Cryptosporidium impacts epithelial turnover and is resistant to induced death of the host cell.

Infection with the apicomplexan parasite Cryptosporidium is a leading cause of diarrheal disease. Cryptosporidiosis is of particular importance in infants and shows a strong association with malnutrition, both as a risk factor and as a consequence. Cryptosporidium invades and replicates within the small intestine epithelial cells. This is a highly dynamic tissue that is developmentally stratified along the villus axis. New cells emerge from a stem cell niche in the crypt and differentiate into mature epithelial cells while moving toward the villus tip, where they are ultimately shed. Here, we studied the impact of Cryptosporidium infection on this dynamic architecture. Tracing DNA synthesis in pulse-chase experiments in vivo, we quantified the genesis and migration of epithelial cells along the villus. We found proliferation and epithelial migration to be elevated in response to Cryptosporidium infection. Infection also resulted in significant cell loss documented by imaging and molecular assays. Consistent with these observations, single-cell RNA sequencing of infected intestines showed a gain of young and a loss of mature cells. Interestingly, enhanced epithelial cell loss was not a function of enhanced apoptosis of infected cells. To the contrary, Cryptosporidium-infected cells were less likely to be apoptotic than bystanders, and experiments in tissue culture demonstrated that infection provided enhanced resistance to chemically induced apoptosis to the host but not bystander cells. Overall, this study suggests that Cryptosporidium may modulate cell apoptosis and documents pronounced changes in tissue homeostasis due to parasite infection, which may contribute to its long-term impact on the developmental and nutritional state of children. IMPORTANCE: The intestine must balance its roles in digestion and nutrient absorption with the maintenance of an effective barrier to colonization and breach by numerous potential pathogens. An important component of this balance is its constant turnover, which is modulated by a gain of cells due to proliferation and loss due to death or extrusion. Here, we report that Cryptosporidium infection changes the dynamics of this process increasing both gain and loss of enterocytes speeding up the villus elevator. This leads to a much more immature epithelium and a reduction of the number of those cells typically found toward the villus apex best equipped to take up key nutrients including carbohydrates and lipids. These changes in the cellular architecture and physiology of the small intestine may be linked to the profound association between cryptosporidiosis and malnutrition.

Protocol for the cryopreservation of Cryptosporidium isolates using enteroids.

A major bottleneck in the progress of Cryptosporidium research is the lack of accessible cryopreservation of Cryptosporidium oocysts. Here, we present a protocol for the cryopreservation of Cryptosporidium isolates using enteroids. We describe the steps for the establishment of enteroid cultures and cryopreservation of C. parvum-infected HCT-8 cultures. We then detail procedures for the recovery and propagation of frozen parasites using enteroids. For complete details on the use and execution of this protocol, please refer to Deng et al.1.

Intestinal cDC1s provide cues required for CD4+ T cell-mediated resistance to Cryptosporidium.

Cryptosporidium is an enteric pathogen and a prominent cause of diarrheal disease worldwide. Control of Cryptosporidium requires CD4+ T cells, but how protective CD4+ T cell responses are generated is poorly understood. Here, Cryptosporidium parasites that express MHCII-restricted model antigens were generated to understand the basis for CD4+ T cell priming and effector function. These studies revealed that parasite-specific CD4+ T cells are primed in the draining mesenteric lymph node but differentiate into Th1 cells in the gut to provide local parasite control. Although type 1 conventional dendritic cells (cDC1s) were dispensable for CD4+ T cell priming, they were required for CD4+ T cell gut homing and were a source of IL-12 at the site of infection that promoted local production of IFN-γ. Thus, cDC1s have distinct roles in shaping CD4+ T cell responses to an enteric infection: first, to promote gut homing from the mesLN, and second, to drive effector responses in the intestine.

Effect of Glycosaminoglycans on Cryptosporidium Oocyst Attachment and Excystation.

