THE COCCIDIA SPECIES PFEIFFERINELLA ELLIPSOIDES EXHIBITS SPATIAL AND SPECIES-SPECIFIC VARIATION IN PREVALENCE AMONG FRESHWATER SNAILS.

Freshwater snails are commonly studied within the context of their role as intermediate hosts for digenetic trematodes. However, there are fundamental data deficiencies related to our understanding of directly transmitted parasites, such as coccidia, for freshwater snails. Because variation in coccidia pathogenicity and transmission among snail species likely has major impacts on snail community structure, we aimed to investigate the spatial distribution and prevalence of coccidia in several freshwater snail species throughout the Ozark and Ouachita Mountains ecoregions in Arkansas. We opportunistically collected 220 freshwater snails from 24 Ozark sites in summer 2022 and scanned fecal slides for the presence of coccidia. In summer 2023, we surveyed an additional 146 snails from 19 Ouachita sites. To test for apparent interactions among coccidia and trematodes, we scanned feces from a subset of snails (Physa and Planorbella in the Ozarks) that did not have concurrent trematode infections and from those that did. We observed oocysts that morphologically conformed to Pfeifferinella ellipsoides in 2 of the 9 snail taxa from 7 of the 43 sites. Planorbella trivolvis was infected at 2 of 6 sites in the Ozarks and 0 of 5 sites in the Ouachitas. Physa species were infected at 6 of 14 sites in the Ozarks and 0 of 12 sites in the Ouachitas. In the Ozarks, Pl. trivolvis had an overall prevalence of 0.13 (6 of 47), whereas individuals in the genus Physa had an overall prevalence of 0.08 (8 of 97). Our chi-square and Fisher exact tests revealed no significant evidence for trematode-coccidia competition or synergism within the two snail species. There were no other species infected, and we did not observe any coccidia in the snails from the Ouachitas. Our survey of 366 snails among 9 taxa and 43 sites represents the largest survey for freshwater snail coccidia to date and indicates that both Pl. trivolvis and Physa spp. may be primary hosts and/or reservoir hosts for Pf. ellipsoides in freshwater snail communities. The highly aggregated distribution of Pf. ellipsoides in northwestern Arkansas requires further investigation. Our results led to proposal of several hypotheses for additional research, including questions regarding the variation of coccidia host specificity and virulence.

Herbal-based compounds: A review on treatments of cryptosporidiosis.

Cryptosporidium, a monoxenous apicomplexan coccidia, is a prevalent diarrhetic and an opportunistic agent, mainly in immunocompromised individuals. As there are few chemotherapeutic compounds that have limited efficacy, we need to identify new compounds or specific parasite targets for designing more potent drugs to treat cryptosporidiosis. Herbal products with low toxicity, environmental compatibility, wide therapeutic potential, and abundant resources can be considered alternatives for treatment. The current review tried to summarize the studies on plants or herbal bioactive constituents with anti-cryptosporidial activities. Based on constituents, plants act via different mechanisms, and further investigations are needed to clarify the exact mechanisms by which they act on the developmental stages of the parasite or host-parasite relationships.

Immune synergistic mechanism of recombinant plasmid adjuvant containing chicken IL-4 and IL-2 fusion genes on chicken coccidia live vaccine.

