Examination of ELISA against PCR for assessing treatment efficacy against Cryptosporidium in a clinical trial context.

BACKGROUND: Cryptosporidium is a gastrointestinal pathogen that presents a serious opportunistic infection in immunocompromised individuals including those living with human immunodeficiency syndrome. The CRYPTOFAZ trial, previously published, was conducted in Malawi to evaluate the efficacy of clofazimine in response to an unmet need for drugs to treat cryptosporidiosis in HIV populations. A combination of rapid diagnostic tests, ELISA, qPCR, and conventional sequencing were employed to detect Cryptosporidium in 586 individuals during pre-screening and monitor oocyst shedding and identify enteric co-pathogens in 22 enrolled/randomized participants during the in-patient period and follow-up visits. METHODOLOGY: Oocyst shedding as measured by qPCR was used to determine primary trial outcomes, however pathogen was detected even at trial days 41-55 in individuals randomized to either clofazimine or placebo arms of the study. Therefore, in this work we re-examine the trial outcomes and conclusions in light of data from the other diagnostics, particularly ELISA. ELISA data was normalized between experiments prior to comparison to qPCR. The amount of all identified enteric pathogens was examined to determine if co-pathogens other than Cryptosporidium were major causative agents to a participant's diarrhea. CONCLUSION: ELISA had higher sample-to-sample variability and proved to be equally or less sensitive than qPCR in detecting Cryptosporidium positive samples. Compared to qPCR, ELISA had equal or greater specificity in detecting Cryptosporidium negative samples. Sequencing identified several Cryptosporidium species including viatorum which has never been identified in Malawi and Southern Africa. In addition to Cryptosporidium, enterotoxigenic E. coli was also identified as a pathogen in diarrheagenic amounts in 4 out of 22 participants.

The Application of 3base™ Technology to Diagnose Eight of the Most Clinically Important Gastrointestinal Protozoan Infections.

Globally, over 3.5 billion people are infected with intestinal parasites each year, resulting in over 200,000 deaths. Three of the most common protozoan pathogens that affect the gastrointestinal tract of humans are Cryptosporidium spp., Giardia intestinalis, and Entamoeba histolytica. Other protozoan agents that have been implicated in gastroenteritis in humans include Cyclospora cayetanensis, Dientamoeba fragilis, Blastocystis hominis, and the microsporidia Enterocytozoon bieneusi and Encephalitozoon intestinalis. Genetic Signatures previously developed a 3base™ multiplexed Real-Time PCR (mRT-PCR) enteric protozoan kit (EP001) for the detection of Giardia intestinalis/lamblia/duodenalis, Cryptosporidium spp., E. histolytica, D. fragilis, and B. hominis. We now describe improvements to this kit to produce a more comprehensive assay, including C. cayetanensis, E. bieneusi, and E. intestinalis, termed EP005. The clinical performance of EP005 was assessed using a set of 380 clinical samples against a commercially available PCR test and other in-house nucleic acid amplification tests where commercial tests were not available. All methods provided at least 90% agreement. EP005 had no cross-reactivity against 82 organisms commonly found in the gut. The EP005 method streamlines the detection of gastrointestinal parasites and addresses the many challenges of traditional microscopic detection, resulting in cost savings and significant improvements in patient care.

PacBio next-generation sequencing uncovers Apicomplexa diversity in different habitats.

The phylum Apicomplexa comprises a large group of intracellular protozoan parasites. These microorganisms are known to infect a variety of vertebrate and invertebrate hosts, leading to significant medical and veterinary conditions such as toxoplasmosis, cryptosporidiosis, theileriosis, and eimeriosis. Despite their importance, comprehensive data on their diversity and distribution, especially in riverine environments, remain scant. To bridge this knowledge gap, we utilized next-generation high-throughput 18S rRNA amplicon sequencing powered by PacBio technology to explore the diversity and composition of the Apicomplexa taxa. Principal component analysis (PCA) and principal coordinate analysis (PCoA) indicated the habitat heterogeneity for the physicochemical parameters and the Apicomplexa community. These results were supported by PERMANOVA (P < 0.001), ANOSIM (P < 0.001), Cluster analysis, and Venn diagram. Dominant genera of Apicomplexa in inlet samples included Gregarina (38.54%), Cryptosporidium (32.29%), and Leidyana (11.90%). In contrast, outlet samples were dominated by Babesia, Cryptosporidium, and Theileria. While surface water samples revealed 16% and 8.33% relative abundance of Toxoplasma and Cryptosporidium, respectively. To our knowledge, the next-generation high throughput sequencing covered a wide range of parasites in Egypt for the first time, which could be useful for legislation of the standards for drinking water and wastewater reuse.

