Histopathologic observations in a coccidiosis model of Eimeria tenella.

BACKGROUND: Species of the genus Eimeria cause coccidiosis in chickens, resulting in a huge burden to the poultry industry worldwide. Eimeria tenella is one of the most prevalent chicken coccidia in China, and E. tenella infection causes hemorrhagic cecitis. METHODS: Using an established model of coccidiosis in chickens combined with necropsy, imaging of pathological tissue sections, and other techniques, we evaluated the gross and microscopic lesions of cecal tissue within 15 days after inoculation with sporulated oocysts and described the endogenetic developmental process and relationship between E. tenella infection and enteritis development in chickens. RESULTS: We observed three generations of merogony and gamogony in E. tenella. We observed gross lesions in the cecum from 84 hpi (hours post inoculation) and microscopic lesions from 60 hpi. The lesions in the cecum mainly exhibited hemorrhagic enteritis. Their severity increased with the onset of the second generation of merogony. The lesions began to alleviate by the end of the endogenous stages of E. tenella. CONCLUSION: We show, for the first time, the complete observation of a series of changes in enteritis caused by 5 × 103 E. tenella oocysts. This study provides reference materials for E. tenella research and pathological diagnosis.

Expression profile of Toll-like receptors and cytokines in the cecal tonsil of chickens challenged with Eimeria tenella.

Chicken coccidiosis, caused by Eimeria spp., seriously affects the development of the poultry breeding industry. Currently, extensive studies of chicken coccidiosis are mostly focused on acquired immune responses, while information about the innate immune response of chicken coccidiosis is lacking. Toll-like receptor (TLR), the key molecule of the innate immune response, connects innate and adaptive immune responses and induces an immune response against various pathogen infections. Therefore, the quantitative real-time PCR was used to characterize the expression profile of chicken TLRs (chTLRs) and associated cytokines in the cecal tonsil of chickens infected with Eimeria tenella. The results showed that the expression of chTLR1a, chTLR2a, and chTLR5 was significantly upregulated at 3 h post-infection, while chTLR1b, chTLR2b, chTLR3, chTLR7, chTLR15 and chTLR21 was significantly downregulated (p < 0.05). In addition, chTLR1a expression rapidly reached the peaked expression at 3 h post-infection, while chTLR2b and chTLR15 peaked at 168 h post-infection, and chTLR2a expression was highest among chTLRs, peaking at 48 h post-infection (p < 0.05). For cytokines, interleukin (IL)-6 and tumor necrosis factor (TNF)-α peaked at 96 h post-infection, IL-4 and IL-12 peaked at 144 h post-infection, and interferon-γ expression was highest among cytokines at 120 h post-infection. In addition, IL-12 and IL-17 were markedly upregulated at 6 h post-infection (p < 0.05). These results provide insight into innate immune molecules during E. tenella infection in chickens and suggest that innate immune responses may mediate resistance to chicken coccidiosis.

Molecular characterization of cDNA coding for 33.5 and 41 kDa oocyst and sporocyst proteins that are differentially regulated in different strains of Eimeria maxima.

Eimeria maxima (APU1 and APU2) differ in virulence for chickens, due in part to the greater fecundity of the former. In a previous study, RNA-seq was used to identify a transcripts upregulated in E. maxima APU1 compared to E. maxima APU2. In this study, 2 of these upregulated genes (EMWEY 23530 and EMWEY 48910) were characterized by first confirming upregulation using quantitative RT-PCR. For both EMWEY 23530 and EMWEY 48910, RNA transcription was fairly consistent during sporulation. The extent of differential expression was about 2-fold log2 higher in APU-1 compared to APU-2 (peaking at 18 h for EMWEY 23530 and 0 h for EMWEY 48910). EMWEY 23530 and EMWEY 48910 cDNA were cloned and expressed as polyHis-fusion proteins in Escherichia coli. The observed size of recombinant EMWEY 23530 was 24 kDa; the observed size of recombinant EMWEY 48910 was 35 kDa, which are consistent with the predicted size based on the coding sequences. Immunostaining 2D gel blots of E. maxima APU1 and APU2 oocyst/sporocyst protein with antisera specific for EMWEY 23530 identified a 33.5 kDa protein with a pH 7.4 isoelectric point (Emax p33.5). Similar 2D gel blot analysis with EMWEY 48910 identified a 41 kDa protein with a pH 7.2 isoelectric point (Emax p41). The intensity of Emax p33.5 and Emax p41 was noticeably greater in oocyst/sporocyst proteins from E. maxima APU1 compared to E. maxima APU2. This was corroborated by ELISA wherein equal amounts of total E. maxima APU1 and APU2 protein were probed with serial dilutions of anti-rEmax p33.5 or anti-rEmax p41. Immunofluorescence (IFA) staining of permeabilized unsporulated E. maxima APU1 and APU2 oocysts revealed Emax p33.5 to be localized in one end of oocysts, while Emax p41 appeared on the surface of oocysts. After sporulation, the p33.5 and p41 antigens appeared loosely associated with sporocysts. Taken together, these data confirm excess expression of two proteins in the E. maxima strain characterized by greater fecundity and virulence, and may provide insight into basis for phenotypic differences among different E. maxima.