The recombinant plasmid pCI-IL-4-IL-2-EGFP containing fusion genes of chicken IL-4 and IL-2 can be used as an adjuvant to enhance the anticoccidiosis effect of the chicken coccidia live vaccine. The chickens were divided into 3 groups: blank control group, vaccine + pCI-IL-4-IL-2-EGFP adjuvant coimmunization group, and vaccine-only group to investigate the immune synergy mechanism of recombinant plasmid adjuvant pCI-IL-4-IL-2-EGFP. The expressions of IL-2, IL-4, TNF-α, and IFN-γ in chicken sera and tissues were detected by ELISA and RT-qPCR, and the proliferation of T and B lymphocytes and antigen presenting cells (APC) in chicken immune organs and intestines were detected by acid alpha-naphthalase (ANAE) staining, methyl green pyronine (MGP) staining, and immunofluorescence (IF) staining, respectively. Results showed that the mRNA expression of IL-2, IL-4, IFN-γ and the number of activated T and B lymphocytes were significantly upregulated in the spleen and cecum tonsils of chickens in vaccine + pCI-IL-4-IL-2-EGFP group compared with the vaccine-only group on 7 d after vaccination (P < 0.05). Protein contents of IL-2, IL-4 and TNF-α in vaccine + pCI-IL-4-IL-2-EGFP group were significantly increased compared to vaccine-only group on 28 d of inoculation (P < 0.05). The number of T and B lymphocytes and APC in chickens of the vaccine+ pCI-IL-4-IL-2-EGFP group was significantly higher than that of the vaccine-only group in cecum tonsils, thymus and spleen after 14 and 28 d of inoculation (P < 0.05). All results revealed that pCI-IL-4-IL-2-EGFP adjuvant enhanced the immune response of chicken coccidia live vaccine by upregulating the expression of IL-2, IL-4, TNF-α, and IFN-γ and promoting the proliferation of T, B lymphocytes and APCs in chicken intestines and immune organ sites. Moreover, our study provides a theoretical basis for the clinical application of cytogenic plasmids as adjuvants.

Morphological and molecular biology identification of Cystoisospora sp. in the blue fox, Alopex lagopus (Linnaeus, 1758).

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

First Record, Distribution and Occurrence of A Protistan Entomopathogen, Adelina mesnili Perez (Coccidia: Adeleidae) in the Indian Meal Moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) Populations in Türkiye.

OBJECTIVE: Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) originating from South America is one of the important insect pests that damages storage products and is found on every continent. There is a new interest in using enthomopathogens for microbial control of P. interpunctella as well as other stored product pests. Coccidia as a group of protistan entomopathogens are host specific and their pathogenic effects on the hosts are more pronounced. Although this pathogenic effect results in increased host mortality or higher susceptibility to insecticides, the suppressive potential of coccidia in natural populations has not been adequately studied. In this study, characterization, distribution and occurrence of a coccidian entomopathogen was aim to show its natural suppressing potential in P. interpunctella populations. METHODS: During the three years (from 2019 to 2021), a total of 3.432 P. interpunctella samples (2.047 dead and 413 living larvae, 932 adults and 40 pupae) were collected from fourteen populations. After macroscopic examination, suspected samples were dissected in Ringer's solution and then prepared wet smears including host fat body were examined for presence of coccidian pathogens under a light microscope at a magnification of 400-1000X. The oocysts of the coccidian were measured and photographed using a microscope with a digital camera and soft imaging system. RESULTS: The pathogen was observed in the fat bodies of the larvae, pupae and adults. Oocysts measured as 29.52±3.32 (25.27- 35.08) µm in diameter and they include 8 sporocysts. Sporocysts measured as 9.11±0.61 (8.90-9.85) µm. Forty-five of 3.432 P. interpunctella larvae, pupae and adults were found to be infected. Coccidian infections have also reached to the levels that can be considered high in some populations, as significant as 29.2%. The infection was observed in the three (21.4%) of the examined fourteen populations. CONCLUSION: The coccidian entomopathogen presented in this study is the first Adeleid coccidian record from P. interpunctella populations in Türkiye. The detection of Adelina mesnili Perez (Coccidia: Adeleidae) in at least three populations and the infection rate reaching 29.2 percent, confirms that this pathogen has a considerable effect P. interpunctella populations that cannot be underestimated. Our results confirm that the coccidian pathogen is very effective in the larval stage.

Host specificity of passerine Lankesterella (Apicomplexa: Coccidia).

Lankesterella parasites are blood coccidians that have recently gained attention as their records in common passerine species emerge. To date, their occurrence has been molecularly confirmed in several passerine genera, mainly among members of the families Paridae and Acrocephalidae. Despite their relatively high prevalence in some host populations, their life cycles remain unclear, mosquitoes or mites being the proposed vectors. The aim of this study was to reveal Lankesterella host specificity, focusing mainly on parasites of tit and warbler species (families Paridae and Acrocephalidae). We have determined the 18S rRNA gene sequences of Lankesterella from 35 individuals belonging to eight different host species. Phylogenetic analysis revealed that passerine Lankesterella are host-specific, with specificity at the host genus or species level. Besides Lankesterella, Isospora sequences were obtained from avian blood as well, pointing out the need for barcoding.

