A novel fragmented mitochondrial genome in the protist pathogen Toxoplasma gondii and related tissue coccidia.

Mitochondrial genome content and structure vary widely across the eukaryotic tree of life, with protists displaying extreme examples. Apicomplexan and dinoflagellate protists have evolved highly reduced mitochondrial genome sequences, mtDNA, consisting of only three cytochrome genes and fragmented rRNA genes. Here, we report the independent evolution of fragmented cytochrome genes in Toxoplasma and related tissue coccidia and evolution of a novel genome architecture consisting minimally of 21 sequence blocks (SBs) totaling 5.9 kb that exist as nonrandom concatemers. Single-molecule Nanopore reads consisting entirely of SBs ranging from 0.1 to 23.6 kb reveal both whole and fragmented cytochrome genes. Full-length cytochrome transcripts including a divergent coxIII are detected. The topology of the mitochondrial genome remains an enigma. Analysis of a cob point mutation reveals that homoplasmy of SBs is maintained. Tissue coccidia are important pathogens of man and animals, and the mitochondrion represents an important therapeutic target. The mtDNA sequence has been elucidated, but a definitive genome architecture remains elusive.

Island hitchhikers: pathogen agents of Madeira and Azores ticks.

Ticks are blood-sucking arthropods that can transmit pathogens to their host. As insular ecosystems can enhance tick-host interactions, this study aimed to understand tick diversity, pathogen presence, and their respective associations in the Azores and Madeira archipelagos. Unfed or partially engorged ticks (n = 120) were collected from 58 cats and dogs in the Azores (n = 41 specimens) and Madeira (n = 79 specimens) from November 2018 to March 2019. Vector identification was based on morphology and molecular criteria. For pathogen sequencing, 18S gene fragment for Babesia/Hepatozoon and gltA for Rickettsia were performed. Sequence data was explored using BLAST and BLAST and phylogenetic inference tools. In the Azores, Ixodes hexagonus, I. ventalloi, and Rhipicephalus sanguineus (n = 6; 14.6%, n = 6; 14.6%, and n = 29; 70.7% respectively) were found and in Madeira I. ricinus and R. sanguineus (n = 78, 98.7%; and n = 1, 1.3%; respectively) were identified. Tick COI markers confirmed species highlighting confirmation of R. sanguineus s.s. and genotype A of I. ventalloi. In the Azores Islands, the detected Rickettsia massiliae was linked to R. sanguineus (dogs and cats) and I. hexagonus (dogs), and in Madeira Island, R. monacensis (dogs) and Hepatozoon silvestris (cats) were found associated with I. ricinus. Further, I. ventalloi presence in the Azores expands west its known range, and Hepatozoon silvestris in Madeira may suggest that I. ricinus could have a role as a potential vector. Finally, as R. massiliae and R. monacensis presence underlines public health risks, surveillance by health authorities is crucial as pathogen-tick interactions may drive disease spread, therefore monitoring remains pivotal for disease prevention.

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.

Molecular detection of coccidian Apicomplexa Parasites isolated from wild crab-eating and pampas foxes through novel TaqMan™ probes: a contribution to their molecular epidemiology.

Neospora caninum, Toxoplasma gondii and Hammondia spp. are coccidian parasites similar in morphology. Molecular techniques are necessary to detect parasite DNA isolated from stool samples in wild canids because they were reported as definitive hosts of N. caninum life cycle. The objective of this study was to develop a highly sensitive and accurate molecular method for the identification of coccidian Apicomplexa parasites in crab-eating fox (Cerdocyon thous) and pampas fox (Lycalopex gymnocercus). Tissue samples from road-killed animals (pampas fox = 46, crab-eating fox = 55) and feces (pampas fox = 84, crab-eating fox = 2) were collected, and species were diagnosed through molecular assay. PCR was used for the amplification of a fragment of the coccidian Apicomplexa nss-rRNA gene. Additionally, we developed a novel real-time PCR TaqMan™ probe approach to detect T. gondii- Hammondia spp. and N. caninum. This is the first report of N. caninum DNA in pampas fox feces (n = 1), thus it was also detected from pampas fox tissues (n = 1). Meanwhile, T. gondii was found in tissues of pampas (n = 1) and crab-eating (n = 1) foxes and H. triffittae in one crab-eating fox tissue. Despite the low percentage (2.5%) of positive samples, the molecular method developed in this study proved to be highly sensitive and accurate allowing to conduct an extensive monitoring analysis for these parasites in wildlife.