Preliminary evaluation of the protective effect of rEi-SAG19 on Eimeria intestinalis infection in rabbits.

Eimeria intestinalis is one of the most pathogenic coccidia species in rabbits. Anticoccidial treaments are the main measures to control rabbit coccidiosis now, but there are drug resistance and residues concerns. Therefore, vaccine has been used as an alternative strategy. The surface antigens (SAGs) of apicomplexan protozoa play a role in adhesion and invasion of host intestinal cells, and are considered to be potential candidate antigens for vaccines. In this study, transcriptional analysis of 5 Ei-SAGs genes at four developmental stages was conducted, then the Ei-SAG19 gene were screened out for prokaryotic expression and the reactogenicity of recombinant SAG19 (rEi-SAG19) was investigated by immunoblotting. To assessment the protective effects of rEi-SAG19, rabbits (n = 40) were randomly divided into four groups (Blank control, PBS-infected, Trx-His-S-Quil-A-infected and rEi-SAG19 immunized groups), the rEi-SAG19 immunized group was subcutaneously immunized with 100 µg rEi-SAG19 in the neck with an interval of two weeks, and challenged with 5×104 homologous oocysts two weeks after the second immunization. Two weeks after the challenge, all rabbits were sacrificed. After that, the level of serum specific IgG antibody was detected weekly and the level of cytokines in serum before the challenge were determined. At the end of the experiment, the weight gain, oocyst reduction rate, lesion score and anticoccidial index (ACI) were calculated. The results showed that rEi-SAG19 has a good reactogenicity. The relative weight gain rate, oocyst reduction rate and ACI of the rabbits in rEi-SAG19 immunized group were 80.51%, 72.6%, and 165.1, respectively, which has a moderate protective effect. The level of serum specific IgG antibody and IL-4 rised significantly (P < 0.05), but the levels of IL-2, IFN-γ and IL-10 had no significant difference (P > 0.05). Our results indicated that rEi-SAG19 could provides moderate protective effect against E. intestinalis infection in rabbits (ACI = 165.1). Therefore, rEi-SAG19 could be used as a vaccine candidate antigen for E. intestinalis.

Eimeria tenella rhoptry neck protein 2 plays a key role in the process of invading the host intestinal epithelium.