Construction of recombinant SAG22 Bacillus subtilis and its effect on immune protection of coccidia.

Avian coccidiosis causes huge economic losses to the global poultry industry. Vaccine is an important means to prevent and control coccidiosis. In this study, Bacillus subtilis was selected as the expression host strain to express anti Eimeria tenella surface protein SAG22. The synthesized surface protein SAG22 gene fragment of E. tenella was ligated with Escherichia coli-bacillus shuttle vector GJ148 to construct the recombinant vector SAG22-GJ148. And then the recombinant Bacillus strain SAG22-DH61 was obtained by electrotransfer. The results of SDS-PAGE and Western Blot showed that the recombinant protein SAG22 was successfully expressed intracellularly. The immunoprotective effect of recombinant Bacillus strain SAG22-DH61 on broiler chickens was evaluated in 3 identically designed animal experiments. The birds were infected with E. tenella on d 14, 21, and 28, respectively. Each batch of experiments was divided into 4 groups: blank control group (NC), blank control group + infected E. tenella (CON), addition of recombinant SAG22-DH61 strain + infected with E. tenella (SAG22-DH61), addition of recombinant empty vector GJ148-DH61 strain + infected with E. tenella (GJ148-DH61). The animal experiments results showed that the average weight gain of the SAG22-DH61 group was higher than that of the infected control group, and the difference was significant in the d 14 and 28 attack tests (P < 0.05); the oocyst reduction rate of the SAG22-DH61 group was much higher than that of the GJ148-DH61 group (P < 0.05); the intestinal lesion count score of the SAG22-DH61 group was much lower than that of the GJ148-DH61 group (P < 0.05). In addition, the SAG22-DH61 group achieved highly effective coccidia resistance in the d 14 attack test and moderately effective coccidia resistance in both the d 21 and 28 attack tests. In summary, recombinant SAG22 B. subtilis has the potential to become one of the technological reserves in the prevention and control of coccidiosis in chickens in production.

Isolation of saprophytic filamentous fungi from avian fecal samples and assessment of its predatory activity on coccidian oocysts.

Fungal strains used in the biocontrol of animal gastrointestinal parasites have been mainly isolated from pasture soil, decaying organic matter, and feces from herbivores and carnivores. However, their isolation from birds and assessment of predatory activity against avian GI parasites has been scarce thus far. This research aimed to isolate filamentous fungi from avian fecal samples and evaluate their predatory activity against coccidia. A pool of 58 fecal samples from chickens, laying hens, and peacocks, previously collected between July 2020-April 2021, were used for isolation of filamentous fungi and assessment of their in vitro predatory activity against coccidian oocysts, using Water-Agar medium and coprocultures. The Willis-flotation technique was also performed to obtain concentrated suspensions of oocysts. A total of seven Mucor isolates was obtained, being the only fungal taxa identified, and all presented lytic activity against coccidia. Isolates FR3, QP2 and SJ1 had significant coccidiostatic efficacies (inhibition of sporulation) higher than 70%, while isolates FR1, QP2 and QP1 had coccidicidal efficacies (destruction of the oocysts) of 22%, 14% and 8%, respectively, after 14 days of incubation, being a gradual and time-dependent process. To our knowledge, this is the first report regarding the isolation of native predatory fungi from avian feces and demonstration of their lytic activity against coccidia.

Effects of feeding spray-dried plasma to broiler breeders and their progeny on broiler performance under stressful rearing conditions of coccidial challenge and heat stress.

