Protozoan parasites of the Eimeria genus have undergone extensive speciation and are now represented by a myriad of species that are specialised to different hosts. These species are highly host-specific and usually parasitise single host species, with only few reported exceptions. Doubts regarding the strict host specificity were frequent in the original literature describing coccidia parasitising domestic turkeys. The availability of pure characterised lines of turkey and chicken Eimeria species along with the recently developed quantitative PCR identification of these species allowed to investigate the issue of host specificity using well-controlled cross-transmission experiments. Seven species of gallinaceous birds (Gallus gallus, Meleagris gallopavo, Alectoris rufa, Perdix perdix, Phasianus colchicus, Numida meleagris and Colinus virginianus) were inoculated with six species and strains of turkey Eimeria and six species of chicken coccidia and production of oocysts was monitored. Turkey Eimeria species E. dispersa, E. innocua and E. meleagridis could complete their development in the hosts from different genera or even different families. Comparison of phylogenetic positions of these Eimeria species according to 18S rDNA and COI showed that the phylogeny cannot explain the observed patterns of host specificity. These findings suggest that the adaptation of Eimeria parasites to foreign hosts is possible and might play a significant role in the evolution and diversification of this genus.

Coccidiosis is a disease caused by apicomplexan parasites of the genus Eimeria, which has a significant economic impact on poultry production. Multiple species infecting the turkey have been described; however, due to the general lack of unambiguous description, their identification and taxonomy is debatable. In this work, a systematic approach was taken to isolate, characterise and compare coccidian species in the turkey. Individual species were tracked according to their unique 18S ribosomal DNA sequence. The single-oocyst isolation technique and passaging of mixed species field isolates in selectively immunised birds enabled the derivation of pure species. Six distinct strains representing five eimerian species that infect the turkey were obtained. It appears highly probable that these species represent all species described in the past with the exception of Eimeria subrotunda. The species were analysed using both traditional methods and DNA sequencing. For each strain the oocyst morphology, prepatent period, gross pathology, pathogenicity, host specificity and endogenous cycle were studied. Antigenic similarity was investigated in multiple cross-immunity experiments. For identification and quantification of each individual species or strain, quantitative real-time PCR markers were also developed. Parallel characterisation of pure strains allowed comprehensive comparison with the original descriptions and assignment of correct species names. The species Eimeria meleagridis, Eimeria dispersa, Eimeria gallopavonis, Eimeria meleagrimitis and Eimeria innocua were identified. Comparison of our data with those of previous studies indicates that Eimeria adenoeides is most probably a synonym for either E. meleagridis or E. gallopavonis, or a description based on a mixture of these species, and thus nomen dubium. The species E. dispersa and E. innocua were also found to infect Bobwhite Quail. Phylogenetic reconstruction based on 18S rDNA and cytochrome c oxidase subunit I gene (COI) sequences showed that these two species form a distinct clade unrelated to other turkey coccidia and point to a polyphyletic origin of the species infecting the turkey.

Life cycle of Eimeria coecicola was studied in experimentally infected rabbits by light microscopy and by transmission and scanning electron microscopy. First and second generation meronts developed in the vermiform appendix; third and fourth generation meronts were located in the epithelium of the ileum. Gametogony developed again in the vermiform appendix. The prepatent period was 9 days. New data were obtained by the study of asexual reproduction. First generation meronts were first observed 4 days post infection (DPI), which is relatively late in comparison with other species of rabbit coccidia. Sporozoites were found in lymphatic follicles of the vermiform appendix at 4 DPI by transmission electron microscopy. This suggests, together with selective location of first generation meronts in the epithelium adjacent to these follicles, that major part of sporozoites enter the epithelium cells through lymphatic follicles and not through the lumen of the vermiform appendix. The process of development of first generation merozoites is similar to endodyogeny. The differences are in formation of apical parts of daughter merozoites which is not coincidental with nuclear division and in formation of the outer membrane of pellicle which arises within the mother cell. Some first generation merozoites have 2-3 nuclei, second and fourth generation merozoites are only uninucleate, while third generation merozoites are only multinucleate. We found that further merozoites are formed in multinucleate third generation merozoites by endopolygeny.

