vacuoles (PVs). Plaque assays demonstrated that parasites lacking this gene could not form plaques. One motif was successfully identified using DAP-Seq technique. Two prokaryotic expression vectors containing the AP2 domain of NcAP2XII-4 were successfully constructed, and two prokaryotic expression proteins, AP2-D1 and AP2-D2, and ROP5 biotinylated probes were prepared. Using EMSA, NcAP2XII-4 was shown to regulate ROP5 transcription by binding to its promoter. CONCLUSIONS: NcAP2XII-4 is an essential gene in N. caninum. This study provides a foundation for further research on transcriptional regulation in N. caninum and identifies a new candidate factor for the development of vaccines against N. caninum.

Anti-coccidial efficacy of Enteromorpha prolifera polysaccharide in indigenous chickens of Northwest Ethiopia.

BACKGROUND: A variety of bioactive compounds isolated from various botanical sources have been found to have therapeutic and immunotherapeutic effects on chicken coccidiosis. AIM: This study aimed to evaluate the anti-coccidial potential of Enteromorpha prolifera polysaccharide (EP) in indigenous chickens in Northwest Ethiopia. MATERIALS AND METHODS: A total of 78 male indigenous chickens were used for this study. The study had two treatment groups: (1) the EP non-supplemented group (those fed on diets without EP and Eimeria oocyst inoculated) and (2) the EP group (those receiving diets supplemented with 400 mg EP/kg diet and Eimeria oocyst inoculated). Each treatment group had five replications. Following fourteen days of EP supplementation, 1.5 × 104 oocysts of mixed Eimeria species were inoculated into individual birds. RESULTS: EP-supplemented chicken showed significantly lower (p < 0.05) oocyst counts compared to non-supplemented ones on 9 and 11 days post-challenge. In addition, chickens in the EP-supplemented group showed less severe lesion scores, with an average score of 1.33. Chickens that received EP showed a maximum of 27.27% protection against lesions. In contrast, the non-supplemented chickens had a lower percentage of protection (19.83%). The maximum anti-coccidial index value (146.98) was obtained from EP-supplemented chickens. Chickens in the EP-supplemented group exhibited a significantly higher (p < 0.05) weight gain. CONCLUSION: Overall, the inclusion of EP in chickens' diets shows promise as a potential anti-coccidial strategy. However, additional research is required to explore the mechanisms by which EP in chickens' diet could involve in increasing the protection ability of chickens against coccidiosis.

Breed variability in the cellular mediated immune response to experimental Neospora caninum infection in heifers.

Protozoan parasite Neospora caninum causes abortion in infected cattle while others remain asymptomatic. Host immunity plays a critical role in the outcome of bovine neosporosis. Despite extensive research, there is a critical gap in therapeutic and preventive measures, and no effective vaccines are available. Both beef and dairy cattle can suffer from N. caninum-induced abortions, but cumulative evidence suggests a breed susceptibility being higher in dairy compared with beef breeds. It has been established that the response to N. caninum infection primarily involves a cell-mediated immune response (CMIR) regulated by T-helper type 1 (Th1) cells and specific cytokines. The delayed-type hypersensitivity (DTH) skin test has been used to measure the ability of livestock to generate CMIR, in the context of breeding for disease resistance and as a method for diagnosis of several diseases. In this study, we evaluated the immune response triggered by an N. caninum-induced DTH skin test between Holstein - a dairy breed intensively selected- and Argentinean Creole heifers - a beef breed with minimal genetic selection- to assess differences in CMIR following experimental N. caninum infection. The immune response, measured through skinfold thickness and histological and immune molecular analysis, revealed variations between the breeds. Our study found an increased CMIR in Argentinean Creole heifers compared to Holstein heifers. Differential gene expression of key cytokines was observed at the DTH skin test site. Argentinean Creole heifers exhibited elevated IFN-γ, IL-12, IL-10, and IL-4, while Holstein heifers only showed higher expression of IL-17. This finding could underscore genetic diversity in response to neosporosis, which could be used in breeding cattle strategies for disease resistance in cattle populations.

The CAMKK/AMPK Pathway Contributes to Besnoitia besnoiti-Induced NETosis in Bovine Polymorphonuclear Neutrophils.