The Apicomplexa parasitic phylum rhoptry neck protein 2 (RON2) plays a key role in the process of invading host cells. Eimeria tenella, an intracellular protozoan shares a similar conserved invasion pattern. However, whether E. tenella RON2 participates in the process of invading the host intestinal epithelium is poorly understood. In this study, the sequence of EtRON2 was analyzed and expressed. The expression of the truncated extracellular N-terminal fragment of EtRON2 (403-700 aa, designated EtRON2403-700) with a molecular mass of 38.3 kDa. EtRON2 in the sporozoite protein was detected at 151.4 kDa by rabbit anti-rEtRON2403-700 antibody. Immunofluorescence results showed that EtRON2 was mainly localized to the nucleus and apex of the E. tenella sporozoite. qPCR results showed that the highest expression level of EtRON2 was detected in sporulated oocysts compared with other developmental stages of E. tenella. In vitro invasion inhibition assays showed that the capacity of sporozoites to invade DF-1 cells was significantly inhibited after pretreatment with the rabbit anti-rEtRON2403-700 antibody. Silencing the EtRON2 gene by RNA interference (RNAi) significantly inhibited EtRON2 expression and significantly reduced the invasion of DF-1 cells by sporozoites. In vivo experiments revealed a significant decrease parasite burden and oocyst outputs in chicks after infection with EtRON2 gene-silenced sporozoites by cloacal inoculation. Recombinant EtRON2403-700 (rEtRON2403-700) immunizes chicks effectively against E. tenella infection by inducing humoral immunity and upregulating IFN-γ and CD8+ T lymphocytes. Furthermore, chicks exhibited increased relative weight gain rates, lower cecum lesion scores, and reduced oocyst outputs during the E. tenella challenge. H&E staining showed that the cecum tissue of chicks immunized with rEtRON2403-700 showed relatively mild histopathological changes. In conclusion, the results of this study demonstrated that EtRON2 plays a key role in E. tenella invasion of the host intestinal epithelium and provides a potential target for vaccines against E. tenella infection.

Organellar genome dynamics of exogenous stages of Eimeria tenella.

BACKGROUND: Coccidia are a group of intracellular protozoal parasites within the phylum Apicomplexa. Eimeria tenella, one of the species that cause intestinal coccidiosis in poultry, can cause significant mortality and morbidity. Diploid oocysts of Eimeria species are shed in the feces of an infected host and must sporulate to achieve infectivity. This process results in eight haploid infectious units, called sporozoites, held within a single oocyst. Each Eimeria spp. parasite possesses a single apicoplast and a single mitochondrion, both of which carry multiple copies of their respective organellar genomes. Reports of copy numbers of organellar genomes have varied widely. METHODS: We report the application of quantitative polymerase chain reaction (qPCR), supported by next-generation sequencing, for the quantification of the extranuclear genomes relative to the nuclear genome over the course of sporulation and following its completion. RESULTS: At 64 elapsed hours, 93.0% of oocysts were fully sporulated; no increase in percent sporulation was observed after this time. Apicoplast relative genome copy number showed several significant shifts up to 72 elapsed hours, after which no significant shifts were observed. Oocysts were shed with approximately 60% the amount of apicoplast DNA present at 72 h, after which point no significant shifts in apicoplast genome relative abundance occurred. Mitogenome relative copy number showed only two significant shifts, from 16 to 24 elapsed hours and from 24 to 32 elapsed hours. Oocysts were shed with approximately 28% the amount of mitochondrial DNA that was present at the time sporulation was deemed morphologically complete, at 64 elapsed hours. CONCLUSIONS: The characterization of the dynamics of genome abundance in exogenous stages sheds new light on the basic biology of Eimeria spp. and supports the use of extranuclear targets for molecular modes of parasite quantification and identification with improved sensitivity and accuracy.

Use of Metallic Nanoparticles Against Eimeria-the Coccidiosis-Causing Agents: A Comprehensive Review.

Coccidiosis is a protozoan disease caused by Eimeria species and is a major threat to the poultry industry. Different anti-coccidial drugs (diclazuril, amprolium, halofuginone, ionophores, sulphaquinoxaline, clopidol, and ethopabate) and vaccines have been used for their control. Still, due to the development of resistance, their efficacy has been limited. It is continuously damaging the economy of the poultry industry because under its control, almost $14 billion is spent, globally. Recent research has been introducing better and more effective control of coccidiosis by using metallic and metallic oxide nanoparticles. Zinc, zinc oxide, copper, copper oxide, silver, iron, and iron oxide are commonly used because of their drug delivery mechanism. These nanoparticles combined with other drugs enhance the effect of these drugs and give their better results. Moreover, by using nanotechnology, the resistance issue is also solved because by using several mechanisms at a time, protozoa cannot evolve and thus resistance cannot develop. Green nanotechnology has been giving better results due to its less toxic effects. Utilization of metallic and metallic oxide nanoparticles may present a new, profitable, and economical method of controlling chicken coccidiosis, thus by changing established treatment approaches and improving the health and production of chickens. Thus, the objective of this review is to discuss about economic burden of avian coccidiosis, zinc, zinc oxide, iron, iron oxide, copper, copper oxide, silver nanoparticles use in the treatment of coccidiosis, their benefits, and toxicity.