Although the validity of the coccidian species, Eimeria mivati, has been questioned by many researchers for a long time there has not been any molecular analysis that would help resolve this issue. Here we report on the discovery of the two types of small ribosomal subunit (18S) gene within the Eimeria mitis genome that correspond to the known 18S sequences of E. mitis and E. mivati, and this is in conflict with the existence of E. mivati as an independent species. We have carried out five single oocyst isolations to obtain five single-oocyst-derived strains of E. mitis and these were analyzed by the sequencing of 18S and mitochondrial cytochrome c oxidase subunit I genes. The two types of 18S gene were found to be present in each strain in roughly equal ratios. This indicates that if the strains carrying only one or the other 18S type exist, they will likely cross-breed and still represent a single species. However, the more probable explanation is that all strains of E. mitis contain two types of 18S gene and that the occasional detection of only one or the other type by sequencing might be caused by insufficient sampling. This is also the first report of the two types of 18S gene in Eimeria, which has already been described in some other apicomplexan species, most notably Plasmodium. We also found that these two types of ribosomal RNA differ significantly in their secondary structure. The biological significance of the two 18S gene variants in E. mitis is not known, however, we hypothesize that these variants might be used in different stages of the parasite's life-cycle as it is in other apicomplexan species investigated so far.

Coccidiosis are the major parasitic diseases in poultry and other domestic animals including the domestic rabbit (Oryctolagus cuniculus). Eleven distinct Eimeria species have been identified in this host, but no PCR-based method has been developed so far for unequivocal species differentiation. In this work, we describe the development of molecular diagnostic assays that allow for the detection and discrimination of the 11 Eimeria species that infect rabbits. We determined the nucleotide sequences of the ITS1 ribosomal DNAs and designed species-specific primers for each species. We performed specificity tests of the assays using heterologous sets of primers and DNA samples, and no cross-specific bands were observed. We obtained a detection limit varying from 500fg to 1pg, which corresponds approximately to 0.8–1.7 sporulated oocysts, respectively. The test reported here showed good reproducibility and presented a consistent sensitivity with three different brands of amplification enzymes. These novel diagnostic assays will permit population surveys to be performed with high sensitivity and specificity, thus contributing to a better understanding of the epidemiology of this important group of coccidian parasites.

This article summarises the current knowledge of the rabbit coccidia and the disease they cause. Various aspects, such as life cycles, localisation in the host, pathology and pathogenicity, immunity and control, are discussed.

To study the immune response to coccidiosis, the suckling rabbits were inoculated with 2,000 oocysts of either Eimeria intestinalis or Eimeria flavescens at 19, 22, 25, 29, and 33 days of age (DA) and in the case of E. intestinalis at 14 and 16 DA as well and sacrificed 14 days later. Another group served as an uninfected control and the rabbits were killed at the same age as their infected counterparts. Unlike the antibody response, the parameters reflecting cellular immunity (total number of leukocytes in mesenteric lymph nodes, lymphocyte proliferation upon stimulation with specific antigen and the dynamics of CD4+ and CD8+ cell proportions in the intestinal epithelium at the specific site of parasite development) were significantly changed from about 25 DA onwards. In contrast to the rabbits infected with weakly immunogenic coccidium E. flavescens, the proportions of CD4+ and CD8+ lymphocytes in intraepithelial lymphocytes from the specific site of parasite development were considerably changed after infection with highly immunogenic species E. intestinalis. As the immune system of sucklings from about 25 DA reacts to the infection, this age may be considered in terms of vaccination against coccidiosis.

Seven- to eight-week-old rabbits were infected with Eimeria intestinalis Cheissin, 1948, a highly immunogenic coccidium, or Eimeria flavescens Marotel et Guilhon, 1941, which is weakly immunogenic. Immune response was investigated at 7, 14 and 21 days post inoculation (DPI). The level of serum immunoglobulins, lymphocyte proliferation stimulated by parasite antigens and weight of mesenteric lymph nodes (MLN) showed similar dynamics in rabbits inoculated with both coccidia species. The amount of serum IgG and IgM, but not IgA, was increased from 14 DPI. The lymphocytes from MLN of infected animals significantly reacted to stimulation with parasite antigen 14 and 21 DPI and MLN were enlarged at 14 DPI. Thus, both parasite species elicited immune response characterized by these parameters in a similar manner despite of their different immunogenicity. The only apparent difference in the responses was in the percentage of CD8+ lymphocytes in the specific site of parasite development (the last third of the small intestine in E. intestinalis, caecum in E. flavescens), which increased in rabbits infected with E. intestinalis but not with E. flavescens. This parameter reflects the status of local immunity and hence the results suggest that the local reaction plays an important role in induction of protective immunity to coccidia in rabbits.