Besnoitia besnoiti is an obligate intracellular apicomplexan parasite and the causal agent of bovine besnoitiosis. Bovine besnoitiosis has a considerable economic impact in Africa and Asia due to reduced milk production, abortions, and bull infertility. In Europe, bovine besnoitiosis is classified as an emerging disease. Polymorphonuclear neutrophils (PMN) are one of the most abundant leukocytes in cattle blood and amongst the first immunological responders toward invading pathogens. In the case of B. besnoiti, bovine PMN produce reactive oxygen species (ROS), release neutrophil extracellular traps (NETs), and show increased autophagic activities upon exposure to tachyzoite stages. In that context, the general processes of NETosis and autophagy were previously reported as associated with AMP-activated protein kinase (AMPK) activation. Here, we study the role of AMPK in B. besnoiti tachyzoite-induced NET formation, thereby expanding the analysis to both upstream proteins, such as the calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK), and downstream signaling and effector molecules, such as the autophagy-related proteins ULK-1 and Beclin-1. Current data revealed early AMPK activation (<30 min) in both B. besnoiti-exposed and AMPK activator (AICAR)-treated bovine PMN. This finding correlated with upstream responses on the level of CAMKK activation. Moreover, these reactions were accompanied by an augmented autophagic activity, as represented by enhanced expression of ULK-1 but not of Beclin-1. Referring to neutrophil effector functions, AICAR treatments induced both AMPK phosphorylation and NET formation, without affecting cell viability. In B. besnoiti tachyzoite-exposed PMN, AICAR treatments failed to affect oxidative responses, but led to enhanced NET formation, thereby indicating that AMPK and autophagic activation synergize with B. besnoiti-driven NETosis.

Efficacy of cordycepin against Neospora caninum infection in vitro and in vivo.

Neospora caninum (N. caninum) is an obligate intracellular Apicomplexa parasite that causes abortions in dairy cows and incurs substantial to significant economic losses in the global dairy farming industry. Cordycepin, a nucleoside antibiotic derived from Chinese medicine Cordyceps militaries, exhibits diverse biological activities. However, it remains unclear whether cordycepin possesses inhibitory effects against N. caninum infection. Therefore, this study aimed to establish both in vivo and in vitro models of N. caninum to investigate the potential impact of cordycepin against N. caninum infection. We successfully established an in vitro model of N. caninum infection in RAW264.7 cells, followed by qRT- PCR analysis to detect the content of N. caninum DNA within the cells. The effects of cordycepin on N. caninum was observed using the Giemsa method on RAW264.7, and the rate of cell infection was calculated. Cordycepin exhibited inhibitory effects on N. caninum tachyzoites in vitro, preserving cellular integrity and reducing the rate of cell infection. In mice, we established an in vivo model of N. caninum infection and detected N. caninum presence in tissues using. Real-time fluorescence quantitative PCR. Histopathological changes were observed through Hematoxylin-eosin staining. Liver function was assessed by using glutamic acid aminotransferase (ALT) and aspartic acid aminotransferase (AST) kits. Oxidative stress status was measured using catalase (CAT), malondialdehyde (MDA), and glutathione (GSH) kits. Compared with the model group, mice treated with cordycepin showed reduced clinical symptoms, increased food intake, and their body weight (P=0.0143, P=0.0068) was significantly higher than those in the model group. Furthermore, cordycepin treatment significantly alleviated hepatic cord disorders, hepatocellular swelling, detachment, and vacuolization; duodenal epithelial detachment and shortening of villi caused by N. caninum infection. Cordycepin administration reduced the increase in ALT (P=0.01, P=0.008) and AST (P<0.001) levels caused by N. caninum infection, while ameliorating hepatocyte swelling, necrosis, and detachment as well as inflammatory cell infiltration within mice liver; it also led to shortened or even disappeared duodenal villi along with and oedema of the submucosa. Analysis of oxidative stress showed that cordycepin ameliorated the damage caused by N. caninum by reducing MDA (P=0.03, P=0.02, P=0.005) and increasing CAT (P=0.004, P<0.001) and GSH (P=0.004, P<0.001) levels. In conclusion, this study reports for the first time on cordycepin's efficacy against N. caninum infection providing a potential candidate drug for neosporosis treatment.