A large-scale study on the prevalence of intestinal parasites in patients referred to medical laboratories in Urmia, Northwest Iran.

INTRODUCTION: Intestinal parasitic infections (IPIs), caused by helminths and protozoans, are among the most prevalent infections in humans in developing countries. This study aimed to determine the prevalence of IPIs in patients referred to three educational and medical centers affiliated with Urmia University of Medical Sciences in Urmia. MATERIALS AND METHODS: In this cross-sectional study, 2845 stool samples, including 2174 (76.4%) males and 671 (23.6%) females, were collected from patients referred to Imam Khomeini and Shahid Motahhari hospitals and Shahid Nikkhah Health Center in Urmia, Northwest Iran, from January 2020 to February 2022. The microscopic examination for IPIs was carried out using the wet mount method, and the hard-to-identify samples were stained by trichrome for accurate identification of protozoa. For diagnosis of infections by coccidian parasites modified Ziehl-Neelsen (mZN) staining was used. RESULTS: Based on the results, two hundred nine intestinal parasites were identified in 184 out of 2845 (6.5%) patients of which 136 out of 2174 males (6.3%) and 48 out of 671 females (7.2%) were positive. Some patients had tested positive for multiple protozoa. The observed intestinal protozoa are as follows: Blastocystis spp. 118 (4.1%), Endolimax Nana 42 (1.5%), Entamoeba coli 24 (0.8%), Giardia lamblia 13 (0.5%), Cryptosporidium spp. 6 (0.2%), Iodamoeba butschlii 3 (0.1%), Chilomastix mesnili 2 (0.1%), and an accidentally detected helminthic infection Enterobius vermicularis 1 (0.05%). CONCLUSION: According to the results, the most prevalent IPIs in West Azerbaijan Province are caused by Blastocystis spp., and Giardia lamblia. Most intestinal protozoa observed in the study were nonpathogenic and commensal, which shows water or food contamination in the area. Thus, medical technologists in the parasitology section must be trained and aware of IPIs in medical laboratories.

Study on the Therapeutic Effect of Coconut Oil Extracts as An Alternative Medicinal Plant in Cryptosporidium Infected Mice.

OBJECTIVE: Cryptosporidiosis caused by Cryptosporidium sp. is a globally spreading disease. Nowadays, new researches are moving towards an effective treatment without side effects, especially for young and immune-compromised patients. The current study was designed to evaluate the therapeutic effect of the coconut oil extracts as an alternative medicinal plant in Cryptosporidium infected immunocompromised mice. METHODS: Sixty white albino mice were classified into six groups; Group I: Infected with Cryptosporidium oocysts treated with Nitazoxanide, Group II: Infected with Cryptosporidium oocysts and treated with coconut water extract, Group III: Infected with Cryptosporidium oocysts and treated with coconut Hexan extract, Group IV: Infected with Cryptosporidium oocysts and treated with coconut ethanol extract, Group V: Positive control, Group VI: Negative control. Stool samples were collected and examined; histopathological and immune-histochemical assessment using anti caspase-3 and anti CDX2 monoclonal antibodies were performed. RESULTS: Coconut oil extracts results revealed a significant decrease of oocyst count, correlated with an amelioration of histopathological and confirmed by immunohistochemical changes in ileal tissue. CONCLUSION: The present study has opened fresh avenues for development of natural therapy like coconut oil extracts, which have a potential therapeutic efficacy against Cryptosporidiosis. That was confirmed by different methodologies, parasitological examination, histopathological examination, and immunohistochemical assays. It paves the way for being a promising anti-parasitic agent for infection eradication. However, further studies are still required to gain more knowledge about different coconut extracts in order to reach the best treatment efficacy.