A total of 216 Cobb 500 broiler breeder hens were randomly distributed across 2 dietary treatments with 0 or 1% spray-dried plasma (SDP) resulting in 27 replications/treatment and 4 birds/replication. In addition, 36 roosters were divided between the same treatments and housed, in individual pens, being each bird considered a replicate. Experimental diets were fed from 26 wk until 65 wk of age. At 29, 45, and 63 wk of age, broiler breeder hens were inseminated, and eggs were incubated. Three progeny studies were performed and hatched birds were allocated in a randomized 2 × 2 factorial design (maternal diet: with or without inclusion of 1% SDP × progeny diet: with or without the inclusion of 2% SDP from 1 to 7 d of age). After 7 d of age, all birds received the same diet until 42 d. In all trials, birds were challenged with coccidiosis vaccine at 7 d of age. Furthermore, in the second experiment, heat stress was also included for 6 h a day during the whole trial. At 42-days posthatch in the first experiment, greater FI, BW, and BWG in chicks hatched from breeders fed 1% dietary SDP was observed. This effect did not extend to the other hatches. In the second trial, a decreased FCR in broilers fed the control diet from breeder hens fed 1 % of SDP was observed, as well as an interaction between the SDP groups, since broilers supplemented with SDP and originated from breeders fed SDP showed higher BW and BWG in comparison to other groups at 42 d. In the third trial, contrary to what was observed in the first study, SDP supplementation did not affect any of the performance indexes. In the 3 studies, no differences were found in carcass characteristics. SDP did not affect hen BW, egg production, fertility, or hatch of fertile. These results suggest that providing dietary SDP to broilers has some beneficial effects on broiler chickens.

Occurrence of Apicomplexa protozoa in wild birds in the Northeast region of Brazil.

Protozoa of the Apicomplexa phylum are worldwide distributed with capacity to infect endothermic animals. The study of these protozoa in wild birds in Brazil is scarce. This study aimed to evaluate the occurrence of apicomplexan protozoa in wild birds in the Northeast of Brazil. From October to December 2019, brain tissue samples were collected from 71 captive birds from the Wild Animal Screening Center of the Pernambuco State (CETRAS-Tangara) and 25 free-living birds from the Caatinga biome in Rio Grande do Norte, totaling 96 animals (41 species). Brain fragments were subjected to molecular diagnosis by nested PCR for the 18s rDNA gene of Apicomplexa parasites, followed by DNA sequencing. This gene was detected in 25% (24/96) of the samples, and it was possible to perform DNA sequencing of 14 samples, confirming three genera: Isospora, Sarcocystis and Toxoplasma from eight bird species (Amazona aestiva, Coereba flaveola, Egretta thula, Paroaria dominicana, Sporophila nigricollis, Cariama cristata, Columbina talpacoti, Crypturellus parvirostris). The occurrence these coccidia in wild birds provides important epidemiological information for the adoption of preventive measures for its conservation. Future studies are needed to better understand the consequence of Apicomplexa infection in birds in Caatinga and Atlantic Forest biomes.

Effect of benzoic acid, Enterococcus faecium, and essential oil complex on intestinal microbiota of laying hens under coccidia and Clostridium perfringens challenge.

The objective of this study was to investigate whether dietary supplementation with benzoic acid, Enterococcus faecium, and essential oil complex (BEC) could help laying hens recover from coccidia and Clostridium perfringens type A challenge. A total of 60 (35-wk-old) Lohmann-laying hens were randomly assigned to 3 experimental groups (10 replicates with 2 hens per replicate): I) control group (CON), II) challenge group (CC), and III) BEC group (2,000 mg/kg BEC). The total experimental period was 8 wk. The results shown that the challenge layers had lower egg-laying rate and average daily feed intake (ADFI) (P < 0.05), and addition of BEC after challenge increased egg-laying rate (P < 0.05). The content of propionic acid (PA) and butyric acid (BA) in short-chain fatty acid (SCFA) was significantly decreased by challenge (P < 0.05). CC and BEC groups had lower villus height to crypt depth ratio (V/C) and higher pathological scores in duodenum (P < 0.05), whereas the BEC group had lower pathological scores in jejunum when compared with the CC group (P < 0.05). The challenge increased the concentration of proinflammatory cytokines (IL-1ß and IL-6) (P < 0.05). An increase in the abundance of Bacteroidoes (genus), Bacteroidaceae (family), Bacteroidoes sp. Marseille P3166 (species), Bacteroidoes caecicola (species) was observed in the CC group, whereas the BEC group had higher abundance of Bacteroides caecigallinarum (species). The genera Faecalibacterium and Asterolplasma were positively correlated with egg-laying rate (r = 0.57, 0.60; P < 0.01); and the genera Bacteroides and Romboutsia were negatively correlated with egg-laying rate (r = -0.58, -0.74; P < 0.01). The genera Bacteroides, Lactobacillus, and Rombutzia were positively correlated with jejunal mucosa proinflammatory factor IL-1ß level (r = 0.61, 0.60, 0.59; P < 0.01), which were negatively correlated with genera Rikenbacteriaceae RC9, Faecalibacterium, and Olsenlla (r = -0.56, -0.57, -0.61; P < 0.01). There genera UCG.005 was positively correlated with proinflammatory factor IL-6 level in jejunal mucosa (r = 0.58; P < 0.01), which was negatively correlated with Rikenbacteriaceae RC9 (r = -0.62; P < 0.01). The experiment results revealed that the addition of BEC to the diet restored the production performance of the laying hens. In addition, supplementation of 2,000 mg/kg BEC modulated gut health by reducing gut damage scores and modulating microbial composition, thereby promoting recovery of laying hens after coccidia and Clostridium perfringens challenge.

