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Despite their wide occurrence, cryptosporidiosis and giardiasis are considered neglected diseases by the World Health Organization. The epidemiology of these diseases and microsporidiosis in humans in developing countries is poorly understood. The high concentration of pathogens in raw sewage makes the characterization of the transmission of these pathogens simple through the genotype and subtype analysis of a small number of samples. The distribution of genotypes and subtypes of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in 386 samples of combined sewer systems from Shanghai, Nanjing and Wuhan and the sewer system in Qingdao in China was determined using PCR-sequencing tools. Eimeria spp. were also genotyped to assess the contribution of domestic animals to Cryptosporidium spp., G. duodenalis, and E. bieneusi in wastewater. The high occurrence of Cryptosporidium spp. (56.2%), G. duodenalis (82.6%), E. bieneusi (87.6%), and Eimeria/Cyclospora (80.3%) made the source attribution possible. As expected, several human-pathogenic species/genotypes, including Cryptosporidium hominis, Cryptosporidium meleagridis, G. duodenalis sub-assemblage A-II, and E. bieneusi genotype D, were the dominant parasites in wastewater. In addition to humans, the common presence of Cryptosporidium spp. and Eimeria spp. from rodents indicated that rodents might have contributed to the occurrence of E. bieneusi genotype D in samples. Likewise, the finding of Eimeria spp. and Cryptosporidium baileyi from birds indicated that C. meleagridis might be of both human and bird origins. The distribution of Cryptosporidium species, G. duodenalis genotypes and subtypes, and E. bieneusi genotypes in urban wastewater indicates that anthroponotic transmission appeared to be important in epidemiology of cryptosporidiosis, giardiasis, and microsporidiosis in the study areas. The finding of different distributions of subtypes between Shanghai and Wuhan was indicative of possible differences in the source of C. hominis among different areas in China.

The emergence of coinfection with Toxoplasma gondii , Neospora caninum , Eimeria stiedai , Giardia lamblia , and Trypanosoma evansi is an important problem that endangers human health, animal quality and public sanitary security. These pathogenic protozoans play important roles in establishment of similar clinical signs of diseases in humans, pigs, sheep, and rabbits, including fever, diarrhea, hepatitis, encephalitis, and reproductive disorders. Therefore, a rapid and specific diagnostic method to simultaneously detect these five pathogens is urgently required. Here, we developed a TaqMan-probe-based quantitative real-time polymerase chain reaction (qPCR) for the simultaneous detection of these five pathogens for the first time. Specific primers and probes were designed targeting the G3PDH gene of Toxoplasma gondii , the NC5 gene of Neospora caninum , the ADF gene of Eimeria stiedai , the GDH gene of Giardia lamblia , the COX1 gene of Trypanosoma evansi , and a TaqMan-probe-based pentaplex qPCR assay capable of simultaneously detecting these five pathogens was developed. The assay showed strong specificity, with no cross-reactivity detected against nucleic acids from other control pathogens. The assay demonstrated high sensitivity, with the lower limit of quantification (LLOQ) of 10 copies per reaction for the recombinant plasmid standards pTgG3PDH, pNcNC5, pEsADF, pGlGDH, pTeCOX1, and the limit of detection (LOD) as low as 1.1 copies. The standard curves exhibited excellent linearity (correlation coefficient values of 0.996, 0.996, 0.996, 0.992, and 0.996, respectively) and high amplification efficiencies (95.534%, 96.203%, 107.818%, 100.851%, and 104.487%, respectively). The assay also exhibited excellent repeatability and reproducibility, with inter- and intra-assay coefficient of variation (CV) ranging from 0.07% to 2.13%. The anti-interference test showed that high-concentration nucleic acids did not interfere with low-concentration nucleic acids, thus ensuring excellent detection results even in complex samples. Among 210 clinical samples, the assay detected Toxoplasma gondii in 2.38%, Neospora caninum in 2.38%, Eimeria stiedai in 10%, and Giardia lamblia in 36.19%. The coinfection rates were 8.1% for Eimeria stiedai & Giardia lamblia . This TaqMan-probe-based pentaplex qPCR assay offers a rapid, sensitive, and specific tool for the simultaneous detection of Toxoplasma gondii , Neospora caninum , Eimeria stiedai , Giardia lamblia , and Trypanosoma . It is of great significance to safeguard human health.