Changing Molecular Profiles of Human Cryptosporidiosis Cases in Scotland as a Result of the Coronavirus Disease, COVID-19 Pandemic.

Cryptosporidium, the most frequently reported parasite in Scotland, causes gastrointestinal illness resulting in diarrhoea, nausea and cramps. Two species are responsible for most cases: Cryptosporidium hominis (C. hominis) and Cryptosporidium parvum (C. parvum). Transmission occurs faecal-orally, through ingestion of contaminated food and water, or direct contact with faeces. In 2020, the COVID-19 pandemic led to global restrictions, including national lockdowns to limit viral transmission. Such interventions led to decreased social mixing, and reduced/no local and international travel, which are factors associated with transmission of multiple communicable diseases, including cryptosporidiosis. This report assessed the impact of the pandemic on Scottish cryptosporidiosis cases, and identified changes in circulating molecular variants of Cryptosporidium species. Molecular data generated using real time PCR and GP60 nested-PCR assays on laboratory-confirmed cryptosporidiosis cases reported during 2018-22 were analysed. The Scottish Microbiology Reference Laboratories (SMiRL), Glasgow, received 774 Cryptosporidium-positive faeces during 2018-22, of which 486 samples were successfully subtyped. During this time period, C. hominis (n = 155; 21%) and C. parvum (n = 572; 77%) were the most commonly detected species. The total number of cases during 2020, which was greatly affected by the pandemic, was markedly lower in comparison to case numbers in the 2 years before and after 2020. The most predominant C. hominis family detected prior to 2020 was the Ib family which shifted to the Ie family during 2022. The most common C. parvum variant during 2018-22 was the IIa family, however a rise in the IId family was observed (n = 6 in 2018 to n = 25 in 2022). The dominant C. hominis subtype IbA10G2, which accounted for 71% of C. hominis subtypes in 2018-19 was superseded by three rare subtypes: IeA11G3T3 (n = 15), IdA16 (n = 8) and IbA9G3 (n = 3) by 2022. Frequently reported C. parvum subtypes in 2018-19 were IIaA15G2R1 and IIaA17G1R1, accounting for 59% of total C. parvum subtypes. By 2022, IIaA15G2R1 remained the most common (n = 28), however three unusual subtypes in Scotland emerged: IIdA24G1 (n = 7), IIaA16G3R1 (n = 7) and IIaA15G1R2 (n = 7). Continuous monitoring of Cryptosporidium variants following the pandemic will be essential to explore further changes and emergence of strains with altered virulence.

Cytotoxic innate intraepithelial lymphocytes control early stages of Cryptosporidium infection.

Background: Intraepithelial lymphocytes (IELs) are the first immune cells to contact and fight intestinal pathogens such as Cryptosporidium, a widespread parasite which infects the gut epithelium. IFN-γ producing CD4+ T IELs provide an efficient and a long-term protection against cryptosporidiosis while intraepithelial type 1 innate lymphoid cells limits pathogen spreading during early stages of infection in immunodeficient individuals. Yet, the role of T-cell like innate IELs, the most frequent subset of innate lymphocytes in the gut, remains unknown. Methods: To better define functions of innate IELs in cryptosporidiosis, we developed a co-culture model with innate IELs isolated from Rag2-/- mice and 3D intestinal organoids infected with C. parvum using microinjection. Results: Thanks to this original model, we demonstrated that innate IELs control parasite proliferation. We further showed that although innate IELs secrete IFN-γ in response to C. parvum, the cytokine was not sufficient to inhibit parasite proliferation at early stages of the infection. The rapid protective effect of innate IELs was in fact mediated by a cytotoxic, granzyme-dependent mechanism. Moreover, transcriptomic analysis of the Cryptosporidium-infected organoids revealed that epithelial cells down regulated Serpinb9b, a granzyme inhibitor, which may increase their sensitivity to cytolytic attack by innate IELs. Conclusion: Based on these data we conclude that innate IELs, most likely T-cell-like innate IELs, provide a rapid protection against C. parvum infection through a perforin/granzymes-dependent mechanism. C. parvum infection. The infection may also increase the sensitivity of intestinal epithelial cells to the innate IEL-mediated cytotoxic attack by decreasing the expression of Serpin genes.

