Taxonomy and molecular epidemiology of Cryptosporidium and Giardia - a 50 year perspective (1971-2021).

The protozoan parasites Cryptosporidium and Giardia are significant causes of diarrhoea worldwide and are responsible for numerous waterborne and foodborne outbreaks of diseases. Over the last 50 years, the development of improved detection and typing tools has facilitated the expanding range of named species. Currently at least 44 Cryptosporidium spp. and >120 genotypes, and nine Giardia spp., are recognised. Many of these Cryptosporidium genotypes will likely be described as species in the future. The phylogenetic placement of Cryptosporidium at the genus level is still unclear and further research is required to better understand its evolutionary origins. Zoonotic transmission has long been known to play an important role in the epidemiology of cryptosporidiosis and giardiasis, and the development and application of next generation sequencing tools is providing evidence for this. Comparative whole genome sequencing is also providing key information on the genetic mechanisms for host specificity and human infectivity, and will enable One Health management of these zoonotic parasites in the future.

Zoonotic Enterocytozoon bieneusi genotypes found in brazilian sheep.

The presence of Enterocytozoon bieneusi in sheep has been reported in only three countries worldwide. The present study has found E. bieneusi in Brazilian sheep for the first time; in 24/125 (19.2%) fecal samples by PCR and on 8/10 (80%) farms from three diverse locations. A significantly greater number of lambs (34.1%) were found infected than older sheep (11.1%) (P=0.0036); most of the lambs were less than 6months of age. Farms with an intensive production system had a lower infection rate (10.5%) of infection than semi-intensive farms (23%), but this difference was not statistically significant. Sequencing analysis of the internal transcribed spacer (ITS) region of the rRNA gene revealed four known E. bieneusi genotypes (BEB6, BEB7, I, and LW1) and two novel genotypes (BEB18 and BEB19). Genotypes LW1 and BEB19 clustered within designated zoonotic Group 1 while genotypes BEB6, BEB7, I, and BEB18, and clustered within Group 2. BEB6 was the most prevalent (45.8%), followed by BEB7 (33.3%). Genotypes BEB6, I, and LW1 are zoonotic and can pose a risk to human health for immunocompromised individuals.

New findings of Enterocytozoon bieneusi in beef and dairy cattle in Brazil.

Microsporidia are widely recognized as important human pathogens with Enterocytozoon bieneusi as the most common species infecting humans and animals, including cattle. Although Brazil has the second largest cattle herd in the world and it is the largest exporter of beef there are no data on the presence or impact of E. bieneusi on this important population. To fill this knowledge gap, fecal specimens were collected from 452 cattle from pre-weaned calves to adult cattle in the state of Rio de Janeiro. Host factors including age, gender, dairy/beef, body composition, and fecal consistency were included in the study. Using molecular methods, E. bieneusi was found in 79/452 (17.5%) fecal specimens. This represents the first report of this parasite in Brazilian cattle. A significantly higher prevalence was found in calves less than 2 months of age (27.6%) and those 3-8 months of age (28.8%) versus heifers (14.1%) and adults (1.4%) (P<0.05). Dairy cattle (26.2%) had a higher prevalence than beef cattle (9.7%) (P<0.001). No correlation was found between infection and gender, body composition, and fecal consistency. Molecular characterization of the internal transcribed spacer (ITS) revealed 12 genotypes; five previously reported in cattle (BEB4, BEB8, D, EbpA and I), and seven novel genotypes (BEB11-BEB17). A phylogenetic analysis showed that 6 genotypes (D, EbpA, BEB12, BEB13, BEB15, and BEB16) identified in 18 animals clustered within the designated zoonotic Group 1 while the other 6 genotypes (I, BEB4, BEB8, BEB11, BEB14, BEB17) identified in 61 animals clustered within Group 2. The identification of genotypes in Brazilian cattle that have previously been reported in humans highlights the potential risk of zoonotic transmission and suggests that the role of cattle in transmission of human infections requires further study.

Sarcocystis heydorni, n. sp. (Apicomplexa: Sarcocystidae) with cattle (Bos taurus) and human (Homo sapiens) cycle.