Cryptosporidium causes severe gastrointestinal disease resulting from the ingestion of oocysts, followed by oocyst excystation in the small intestine and the release of infective sporozoites. An understudied strategy for Cryptosporidium inactivation is purposeful oocyst excystation, as sporozoites do not survive long in the environment. This study showed that C. parvum oocyst excystation was induced by direct contact with various glycosaminoglycans (GAGs), including heparin (Hep), chondroitin sulfate A (CSA), and hyaluronan (HA), assembled on polydopamine (PD)-functionalized surfaces. PD surfaces elicited 97.9 ± 3.6% oocyst attachment, with some of the attached oocysts partially (7.3 ± 1.3%) or fully (4.0 ± 0.6%) excysted after 4 days. The PD-GAG surfaces (GAG concentration = 2 mg/mL) elicited similarly high attachment (>97%) and higher oocyst excystation efficiencies after 4 days. The PD-Hep surfaces elicited the highest number of attached excysted oocysts (11.8 ± 0.63% partially excysted; 11.9 ± 0.49% fully excysted), and the PD-HA surfaces elicited the lowest (8.8 ± 2.1% partially excysted; 7.8 ± 1.2% fully excysted). Surface characterization revealed that the addition of GAGs to the PD surface changed both the surface roughness as well as the surface wettability. Treatment of oocysts with an enzyme that degraded the surface glycocalyx markedly reduced excystation (to <2%) of the oocysts attached to the PD and PD-GAG surfaces. These findings suggest that GAGs provide an important local signal for the excystation of C. parvum oocysts and that certain surface-expressed oocyst receptors are necessary for efficient excystation. These oocyst-receptor relationships may be useful in the design of functionalized surfaces for the purposeful inactivation of oocysts in the environment or in water treatment systems. IMPORTANCE Polydopamine surfaces functionalized with glycosaminoglycans were shown to facilitate the attachment and excystation of Cryptosporidium parvum oocysts. Our findings suggest that a surface-expressed receptor on the oocyst wall plays a key role in excystation, with glycosaminoglycans serving as ligands that trigger the initiation of the process. Future technologies and treatment strategies designed to promote premature excystation of oocysts will minimize the ingestion of sporozoites that initiate infection. Therefore, the results from this study have important implications for the protection of public health from waterborne cryptosporidiosis and may serve as a foundation for engineered surfaces designed to remove oocysts from surface waters or inactivate oocysts in water treatment systems.

A productive immunocompetent mouse model of cryptosporidiosis with long oocyst shedding duration for immunological studies.

OBJECTIVES: Studies on the pathogenesis and immune responses of Cryptosporidium infection and development of drugs and vaccines use mostly immunocompromised mouse models. In this study, we establish an immunocompetent mouse model of cryptosporidiosis with high intensity and long duration of infection. METHODS: We have obtained a Cryptosporidium tyzzeri isolate from laboratory mice, and infect adult C57BL/6 J mice experimentally with the isolate for determinations of infectivity, infection patterns, pathological changes, and transcriptomic responses. RESULTS: The isolate has an ID50 of 5.2 oocysts, with oocyst shedding lasting at high levels for >2 months. The oocyst shedding is boosted by immunosuppression of animals and suppressed by paromomycin treatment. The isolate induces strong inflammatory and acquired immune responses, but down-regulates the expression of α-defensins in epithelium. Comparative genomics analysis has revealed significant sequence differences from other isolates in subtelomeric genes. The down-regulation of the expression of α-defensins may be responsible for the high-intensity and long-lasting infection in this animal model. CONCLUSIONS: The immunocompetent mouse model of cryptosporidiosis developed has the advantages of high oocyst shedding intensity and long oocyst shedding duration. It provides an effective mechanism for the propagation of Cryptosporidium, evaluations of potential therapeutics, and studies of pathogen biology and immune responses.

Molecular characterization of Cryptosporidium spp. in dogs and cats in the city of Rio de Janeiro, Brazil, reveals potentially zoonotic species and genotype.