Prevention and control of chicken coccidiosis construction of recombinant Lactococcus lactis expressing chicken IL-4 and IL-2 fusion protein and its immune synergistic effect on chicken coccidia live vaccine.

Intestinal mucosa injury and loss of weight gain are unavoidable while using live vaccine strain to prevent chicken coccidiosis. In this study, recombinant Lactococcus lactis NZ3900/pNZ8149-IL-4-IL-2, expressing the fusion protein of chicken IL-4 and IL-2, was constructed using food-grade NICE expression system, trying to develop a possible oral immune adjuvant to enhance the immune effect of the live vaccine against chicken coccidiosis and minimize its adverse effects. Chickens were given different doses of recombinant L. lactis together with the live vaccine, then experimently attacked with coccidia virulent strains. Results showed that weight gains of co-immunization groups, given both 1 × 109 or 1 × 1010 CFU recombinant L. lactis and the live vaccine, were significantly higher than the vaccine-only group (P<0.05), while intestinal lesion scores of duodenum, jejunum, and cecum were significantly lower than the vaccine-only group (P<0.05), so was the oocyst shedding. The anticoccidial indexes (ACI) of the co-immunized groups given 1 × 109 and 1 × 1010 CFU recombinant L. lactis were 187.85 and 193.33, respectively, higher than 174.61 of the vaccine-only group. In addition, chickens in co-immunization groups gained more body weight than the vaccine-only group before being challenged with the virulent strains (P<0.05). All the results indicated that the constructed recombinant L. lactis NZ3900/ pNZ8149-IL-4-IL-2 exhibited an immune synergistic function to coccidiosis live vaccine, and could alleviate its adverse effect affecting weight gain. The application of the recombinant L. lactis showed the potency to lift the anticoccidial efficiency of the live vaccine from a medium level to a high level.

First report of gastrointestinal nematodes and coccidia parasites from free-range chickens in Mafeteng district, Lesotho.

Free-range chickens are an integral part of poultry production in many developing countries. In the Mountain Kingdom of Lesotho, the majority of the population own free-range chickens, which serve a variety of purposes including being a source of meat, eggs and use for cultural rituals amongst others. However, there is lack of scientific studies on occurrence of parasitic infections on free-range chickens in Lesotho. The aim of this study was to document common gastrointestinal parasites infecting free-range chickens in four villages of Mafeteng District in Lesotho. A total number of 462 pooled faecal samples were collected from various households in HaKubutu (n = 114), HaMatjeka (n = 120), HaMpalipali (n = 120) and Thabang Villages (n = 108) which were subjected to microscopic examination using McMaster technique. The prevalence of gastrointestinal parasite infection was as follows: Eimeria tenella (12.8%), Ascaridia galli (10.4%) and Heterakis gallinarum (5%). The prevalence for H. gallinarum and Ascaridia galli were comparatively higher during the hot-wet season (7.1% and 2.8% respectively) than the cold-dry season (3.2% and 1.9% respectively) and varied significantly (P < 0.05). For E. tenella, the oocysts per gram were slightly higher in the cold-dry season than the hot-wet season. Polymerase chain reaction only amplified DNA from six (29%) adult A. galli worms of which two amplicons were successfully sequenced. The obtained cytochrome C oxidase subunit 1 partial gene sequences displayed 98-100% identity with South African A. galli isolates. This is the first scientific study on prevalence and molecular characterization of nematodes and coccidia species infecting free-range village chickens in Lesotho. The findings can be used to review management of gastrointestinal nematodes and protozoal parasites of free-range chickens in Lesotho.