Acute phase response and oxidative stress in coccidiosis: A review in domestic animals.

Coccidiosis is a highly significant disease in domestic animals due to its global distribution and economic impact. The occurrence of oxidative stress (OS) and the acute phase response (APR) play crucial roles in the development of coccidiosis, thereby contributing to the pathogenicity of coccidia. A range of triggers including parasitic infection, can induce the APR. This response encompasses a set of hormonal and metabolic changes to restore body stability and improve the body's healing capabilities. Ovine coccidiosis has the potential to cause OS, which can be prevented and treated through the use of dietary additives. By including Curcuma longa in the diets of infected sheep, it is possible to reduce lipid peroxidation (LPO) and nitric oxide (NO) production, while simultaneously improving serum antioxidant capacity and interleukin-10 (IL-10) levels. Caprine coccidiosis can activate the APR. Research indicated that goats suffering from coccidiosis exhibited elevated concentrations of malondialdehyde (MDA) and total homocysteine, along with reduced levels of some enzymes such as superoxide dismutase (SOD) and glutathione reductase (GR), as well as decreased levels of zinc (Zn), manganese (Mn), selenium (Se), vitamin C, and total antioxidant capacity (TAC). Bovine coccidiosis is linked to elevated MDA concentrations and reduced serum glutathione (GSH) and TAC levels. Eimeria can induce OS and inflammatory damage in infected birds by releasing pro-inflammatory mediators from cells, resulting in a significant increase in CAT and SOD activity, lipid peroxidation and damage to the intestinal epithelium. To promote the antioxidant system of infected birds, some herbal food additives such as grape seed proanthocyanidine extract, Curcuma longa and Rumex nervosus leaf extract, can be used. Research on the APR in birds is not as extensive as in ruminants. Currently, there is a lack of studies on the occurrence of OS and APR in camels, horses, dogs, and cats with coccidiosis.

Neospora caninum is not transmissible via embryo transfer, but affects the quality of embryos in dairy cows.

The aim of this study was to evaluate embryo transfer in cattle as a pathway of transmitting neosporosis and to quantitatively and qualitatively compare the embryo production from seropositive and seronegative donors. Superovulatory treatments were performed on eight Girolando donors (four seropositive and four seronegative for N. caninum), resulting in the recovery of ninety-one structures (embryos and non-fertilized structures). Embryos collected and classified as viable were transferred to seronegative recipients and analyzed by PCR to identify N. caninum. No difference was observed in the number of structures collected from seropositive and seronegative donors (40 and 51; P = 0.64). There was a significant difference in the percentages of freezable (25 % and 74 %, P = 0.04) and transferable (27.5 % and 78.4 %, P = 0.003) embryos. The recipients had similar pregnancy rates (60 % and 57.1 %, P = 0.97) and pregnancy loss (50 % and 43.8 %, P = 0.58). There was no change in the serological status of the recipients, and no protozoan DNA was identified in none of the samples. It was concluded that the embryo transfer technique is safe regarding the transmission of neosporosis; however, the quality of embryo production was compromised from donors seropositive for N. caninum.

Traditional Chinese medicine formulation ChangQing compound has significant therapeutic effects on chickens infected with Eimeria tenella.

Coccidiosis poses a significant challenge to the poultry industry. However, the excessive and improper use of anticoccidial drugs and vaccines has led to resistance and food safety concerns. Consequently, traditional Chinese herbs have garnered attention as a potentially safer and more effective alternative. ChangQing compound derived from various Chinese herbal medicines is a promising anticoccidiosis agent, but its therapeutic effects have not been comprehensively evaluated. This study aimed to assess the therapeutic efficacy of ChangQing Compound against Eimeria tenella-induced coccidiosis in chickens on the basis of physiological indicators, cecum lesions, and changes in microbial diversity. The comparison with the positive control group revealed the average weight gain (AWG) and anticoccidial index (ACI) of the chicks were significantly higher, in contrast, the feed conversion ratio (FCR), cecal lesion score (CLS), and oocyst count per gram of cecal content (OPG) were significantly lower (P<0.05). Notably, AWG (138.87 g), OPG (0.57 × 106), ACI (177.92), and FCR (2.51) reflected the significant therapeutic effect of the 2.5 g/L ChangQing compound treatment (CQM). Histological sections showed that the cecal villus damage and intestinal wall swelling were minimal in the CQM, consistent with the CLS (0.73). Additionally, the 2.5 g/L ChangQing compound treatment effectively prevented the decrease of red blood cells, platelets, and hemoglobin, while promoting the release of anti-inflammatory factors interleukin-10 and interleukin-4, and inhibiting the pro-inflammatory factors interferon-γ and interleukin-17. The microbial community structure in the CQM was most similar to that of the negative control group. In summary, ChangQing compound had multiple positive effects (e.g., promoting weight gain, alleviating anemia, suppressing coccidial proliferation, reducing intestinal damage, modulating immunity, and maintaining intestinal microbiota homeostasis). The study results may be relevant to developing a novel strategy for the clinical management of coccidiosis.