Occurence and genotype distributionof Cryptosporidium spp.,and Giardia duodenalis in sheep in Siirt, Turkey.

Cryptosporidium spp., and Giardia duodenalis are intestinal protozoan parasites known to infect humans and various animals and cause diarrhea. This study aimed at determining the prevalence and genotype of Cryptosporidium spp. and Giardia duodenalis in sheep in different locations of Siirt province. The fecal material for this study was collected from 500 sheep in different locations of Siirt province, Turkey. Fecal samples obtained from sheep were examined for Cryptosporidium spp. by Kinyoun Acid Fast staining and the Nested PCR method. Microscopic and Nested PCR methods revealed a prevalence of 2.4% (12/500) and 3.6% (18/500), respectively. Sequence analysis revealed the presence of C. ryanae, C. andersoni, and zoonotic C. parvum. In terms of Giardia duodenalis, 8.4% (42/500) and 10.2% (51/500) prevalence was determined using Nativ-Lugol and Nested PCR methods, respectively. Using sequence analysis, zoonotic assemblages A and B as well as assemblages E and D were detected. As a result of this study, both the prevalence of Cryptosporidium spp. and Giardia duodenalis and the presence of species that appear to be host-specific, as well as those known to be zoonotic, were revealed. A large-scale study is needed to understand the impact of these agents on sheep farming and their consequences on human health.

Origin and arrangement of actin filaments for gliding motility in apicomplexan parasites revealed by cryo-electron tomography.

The phylum Apicomplexa comprises important eukaryotic parasites that invade host tissues and cells using a unique mechanism of gliding motility. Gliding is powered by actomyosin motors that translocate host-attached surface adhesins along the parasite cell body. Actin filaments (F-actin) generated by Formin1 play a central role in this critical parasitic activity. However, their subcellular origin, path and ultrastructural arrangement are poorly understood. Here we used cryo-electron tomography to image motile Cryptosporidium parvum sporozoites and reveal the cellular architecture of F-actin at nanometer-scale resolution. We demonstrate that F-actin nucleates at the apically positioned preconoidal rings and is channeled into the pellicular space between the parasite plasma membrane and the inner membrane complex in a conoid extrusion-dependent manner. Within the pellicular space, filaments on the inner membrane complex surface appear to guide the apico-basal flux of F-actin. F-actin concordantly accumulates at the basal end of the parasite. Finally, analyzing a Formin1-depleted Toxoplasma gondii mutant pinpoints the upper preconoidal ring as the conserved nucleation hub for F-actin in Cryptosporidium and Toxoplasma. Together, we provide an ultrastructural model for the life cycle of F-actin for apicomplexan gliding motility.

European ground squirrels Spermophilus citellus (Linnaeus) do not share identical Cryptosporidium spp. with North American ground squirrels.

Cryptosporidium Tyzzer, 1910 is one of the most common protistan parasites of vertebrates. The results of this study provide the first data on Cryptosporidium diversity in the European ground squirrel Spermophilus citellus (Linnaeus). A total of 128 faecal samples of European ground squirrels from 39 localities in the Czech Republic were analysed for the presence of Cryptosporidium spp. by microscopy and PCR/sequence analysis of small subunit ribosomal RNA (SSU) and the actin gene. While the microscopical examination did not reveal the presence of any Cryptosporidium oocysts, eight samples from six localities were PCR-positive. Phylogenetic analyses revealed the presence of five different Cryptosporidium spp. isolates. Four isolates, designated as Cryptosporidium sp. isolate Sc01-04, detected in wild populations and never recorded before, clustered closely to Cryptosporidium genotypes that have previously been found in North American ground squirrels' species. Cryptosporidium sciurinum Prediger, Jezková, Holubová, Sak, Konecný, Rost, McEvoy, Rajský et Kvác, 2021 was found in an animal sanctuary. Because C. sciurinum had previously been detected in Eurasian red squirrels Sciurus vulgaris Linnaeus at the same facility, it can be concluded that this Cryptosporidium was transmitted from tree squirrels to ground squirrels within the animal sanctuary. The results indicate that populations of European and North American ground squirrels are parasitised by different Cryptosporidium spp. At the same time, this is the first description of the occurrence of C. sciurinum in ground squirrels.