Combination of herbal components (curcumin, carvacrol, thymol, cinnamaldehyde) in broiler chicken feed: Impacts on response parameters, performance, fatty acid profiles, meat quality and control of coccidia and bacteria.

The objective of this study was to determine whether curcumin and a commercial microencapsulated phytogenic supplement containing thymol, cinnamaldehyde and carvacrol in broiler chicken feed would improve health and meat quality (fatty acid profile), as well as to determine the coccidiostatic and bactericidal potential of the additives. The broiler chickens were divided into five groups: NC - negative control feed; PC - positive control; CU - with 50 mg/kg of curcumin, PHY - 100 mg/kg phytogenic; and PHY + CU, a combination of both additives at 50 mg/kg (curcumin) and 100 mg/kg (phytogenic). We observed significantly higher levels of total proteins associated with increased circulating globulins, as well as lower levels of uric acid, cholesterol and triglycerides in the PHY + CU group than in the NC. There were significantly fewer oocysts in birds supplemented with additives in the NC group on day 21; on day 35, the NC, PHY and PHY + CU groups had significantly lower counts than the PC and CU groups; however, at 44 days, the lowest counts were in PC group. The bacterial counts were significantly lower on day 21 in all groups that received additives than those of the control group; however, at 44 days, the bacterial and Escherichia coli counts in these groups were significantly higher than those of the control. Curcumin with or without phytogenic agent improved meat quality, with increased antioxidant levels and reduction of lipid peroxidation. There were significantly lower total saturated fatty acid levels and significantly greater monounsaturated/polyunsaturated fatty acid levels in broilers that consumed additives individually and in combination. The combination of additives significantly increased the crypt/villus ratio, a marker of improved intestinal health and performance. Additives potentiated their individual effects, suggesting they can replace conventional growth promoters without compromising health, intestinal mucosa or meat quality.

Molecular detection of Apicomplexan hemoparasites in anurans from Brazil.

Amphibians are among the most threatened vertebrate groups in the world, and the main causes include climate change, habitat destruction, and emerging diseases. Herein, we investigated the occurrence and characterized molecularly Apicomplexa in anurans from southeastern Brazil. Forty individuals from seven anuran species were sampled in São Paulo state. In the molecular analyses, one Leptodactylus latrans and one Rhinella diptycha were positive in PCR assays for species of Hepatozoon. Two L. latrans were also positive for coccidian infections (Lankesterella sp. and an unidentified coccidian species). Phylogenetic analysis based on 18S rDNA clustered the sequences detected in anurans from the present study with Hepatozoon spp. detected in reptiles and other anurans from Brazil, albeit they were separate from Hepatozoon haplotypes detected in frogs from Africa and North America. Our study showed, for the first time, the molecular detection of Lankesterella sp. and another coccidian in L. latrans. Additionally, co-infection by different species of Hepatozoon haplotypes and an unidentified coccidian in anurans from Brazil was documented.

Mysteries of host switching: Diversification and host specificity in rodent-coccidia associations.

Recent studies show that host switching is much more frequent than originally believed and constitutes an important driver in evolution of host-parasite associations. However, its frequency and ecological mechanisms at the population level have been rarely investigated. We address this issue by analyzing phylogeny and population genetics of an extensive sample, from a broad geographic area, for commonly occurring parasites of the genus Eimeria within the abundant rodent genera Apodemus, Microtus and Myodes, using two molecular markers. At the most basal level, we demonstrate polyphyletic arrangement, i.e. multiple origin, of the rodent-specific clusters within the Eimeria phylogeny, and strong genetic/phylogenetic structure within these lineages determined at least partially by specificities to different host groups. However, a novel and the most important observation is a repeated occurrence of host switches among closely related genetic lineages which may become rapidly fixed. Within the studied model, this phenomenon applies particularly to the switches between the eimerians from Apodemus flavicollis/Apodemus sylvaticus and Apodemus agrarius groups. We show that genetic differentiation and isolation between A. flavicollis/A. sylvaticus and A. agrarius faunas is a secondary recent event and does not reflect host-parasite coevolutionary history. Rather, it provides an example of rapid ecology-based differentiation in the parasite population.