Intestinal cryptosporidiosis is a diarrheal disease caused by protists of genus Cryptosporidium that infect a wide variety of hosts, primarily vertebrates. Due to the close contact between humans and their companion animals, especially dogs and cats, there is concern about the potential for zoonotic transmission of this enteric protozoan parasite by infected animals. This study aimed to perform a microscopic and molecular diagnosis of Cryptosporidium spp. in fecal samples from domiciled dogs and cats. One hundred and nineteen fecal samples were processed using sugar centrifugal flotation followed by molecular detection of Cryptosporidium spp. DNA using nested PCR. Subtyping of isolates positive for C. parvum was performed by sequence analysis of the 60 kDa glycoprotein gene (GP60). Cryptosporidium oocysts were detected in 7.8% (5/64) and 5.4% (3/55) of the fecal samples from dogs and cats, respectively. Cryptosporidium canis (n = 3) and C. parvum (n = 2) were the main species found in dogs, whereas C. felis (n = 3) was prevalent in cats. Subtype IIaA17G2R2 (potentially zoonotic) was identified in samples positive for C. parvum. Despite the low prevalence of Cryptosporidium observed in the domiciled dogs and cats, the presence of potentially zoonotic C. parvum in dogs evidences a public health concern. Further research is needed to better understand the epidemiology, source, and potential impacts of Cryptosporidium infection in cats and dogs.

Serum metabolomics in chickens infected with Cryptosporidium baileyi.

BACKGROUND: Cryptosporidium baileyi is an economically important zoonotic pathogen that causes serious respiratory symptoms in chickens for which no effective control measures are currently available. An accumulating body of evidence indicates the potential and usefulness of metabolomics to further our understanding of the interaction between pathogens and hosts, and to search for new diagnostic or pharmacological biomarkers of complex microorganisms. The aim of this study was to identify the impact of C. baileyi infection on the serum metabolism of chickens and to assess several metabolites as potential diagnostic biomarkers for C. baileyi infection. METHODS: Ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) and subsequent multivariate statistical analysis were applied to investigate metabolomics profiles in the serum samples of chickens infected with C. baileyi, and to identify potential metabolites that can be used to distinguish chickens infected with C. baileyi from non-infected birds. RESULTS: Multivariate statistical analysis identified 138 differential serum metabolites between mock- and C. baileyi-infected chickens at 5 days post-infection (dpi), including 115 upregulated and 23 downregulated compounds. These metabolites were significantly enriched into six pathways, of which two pathways associated with energy and lipid metabolism, namely glycerophospholipid metabolism and sphingolipid metabolism, respectively, were the most enriched. Interestingly, some important immune-related pathways were also significantly enriched, including the intestinal immune network for IgA production, autophagy and cellular senescence. Nine potential C. baileyi-responsive metabolites were identified, including choline, sirolimus, all-trans retinoic acid, PC(14:0/22:1(13Z)), PC(15:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(16:1(9Z)/24:1(15Z)), phosphocholine, SM(d18:0/16:1(9Z)(OH)) and sphinganine. CONCLUSIONS: This is the first report on serum metabolic profiling of chickens with early-stage C. baileyi infection. The results provide novel insights into the pathophysiological mechanisms of C. baileyi in chickens.

Stem cell-derived enteroid cultures as a tool for dissecting host-parasite interactions in the small intestinal epithelium.

Toxoplasma gondii and Cryptosporidium spp. can cause devastating pathological effects in humans and livestock, and in particular to young or immunocompromised individuals. The current treatment plans for these enteric parasites are limited due to long drug courses, severe side effects or simply a lack of efficacy. The study of the early interactions between the parasites and the site of infection in the small intestinal epithelium has been thwarted by the lack of accessible, physiologically relevant and species-specific models. Increasingly, 3D stem cell-derived enteroid models are being refined and developed into sophisticated models of infectious disease. In this review, we shall illustrate the use of enteroids to spearhead research into enteric parasitic infections, bridging the gap between cell line cultures and in vivo experiments.

Influence of weather and climate on cryptosporidiosis-A review.