Mining for crypto protection: a search for Cryptosporidium antibodies reveals antigens associated with immunity.

Infectious diarrhea is a major cause of morbidity and mortality, particularly for children in low- and middle-income countries. Cryptosporidium is a diarrheal pathogen for which there is no vaccine and current therapies are only partially effective. In this issue of the JCI, Gilchrist, Campo, and colleagues surveyed a large cohort of Bangladeshi children to profile antibody responses against an array of Cryptosporidium proteins. They discovered 233 proteins to which children developed antibodies, identified seven as being associated with protection from reinfection, and provided insights regarding the longevity of Cryptosporidium antibodies and the development of antibody breadth. In this commentary, we discuss the burden of disease caused by Cryptosporidium and how these studies highlight the strategies to better manage this parasite.

Biosensors as early warning detection systems for waterborne Cryptosporidium.

Waterborne disease is a global health threat contributing to a high burden of diarrhoeal disease, and growing evidence indicates a prospective increase in incidence coinciding with the profound effects of climate change. A major causative agent of gastrointestinal disease is Cryptosporidium, a protozoan waterborne parasite identified in over 70 countries. Cryptosporidium is a cause of high disease morbidity in children and the immunocompromised with limited treatment options for patients at risk of severe illness. The hardy nature of the organism leads to its persistence in various water sources, with certain water treatment procedures proving inefficient for its complete removal. While diagnostic methods for Cryptosporidium are well-defined in the clinical sphere, detection of Cryptosporidium in water sources remains suboptimal due to low dispersion of organisms in large sample volumes, lengthy processing times and high costs of equipment and reagents. A need for improvement exists to identify the organism as an emerging threat in domestic water systems, and the technological advantages that biosensors offer over current analytical methods may provide a preventative approach to outbreaks of Cryptosporidium. Biosensors are innovative, versatile and adaptable analytical tools that could provide highly sensitive, rapid, on-site analysis needed for Cryptosporidium detection in low-resource settings.

Interactions between free-living amoebae and Cryptosporidium parvum: an experimental study.

Free-Living Amebae (FLA) and Cryptosporidium oocysts occasionally share the same environment. From 2004 to 2016, Cryptosporidium was responsible for 60% of 905 worldwide waterborne outbreaks caused by protozoan parasites. The aim of this study was to evaluate interactions between C. parvum oocysts and two common FLAs (Acanthamoeba castellanii and Vermamoeba vermiformis) in a water environment. Encystment and survival of FLAs were evaluated by microscopy using trypan blue vital coloration. Oocysts were numerated on microscopy. Interactions were studied over time in conditions both unfavorable and favorable to phagocytosis. Potential phagocytosis was directly evaluated by several microscopic approaches and indirectly by numeration of microorganisms and oocyst infectivity evaluation. Occasional phagocytosis of C. parvum by FLAs was documented. However, oocyst concentrations did not decrease significantly, suggesting resistance of oocysts to phagocytosis. A temporary decrease of oocyst infectivity was observed in the presence of A. castellanii. The effect of these interactions on C. parvum infectivity is particularly interesting. The biofilm condition could favor the persistence or even the proliferation of oocysts over time. This study demonstrated interactions between C. parvum and FLAs. Further knowledge of the mechanisms involved in the decrease of oocyst infectivity in the presence of A. castellanii could facilitate the development of new therapeutic approaches.