An optimized DNA extraction method for molecular identification of coccidian species.

Molecular identification of Eimeria parasites infecting poultry and livestock has been commonly used for more than 20 years. An important step of the molecular identification technique is the rupturing of the oocyst wall for DNA extraction. Previously, DNA extraction methods included pre-treatment with sodium hypochlorite and osmotic shock with saturated salt solution. Here, we present a modification of this technique for a more sensitive and efficient identification of Eimeria spp. in field samples. The disruption extent of the oocyst walls, yield of DNA extraction, and identification of species-specific DNA sequences by PCR were used to evaluate this optimized method. Incubation of oocysts in sodium hypochlorite for 1.5 h at 4 °C followed by treatment with a saturated salt solution for 1 h at 55 °C broke up the walls of most Eimeria tenella oocysts, as well as other coccidian species of chicken and rabbit, such as Eimeria intestinalis and even Cryptosporidium cuniculus. Notably, polymerase chain reaction (PCR) amplification of the intervening transcribed sequence 1 (ITS-1) was successfully performed with genomic DNA extracted from just 50 oocysts using this optimized method. Our findings will greatly promote the development of molecular diagnosis methods of coccidiosis and simplify coccidian species identification and categorization as well as infection prevalence, providing a significant advancement in the development of techniques for coccidiosis control and prevention.

Identification of Cystoisospora ohioensis in a Diarrheal Dog in Korea.

A 3-month-old female Maltese puppy was hospitalized with persistent diarrhea in a local veterinary clinic. Blood chemistry and hematology profile were analyzed and fecal smear was examined. Diarrheal stools were examined in a diagnostic laboratory, using multiplex real-time polymerase chain reaction (PCR) against 23 diarrheal pathogens. Sequence analysis was performed using nested PCR amplicon of 18S ribosomal RNA. Coccidian oocysts were identified in the fecal smear. Although multiplex real-time PCR was positive for Cyclospora cayetanensis, the final diagnosis was Cystoisospora ohioensis infection, confirmed by phylogenetic analysis of 18S rRNA. To our knowledge, this the first case report of C. ohioensis in Korea, using microscopic examination and phylogenetic analysis.

Multiyear Survey of Coccidia, Cryptosporidia, Microsporidia, Histomona, and Hematozoa in Wild Quail in the Rolling Plains Ecoregion of Texas and Oklahoma, USA.

We developed nested PCR protocols and performed a multiyear survey on the prevalence of several protozoan parasites in wild northern bobwhite (Colinus virginianus) and scaled quail (Callipepla squamata) in the Rolling Plains ecoregion of Texas and Oklahoma (i.e. fecal pellets, bird intestines and blood smears collected between 2010 and 2013). Coccidia, cryptosporidia, and microsporidia were detected in 46.2%, 11.7%, and 44.0% of the samples (n = 687), whereas histomona and hematozoa were undetected. Coccidia consisted of one major and two minor Eimeria species. Cryptosporidia were represented by a major unknown Cryptosporidium species and Cryptosporidium baileyi. Detected microsporidia species were highly diverse, in which only 11% were native avian parasites including Encephalitozoon hellem and Encephalitozoon cuniculi, whereas 33% were closely related to species from insects (e.g. Antonospora, Liebermannia, and Sporanauta). This survey suggests that coccidia infections are a significant risk factor in the health of wild quail while cryptosporidia and microsporidia may be much less significant than coccidiosis. In addition, the presence of E. hellem and E. cuniculi (known to cause opportunistic infections in humans) suggests that wild quail could serve as a reservoir for human microsporidian pathogens, and individuals with compromised or weakened immunity should probably take precautions while directly handling wild quail.

Widespread occurrence of organelle genome-encoded 5S rRNAs including permuted molecules.