Studies have shown that climatic factors can significantly influence transmission of many waterborne diseases. However, knowledge of the impact of climate variability on cryptosporidiosis is much less certain. Associations between the incidence of cryptosporidiosis and climatic variables have been reported in several countries. Given that the identified relationships were not consistently reported across studies, it is not known whether these were country-specific observations or can be considered more globally. Variation in the disease risk in both low- and middle-income countries and high-income countries presents new challenges and opportunities to enact responsive changes in research and public health policies. Available epidemiological evidence of the influence of weather and climate on cryptosporidiosis is reviewed. Fourteen studies met the inclusion criteria, and most studies showed that the incidence of cryptosporidiosis is highly sensitive to climatic conditions, especially temperature, rainfall and relative humidity. The identified associations varied across studies, with different conditions of importance and lag times across different locations. Therefore, there is a need for countries at risk to assess Cryptosporidium transmission routes based on the spatiotemporal patterns of the disease and what role climate and other socio-ecological changes play in the transmission. Information gathering will then allow us to provide information for evidence-based control strategies and mitigation of transmission. This review offers new perspectives on the role of climate variability on Cryptosporidium transmission. It highlights different epidemiological approaches adopted and provides the potential for future research and surveillance to reduce the disease burden. By evaluating the epidemiological transmission of this organism in high-income countries, all mitigation strategies, for example filtration and water catchment management, can be used as exemplars of preventing infection in low- to middle-income countries.

The intestinal parasite Cryptosporidium is controlled by an enterocyte intrinsic inflammasome that depends on NLRP6.

The apicomplexan parasite Cryptosporidium infects the intestinal epithelium. While infection is widespread around the world, children in resource-poor settings suffer a disproportionate disease burden. Cryptosporidiosis is a leading cause of diarrheal disease, responsible for mortality and stunted growth in children. CD4 T cells are required to resolve this infection, but powerful innate mechanisms control the parasite prior to the onset of adaptive immunity. Here, we use the natural mouse pathogen Cryptosporidium tyzzeri to demonstrate that the inflammasome plays a critical role in initiating this early response. Mice lacking core inflammasome components, including caspase-1 and apoptosis-associated speck-like protein, show increased parasite burden and caspase 1 deletion solely in enterocytes phenocopies whole-body knockout (KO). This response was fully functional in germfree mice and sufficient to control Cryptosporidium infection. Inflammasome activation leads to the release of IL-18, and mice that lack IL-18 are more susceptible to infection. Treatment of infected caspase 1 KO mice with recombinant IL-18 is remarkably efficient in rescuing parasite control. Notably, NOD-like receptor family pyrin domain containing 6 (NLRP6) was the only NLR required for innate parasite control. Taken together, these data support a model of innate recognition of Cryptosporidium infection through an NLRP6-dependent and enterocyte-intrinsic inflammasome that leads to the release of IL-18 required for parasite control.

Intestinal organoid/enteroid-based models for Cryptosporidium.

Cryptosporidium is a leading cause of diarrhea and death in young children and untreated AIDS patients in resource-poor settings, and of waterborne outbreaks of disease in developed countries. However, there is no consistently effective treatment for vulnerable populations. Progress towards development of therapeutics for cryptosporidiosis has been hampered by lack of optimal culture systems to study it. New advances in organoid/enteroid technology have contributed to improved platforms to culture and propagate Cryptosporidium. Here we discuss recent breakthroughs in the field and highlight different models for functional ex vivo organoid or enteroidderived culture systems. These systems will lead to a better understanding of the mechanisms of host-parasite interactions in vivo.

The Biology of the Intestinal Intracellular Parasite Cryptosporidium.

Cryptosporidium emerged as a leading global cause of severe diarrheal disease in children. The parasite occupies a unique intracellular niche at the brush border of intestinal epithelial cells, where it undergoes a complex sexual life cycle. How this life cycle unfolds and how host and parasite interact remain largely to be discovered. A series of technical advances now offer genetic and immunological tools for mechanistic investigation of the parasite. Here we introduce the pathogen and disease and highlight important questions to tackle onward. We invite scientists to consider this versatile parasite model to probe the biology and immunology of the intestine.