Temporal Changes in Jejunal and Ileal Microbiota of Broiler Chickens with Clinical Coccidiosis (Eimeria maxima).

Coccidiosis in broiler chickens continues to be a major disease of the gastrointestinal tract, causing economic losses to the poultry industry worldwide. The goal of this study was to generate a symptomatic Eimeria maxima (1000 oocysts) infection to determine its effect on the luminal and mucosal microbiota populations (L and M) in the jejunum and ileum (J and IL). Samples were taken from day 0 to 14 post-infection, and sequencing of 16S rRNA was performed using Illumina technology. Infected birds had significantly (p < 0.0001) lower body weight gain (BWG), higher feed conversion ratio (FCR) (p = 0.0015), increased crypt depth, and decreased villus height (p < 0.05). The significant differences in alpha and beta diversity were observed primarily at height of infection (D7). Analysis of taxonomy indicated that J-L and M were dominated by Lactobacillus, and in IL-M, changeover from Candidatus Arthromitus to Lactobacillus as the major taxon was observed, which occurred quicky in infected animals. LEfSe analysis found that in the J-M of infected chickens, Lactobacillus was significantly more abundant in infected (IF) chickens. These findings show that E. maxima infection affects the microbiota of the small intestine in a time-dependent manner, with different effects on the luminal and mucosal populations.

Molecular identification and biological characterization of Eimeria columbarum from domestic pigeons (Columba livia domestica) in Guangdong, China.

Pigeon coccidiosis caused by Eimeria spp. is an important veterinary disease with a significant economic impact on the pigeon industry. Preventive measures for Eimeria columbarum in pigeons have been hampered by the lack of extensive genetic, morphological, and biological data on the oocysts. In this study, we examined the prevalence and identity of Eimeria spp. in domestic pigeons from seven cities in Guangdong Province, China. Data show that coccidiosis was prevalent in domestic pigeons in Guangdong Province, with an overall Eimeria spp. detection rate of 73.4%. Five Eimeria species were identified, including E. columbarum (73.4%), Eimeria kapotei (25.6%), Eimeria labbeana (19.6%), Eimeria duculai (19.6%), and Eimeria tropicalis (6.7%). We obtained single oocyst-derived lines of the dominant E. columbarum from fecal specimens. E. columbarum oocysts measured 20.06 ± 0.69 µm × 18.63 ± 1.03 µm, and sporocysts measured 10.29 ± 0.82 µm × 85.38 ± 0.46 µm. In infection experiment using obtained E. columbarum isolates, 60-day-old coccidia-free pigeons exhibited a prepatent period of 105 h and patent period of 9-10 days followed by severe diarrhea, depression, anorexia, and emaciation. Endogenous development of the parasite was observed mainly in the cytoplasm of epithelial cells in the duodenum, jejunum, ileum, and rectum. Two generations of meronts developed on days 3 and 4 after infection, respectively, while gamont and gamete developed on day 5 after infection. The morphological, genetic, and biological data are expected to be useful in elucidating the biological characterization of pigeon coccidiosis to develop measures against the treatment and containment of this disease.

Efficacy and safety of diclazuril nanoemulsion in control of Eimeria tenella in broilers.