Cattle (Bos taurus) are intermediate hosts for four species of Sarcocystis, namely Sarcocystis cruzi, Sarcocystis hirsuta, Sarcocystis hominis, and Sarcocystis rommeli. Of these four species, mature sarcocysts of S. cruzi are thin-walled (<1 µm), whereas S. hirsuta, S. hominis, and S. rommeli have thick walls (4 µm or more). Here, we describe a new species of Sarcocystis with thin-walled sarcocysts in cattle. Two newborn calves were fed with sporocysts from the feces of a human volunteer who had ingested raw beef. The calves were killed 111 and 222 days later. In addition to thick-walled sarcocysts of S. hominis, both calves were coinfected with a Sarcocystis species that had a thin-walled sarcocysts, distinct from S. cruzi. The sarcocysts were mature, microscopic, up to 80 µm wide, and up to 1060 µm long. By light microscopy, the sarcocyst wall was thin (<1 µm thick) and had minute protrusions. By transmission electron microscopy, the sarcocyst wall had short, conical villar protrusions (vp) that were up to 0.5 µm long and up to 0.5 µm wide, similar to type 29. The vp on the sarcocyst wall lacked microtubules but had six or more disc-shaped plaques. The ground substance layer was smooth, approximately 0.5 µm thick, and without microtubules. The bradyzoites were 8-11 µm long. The structure of the sarcocyst wall was distinct from any species of Sarcocystis reported from livestock. This unique species is named in honor of Dr. Alfred Otto Heydorn who provided the sporocysts.

Human infections with Sarcocystis species.

Recurrent outbreaks of muscular sarcocystosis among tourists visiting islands in Malaysia have focused international attention on sarcocystosis, a disease once considered rare in humans. Sarcocystis species require two hosts, definitive and intermediate, to complete their life cycle. Humans can serve as definitive hosts, with intestinal sarcocystosis for two species acquired from eating undercooked meat: Sarcocystis hominis, from beef, and Sarcocystis suihominis, from pork. Symptoms such as nausea, stomachache, and diarrhea vary widely depending on the number of cysts ingested but appear more severe with pork than with beef. Humans serve as intermediate hosts for Sarcocystis nesbitti, a species with a reptilian definitive host, and possibly other unidentified species, acquired by ingesting sporocysts from feces-contaminated food or water and the environment; infections have an early phase of development in vascular endothelium, with illness that is difficult to diagnose; clinical signs include fever, headache, and myalgia. Subsequent development of intramuscular cysts is characterized by myositis. Presumptive diagnosis based on travel history to tropical regions, elevated serum enzyme levels, and eosinophilia is confirmed by finding sarcocysts in muscle biopsy specimens. There is no vaccine or confirmed effective antiparasitic drug for muscular sarcocystosis, but anti-inflammatory drugs may reduce symptoms. Prevention strategies are also discussed.

A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for cryptosporidium.

Cryptosporidium spp are well recognised as causes of diarrhoeal disease during waterborne epidemics and in immunocompromised hosts. Studies have also drawn attention to an underestimated global burden and suggest major gaps in optimum diagnosis, treatment, and immunisation. Cryptosporidiosis is increasingly identified as an important cause of morbidity and mortality worldwide. Studies in low-resource settings and high-income countries have confirmed the importance of cryptosporidium as a cause of diarrhoea and childhood malnutrition. Diagnostic tests for cryptosporidium infection are suboptimum, necessitating specialised tests that are often insensitive. Antigen-detection and PCR improve sensitivity, and multiplexed antigen detection and molecular assays are underused. Therapy has some effect in healthy hosts and no proven efficacy in patients with AIDS. Use of cryptosporidium genomes has helped to identify promising therapeutic targets, and drugs are in development, but methods to assess the efficacy in vitro and in animals are not well standardised. Partial immunity after exposure suggests the potential for successful vaccines, and several are in development; however, surrogates of protection are not well defined. Improved methods for propagation and genetic manipulation of the organism would be significant advances.

Enterocytozoon bieneusi, giardia, and Cryptosporidium infecting white-tailed deer.