5S Ribosomal RNA (5S rRNA) is a universal component of ribosomes, and the corresponding gene is easily identified in archaeal, bacterial and nuclear genome sequences. However, organelle gene homologs (rrn5) appear to be absent from most mitochondrial and several chloroplast genomes. Here, we re-examine the distribution of organelle rrn5 by building mitochondrion- and plastid-specific covariance models (CMs) with which we screened organelle genome sequences. We not only recover all organelle rrn5 genes annotated in GenBank records, but also identify more than 50 previously unrecognized homologs in mitochondrial genomes of various stramenopiles, red algae, cryptomonads, malawimonads and apusozoans, and surprisingly, in the apicoplast (highly derived plastid) genomes of the coccidian pathogens Toxoplasma gondii and Eimeria tenella. Comparative modeling of RNA secondary structure reveals that mitochondrial 5S rRNAs from brown algae adopt a permuted triskelion shape that has not been seen elsewhere. Expression of the newly predicted rrn5 genes is confirmed experimentally in 10 instances, based on our own and published RNA-Seq data. This study establishes that particularly mitochondrial 5S rRNA has a much broader taxonomic distribution and a much larger structural variability than previously thought. The newly developed CMs will be made available via the Rfam database and the MFannot organelle genome annotator.

Disease surveillance of Atlantic herring: molecular characterization of hepatic coccidiosis and a morphological report of a novel intestinal coccidian.

Surveillance for pathogens of Atlantic herring, including viral hemorrhagic septicemia virus (VHSV), Ichthyophonus hoferi, and hepatic and intestinal coccidians, was conducted from 2012 to 2016 in the NW Atlantic Ocean, New Jersey, USA. Neither VHSV nor I. hoferi was detected in any sample. Goussia clupearum was found in the livers of 40 to 78% of adult herring in varying parasite loads; however, associated pathological changes were negligible. Phylogenetic analysis based on small subunit 18S rRNA gene sequences placed G. clupearum most closely with other extraintestinal liver coccidia from the genus Calyptospora, though the G. clupearum isolates had a unique nucleotide insertion between 604 and 729 bp that did not occur in any other coccidian species. G. clupearum oocysts from Atlantic and Pacific herring were morphologically similar, though differences occurred in oocyst dimensions. Comparison of G. clupearum genetic sequences from Atlantic and Pacific herring revealed 4 nucleotide substitutions and 2 gaps in a 1749 bp region, indicating some divergence in the geographically separate populations. Pacific G. clupearum oocysts were not directly infective, suggesting that a heteroxenous life cycle is likely. Intestinal coccidiosis was described for the first time from juvenile and adult Atlantic herring. A novel intestinal coccidian species was detected based on morphological characteristics of exogenously sporulated oocysts. A unique feature in these oocysts was the presence of 3 long (15.1 ± 5.1 µm, mean ±SD) spiny projections on both ends of the oocyst. The novel morphology of this coccidian led us to tentatively name this parasite G. echinata n. sp.

Canine hepatozoonosis in southeastern Bahia, Brazil.

In Brazil, canine hepatozoonosis is a tick-borne subclinical hemoparasitosis caused by a protozoa Hepatozoon canis and is highly prevalent in dogs in rural areas. An epizootiological study was conducted to investigate the prevalence of H. canis in the canine population of Ituberá, Bahia, and to analyze any associated risk factors. Blood samples were collected from 380 dogs and determined the presence of the protozoan by performing capillary blood smear and polymerase chain reaction (PCR). Epizootiological data were collected by asking dog owners to answer a structured questionnaire. H. canis gamonts were not detected in the blood smears. However, PCR detected H. canis in 163/380 (42.9%) dogs examined. Physical examination and anamnesis indicated 105 (64.4%) positive asymptomatic dogs. Hematological alterations were observed in 115 (70.5%) infected dogs. No clinical, hematological, or epizootiological variable was found to be significantly associated to the infection. In conclusion, the high prevalence of H. canis infection in local dogs may be because of the peri-urban features of this municipality. Moreover, to the best of our knowledge, this study the first study to report H. canis infection in the State of Bahia.

Molecular phylogenetics of eimeriid coccidia (Eimeriidae, Eimeriorina, Apicomplexa, Alveolata): A preliminary multi-gene and multi-genome approach.