BACKGROUND: Nanotechnology has the potential to reduce drug dosage while increasing efficacy; thus, the current work intends to synthesize diclazuril nanoemulsion and assess its performance against experimental coccidiosis in broilers. METHODS: Diclazuril nanoemulsion (DZN) was formulated and characterized by zeta seizer and zeta potential. The formulated DZN was evaluated in vivo against Eimeria tenella infected chicks. DZN and DZ were used in 2 programs; therapeutic and prophylactic. A total of 210 one-day-old broiler chicks were distributed equally into six groups. The controls were negative uninfected untreated and positive infected untreated (G1 & G2). Therapeutic groups (G3 & G4) treated by DZ and DZN after appearance of the clinical signs of coccidiosis and continued for 5 days. Prophylaxis groups (G5 & G6) received DZ and DZN at 3 days before challenge and continued for 5 days after infection. The treatments dosages were 10 mg/mL for DZ of commercial origin and 2.5 mg/mL for the prepared DZN. All groups (except negative control) orally infected then followed up for clinical signs of coccidiosis, mortality rate, oocysts count, performance, hematological and biochemical parameters in addition to histopathological lesions. RESULTS: The therapeutic groups showed that both treated groups (DZ and DZN) revealed similar results including good body weight gain, a low lesion caecal score, a low daily and total oocyst shedding count, and a low mortality rate. Regarding the biochemical parameters, all parameters were affected during infection then restored after the 12th day post infection. However, in the prophylactic groups, showed mild clinical signs and the blood pictures and biochemical parameters were nearly like the control negative without infection. CONCLUSION: DZN at a quarter dose of standard DZ produced the same outcomes as DZ at 10 mg/mL. Furthermore, DZN does not impair the typical safety of diclazuril in treated chicks.

Dietary prebiotic alleviates experimentally induced coccidiosis in broilers.

An experiment was conducted to determine the effects of prebiotic on growth performance and coccidiosis prevention in challenged broilers with Eimeria. A total of 420 1-d-old male Ross 308 chicks were used in a completely randomized design with 7 treatments and 5 replicates with 12 birds in each replication. Dietary treatments were: 1) negative control (without prebiotic and without challenge), 2) positive control (challenged with sporulated oocysts of Eimeria (SOE) without prebiotic), 3) 0.2 % prebiotic in starter, 0.1 % in grower and 0.05 % in finisher challenged with SOE, 4) 0.2 % prebiotic in starter,0.1 % in grower and 0.05 % in finisher without challenge, 5) 0.2 % prebiotic in the whole rearing period challenged with SOE, 6) 0.2 % prebiotic in the whole rearing period without challenge and 7) and Salinomycin (0.05 % of diet). At 7 d of age, treatments were challenged with 20-fold dose of the EIMERIAVAX 4 m as a trivalent live attenuated coccidiosis vaccine. On d 28, intestinal coccidiosis lesions and dropping were scored in the scale of 0-3 and 0-4, respectively, and oocysts per gram feces (OPG) were measured. Prebiotic at either supplementation rate increased body weight gain and improved feed conversion ratio compared with PC group. Challenged broilers fed fixed level of prebiotic displayed lower OPG, dropping scores and coccidiosis lesions scores in upper and middle regions of intestine than PC group, with the effect being similar to unchallenged birds.

Effect of ethanol extract from Enterolobium cyclocarpum fruit on Leghorn chickens exposed to Eimeria.

There are concerns about residues of drugs in meat that are used to prevent coccidiosis in chickens. Natural compounds are an alternative to drugs. Two studies investigated the effect of an extract of Enterolobium cyclocarpum fruits (EEC) in the feed of male Leghorn chickens exposed to Eimeria spp. In the first experiment, the administration of EEC after infection with Eimeria spp. was investigated over 16 days. One thousand chickens were randomly housed in 20 pens of 1 m2 each. The pens were randomly assigned to each treatment. Five treatments were administered, containing 150, 300 and 450 mg/kg of EEC in the feed, the fourth treatment (C) contained 0.5 g/kg of a commercial anticoccidial, and the fifth treatment provided no treatment (WA). The second experiment lasted 18 days. Administration of the EEC began five days before the chickens were infected with Eimeria spp. Four hundred and eighty chickens were randomly allocated to 24 pens of 1 m2. The pens were randomly assigned to each treatment. In the second experiment, the same five treatments were tested and one additional treatment containing 300 mg EEC plus 1 g of polyethylene glycol (PEG)/kg of feed (E300PEG). In the experiment one chickens in the EEC treatments had lower faecal oocyst excretion (OE) on day 14 post infection with Eimeria spp., than chickens in the WA treatment (P < 0.05). A reduction in live weight gain (LWG) was observed in the EEC treatments (P < 0.05). In the second experiment, the excretion of oocysts in chickens from the EEC and E300PEG treatments on day 13 post-infection with Eimeria spp. was the same as in the C treatment and lower than in the WA treatment (P < 0.05). LWG was lower in the EEC treatments than in the C treatment (P < 0.05). However, the Chickens in the E300PEG and C treatments had similar LWG (P > 0.05) suggesting that PEG inhibits the negative effect of EEC tannins on LWG. In conclusion, the addition of EEC to chicken feed reduced both OE and LWG. Treatment with EEC and PEG (E300PEG) reduced the excretion of oocysts without negative effects on LWG.