Title: Interactions entre amibes libres et Cryptosporidium parvum : étude expérimentale. Abstract: Les amibes libres et les oocystes de Cryptosporidium partagent parfois le même environnement. Entre 2004 et 2016, Cryptosporidium a été responsable de 60 % des 905 épidémies d'origine hydrique dans le monde causées par des parasites protozoaires. Le but de cette étude était d'évaluer les interactions entre les oocystes de C. parvum et deux espèces d'amibes libres communes (Acanthamoeba castellanii et Vermamoeba vermiformis) en environnement aquatique. L'enkystement et la survie des amibes libres ont été évalués par microscopie en utilisant une coloration vitale au bleu trypan. Les oocystes ont été comptés au microscope. Les interactions ont été étudiées au cours du temps dans des conditions à la fois défavorables et favorables à la phagocytose. La phagocytose potentielle a été évaluée directement par plusieurs approches microscopiques et indirectement par la numération des micro-organismes et l'évaluation de l'infectiosité des oocystes. Une phagocytose occasionnelle de C. parvum par amibes libre a été documentée. Cependant, les concentrations d'oocystes n'ont pas diminué de manière significative, ce qui suggère une résistance des oocystes à la phagocytose. Une diminution temporaire de l'infectivité des oocystes a été observée en présence d'A. castellanii. L'effet de ces interactions sur l'infectiosité de C. parvum est particulièrement intéressant. La condition biofilm pourrait favoriser la persistance ou même la prolifération des oocystes de C. parvum au fil du temps. Cette étude a démontré des interactions entre C. parvum et amibes libres. Une meilleure connaissance des mécanismes impliqués dans la diminution de l'infectiosité des oocystes en présence d'A. castellanii pourrait faciliter le développement de nouvelles approches thérapeutiques.

A highly antigenic fragment within the zoonotic Cryptosporidium parvum Gp900 glycoprotein (Domain 3) is absent in human restricted Cryptosporidium species.

We identified a fragment (Domain 3-D3) of the immunodominant sporozoite surface glycoprotein of the zoonotic parasite Cryptosporidium gp900, which is absent C. hominis and C. parvum anthroponosum. The fragment is highly antigenic and is able to effectively differentiate between zoonotic C. parvum and species/genotypes that infect preferentially humans. D3 detection provides a serological tool to determine whether the source of human cryptosporidiosis is of animal or human origin. We demonstrate this in experimentally challenged piglets, mice, rats, and alpaca. We speculate that the absence of this fragment from the C. hominis and C. parvum anthroponosum gp900 protein may play a key role in their host restriction.

Molecular Detection and Genotyping of Cryptosporidium spp. Isolates from Bats in Colombia.

PURPOSE: Cryptosporidiosis is a zoonotic infectious disease caused by the protozoan parasite Cryptosporidium spp., frequently found in several animal species, including bats. Several Cryptosporidium genotypes have been described in bats worldwide, suggesting that bats are infected by host-specific Cryptosporidium spp. To date, there are no published reports about Cryptosporidium spp. in bats from Colombia. Therefore, this study aimed to determine the presence and molecular diversity of Cryptosporidium spp. in Colombian bats. METHODS: A total of 63 gut samples from three bat species served for molecular detection of Cryptosporidium spp. 18S rDNA gene by qPCR. The sequenced amplicons were used in subsequent phylogenetic analyses to identify them as species or genotypes. RESULTS: Cryptosporidium spp. qPCR detection occurred in 9.5% (6/63) of bat intestines, and four sequences represented two new genotypes, called Cryptosporidium bat genotypes XIX and XX, were identified. CONCLUSIONS: This study describes the detection of two novel Cryptosporidium bat genotypes, in two species of bats from a region of Colombia, requiring further studies to determine the relationhip between Cryptosporidium and bats in Colombia.

First identification of Cryptosporidium parvum virus 1 (CSpV1) in various subtypes of Cryptosporidium parvum from diarrheic calves, lambs and goat kids from France.