SUMMARY Monophyly of all 11 valid Eimeria species from rabbits ( Oryctolagus cuniculus Linnaeus, 1758) was revealed based on nuclear 18S rDNA sequence data. This finding implies that these species, which vary considerably in terms of their morphology and biology, diversified on a single host or several closely related species. Phylogenetic analysis divided rabbit Eimeria species into 2 sister lineages, corresponding to the presence/absence of the oocyst residuum. Other morphological or biological traits (oocyst shape and size, presence/absence of oocyst inner structures, pathogenicity, infection site, pre-patent and patent periods, sporulation time, and number of asexual generations) do not explicitly correlate with the phylogeny of rabbit coccidia.

Freshly defecated unsporulated oocysts of Eimeria piriformis from rabbit were treated with various concentrations (1%, 2.5%, 5%, and 10%) of chromium compounds, potassium dichromate, potassium chromate, chromium oxide and chromium nitrate, to examine their effect on sporulation. The sporulation time of oocysts treated with 1 to 10% K2Cr2O7 was 28 h. However, much longer sporulation times of about 60 h were required for oocysts treated with 2.5% CrO3 and Cr(NO3)3. Moreover, for oocysts treated with distilled water, 1% K2CrO4 and 10% K2CrO4, the sporulation times required were 216, 156 and 96 h, respectively. Thus, potassium dichromate was found to have higher catalytic activity for the sporulation of E. piriformis oocysts than other chromium compounds.

The coccidium Eimeria exigua from the tame rabbit (Oryctolagus cuniculus) has been neglected so far since it was considered to be an invalid species. Indeed, little is known about this coccidium. We have studied its endogenous development in domestic rabbits by means of light and electron microscopy. The first meronts appeared 72h post-inoculation (h.p.i.), but a total of four asexual generations developed from 72 to 144h.p.i. Meronts and gamonts were localized in the small intestine and the asexual stages successively moved from the duodenum to ileum. All parasite stages were found exclusively in the epithelium of the walls and tops of the villi. Two types of meronts developing in parallel during the asexual phase were observed from at least the 2nd generation. As in other rabbit coccidia, these forms corresponded to type A, which usually develops two polynucleate merozoites where endomerogony occurs, and type B, characterized by more numerous uninucleate merozoites arising by ectomerogony. This report is the first description of the endogenous stages of E. exigua.

Cervical cancer is one of the leading causes of female-specific cancer death. Tumor region segmentation plays a pivotal role in both the clinical analysis and treatment planning of cervical cancer. Due to the heterogeneity and low contrast of biomedical images, current state-of-the-art tumor segmentation approaches are facing the challenge of the insensitive detection of small lesion regions. To tackle this problem, this paper proposes an augmented multiscale network (AugMS-Net) based on 3D U-Net to automatically segment cervical Magnetic Resonance Imaging (MRI) volumes. Since a multiscale strategy is considered one of the most promising algorithms to tackle small object recognition, we introduce a novel 3D module to explore more granular multiscale representations. Besides, we employ a deep multiscale supervision strategy to doubly supervise the side outputs hierarchically. To demonstrate the generalization of our model, we evaluated AugMS-Net on both a cervical dataset from MRI volumes and a liver dataset from Computerized Tomography (CT) volumes. Our proposed AugMS-Net shows superior performance over baseline models, yielding high accuracy while reducing the number of model parameters by nearly 20%. The source code and trained models are available at https://github.com/Cassieyy/AugMS-Net.

Naive and immune specific-pathogen-free rabbits were inoculated in the duodenum with sporocysts of Eimeria coecicola or Eimeria intestinalis. Samples were taken from the following tissues: duodenum (site of penetration of sporozoites), ileum (specific target site of the endogenous development of E. intestinalis), vermiform appendix (target site of E. coecicola) and two extraintestinal sites, mesenteric lymph nodes (MLNs), and spleen. The presence of sporozoites was checked by immunohistochemistry. In rabbits primary-infected with E. coecicola, large numbers of sporozoites were detected in the duodenum, extraintestinal sites, and vermiform appendix. The abundance of sporozoites in the spleen, MLN, and appendix was significantly reduced in the immune rabbits, and the migration seemed impeded. In the rabbits infected with E. intestinalis, sporozoites were absent in the spleen and MLN, indicating that the route of migration is different from that of E. coecicola. The number of sporozoites in the crypts of the ileum was markedly reduced in the immune animals.