Multicentric Study on Enteric Protists Occurrence in Zoological Parks in Portugal.

Parasitic infections of the gastrointestinal tract of domestic animals play a major role in the transmission of disease, which in turn may result in financial and productive losses. Notwithstanding, studies on the burden and distribution of diarrheagenic protists in zoological gardens are still insufficient. Given the close animal-animal and animal-human interaction in these settings, Public Health concerns under the One Health context are raised. Using molecular detection tools and phylogenetic analysis, the goal of this study was to assess the occurrence of four potentially zoonotic protists-Balantioides coli, Blastocystis sp., Cryptosporidium spp. and Giardia spp.-in animals residing in zoological parks in Portugal. Occurrence of Eimeria spp. was also assessed because of its veterinary relevance. Although Blastocystis sp. represents most of the positive samples obtained (11.6%; 95% CI: 0.08-0.17), all parasites were detected (B. coli (2.9%; 95% CI: 0.01-0.06), and Cryptosporidium spp., Eimeria spp. and Giardia spp. presented the same prevalence (0.5%; 95% CI: 0.00-0.03)). We also describe the first molecular detection of B. coli in a collared peccary (Tayassu tajacu), of Blastocystis sp. in three different python species, and G. muris in a central bearded dragon (Pogona vitticeps), suggesting the broadening of the host range for these parasites.

Comparison of the effect of anticoccidial drug, probiotic, synbiotic, phytochemicals and vaccine in prevention and control of coccidiosis in broiler chickens challenged with Eimeria spp.

The objective of this study was to investigate the effects of an anti-coccidiosis drug, vaccine, probiotic, symbiotic, and phytochemicals in the prevention and control of coccidia infection in broilers. A total of 525 one-day-old Ross 308 chicks were randomly allocated to 7 experimental diets with 5 replicates of 15 birds each in a completely randomized design. Experimental diets consisted of negative control (NC) without any additives and not challenged. The other 6 groups were challenged with mixed Eimeria and fed the basal diet with no additives (Positive Control, PC) or supplemented with Coxidine 100 (1 g / 1 kg), probiotic, synbiotic, Livacox T vaccine and phytobiotic additives based on the manufacturer's recommended dose. Body weight gain (WG), feed intake (FI) and feed conversion ratio (FCR) were recorded weekly. Oocysts per gram of excreta (OPG) were determined on d 25 to 33 and 42. One bird per cage was euthanized to analyze lesion score and jejunum and ileum inflammatory genes expression. Coccidial challenge reduced WG (P < 0.05) during 15 to 28 d and vaccine treatment was more effective in improving WG and FCR on d 29 to 42 and 1 to 42 (P < 0.05) than other treatments. Birds in the PC group had higher (P < 0.05) OPG than NC group for all days and the vaccine treatment resulted in the lowest rate of OPG compared to other treatments (P < 0.05) at 27, 28, 29, 30, 32, and 33 d of age and overall average. Relative mRNA levels of IFN-γ, IL-1ß and IL-10 were significantly upregulated among treatments under coccidiosis challenge in jejunum and ileum except for IL-1ß expression in the ileum. In conclusion, based on the results of this study the individual characteristics of feed additives for the prevention of coccidiosis can be different depending on the type and source of feed additives, duration, and amount used, levels of oocyst inoculation and Eimeria types.

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.

Phytase, 25-hydroxyvitamin D3 and cocci vaccination to broilers fed a calcium and phosphorus-reduced diet under Eimeria spp. challenge: effects on growth performance and intestinal health.