Despite a white-tailed deer (WTD) population in the United States of approximately 32 million animals extremely little is known of the prevalence and species of the protists that infect these animals. This study was undertaken to determine the presence of potential human protist pathogens in culled WTD in central Maryland. Feces from fawns to adults were examined by molecular methods. The prevalence of Enterocytozoon bieneusi, Cryptosporidium, and Giardia was determined by PCR. All PCR-positive specimens were sequenced to determine the species and genotype(s). Of specimens from 80 WTD, 26 (32.5%) contained 17 genotypes of E. bieneusi. Four genotypes were previously reported (I, J, WL4, LW1) and 13 novel genotypes were identified and named DeerEb1-DeerEb13. Genotypes I, J, and LW1 are known to infect humans. Ten (12.5%) specimens contained the Cryptosporidium deer genotype, and one (1.25%) contained Giardia duodenalis Assemblage A. The identification zoonotic G. duodenalis Assemblage A as well as four E. bieneusi genotypes previously identified in humans suggest that WTD could play a role in the transmission of those parasites to humans.

Acute muscular sarcocystosis: an international investigation among ill travelers returning from Tioman Island, Malaysia, 2011-2012.

BACKGROUND: Through 2 international traveler-focused surveillance networks (GeoSentinel and TropNet), we identified and investigated a large outbreak of acute muscular sarcocystosis (AMS), a rarely reported zoonosis caused by a protozoan parasite of the genus Sarcocystis, associated with travel to Tioman Island, Malaysia, during 2011-2012. METHODS: Clinicians reporting patients with suspected AMS to GeoSentinel submitted demographic, clinical, itinerary, and exposure data. We defined a probable case as travel to Tioman Island after 1 March 2011, eosinophilia (>5%), clinical or laboratory-supported myositis, and negative trichinellosis serology. Case confirmation required histologic observation of sarcocysts or isolation of Sarcocystis species DNA from muscle biopsy. RESULTS: Sixty-eight patients met the case definition (62 probable and 6 confirmed). All but 2 resided in Europe; all were tourists and traveled mostly during the summer months. The most frequent symptoms reported were myalgia (100%), fatigue (91%), fever (82%), headache (59%), and arthralgia (29%); onset clustered during 2 distinct periods: "early" during the second and "late" during the sixth week after departure from the island. Blood eosinophilia and elevated serum creatinine phosphokinase (CPK) levels were observed beginning during the fifth week after departure. Sarcocystis nesbitti DNA was recovered from 1 muscle biopsy. CONCLUSIONS: Clinicians evaluating travelers returning ill from Malaysia with myalgia, with or without fever, should consider AMS, noting the apparent biphasic aspect of the disease, the later onset of elevated CPK and eosinophilia, and the possibility for relapses. The exact source of infection among travelers to Tioman Island remains unclear but needs to be determined to prevent future illnesses.

Cryptosporidium species in humans and animals: current understanding and research needs.

Cryptosporidium is increasingly recognized as one of the major causes of moderate to severe diarrhoea in developing countries. With treatment options limited, control relies on knowledge of the biology and transmission of the members of the genus responsible for disease. Currently, 26 species are recognized as valid on the basis of morphological, biological and molecular data. Of the nearly 20 Cryptosporidium species and genotypes that have been reported in humans, Cryptosporidium hominis and Cryptosporidium parvum are responsible for the majority of infections. Livestock, particularly cattle, are one of the most important reservoirs of zoonotic infections. Domesticated and wild animals can each be infected with several Cryptosporidium species or genotypes that have only a narrow host range and therefore have no major public health significance. Recent advances in next-generation sequencing techniques will significantly improve our understanding of the taxonomy and transmission of Cryptosporidium species, and the investigation of outbreaks and monitoring of emerging and virulent subtypes. Important research gaps remain including a lack of subtyping tools for many Cryptosporidium species of public and veterinary health importance, and poor understanding of the genetic determinants of host specificity of Cryptosporidium species and impact of climate change on the transmission of Cryptosporidium.

A highly divergent 33 kDa Cryptosporidium parvum antigen.

Previous studies comparing the genome sequences of Cryptosporidium parvum with Cryptosporidium hominis identified a number of highly divergent genes that might reflect positive selection for host specificity. In the present study, the C. parvum DNA sequence cgd8-5370, which encodes a protein whose amino acid sequence differs appreciably from its homologue in C. hominis , was cloned by PCR and expressed as a recombinant protein in Escherichia coli . Antisera raised against the recombinant cgd8-5370 antigen strongly recognized a unique 33 kDa protein in immunoblots from reducing and non-reducing SDS-PAGE of native C. parvum protein. However, anti-Cp33 sera did not recognize the native 33 kDa homologue in C. hominis . In an immunofluorescence assay (IFA), anti-Cp33 serum recognized an antigen in the anterior end of air-dried C. parvum sporozoites but failed to bind at any sites in C. hominis sporozoites, indicating its specificity for C. parvum . IFA staining of live C. parvum sporozoites with anti-Cp33 serum failed to bind to the parasite, indicating that the CP33 antigen is not on the sporozoite surface, which is consistent with topology predictions based on the encoded amino acid sequence. RT-PCR analysis of cgd8-5370 mRNA before or during C. parvum oocyst excystation revealed transcripts only in excysting sporozoites. Thus, Cp33 represents one of a small number of proteins shown to differentiate C. parvum from C. hominis sporozoites and oocysts.