A colorimetric assay for Neospora caninum utilizing the loop-mediated isothermal amplification technique.

Neospora caninum (N. caninum) is a protozoan parasite that poses a serious risk to livestock by infecting various domestic and wild animals. Loop-Mediated Isothermal Amplification (LAMP) offers a cost-effective, highly sensitive, and specific method for detecting protozoan parasites. This study aims to develop a precise, rapid, and visually assessable colorimetric LAMP method, improving on traditional techniques. We employed a rigorous screening process to identify the optimal primer set for this experiment. Subsequently, we fine-tuned the LAMP reaction at 65 °C for 40 min with 270 µmol/L neutral red. We then confirmed the specificity of primers for N. caninum through experimental validation. The LAMP method demonstrated a lower detection limit compared to traditional Polymerase Chain Reaction (PCR) techniques. While LAMP offers clear advantages, the prevalence of DNA detected in 89 sheep serum and 59 bovine serum samples using the nested PCR method was 3.37 % (3/89) and 1.69 % (1/59), respectively. In contrast, when the LAMP method was employed, the prevalence of detected DNA rose to 5.61 % (5/89) for sheep and 3.38 % (2 /59) for bovine. A comparison of two molecular assays using the intragroup correlation coefficient (ICC) resulted in a value of 0.999 (95 % CI: 0.993-0.996, p < 0.001), indicating the LAMP method is in the "better" range according to James Lee's categorization. The LAMP technique, optimized with specific primers of N. caninum and neutral red dye, not only exhibited higher sensitivity but also provided convenience over conventional PCR methods, highlighting its potential for on-site applications and cost-effective field detection.

Molecular and serological prevalence rates of Neospora caninum infection in dogs from Jordan.

Neosporosis is a proven disease of farm animals and dogs caused by Neospora caninum. This cross-sectional study investigates N. caninum prevalence and seroprevalence among 268 dogs. Nc5 gene PCR was carried out on dog faeces and confirmed by sequencing. Seroprevalence was detected using an indirect fluorescent antibody test (IFAT). Three age groups, gender, locality (Amman, Irbid, and Zarqa Governorates), dog type (stray, pet, and breeding), place of living (indoor/outdoor), food type (raw/cooked), having diarrhoea, having abortion in the area, and having animals nearby were tested as independent variables for associations with positivity to N. caninum using univariate and multivariable logistic regression analyses. The true prevalence of N. caninum was 34.3% (95% CI 28.4, 40.5) using the Nc5-PCR test. The true seroprevalence rate of N. caninum among dogs in Jordan was 47.9% (95% CI 41.4, 54.5) using IFAT. The sequenced isolates of Nc5-PCR products (n = 85) matched three N. caninum strains, namely, NcHareGre (n = 70, 82.4%, 95% CI 72.6-89), NC MS2 (n = 14, 16.5%, 95% CI 9.3-26.1), and L218 (n = 1, 1.2%, 95% CI 0.03-6.4). The three strains were isolated previously from three different countries and continents. N. caninum shedding is associated with abortion among dogs and animals in the area (odds ratio = 3.6). In Amman and Zarqa, living indoors reduced seroprevalence at 0.45, 0.24, and 0.02 odds ratios, respectively. Jordan shares three molecular N. caninum strains with three different countries and continents.