Biofilm Sampling for Detection of Cryptosporidium Oocysts in a Southeastern Pennsylvania Watershed.

This study investigated the use of biofilms to monitor Cryptosporidium in water. Benthic rock and submersible slide biofilms were sampled upstream and downstream of point sources in a suburban watershed in southeastern Pennsylvania. More oocysts were detected in biofilms scraped from rocks downstream than upstream of a wastewater treatment plant (WWTP) (19 versus 5, respectively; n = 1). Although not statistically significant, Cryptosporidium oocysts were detected more frequently, and in greater numbers, in biofilms grown on slides downstream than upstream of this same WWTP (83.3% positive samples [n = 12] versus 45.5% positive samples [n = 11], respectively; P = 0.0567). Similarly, Cryptosporidium oocysts were detected more frequently, and in greater numbers, in rock biofilms collected downstream than upstream of a stormwater outfall impacted by defective sewer laterals (50% positive samples downstream and 17% positive samples upstream; n = 6; P = 0.2207). While oocyst detection data obtained by slide biofilms versus filters did not necessarily agree on a given day, there was no seasonal difference in the frequency of oocyst detection (P > 0.05) or numbers of oocysts detected (P > 0.05) whether the water was monitored by filtration or slide biofilm sampling. Within any given season, there was no difference in the frequency of oocyst detection (P > 0.05) or the numbers of oocysts detected (P > 0.05) whether the water was monitored by filtration or slide biofilm sampling. These data show that oocyst detection in biofilms is comparable to oocyst detection in filtered water samples. Biofilm sampling offers significant cost savings compared to the filtration-based EPA Method 1623.1 and could be used to identify watershed locations at potential risk for increased oocyst loads.IMPORTANCE Monitoring Cryptosporidium occurrence in watersheds that provide drinking water is necessary to determine where limited resources should most effectively be directed to protect consumers from waterborne exposure to pathogenic oocysts. Biofilms are a useful tool to monitor complex watersheds and identify point sources of Cryptosporidium oocyst contamination that need to be managed to protect public health. Compared to EPA Method 1623.1, the cost benefit of using biofilms to monitor for Cryptosporidium contamination will enable utilities to sample water supplies more frequently, and at more locations, than is currently possible given limited operating budgets. Biofilm sampling could be used to identify high-risk regions within a large, complex watershed and the associated water treatment plants at potential risk for increased oocyst loads in the water supply; this information could then be used to select the locations within the watershed where the more expensive EPA Method 1623.1 is warranted.

Global prevalence of Cryptosporidium infection in rodents: A systematic review and meta-analysis.

The One-Health approach implies that the health of people is connected to the health of animals and the environment. Rodents, which are abundant and widespread, have been considered the major reservoirs of Cryptosporidium infection in humans and other animals. However, there is a paucity of information about global patterns of occurrence of Cryptosporidium in rodents. This systematic review and meta-analysis aimed to estimate the pooled global prevalence of Cryptosporidium infection and associated risk factors in rodents. International databases (PubMed, Web of Science, Scopus, and Google scholar) were systematically searched to identify relevant studies. A random-effects meta-analysis model was used to estimate the overall and the subgroup-pooled prevalence of Cryptosporidium across studies, and the variance between studies (heterogeneity) were quantified by I2 index. The data were classified according to WHO-region, type of diagnostic methods, different kinds of rodents, and specific risk factors, including geographical and climate parameters. Eighty-four articles (including 92 datasets), from 29 countries met eligibility criteria for analysis. The pooled global prevalence (95% CI) of Cryptosporidium infection in rodents was 17% (13-20%), being highest in North American and Caribbean regions 27% (16-40%) and lowest in South American 5% (0-16%) countries. Considering the detection methods, the pooled prevalence was estimated to be 25% (13-39%) using coproantigen detection methods, 17% (13-23%) using molecular detection methods, and 14% (9-20%) using microscopic detection methods. The highest prevalence of Cryptosporidium was found in muskrats 47% (27-68%), while Meriones persicus 1% (0-3%) had the lowest prevalence. These results emphasize the role of rodents as reservoir hosts for human-infecting Cryptosporidium. This highlights the need for an increased focus on implementing affordable, appropriate control programs to reduce the public health threat of cryptosporidiosis as a zoonosis of global importance.