Cryptosporidium parvum GP60 subtypes in dairy cattle from Buenos Aires, Argentina.

Cryptosporidium parvum from 73 dairy calves less than two months old from Buenos Aires province (Argentina) were molecularly characterized using sequence analysis of the GP60 gene. Seventy-five sequences were obtained, and seven different subtypes were identified, all belonging to the IIa subtype family. The most common subtypes were IIaA20G1R1 (27/75), IIaA22G1R1 (16/75), and IIaA18G1R1 (13/75). Subtypes IIaA21G1R1, IIaA23G1R1, IIaA16G1R1 and IIaA19G1R1 were found sporadically. Two samples contained mixed infections with IIaA21G1R1 and IIaA22G1R1. A significant association was found between subtypes and geographic location, whereas there was no relation between subtypes and presence of diarrhea. Three of the subtypes found in this study (IIaA16G1R1, IIaA18G1R1, and IIaA19G1R1) were previously identified in humans. These findings suggest that cattle could play an important role in the transmission of cryptosporidiosis to humans in Buenos Aires province.

Subtyping Cryptosporidium ubiquitum,a zoonotic pathogen emerging in humans.

Cryptosporidium ubiquitum is an emerging zoonotic pathogen. In the past, it was not possible to identify an association between cases of human and animal infection. We conducted a genomic survey of the species, developed a subtyping tool targeting the 60-kDa glycoprotein (gp60) gene, and identified 6 subtype families (XIIa-XIIf) of C. ubiquitum. Host adaptation was apparent at the gp60 locus; subtype XIIa was found in ruminants worldwide, subtype families XIIb-XIId were found in rodents in the United States, and XIIe and XIIf were found in rodents in the Slovak Republic. Humans in the United States were infected with isolates of subtypes XIIb-XIId, whereas those in other areas were infected primarily with subtype XIIa isolates. In addition, subtype families XIIb and XIId were detected in drinking source water in the United States. Contact with C. ubiquitum-infected sheep and drinking water contaminated by infected wildlife could be sources of human infections.

A large scale molecular study of Giardia duodenalis in horses from Colombia.

The prevalence of Giardia duodenalis assemblages in horses is poorly known. The present study examined feces from 195 horses, 1 month-17 years of age, in 4 locations in Colombia. Prevalence of infection was determined by PCR and all positives were sequenced to determine the genotypes. Thirty four (17.4%) horses were found positive. This is the first report of G. duodenalis in horses from Colombia. Prevalence in female and male horses was 18.9% and 15.1%, respectively. Prevalence in horses <1 year of age and horses >1 year of age was 21.1% and 15.1%, respectively. Molecular characterization using the beta giardin (bg), glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), and small subunit ribosomal RNA (ssurRNA) genes identified G. duodenalis Assemblages A and B, the assemblages regarded as zoonotic.

Adhesive-tape recovery combined with molecular and microscopic testing for the detection of Cryptosporidium oocysts on experimentally contaminated fresh produce and a food preparation surface.