The SPF rabbits were inoculated with oocysts of Eimeria flavescens and the first newly developed oocysts were recovered. They were used for inoculation of other rabbits which conseqeuntly excreted oocysts sooner than in the previous passage. By repeated use of this method, the prepatent period was shortened after 18 passages by more than 60 h. The endogenous development of this precocious line (PL) differed from that of the original strain (OS). Compared to OS, two asexual generations, second (or third) and fourth, were absent in PL. The first merogony took place in the jejunum and ileum in OS and, in contrast, in the large intestine in PL. Like in other rabbit coccidia, two types of meronts (A and B) were seen in each generation. However, the ratio of B: A meronts in the last (fifth) asexual generation as well as ratio of microgamonts:macrogamonts differs in OS and PL.

The endogenous cycle of Eimeria flavescens was studied in specific pathogen-free rabbits by means of histology and transmission electron microscopy. In total, five asexual generations were observed and two types of meronts and merozoites were found in each generation. Type A gave rise to a smaller number of thick polynucleate merozoites in which daughter merozoites were formed by endomerogony, while in the type B meronts slender uninucleate merozoites arose from ectomerogony. The first generation meronts were found in the crypts and proximal part of the villi of the duodenum and jejunum, whereas the three following generations developed in the superficial epithelium of the large intestine (cecum, vermiform appendix and colon). The last merogony as well as gamogony took place in crypts of the large intestine.

An electron microscopic study of the endogenous development of Eimeria mulardi Chauve, Reynaud and Gounel, 1994 was carried out in mule ducks which are hybrids of the domestic duck (Anas platyrhynchos) and the muscovy duck (Cairina moschata). All of the endogenous stages were seen within the nucleus of the host cell. Merozoites arose from ectomerogony and three mutually similar merogonies were noted. The asexual stages were found in leukocyte-like cells in the lamina propria of the jejunum, ileum and caecum, while the gamonts developed in glandular epithelial cells in the same part of the intestine.

The fine structure of sporocysts and sporozoites of parent strains and precocious lines of rabbit coccidia Eimeria intestinalis, E. magna and E. media was studied. The parent strains and precocious lines differ only in the shape and size of refractile bodies (RB). In the sporocysts of precocious lines of E. magna and E. media, one extremely large RB was seen, either inside one of the sporozoites, or free in the sporocyst. In the oocysts of the precocious strain of E. intestinalis, two sporocysts resembled those of the precocious lines of E. magna and E. media, whereas the other two sporocysts did not harbour any RB. Sporozoites of all the precocious lines contained no, or very small, RB after in vitro excystation.

This study was designed to identify an extraintestinal route of migration of Eimeria coecicola sporozoites and the types of cell harbouring the parasite during the invasion of the intestine. The presence of E. coecicola in blood, spleen and mesenteric lymph nodes of infected donor rabbits was demonstrated by immunohistology on donor organs and measurement of oocyst excretion by coccidia-free recipient rabbits injected with whole-cell suspensions prepared from donor tissues. Two types of donor lymphocyte, B (IgM(+)) and T (CD5(+)), were labelled using a two-colour immunofluorescence-labelling technique and separated with a cell-sorter (FACStar(plus)). The presence of parasites in the sorted cells was assessed by direct examination and by using the same in vivo test after intravenous injection of IgM(+) B or CD5(+) T lymphocytes collected from donors at different times after inoculation. This test provided evidence that the parasites were alive and still infectious within the sorted lymphocytes. It was demonstrated that both B and T lymphocytes were infected.

A total of 359 faecal samples were collected from ostriches of various age groups (2 weeks, less than 1 month, 2-3 months, 3-7 months, 7-12 months, adult individuals) in a farm breeding in Multip Moravia, Studenka. The samples were examined by four different methods: flotation using Sheather's sugar solution, staining with Heidenhain's iron haematoxylin using the method of Noller-Westphal-Gonnert, cultivation in egg-slant medium (LES) and Diamond's TYM medium. High prevalence of Blastocystis sp. was observed in all the age groups (Tab. I), but the intensity of infection was mostly inappreciable. A very small number of oocysts of a coccidium of the genus Isospora was found in two birds younger than one month. No other parasites were found and therefore we believe that the intestinal parasites do not represent any important obstacle to a farm breeding of African ostrich.