The purpose of this study was to determine if Eimeria oocyst concentrations and species composition in commercial broiler house litter changed during different cycles of anticoccidial drug (ACD) or live Eimeria oocyst vaccine (VAC) control programs and if there was a correlation between Eimeria oocyst levels and broiler performance. Litter samples were collected from a total of 15 different broiler farms encompassing a total of 45 individual houses during at least one complete grow-out cycle over a 21-mo period. Of these 15 broiler farms, three were followed for the entire 21-mo period spanning three ACD and four VAC cycles. Samples were collected at 2, 4, and 7–8 wk of grow-out corresponding to starter, grower, and withdraw periods of the ACD cycle. On a number of occasions, litter samples were obtained just prior to chick placement. Eimeria oocysts were isolated from all samples, counted by microscopy, and extracted for DNA to identify Eimeria species by ITS1 PCR. In general, Eimeria oocyst concentration in litter reached peak levels at 2–4 wk of grow-out regardless of coccidiosis control measure being used. However, peak oocyst numbers were sometimes delayed until 7–8 wk, indicating some level of Eimeria spp. drug resistance or incomplete vaccine coverage. Eimeria maxima, Eimeria acervulina, Eimeria praecox, and Eimeria tenella were generally present in all samples, and no difference in the species composition was noted between houses on a particular farm. While Eimeria species composition was similar among houses, Eimeria spp. oocyst levels exhibited sporadic peaks in one house of a given location's houses. Of particular interest was the observed correlation between E. maxima oocyst abundance and chick mortality. However, no correlation was observed in E. maxima oocyst levels, and the performance parameters adjusted feed conversion ratio and average daily weight gain. This study showed that understanding the dynamics of Eimeria spp. oocyst levels and species composition in litter during ACD or VAC programs may provide insight into the effectiveness of coccidiosis control measures in commercial broiler production.

Swine coccidiosis is a host-specific protozoan disease caused by Cystoisospora suis and various Eimeria species, leading to diarrhea or subclinical signs in pigs. In this study, 3296 fecal samples from 55 farms across six provinces in China were collected and examined to determine the prevalence and molecular characteristics of swine coccidia. The single oocyst isolation technique (SOIT) and molecular characterization identified nine coccidian species, with an overall infection prevalence of 13.83%. Infection rates varied by locations, host age groups, and sampling seasons. Among the positive swine coccidia samples, Cystoisospora suis showed the highest prevalence at 58.77%, followed by eight Eimeria spp . species. SOIT along with molecular characterization of C. suis at the SSU rRNA and ITS rRNA gene loci revealed the sequence homology from 99.8 to 100.0%. Additionally, the eight species of Eimeria . exhibited a high degree of sequence homology at the SSU rRNA and COX I gene loci. Due to the limitation of single molecular marker genes, this study introduced SOIT for DNA extraction, followed by nested PCR amplification of the SSU rRNA, ITS rRNA, and COX I gene loci to identify swine coccidia. This study is the first to systematically evaluate the prevalence and genetic diversity of coccidia in Chinese pig farms by SOIT, offering a method for accurately identifying swine coccidiosis and a scientific foundation for its effective prevention and control. Graphical Abstract

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.

The genera Cryptosporidium , Eimeria , and Cystoisospora cause gastrointestinal diseases in pigs that can lead to economic losses in the pig industry. Despite their importance, the molecular epidemiology and species diversity of these parasites remain poorly understood. Therefore, this study aimed to investigate the distribution and diversity of these genera Cryptosporidium , Eimeria , and Cystoisospora in pigs in Korea and to evaluate their potential influencing factors, including geographical location and season. A total of 700 fecal samples were collected from 103 pig farms between May 2020 and February 2023. PCR identified the genera Cryptosporidium , Eimeria , and Cystoisospora in 49 (7.0%), 24 (3.4%), and 6 (0.9%) samples, respectively. At the farm level, 43 (41.8%) out of 103 farms had at least one pig infected with these parasites. According to the region, Eimeria spp. showed the highest prevalence in Gyeongsangnam-do (8.5%; 17/200) with a statistically significant difference. Seasonal analysis revealed a statistically significant difference for Eimeria spp. with higher prevalence in summer (6.4%; 15/233) and winter (4.7%; 7/149). Phylogenetic analyses revealed Cryptosporidium ( Cr. ) scrofarum and Cr. suis , and confirmed the presence of Eimeria ( E. ) debliecki, E. perminuta, E. spinosa, and E. suis, as well as Eimeria sp. genotype 1–4. All Cystoisospora ( Cy. ) positive samples were confirmed as Cy. suis . This study examined the nationwide distribution of the genera Cryptosporidium , Eimeria , and Cystoisospora in pigs in Korea, providing molecular evidence of these parasites. The results improve our understanding of the distribution and diversity of apicomplexan protozoa in pigs in Korea. Notably, Cr. scrofarum and Cr. suis identified in this study are known to infect humans, indicating potential zoonotic risks. These findings highlight the importance of continued surveillance to mitigate economic losses on the pig industry and to address public health concerns.