Inhibition of sexual stage-specific proteins results in reduced numbers of sexual stages and oocysts of Cystoisospora suis (Apicomplexa: Coccidia) in vitro.

Parasites of the order Coccidia (phylum: Alveolata, subphylum: Apicomplexa) have sophisticated life cycles that include a switch from asexual to sexual development, characterised by distinct cell types. During the development of gametes (gamogony), substantial changes occur at the cellular and subcellular levels, leading to cell fusion of micro- and microgametes, and the development of a zygote that forms a protective outer layer for environmental survival as an oocyst, the transmissible stage. Studies on the porcine coccidian Cystoisospora suis already identified changes in transcription profiles during different time points in the parasite's development and identified proteins with potential roles in the sexual development of this parasite. Here, we focus on three proteins that are possibly involved in the sexual development of C. suis. Enkurin and hapless protein 2 (HAP2) play important roles in signal transduction and gamete fusion during the fertilisation process, and oocyst wall forming protein 1 (OWP1) is a homologue of oocyst wall forming proteins of related parasites. We evaluated their locations in the different life cycle stages of C. suis and their inhibition by specific antibodies in vitro. Immunolocalization detected enkurin in merozoites and sporulated oocysts, HAP2 in merozoites and microgamonts, and OWP2 in merozoites, macrogamonts, oocysts and sporozoites. Up to 100% inhibition of the development of sexual stages and oocyst formation with purified chicken immunoglobulin IgY sera against recombinant enkurin, HAP2, and especially OWP1, were demonstrated. We conclude that the three investigated sexual stage-specific proteins constitute targets for in vivo intervention strategies to interrupt parasite development and transmission to susceptible hosts.

A Coccidia-Specific Phosphate Transporter Is Essential for the Growth of Toxoplasma gondii Parasites.

Toxoplasma gondii is an obligate intracellular parasite that acquires all necessary nutrients from the hosts, but the exact nutrient acquisition mechanisms are poorly understood. Here, we identified three putative phosphate transporters in T. gondii. TgPiT and TgPT2 are mainly on the plasma membrane, whereas TgmPT is localized to the mitochondrion. TgPiT and TgmPT are widely present and conserved in apicomplexan parasites that include Plasmodium and Eimeria species. Nonetheless, they are dispensable for the growth and virulence of Toxoplasma. TgPT2, on the other hand, is restricted to coccidia parasites and is essential for Toxoplasma survival. TgPT2 depletion led to reduced motility and invasion, as well as growth arrest of the parasites both in vitro and in vivo. Both TgPiT and TgPT2 have phosphate transport activities and contribute to parasites' inorganic phosphate (Pi) absorption. Interestingly, the Pi importing activity of Toxoplasma parasites could be competitively inhibited by ATP and AMP. Furthermore, direct uptake of 32P-ATP was also observed, indicating the parasites' ability to scavenge host ATP. Nonetheless, ATP/AMP import is not mediated by TgPiT or TgPT2, suggesting additional mechanisms. Together, these results show the complex pathways of phosphate transport in Toxoplasma, and TgPT2 is a potential target for antitoxoplasmic intervention design due to its essential role in parasite growth. IMPORTANCE To grow and survive within host cells, Toxoplasma must scavenge necessary nutrients from hosts to support its parasitism. Transporters located in the plasma membrane of the parasites play critical roles in nutrient acquisition. Toxoplasma encodes a large number of transporters, but so far, only a few have been characterized. In this study, we identified two phosphate transporters, TgPiT and TgPT2, to localize to the plasma membrane of Toxoplasma. Although both TgPiT and TgPT2 possess phosphate transport activities, only the novel transporter TgPT2 was essential for parasite growth, both in vitro and in vivo. In addition, TgPT2 and its orthologs are only present in coccidia parasites. As such, TgPT2 represents a potential target for drug design against toxoplasmosis. In addition, our data indicated that Toxoplasma can take up ATP and AMP from the environment, providing new insights into the energy metabolism of Toxoplasma.