Coccidia possess three distinct genomes: nuclear, mitochondrial, and plastid. Sequences from five genes located on these three genomes were used to reconstruct the phylogenetic relationships of members of the phylum Apicomplexa: 18S rDNA sequences from the nuclear (nu) genome, partial cytochrome c oxidase subunit I sequences from the mitochondrial (mt) genome, and partial 16S and 23S rDNA sequences and RNA polymerase B sequences from plastid (pl) genomes. Maximum parsimony, maximum likelihood, and Bayesian inference were used in conjunction with nuclear substitution models generated from data subsets in the analyses. Major groups within the Apicomplexa were well supported with the mitochondrial, nuclear, and a combination of mitochondrial, nuclear and concatenated plastid gene sequences. However, the genus Eimeria was paraphyletic in phylogenetic trees based on the nuclear gene. Analyses using the individual genes (18S rDNA and cytochrome c oxidase subunit I) resolved the various apicomplexan groups with high Bayesian posterior probabilities. The multi-gene, multi-genome analyses based on concatenated nu 18S rDNA, pl 16S, pl 23S, pl rPoB, pl rPoB1, and mt COI sequences appeared useful in resolving phylogenetic relationships within the phylum Apicomplexa. Genus-level relationships, or higher, appear best supported by 18S rDNA analyses, and species-level analyses are best investigated using mt COI sequences; for parasites for which both loci are available, nuclear 18S rDNA sequences combined with mitochondrial COI sequences provide a compact and informative molecular dataset for inferring the evolutionary relationships taxa in the Apicomplexa.

Numerical and functional responses to the presence of a competitor--the case of Aggregata sp. (Apicomplexa: Aggregatidae) and Octopicola superba (Copepoda: Octopicolidae).

Evidence of interference competition between the eimeriorin coccidian Aggregata sp. and the octopicolid copepod Octopicola superba at the level of the gills of naturally infected Octopus vulgaris is evaluated. Numerical and functional responses are considered for analysis, and the fundamental and realized spatial niches (FSNs and RSNs) are measured as part of the study. While it was not possible to measure the FSN of Aggregata sp., the analysis of the infection levels of O. superba recorded for non-concomitantly and concomitantly infected hosts suggests that the gills and body skin constitute, respectively, the main and accessory sites of infection of the parasite. According to the evidence found, the gills function mainly as an accessory site of infection of Aggregata sp., in specimens in which the caecum and intestine are massively infected. Evidence for a negative interaction between Aggregata sp. and O. superba has been found while controlling for a potential confounding effect of host size. Furthermore, the presence of O. superba on gill lamellae appears to have been negatively affected by the presence of Aggregata sp., while this latter remained mostly undisturbed. The mean number of oocysts of Aggregata sp. in the gills was higher in spring and summer, which were also the seasons presenting the broadest RSN for O. superba.

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.

Is there only one species of Hepatozoon infecting Brazilian caimans? Integrative taxonomy unveiling the parasite's diversity.

Hepatozoon spp. are the most common haemoparasites reported from reptiles around the world, however, only six species have been described infecting crocodilians. In Brazil, Hepatozoon caimani Carini, 1909 is currently the only recognized species from the caiman hosts. This study provides new data on the diversity of species of Hepatozoon infecting Caiman crocodilus (Linnaeus) using molecular data and phylogenetic analysis, with additional support of morphological data of developmental stages from host blood and tissue. Forty-four individuals were collected and screened for haemogregarines, and blood and tissue samples were analysed by light microscopy with 31 (70.45%) infected. Hepatozoon spp. blood developmental stages included immature and mature gamonts with or without cytoplasmic vacuoles and free gamonts. Additionally, merogonic developmental stages were found in the liver and spleen of infected hosts. Based on the morphological and molecular data, this study identified two possible different species of Hepatozoon, being one of them the H. caimani with intragenotypic divergence.

Evaluation of coccidia DNA in irrigation pond water and wastewater sludge associated with Cyclospora cayetanensis 18S rRNA gene qPCR detections.