Cryptosporidium spp. remain a major cause of waterborne diarrhea and illness in developing countries and represent a significant burden to farmers worldwide. Cryptosporidium parvum virus 1 (CSpV1), of the genus Cryspovirus, was first reported to be present in the cytoplasm of C. parvum in 1997. Full-length genome sequences have been obtained from C. parvum from Iowa (Iowa), Kansas (KSU) and China. We aimed at characterizing the genome of CSpV1 from France and used sequence analysis from Cryptosporidium isolates to explore whether CSpV1 genome diversity varies over time, with geographical sampling location, C. parvum genetic diversity, or ruminant host species. A total of 123 fecal samples of cattle, sheep and goats were collected from 17 different French departments (57 diseased animal fecal samples and 66 healthy animal fecal samples). Subtyping analysis of the C. parvum isolates revealed the presence of two zoonotic subtype families IIa and IId. Sequence analysis of CSpV1 revealed that all CSpV1 from France, regardless of the subtype of C. parvum (IIaA15G2R1, IIaA17G2R1 and IIdA18G1R1) are more closely related to CSpV1 from Turkey, and cluster on a distinct branch from CSpV1 collected from C. parvum subtype IIaA15G2R1 from Asia and North America. We also found that samples collected on a given year or successive years in a given location are more likely to host the same subtype of C. parvum and the same CSpV1 strain. Yet, there is no distinct clustering of CSpV1 per French department or ruminants, probably due to trade, and transmission of C. parvum among host species. Our results point towards (i) a close association between CSpV1 movement and C. parvum movement, (ii) recent migrations of C. parvum among distantly located departments and (iii) incidental transmission of C. parvum between ruminants. All together, these results provide insightful information regarding CSpV1 evolution and suggest the virus might be used as an epidemiological tracer for C. parvum. Future studies need to investigate CSpV1's role in C. parvum virulence and on subtype ability to infect different species.

Prevalence and molecular characterization of Cryptosporidium spp. in dairy cattle in Central Inner Mongolia, Northern China.

BACKGROUND: Cryptosporidium is a gastrointestinal protozoan that widely exists in nature, it is an established zoonotic pathogen. Infected cattle are considered to be associated with cryptosporidiosis outbreaks in humans. In the present study, we aimed to assess the prevalence and species distribution of Cryptosporidium in dairy cattle in Central Inner Mongolia. METHODS: We focused on the small subunit ribosomal RNA gene (SSU rRNA) of Cryptosporidium and 60-kDa glycoprotein gene (gp60) of Cryptosporidium parvum. We collected 505 dairy cattle manure samples from 6 sampling sites in Inner Mongolia in 2021; the samples were divided into 4 groups based on age. DNA extraction, polymerase chain reaction (PCR), sequence analysis, and restriction fragment length polymorphism (RFLP) using SspI and MboII restriction endonucleases were performed. RFLP analysis was performed to determine the prevalence and species distribution of Cryptosporidium. RESULTS: SSU rRNA PCR revealed that the overall prevalence of Cryptosporidium infection was 29.90% (151/505), with a prevalence of 37.67% (55/146) and 26.74% (96/359) in diarrheal and nondiarrheal samples, respectively; these differences were significant. The overall prevalence of Cryptosporidium infection at the 6 sampling sites ranged from 0 to 47.06% and that among the 4 age groups ranged from 18.50 to 43.81%. SSU rRNA sequence analysis and RFLP analysis revealed the presence of 4 Cryptosporidium species, namely, C. bovis (44.37%), C. andersoni (35.10%), C. ryanae (21.85%), and C. parvum (11.92%), along with a mixed infection involving two or three Cryptosporidium species. Cryptosporidium bovis or C. andersoni was the most common cause of infection in the four age groups. The subtype of C. parvum was successfully identified as IIdA via gp60 analysis; all isolates were identified as the subtype IIdA19G1. CONCLUSIONS: To the best of our knowledge, this is the first report of dairy cattle infected with four Cryptosporidium species in Inner Mongolia, China, along with a mixed infection involving two or three Cryptosporidium species, with C. bovis and C. andersoni as the dominant species. Moreover, this is the first study to identify C. parvum subtype IIdA19G1 in cattle in Inner Mongolia. Our study findings provide detailed information on molecular epidemiological investigation of bovine cryptosporidiosis in Inner Mongolia, suggesting that dairy cattle in this region are at risk of transmitting cryptosporidiosis to humans.