Long-term temporal trends in gastrointestinal parasite infection in wild Soay sheep.

Monitoring the prevalence and abundance of parasites over time is important for addressing their potential impact on host life histories, immunological profiles and their influence as a selective force. Only long-term ecological studies have the potential to shed light on both the temporal trends in infection prevalence and abundance and the drivers of such trends, because of their ability to dissect drivers that may be confounded over shorter time scales. Despite this, only a relatively small number of such studies exist. Here, we analysed changes in the prevalence and abundance of gastrointestinal parasites in the wild Soay sheep population of St. Kilda across 31 years. The host population density (PD) has increased across the study, and PD is known to increase parasite transmission, but we found that PD and year explained temporal variation in parasite prevalence and abundance independently. Prevalence of both strongyle nematodes and coccidian microparasites increased during the study, and this effect varied between lambs, yearlings and adults. Meanwhile, abundance of strongyles was more strongly linked to host PD than to temporal (yearly) dynamics, while abundance of coccidia showed a strong temporal trend without any influence of PD. Strikingly, coccidian abundance increased 3-fold across the course of the study in lambs, while increases in yearlings and adults were negligible. Our decades-long, intensive, individual-based study will enable the role of environmental change and selection pressures in driving these dynamics to be determined, potentially providing unparalleled insight into the drivers of temporal variation in parasite dynamics in the wild.

Effects of phytase and coccidial vaccine on growth performance, nutrient digestibility, bone mineralization, and intestinal gene expression of broilers.

A study was conducted to evaluate effects of phytase and coccidial vaccine on growth performance, bone mineralization, nutrient digestibility, and intestinal gene expression of broiler chickens. The experiment was conducted in a 2 × 4 completely randomized factorial arrangement with 6 replicates per treatment and 10 birds each. Applications of coccidiosis vaccine and different dietary treatments were the 2 main factors in the current study. The dietary treatments included 1) a positive control (PC; 0.90% Ca and 0.45% available P: avP); 2) a negative control (NC; 0.75% Ca and 0.30% AvP); 3) NC + 500 FTU/kg of phytase (NC + 500PHY); and 4) NC + 1500 FTU/kg of phytase (NC + 1500PHY). Data were analyzed using SAS by 2-way ANOVA via GLM procedure. The statistical significance was set at P ≤ 0.05, and means were further separated using Tukey's Test. The results indicated that vaccination had no effect on growth performance except for feed intake from 0 to 14 d but negatively (P < 0.05) regulated bone ash and Ca digestibility. Birds fed with the Ca and P-reduced diet (NC) showed a lower BWG and bone ash compared to birds fed with the normal diet (PC), but supplementing phytase mitigated the negative effects on those birds. Broilers fed the NC diet had higher (P < 0.05) total Ca and P digestibility, and phytate degradation; supplementing phytase further increased P digestibility and phytate degradation of the broilers. A significant interaction (P < 0.05) between phytase and vaccination was observed, suggesting the vaccinated birds fed the PC diet and the unvaccinated birds fed the NC + 1500PHY increased calcium-sensing receptor gene expression compared with the unvaccinated birds fed the PC diet. In conclusion, in spite of coccidiosis vaccine, supplementing phytase at 1,500 FTU/kg alleviated the negative effects on growth performance, bone mineralization, and apparent ileal digestibility of P and phytate.

First report of Caryospora sp. infection in free-living green turtles (Chelonia mydas) in Northeastern Brazil.