A study evaluated the effects of phytase, 25-hydroxyvitamin D3 (25OHD), and cocci vaccination on broilers fed a diet reduced in calcium (Ca) and available phosphorus (avP) under Eimeria challenge. A total of 840 one-day-old male broilers were assigned to a 2 × 5 factorial arrangement based on cocci vaccination and dietary treatments. Half of the birds were vaccinated against coccidia on d 1, and all birds were orally challenged with Eimeria spp. (sporulated oocysts: 12,500 of E. maxima, 12,500 of E. tenella, and 62,500 of E. acervulina) on d 14. Dietary treatments included: 1) a nutrient adequate diet (PC); 2) a diet reduced by 0.2% in Ca and avP (NC); 3) NC plus 1,500 FTU/kg of phytase (NC+PHY); 4) NC plus 3,000 IU/kg of 25OHD (NC+25OHD); 5) NC with both PHY and 25OHD (NC+PHY+25OHD). SAS was used for data analysis, with significance set at P ≤ 0.05. Pre-infection growth performance was comparable across the treatments. However, vaccinated birds exhibited higher body weight (BW) and body weight gain (BWG) from 0 to 6 d postinoculation (DPI; P < 0.05). The NC diet reduced BWG from 6 to 12 DPI and increased the feed conversion ratio (FCR) during 6 to 12 DPI and the overall period (0-26 d) compared to the PC birds. In contrast, the supplementation with phytase, 25OHD, or both, returned BWG and FCR to levels seen with the PC diet (P < 0.01). Vaccinated birds also had reduced gut permeability at 5 DPI, increased intestinal villus height, and lower expression levels of the tight junction proteins junctional adhesion molecule 2 (JAM2) and occludin (OCLN) at 6 DPI (P < 0.05). Interestingly, the cocci vaccine resulted in lower E. acervulina but higher E. tenella oocyst shedding at 6 DPI (P < 0.01). Interaction effects were observed for duodenal lesion scores and ileal crypt depth at 6 DPI (P < 0.05). In conclusion, coccidial vaccination improved growth performance, decreased intestinal permeability, enhanced intestinal morphology, and modulated tight junction protein gene expression under Eimeria infection. Reducing dietary Ca and avP levels adversely affected growth performance and FI during the recovery phase, but these negative effects could be mitigated by supplementing with phytase or 25OHD.

Dynamic response of the intestinal microbiome to Eimeria maxima-induced coccidiosis in chickens.

Eimeria maxima is a major cause of coccidiosis in chickens and a key predisposing factor for other economically significant diseases such as necrotic enteritis. However, a detailed understanding of the intestinal microbiome response to E. maxima infection is still lacking. This study aimed to comprehensively investigate the dynamic changes of the intestinal microbiome for 14 days post-infection (dpi) with E. maxima. Bacterial 16S rRNA gene sequencing was performed with the ileal and cecal digesta collected from mock and E. maxima-infected chickens at the prepatent (3 dpi), acute (5 and 7 dpi), and recovery phases (10 and 14 dpi) of infection. Although no notable changes were observed at 3 dpi, significant alterations of the microbiota occurred in both the ileum and cecum at 5 and 7 dpi. By 14 dpi, the intestinal microbiota tended to return to a healthy state. Notably, Lactobacillus was enriched in response to E. maxima infection in both the ileum and cecum, although individual Lactobacillus, Ligilactobacillus, and Limosilactobacillus species varied in the temporal pattern of response. Concurrently, major short-chain fatty acid-producing bacteria, such as Faecalibacterium, were progressively suppressed by E. maxima in the cecum. On the other hand, opportunistic pathogens such as Escherichia, Enterococcus, and Staphylococcus were significantly enriched in the ileum during acute infection. IMPORTANCE: We have observed for the first time the dynamic response of the intestinal microbiota to Eimeria maxima infection, synchronized with its life cycle. Minimal changes occur in both the ileal and cecal microbiota during early infection, while significant alterations coincide with acute infection and disruption of the intestinal mucosal lining. As animals recover from coccidiosis, the intestinal microbiota largely returns to normal. E. maxima-induced intestinal inflammation likely creates an environment conducive to the growth of aerotolerant anaerobes such as Lactobacillus, as well as facultative anaerobes such as Escherichia, Enterococcus, and Staphylococcus, while suppressing the growth of obligate anaerobes such as short-chain fatty acid-producing bacteria. These findings expand our understanding of the temporal dynamics of the microbiota structure during Eimeria infection and offer insights into the pathogenesis of coccidiosis, supporting the rationale for microbiome-based strategies in the control and prevention of this condition.