Mass Spectrometry Imaging of In Vitro Cryptosporidium parvum-Infected Cells and Host Tissue.

Cryptosporidium parvum is a zoonotic-relevant parasite belonging to the phylum Alveolata (subphylum Apicomplexa). One of the most zoonotic-relevant etiologies of cryptosporidiosis is the species C. parvum, infecting humans, cattle and wildlife. C. parvum-infected intestinal mucosa as well as host cells infected in vitro have not yet been the subject of extensive biochemical investigation. Efficient treatment options or vaccines against cryptosporidiosis are currently not available. Human cryptosporidiosis is currently known as a neglected poverty-related disease (PRD), being potentially fatal in young children or immunocompromised patients. In this study, we used a combination of atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) and liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to determine and locate molecular biomarkers in in vitro C. parvum-infected host cells as well as parasitized neonatal calf intestines. Sections of C. parvum-infected and non-infected host cell pellets and infected intestines were examined to determine potential biomarkers. Human ileocecal adenocarcinoma cells (HCT-8) were used as a suitable in vitro host cell system. More than a thousand different molecular signals were found in both positive- and negative-ion mode, which were significantly increased in C. parvum-infected material. A database search in combination with HPLC-MS/MS experiments was employed for the structural verification of markers. Our results demonstrate some overlap between the identified markers and data obtained from earlier studies on other apicomplexan parasites. Statistically relevant biomarkers were imaged in cell layers of C. parvum-infected and non-infected host cells with 5 µm pixel size and in bovine intestinal tissue with 10 µm pixel size. This allowed us to substantiate their relevance once again. Taken together, the present approach delivers novel metabolic insights on neglected cryptosporidiosis affecting mainly children in developing countries.

Non-potable water reuse and the public health risks from protozoa and helminths: a case study from a city with a semi-arid climate.

The study estimated the risk due to Cryptosporidium, Giardia, and Ascaris, associated with non-potable water reuse in the city of Jaipur, India. The study first determined the exposure dose of Cryptosporidium, Giardia, and Ascaris based on various wastewater treatment technologies for various scenarios of reuse for six wastewater treatment plants (WWTPs) in the city. The exposure scenarios considered were (1) garden irrigation; (2) working and lounging in the garden; and (3) consumption of crops irrigated with recycled water. The estimated annual risk of infection varied between 8.57 × 10-7 and 1.0 for protozoa and helminths, respectively. The order of treatment processes, in decreasing order of annual risk of infection, was found to be: moving-bed bioreactor (MBBR) technology > activated sludge process (ASP) technology > sequencing batch reactor (SBR) technology. The estimated annual risk was found to be in this order: Ascaris > Giardia > Cryptosporidium. The study also estimated the maximum allowable concentration (Cmax) of pathogen in the effluent for a benchmark value of annual infection of risk equal to 1:10,000, the acceptable level of risk used for drinking water. The estimated Cmax values were found to be 6.54 × 10-5, 1.37 × 10-5, and 2.89 × 10-6 (oo) cysts/mL for Cryptosporidium, Giardia, and Ascaris, respectively.

Supporting the implementation of drinking water management systems in New South Wales, Australia.

Since 2010, New South Wales (NSW) Health has assisted local water utilities to develop and implement risk-based drinking water management systems based on the Australian Drinking Water Guidelines Framework for Management of Drinking Water Quality. This support has benefited regional communities, and especially smaller utilities, by helping to identify and control risks. NSW Health's support projects have resulted in statistically significant improvements across many elements of drinking water management system implementation. Through this program of support, NSW Health has identified possible infrastructure and operational needs and assessed implementation of drinking water management systems. In parallel, NSW Health has worked to assess the risk from Cryptosporidium in drinking water supplies and to develop a formal audit program. Findings from the NSW Health support program informed the development of two NSW Government programs and the commitment of more than $1 billion to help local water utilities address public health and other critical needs. The introduction of risk-based drinking water management systems has driven incremental improvement in drinking water quality management across the state of NSW.