Infection by Caryospora cheloniae has been reported to be responsible for green turtle strandings with high morbidity and mortality rates worldwide. Although studies have already shown the pathogenesis of these infections, many aspects of this protozoan are still poorly understood, including their life cycle and infection dynamics in free-living sea turtle populations. Due to the lack of information about the infection by this protozoan in sea turtles in Northeastern Brazil, our study aims to describe Caryospora sp. infection and its pathological findings in free-living Chelonia mydas found on the north coast of the Bahia state. Between 2018 and 2019, 64 specimens of green turtles were necropsied in partnership with Fundação Projeto Tamar; among these, 10 (1.56%) had oocysts morphologically compatible with Caryospora cheloniae in the evaluation of fecal samples and histopathological examination of intestinal samples. The infected animals were juvenile green turtles that were found stranded on the beaches of the north coast of Bahia. The pathological findings were restricted to the lower gastrointestinal tract, with different presentations and intensities. About 70% of the animals with coccidial infection exhibited erosive and ulcerative fibrinous enteritis. This is the first report of coccidiosis in green turtles on the north coast of Bahia.

The transcriptome from asexual to sexual in vitro development of Cystoisospora suis (Apicomplexa: Coccidia).

The apicomplexan parasite Cystoisospora suis is an enteropathogen of suckling piglets with woldwide distribution. As with all coccidian parasites, its lifecycle is characterized by asexual multiplication followed by sexual development with two morphologically distinct cell types that presumably fuse to form a zygote from which the oocyst arises. However, knowledge of the sexual development of C. suis is still limited. To complement previous in vitro studies, we analysed transcriptional profiles at three different time points of development (corresponding to asexual, immature and mature sexual stages) in vitro via RNASeq. Overall, transcription of genes encoding proteins with important roles in gametes biology, oocyst wall biosynthesis, DNA replication and axonema formation as well as proteins with important roles in merozoite biology was identified. A homologue of an oocyst wall tyrosine rich protein of Toxoplasma gondii was expressed in macrogametes and oocysts of C. suis. We evaluated inhibition of sexual development in a host-free culture for C. suis by antiserum specific to this protein to evaluate whether it could be exploited as a candidate for control strategies against C. suis. Based on these data, targets can be defined for future strategies to interrupt parasite transmission during sexual development.

Alleviating effect of dietary supplementation of benzoic acid, Enterococcus faecium and essential oil complex on coccidia and Clostridium perfringens challenge in laying hens.

The purpose of this experiment is to explore the effects of dietary supplementation of benzoic acid, Enterococcus faecium, and essential oil complex (BEC) on coccidia and Clostridium perfringens challenge in laying hens. A total of 80 Lohmann gray laying hens (35 wk old) were allocated to 4 treatments in a 2 × 2 factorial arrangement with the main effects of Clostridium perfringens type A (CP) and coccidia challenge (with or without challenge) and 2 BEC levels (0 and 1,000 mg/kg). The total experimental period was 6 wk. The results showed that: the challenge group significantly decreased the laying rate and average daily feed intake (ADFI) of laying hens (PChallenge < 0.01). The BEC + challenge group significantly increased the laying rate and decreased the feed conversion ratio (FCR) of laying hens (PBEC < 0.05). The challenge significantly decreased the thickness, strength, and relative weight of eggshell (PChallenge < 0.05). The BCE + challenge group significantly increased the relative weight and strength of the eggshell (PBEC < 0.05). The challenge significantly increased the crypt depth of the duodenum, jejunum and ileum, and decreased the villus-to-crypt ratio (V/C) (PChallenge < 0.01). The BEC + challenge group decreased the crypt depth of the duodenum and jejunum, and increased the V/C of the duodenum (PBEC < 0.01). The pathological scores of duodenum and jejunum of the challenge group were significantly higher than other groups (PChallenge < 0.01), while the BEC + challenge group had lower pathological scores of jejunum (PBEC < 0.01). The challenge significantly decreased the mRNA expression of Occludin, Mucin-2, Zonula occluden-1 (ZO-1) (Pchallenge < 0.05); whereas the BEC group significantly increased the expression of Occludin, Mucin-2, and Claudin-1 mRNA (PBEC < 0.05). The challenge significantly increased the level of interleukin 1ß (IL-1ß) in the jejunum (PChallenge < 0.05). Taken together, adding BEC to the diet can improved production performance and egg quality of layers, by protecting intestinal health against Clostridium perfringens type A (CP) and coccidia challenge.