A cross-sectional study was conducted in Bangladesh to determine the prevalence, molecular detection, and risk factors of Eimeria spp. infection in native and exotic chickens under various management systems. A total of 1,500 fecal samples were collected from different breeds, age groups, and sexes across multiple districts. Fecal examination using flotation and McMaster techniques identified positive cases, followed by molecular detection of Eimeria species. A questionnaire survey was also conducted to assess potential risk factors. Among the 1,500 chickens, 87 (5.8%) were positive for Eimeria oocysts, with higher prevalence in exotic breeds (7.96%) than native breeds (4.13%). The prevalence rates were 18.40%, 13.98%, 12.09%, and 3.40% in Aseel, Broiler, Sonali, and Deshi chickens, respectively, with no infection found in Naked Neck, Hilly, or Fayoumi breeds. Molecular analysis detected six Eimeria species: E. tenella was detected in 64 samples (62.07%) and in all breeds with the highest occurrence in Aseel. E. acervulina was the second most common species (25.28%), found in 23 samples from Deshi, Broiler and Sonali breeds. Other species, including E. brunetti , E. mitis , E. necatrix , and E. maxima , were rare and sparsely distributed. Chickens fed commercial feed (7.88%) had significantly higher infection rates (p < 0.0013) than those on local feed (3.99%). Intensive rearing systems (15.27%) showed higher infection rates compared to free-ranging systems, but no infection occurred in intensive systems without litter or semi-intensive systems. This is the first comprehensive report on infection status of Eimeria in chickens including all native breeds rearing in different management system in Bangladesh.

Coccidiosis is a common disease of camels, and camels are important for the economy of Asia and the Arabian Peninsula. Little is known regarding the prevalence of coccidian parasites in camels in Egypt. Fecal samples collected from the rectums of 200 camels at the Cairo slaughterhouse were processed using the sucrose flotation technique. Eimeria species oocysts were found in 38%. Three Eimeria species were identified: Eimeria cameli–like in 31%, Eimeria rajasthani in 18%, and Eimeria dromedarii in 14%. The morphology of E. rajasthani and E. dromedarii oocysts was identical to that in literature. However, there was great variability in size and structure among E. cameli oocysts; oocysts were 70–100 lm long and truncate to ovoid. Four morphotypes (types 1 to 4) were recognized. Types 1 and 2 oocysts had similar truncate ovoid shape and were dark brown, but their shape indices were different. Both types could be easily distinguished from type 3 (elongate ovoid and light brown). All oocysts were enclosed in a transparent outer covering (capsule), although this capsule was barely seen in types 3 and 4. An extension from the capsule situated in front of the micropyle, referred to as polar cap–like structure (PCL), was characteristic for types 1 and 2. The PCL of type 1 resembled the crown, while in type 2 it looked like a small thickening with a smooth top. The PCL was absent in types 3 and 4 oocysts. The latter was found only in a single oocyst. Experimental infections with E. cameli oocysts and molecular studies are needed to determine whether the monotypes described here are different species or strain variations or both.

Juvenile kiwi ( Apteryx spp.) within captive-rearing programmes commonly suffer from coccidiosis, which primarily affects the intestine but can also impact other organs, such as the kidneys, liver, lung, and spleen (Morgan  et al. Avian Pathol 42:137–146 2013 ). In some immune-compromised birds, disease causes significant morbidity and, occasionally, mortality (Morgan et al. NZVJ 62:315–320 2014 ); however, understanding of the biology of disease-causing Eimeria species in kiwi is limited. A probe-based qPCR assay targeting a 115-bp fragment of the Eimeria mitochondrial cytochrome c oxidase I (CO1) gene was developed to identify three distinct kiwi Eimeria species: the two species most commonly recovered from faeces, Eimeria kiwii and Eimeria apteryxii , as well as the newly described species, Eimeria koka (Scheltema et al. Syst Parasitol 102:30  2025 ). The qPCR assay was then applied to retrospectively analyse formalin-fixed paraffin-embedded intestine, kidney, liver, lung, and spleen tissues from ten historic post-mortem cases from kiwi diagnosed with extraintestinal coccidiosis. This novel assay detected infection more often (33/47 tissues) than manual histopathological identification (25/47 tissues). Only one species, E. koka , was detected in extraintestinal tissues with the highest prevalence (9/10) in kidney tissues. In contrast, E. kiwii was reliably detected in 8/9 intestinal tissues but was not detected in the other tissues tested. E. apteryxii was not detected in any of the tissues analysed. These findings suggest that kiwi are infected by at least one intestinal and one renal-specific species, the latter of which is suspected to disseminate under certain conditions to other organs of the body.

Forty-nine olive-backed pocket mice, Perognathus fasciatus were collected during 2011 and 2012 from 4 sites in Wyoming and examined for coccidian parasites. Fifteen (31%) were found to be passing oocysts of a new species of Eimeria Schneider, 1875. Sporulated oocysts of Eimeria fasciata n. sp. are ellipsoidal to ovoidal, 23.3 × 20.7 (19–27 × 17–25) µm, with a shape index of 1.1; they typically contain a single, smooth, bubble-like oocyst residuum. Oocysts possess 1–2 polar granules, lack a micropyle, and are bilayered with a thickness of 1.3 µm. Sporocysts are ovoidal, 10.0 × 8.2 (8–12 × 7–10) µm, with a shape index of 1.2; they contain a sporocyst residuum that appears similar to a cluster of 1–8 grapes. The Stieda body is small, appearing flattened to knobby, and there are no subStieda or paraStieda bodies. This new eimerian represents the only coccidian, to date, reported from P. fasciatus, as well as the only species from any heteromyid rodent in Wyoming.