A proof of concept study was conducted to determine if transparent double-sided adhesive tape could be used to recover and detect [by polymerase chain reaction (PCR) and immunofluorescence microscopy (IFA)] Cryptosporidium parvum oocysts on fresh produce and on a food preparation surface. Oocysts were applied on the surface of ten apples, ten peaches, eight cucumbers, and eight tomatoes within circles drawn with a permanent marker. Approximately 18 h later, skin excised from three uncontaminated and three contaminated circles from each piece of produce was subjected to PCR. Pieces of transparent double-sided adhesive tape were lightly pressed onto the surface of three other contaminated circles and examined by PCR. Other pieces of adhesive tape were pressed against the surfaces of three other circles and examined by IFA. At concentrations of 100 and 50 oocysts per circle, every produce item examined by PCR of contaminated excised skin was found positive, and every item examined by adhesive tape subjected to PCR and IFA was found positive, except one. At ten oocysts per circle, every produce item was found positive by PCR of contaminated excised skin, and all apples, cucumbers, and tomatoes were found positive by adhesive tape subjected to IFA. Detection of low numbers of oocysts on peaches by IFA examination of adhesive tape was problematic because trichomes that cover peaches and impart the fuzzy surface partially restrict the tape from reaching some areas where oocysts adhere. Tape combined with IFA was successful in recovering and identifying oocysts from six areas of laminate countertop where the oocysts had been applied and allowed to dry for 30-60 min. These are the first findings to demonstrate that adhesive tape can be used to recover and identify a protozoan parasite from fresh produce and from a laminate food preparation surface.

Detection of concurrent infection of dairy cattle with Blastocystis, Cryptosporidium, Giardia, and Enterocytozoon by molecular and microscopic methods.

Of fecal specimens examined from 47 dairy cattle ranging in age from neonates to multiparous cows, 9, 10, 24, and 17 were positive for Blastocystis spp., Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi, respectively, as determined by PCR. Eight 3- to 5-month-old cattle were concurrently infected with three or four of these parasites. This is the first report to identify multiple concurrent infections with these four potentially zoonotic protist pathogens in cattle. None of the cattle exhibited signs of illness or effects of infection on growth and are regarded as healthy carriers. A commercially available immunofluorescence (IFA) microscopic test confirmed six of seven available PCR-positive Blastocystis specimens and identified one IFA-positive cow that was PCR negative.

CD40 agonist antibody mediated improvement of chronic Cryptosporidium infection in patients with X-linked hyper IgM syndrome.

X-linked hyper-IgM syndrome (XHM) is a combined immune deficiency disorder caused by mutations in CD40 ligand. We tested CP-870,893, a human CD40 agonist monoclonal antibody, in the treatment of two XHM patients with biliary Cryptosporidiosis. CP-870,893 activated B cells and APCs in vitro, restoring class switch recombination in XHM B cells and inducing cytokine secretion by monocytes. CP-870,893 infusions were well tolerated and showed significant activity in vivo, decreasing leukocyte concentration in peripheral blood. Although specific antibody responses were lacking, frequent dosing in one subject primed T cells to secrete IFN-g and suppressed oocyst shedding in the stool. Nevertheless, relapse occurred after discontinuation of therapy. The CD40 receptor was rapidly internalized following binding with CP-870,893, potentially explaining the limited capacity of CP-870,893 to mediate immune reconstitution. This study demonstrates that CP-870,893 suppressed oocysts shedding in XHM patients with biliary cryptosporidiosis. The continued study of CD40 agonists in XHM is warranted.

Immunolocalization of ß- and δ-giardin within the ventral disk in trophozoites of Giardia duodenalis using multiplex laser scanning confocal microscopy.

Immunolocalization of ß- and δ-giardin in Giardia duodenalis trophozoites revealed that both giardins are strictly associated with the ventral disk (VD). Optical sectioning of the immunolabeled VD, together with quantitative colocalization of δ- and ß-giardin immunoreactivity, demonstrated that δ-giardin is primarily localized to the ventral side, and ß-giardin is localized to the dorsal side of the VD.

Multilocus genotyping of Giardia duodenalis in lambs from Spain reveals a high heterogeneity.

Fecal specimens from 120 lambs in Valencia (Spain) were analyzed for Giardia duodenalis by IFA and nested-PCR using the beta giardin (bg), glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi) and small subunit ribosomal RNA (ssurRNA) genes. The highest prevalence was obtained using the ssurRNA gene (89.2%), whereas values from other techniques ranged from 64.1% to 69.2%. Sequences of the ssurRNA showed a high proportion of assemblage A or mixed assemblage A/E samples (55.1% and 25.2%, respectively). When the other 3 loci were analyzed, between 6.5% and 15.4% were found to be assemblage A or A/E, respectively. Nested PCR for the tpi gene was the most variable of the targets employed. Twelve new sequences of gdh and tpi for G. duodenalis from sheep were found. Multilocus genotyping resulted in 63 patterns from the 71 samples sequenced at the four loci. This high variability among isolates possibly reflects the high frequency of mixed infections.