Cryptosporidium bollandi n. sp. (Apicomplexa: Cryptosporidiiae) from angelfish (Pterophyllum scalare) and Oscar fish (Astronotus ocellatus).

The species name Cryptosporidium bollandi n. sp. is proposed for Cryptosporidium piscine genotype 2 based on morphological, biological and molecular characterisation. Phylogenetic analyses of 18S rRNA (18S) sequences revealed that C. bollandi n. sp. was most closely related to piscine genotype 4 (5.1% genetic distance) and exhibited genetic distances of 10.0%, 12.2% and 25.2% from Cryptosporidium molnari, Cryptosporidium huwi and Cryptosporidium scophthtalmi, respectively. At the actin locus, C. bollandi n. sp. was again most closely related to piscine genotype 4 (6.8% genetic distance) and exhibited 15.5% (C. molnari), 18.4% (C. huwi), 22.9% (C. scophthalmi) and up to 27.5% genetic distance from other Cryptosporidium spp. (Cryptosporidium felis). Phylogenetic analysis of concatenated 18S and actin sequences showed that C. bollandi n. sp. exhibited 12.9% (C. molnari) to 21.1% (C. canis) genetic distance from all other Cryptosporidium spp. Genetic data as well as previous histological analysis clearly supports the validity of C. bollandi n. sp. as a separate species.

Molecular detection of genotypes and subtypes of Cryptosporidium infection in diarrheic calves, lambs, and goat kids from Turkey.

The studies on Cryptosporidium infections of animals in Turkey mostly rely on microscopic observation. Few data are available regarding the prevalence of Cryptosporidium genotypes and subtypes infection. The aim of this study is to analyse the detection of Cryptosporidium genotypes and subtypes from young ruminants. A total of 415 diarrheic fecal specimens from young ruminants were examined for the Cryptosporidium detection by use of nested PCR of the small subunit ribosomal RNA (SSU rRNA) gene and the highly polymorphic 60 kDa glycoprotein (gp60) gene followed by sequence analyses. The results of this study revealed that 25.6% (106 of 415) of the specimens were positive for Cryptosporidium spp. infection. We identified 27.4% (91/333), 19.4% (13/67), and 13.4% (2/15) of positivity in calves, lambs and goat kids, respectively. Genotyping of the SSU rRNA indicated that almost all positive specimens were of C. parvum, except for one calf which was of C. bovis. Sequence analysis of the gp60 gene revealed the most common zoonotic subtypes (IIa and IId) of C. parvum. We detected 11 subtypes (IIaA11G2R1, IIaA11G3R1, IIaA12G3R1, IIaA13G2R1, IIaA13G4R1, IIaA14G1R1, IIaA14G3R1, IIaA15G2R1, IIdA16G1, IIdA18G1, IIdA22G1); three of them (IIaA12G3R1, IIaA11G3R1 and IIaA13G4R1) was novel subtypes found in calves and lambs. Additionally, three subtypes (IIaA11G2R1, IIaA14G3R1, and IIdA16G1) were detected in young ruminants for the first time in Turkey. These results indicate the high infection of Cryptosporidium in Turkey and propose that young ruminants are likely a major reservoir of C. parvum and a potential source of zoonotic transmission.

The global prevalence of Cryptosporidium infection in dogs: A systematic review and meta-analysis.

Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium spp., is an important zoonotic disease and is considered a global public health concern. Dogs are suggested as one of potential reservoirs for transmitting the Cryptosporidium infection to humans. However, there is a paucity of information about global patterns of occurrence of Cryptosporidium in dogs. A systematic review and meta-analysis were carried out to evaluate the global prevalence of Cryptosporidium infection among dogs. In this study, PubMed, Scopus, Web of Science and Google Scholar databases were systematically searched for relevant studies up until October 30, 2019. Finally, 127 articles (including 160 datasets) were eligible for inclusion in the systematic review and meta-analysis. The overall prevalence of Cryptosporidium infection was estimated at 8% (95 % CI: 5-11 %) using microscopic methods, 7% (95 % CI: 4-10%) using coprogantigenic methods and 6% (95 % CI: 4-9%) using molecular diagnostic methods. Molecular methods revealed that dogs were most frequently infected by C. canis (3.64 %) and C. parvum (1.28 %). The pooled prevalence different of subgroups (WHO regions, geographic and climate parameters, diagnostic methods, type of dog) were analyzed separately. The pooled odds ratio (OR) of Cryptosporidium was significantly higher than one for diarrhea status, with dogs suffering from diarrhea having a higher likelihood of Cryptosporidium infection, compared to dogs without diarrhea (OR; 3.61 95 % CI: 1.89-6.90%). The present study is the first systematic review and meta-analysis providing a comprehensive view of the global prevalence of Cryptosporidium in dogs and its related risk factors. Awareness of Cryptosporidium prevalence, risk factors, and disease complications for the health authorities, physicians, veterinarians and dog's owners is important for developing effective strategies to prevent infection.

Selected Emerging Infectious Diseases of Squamata: An Update.

This article details emerging infectious diseases that have devastating impacts on captive and wild squamates. Treatment advances have been attempted for Cryptosporidium infections in squamates. Gram-positive bacteria, Devriesea agamarum and Austwickia chelonae, are contributing to severe disease in captive and now in wild reptiles, some critically endangered. Nannizziposis, Paranannizziopsis, and Ophidiomyces continue to cause fatal disease as primary pathogens in wild and captive populations of squamates and sphenodontids. Nidovirus, bornavirus, paramyxovirus, sunshine virus, and arenavirus have emerged to be significant causes of neurorespiratory disease in snakes. Controlled studies evaluating environmental stability, disinfection, transmission control, and treatment are lacking.

Waterborne protozoan pathogens in environmental aquatic biofilms: Implications for water quality assessment strategies.

Biofilms containing pathogenic organisms from the water supply are a potential source of protozoan parasite outbreaks and a significant public health concern. The aim of the present study was to demonstrate the simultaneous and multi-spatial occurrence of waterborne protozoan pathogens (WBPP) in substrate-associated biofilms (SAB) and compare it to surface water (SW) and sediments with bottom water (BW) counterparts using manual filtration and elution from low-volume samples. For scenario purposes, simulated environmental biofilm contamination was created from in-situ grown one-month-old SAB (OM-SAB) that were spiked with Cryptosporidium parvum oocysts. Samples were collected from the largest freshwater reservoirs in Luzon, Philippines and a University Lake in Thailand. A total of 69 samples (23 SAB, 23 SW, and 23 BW) were evaluated using traditional staining techniques for Cryptosporidium, and Immunofluorescence staining for the simultaneous detection of Cryptosporidium and Giardia. WBPP were found in 43% SAB, 39% SW, and 39% BW of the samples tested in the present study with SAB results reflecting SW and BW results. Further highlights were demonstrated in the potential of using low-volume samples for the detection of parasites in source water. Scanning electron microscopy of OM-SAB samples revealed a naturally-associated testate amoeba shell, while Cryptosporidium oocysts spiked samples provided a visual profile of what can be expected from naturally contaminated biofilms. This study provides the first evidence for the simultaneous and multi-spatial occurrence of waterborne protozoan pathogens in low-volume aquatic matrices and further warrants SAB testing along with SW and BW matrices for improved water quality assessment strategies (iWQAS).