The coccidian parasite Cyclospora cayetanensis is the causative agent for foodborne outbreaks of cyclosporiasis disease and multiple annual fresh produce recalls. The aim of this study was to identify potential cross-reacting species for the C. cayetanensis 18S rRNA and MIT1C gene target real-time quantitative polymerase chain reaction (qPCR) assays. The environmental samples evaluated were irrigation pond water, produce wash water, and wastewater treatment sludge from a previous study with qPCR detections of C. cayetanensis by the 18S rRNA gene target qPCR. From these samples, longer regions of the 18S rRNA gene and the mitochondrial cytochrome c oxidase subunit III gene (cox3) were sequenced. Of 65 irrigation pond water samples with positive test results using the C. cayetanensis 18S rRNA gene qPCR assay, none had MIT1C qPCR assay detections or sequences that clustered with C. cayetanensis based on sequencing of the cox3 and 18S rRNA gene. Sequences from these samples clustered around coccidia sequences found in bird, fish, reptile, and amphibian hosts. Of 26 sludge samples showing detections by either qPCR assay, 14 (54%) could be confirmed as containing C. cayetanensis by sequencing of cox3 and 18S rRNA gene regions. In three of the remaining sludge samples, sequenced reads clustered with coccidia from rodents. This study demonstrated that caution should be taken when interpreting qPCR C. cayetanensis detection data in environmental samples and sequencing steps will likely be needed for confirmation. IMPORTANCE: Fresh produce is a leading transmission source in cyclosporiasis outbreaks. It is therefore essential to understand the role that produce-growing environments play in the spread of this disease. To accomplish this, sensitive and specific tests for environmental and irrigation waters must be developed. Potential cross-reactions of Cyclospora cayetanensis real-time quantitative polymerase chain reaction (qPCR) assays have been identified, hindering the ability to accurately identify this parasite in the environment. Amplicon sequencing of the cox3 and 18S rRNA genes revealed that all irrigation pond water and two sludge samples that initially detected C. cayetanensis by qPCR were most likely cross-reactions with related coccidian organisms shed from birds, fish, reptiles, amphibians, and rodents. These results support that a single testing method for environmental samples is likely not adequate for sensitive and specific detection of C. cayetanensis.

Phylogenetic analysis based on 18S rRNA gene sequences of Schellackia parasites (Apicomplexa: Lankesterellidae) reveals their close relationship to the genus Eimeria.

In the present study we detected Schellackia haemoparasites infecting the blood cells of Lacerta schreiberi and Podarcis hispanica, two species of lacertid lizards from central Spain. The parasite morphometry, the presence of a refractile body, the type of infected blood cells, the kind of host species, and the lack of oocysts in the fecal samples clearly indicated these blood parasites belong to the genus Schellackia. Until now, the species of this genus have never been genetically characterized and its taxonomic position under the Lankesterellidae family is based on the lack of the exogenous oocyst stage. However, the phylogenetic analysis performed on the basis of the 18S rRNA gene sequence revealed that species of the genus Schellackia are clustered with Eimeria species isolated from a snake and an amphibian species but not with Lankesterella species. The phylogenetic analysis rejects that both genera share a recent common ancestor. Based on these results we suggest a revision of the taxonomic status of the family Lankesterellidae.

Description of three new species of Hepatozoon (Apicomplexa, Hepatozoidae) from Rattlesnakes (Crotalus durissus terrificus) based on molecular, morphometric and morphologic characters.

Hepatozoon spp. are commonly found infecting snakes. Since the latter are parasitized by diverse forms and data in the literature show divergence, we studied Hepatozoon spp. diversity on Crotalus durissus terrificus snakes using both molecular and morphological approaches. Naturally infected animals were employed. Blood was collected, blood smears were prepared and an aliquot was stored at -20°C for DNA extraction. Five specimens of C. durissus terrificus were selected, each of them infected with one gamont type. Morphological and morphometric analyses of the found gamonts led to their grouping into three populations. For molecular characterization, seven oligonucleotide pairs that amplify distinct regions of rDNA gene were tested by adopting the PCR technique. Only the oligonucleotide pairs HepF300/Hep900 and HEMO1/HEMO2 were efficient in amplifying and distinguishing different isolates of Hepatozoon spp. from snakes. The better results were obtained when both oligonucleotide pairs were used in association. Based on the molecular and morphologic differences, three new species were proposed: Hepatozoon cuestensis sp. nov.; Hepatozoon cevapii sp. nov. and Hepatozoon massardii sp. nov. This is the first description of new Hepatozoon species from snakes, based on molecular characterization and morphological data, in South America.