Research Note: Dynamics of black grouse (Tetrao tetrix) infestation with various Eimeria species.

The black grouse (Tetrao tetrix) is an endangered species facing challenges in breeding and reintroduction programs, including parasitic infestations. This study aimed to assess natural infestations by various Eimeria species and infestation dynamics in female, male, and young black grouse kept in a stationary aviary. Faecal samples were collected from adult grouses between April and the time of chicks' hatching and rearing (September). Faecal samples from young birds were collected from the hatching for a period of 1 year. The prevalence of Eimeria spp. was determined by a qualitative method (Fulleborn's flotation) and a quantitative method (McMaster's method with Raynaud's modification). The following Eimeria species were identified: E. lyruri, E. nadsoni and E. nonbrumpti. The average percentages of Eimeria spp. in the cock were 80.52%, 9.27%, and 10.21%, respectively; in the hen, they were 86.19%, 9.28%, and 4.53%, respectively; in the young black grouses they were 84.60%, 9.34% and 6.06%, respectively. The highest E. lyruri infestation was observed in the cock in June (144227 OPG) and July (129365 OPG). In the hen, the infestation intensity increased in May (304302 OPG) and then decreased in June (39583 OPG). Furthermore, an additional increase was observed in July (216533 OPG). Two increases in infestation intensity were also observed in young birds, with peaks in January (91387 OPG) and July (126178 OPG). A positive strong correlation was identified between Eimeria spp. in the cock and the young birds. A statistically significant positive correlation was identified in the hen between E. lyruri and E. nadsoni. No correlation was demonstrated between the infestation intensity and the age of the birds or season of the year in all the grouses under study. Despite some attempts, a comprehensive approach to the issue of coccidiosis in the black grouse as a disease that may affect the success of reintroduction has yet to be established. It seems crucial to monitor the level of Eimeria spp. invasion, and the proposed faecal sampling scheme is an important tool for achieving this goal.

Filters comprised of sand and Zero Valent Iron hold promise as tools to mitigate risk posed by Cyclospora cayetanensis oocysts.

Irrigation water contaminated by human fecal material may elevate the risk of produce contamination with the enteric parasite Cyclospora cayetanensis. Oocysts of C. cayetanensis are resistant to commonly used disinfectants and a method of removing C. cayetanensis from irrigation water would mitigate this risk. We evaluated zero valent iron (ZVI) sand filtration as one such method. We sought to determine if sand filters containing ZVI outperformed those without ZVI. We first evaluated the abundant poultry parasites Eimeria maxima, E. tenella and E. acervulina as surrogates for C. cayetanensis. We determined if a miniaturized gravity fed ZVI-sand filter, scaled to evaluate scarce supplies of C. cayetanensis oocysts, provided useful information about the performance of larger filtration systems. Filters were inoculated with oocysts, rinsed, and the resulting filtrate examined microscopically for oocysts. We performed experiments to measure the effect of varying ZVI concentrations, repeated filter use, simulated agricultural water, and oocyst size and condition. We then compared the performance of mini filters to that of larger, gravity-fed pool filters and found that ZVI-sand filtration was far more effective at removing Eimeria spp. from water when compared to sand filtration, at both scales. Sand mini filters retained 13-54 % of E. acervulina oocysts, and pool filters retained 82 %, but when combined with 50 % (mini filter) or 35 % (pool filter) v/v ZVI, mini filters retained 89-99 % of oocysts and pool filters retained >99 %. The effectiveness of the mini filters increased with increasing ZVI concentration, and the addition of ZVI far outweighed the influence of any other measured variable. We then performed experiments including C. cayetanensis, which provided similar results to those utilizing Eimeria; 59 % of inoculated C. cayetanensis oocysts were retained in sand mini filters, and 97 % in mini filters composed of 35 % v/v ZVI. In sum, ZVI is highly effective in removing oocysts from water and Eimeria is a useful surrogate for C. cayetanensis to assess filtration. ZVI-sand filtration shows promise as a tool to mitigate the risk of C. cayetanensis contamination of irrigation water. Further studies should evaluate the performance of ZVI-sand in pressurized fast filtration systems under a range of field conditions.