Cryptosporidium--biotechnological advances in the detection, diagnosis and analysis of genetic variation

Cryptosporidiosis is predominantly a gastrointestinal disease of humans and other animals, caused by various species of protozoan parasites representing the genus Cryptosporidium. This disease, transmitted mainly via the faecal-oral route (in water or food), is of major socioeconomic importance worldwide. The diagnosis and genetic characterization of the different species and population variants (usually recognised as "genotypes" or "subgenotypes") of Cryptosporidium is central to the prevention, surveillance and control of cryptosporidiosis, particularly given that there is presently no broadly applicable treatment regimen for this disease. Although traditional phenotypic techniques have had major limitations in the specific diagnosis of cryptosporidiosis, there have been major advances in the development of molecular analytical and diagnostic tools. This article provides a concise account of Cryptosporidium and cryptosporidiosis, and focuses mainly on recent advances in nucleic acid-based approaches for the diagnosis of cryptosporidiosis and analysis of genetic variation within and among species of Cryptosporidium. These advances represent a significant step toward an improved understanding of the epidemiology as well as the prevention and control of cryptosporidiosis.     

Variation in Cryptosporidium: towards a taxonomic revision of the genus

Cryptosporidium is an important cause of enteric disease in humans and other animals. Limitations associated with conventional diagnostic methods for cryptosporidiosis based on morphological features, coupled with the difficulty of characterising parasites isolated in the laboratory, have restricted our ability to clearly identify species. The application of sensitive molecular approaches has obviated the necessity for laboratory amplification. Such studies have found considerable evidence of genetic heterogeneity among isolates of Cryptosporidium from different species of vertebrate, and there is now mounting evidence suggesting that a series of host-adapted genotypes/strains/species of the parasite exist. In this article, studies on the molecular characterisation of Cryptosporidium during the last 5 years are reviewed and put into perspective with the past and present taxonomy of the genus. The predictive value of achieving a sound taxonomy for the genus Cryptosporidium with respect to understanding its epidemiology and transmission and controlling outbreaks of the disease is also discussed.     

A review of the biology and epidemiology of cryptosporidiosis in humans and animals

The epidemiology of cryptosporidiosis, an infection caused by several genotypically and phenotypically diverse Cryptosporidium species, has been dynamically changing over the past decade from that of a rare, largely asymptomatic infection to an acute enteric disease of animals and humans. In this review, the current understanding of factors (biology and epidemiology) contributing to the emergence of cryptosporidiosis in animals, including parasite biology, genetic diversity, environmental spread, livestock production trends, presence of the parasite in livestock and companion animals, and potential risk of transmission from animals to humans is highlighted. Potential control measures and the role of veterinary and medical professionals in the prevention of cryptosporidiosis are also discussed.     

Morphologic, host specificity, and genetic characterization of a European Cryptosporidium andersoni isolate

This study was undertaken in order to characterize a Cryptosporidium muris-like parasite isolated from cattle in Hungary and to compare this strain with other Cryptosporidium species. To date, the large-type oocysts isolated from cattle were considered as C. muris described from several mammals. The size, form, and structure of the oocysts of the Hungarian strain were identical with those described by others from cattle. An apparent difference between the morphometric data of C. muris-like parasites isolated from cattle or other mammals was noted, which is similar in magnitude to the differences between Cryptosporidium meleagridis and Cryptosporidium felis or between Cryptosporidium serpentis and Cryptosporidium baileyi. The cross-transmission experiments confirmed the findings of others, as C. muris-like oocysts isolated from cattle fail to infect other mammals. The sequence of the variable region of small subunit (SSU) rRNA gene of the strain was 100% identical with that of the U.S. Cryptosporidium andersoni and C. andersoni-like isolates from cattle. The difference between the SSU rRNA sequence of bovine strains and C. muris is similar in magnitude to the differences between C. meleagridis and Cryptosporidium parvum anthroponotic genotype or between Cryptosporidium wrairi and C. parvum zoonotic genotype. Our findings confirm that the Cryptosporidium species responsible for abomasal cryptosporidiosis and economic losses in the cattle industry should be considered a distinct species, C. andersoni Lindsay, Upton, Owens, Morgan, Mead, and Blagburn, 2000.     

Worldwide human zoonotic cryptosporidiosis caused by Cryptosporidium felis

Cryptosporidium is an important enteric pathogen worldwide distributed causing diarrhoeal illness in humans and animals. Identifying Cryptosporidium species using conventional criteria, such as oocyst morphology, is inadequate. The advent of molecular techniques has conducted to characterize different species and genotypes of Cryptosporidium infecting humans. The vast majority of human cases of cryptosporidiosis in the world are caused by both species, Cryptosporidium hominis and Cryptosporidium parvum. However other species including Cryptosporidium felis can infect humans too. In this review, we analyse 58 reported cases of human C. felis infection in different parts of the world. To date this emerging protozoan disease is present in humans around the world, except in Australia and Oceania. Adults and children are infected, more often when immunocompromised by HIV infection (83 % of reported cases). Apparently immunocompetent individuals are also infected by C. felis. In developing countries, inhabitants are more likely infected by C. felis probably through the oocyst contamination of drinking or recreational water. The public health importance of C. felis infection in tropical countries remains to be evaluated.     

Molecular epidemiology of human cryptosporidiosis

Species within the genus Cryptosporidium are protozoan parasites that infect a wide range of vertebrates, and represent a significant cause of morbidity and mortality in those animals. In humans, cryptosporidiosis is a common cause of diarrhoeal disease with a global distribution. Unravelling the epidemiology of human infection has proven to be difficult, due to the existence of multiple transmission routes (person-to-person, animal-to-person, waterborne, foodborne and airborne transmission), and to the difficulties in identifying the different species using conventional criteria, such as oocyst morphology. The advent of molecular techniques has had a remarkable impact on the way the epidemiology of cryptosporidiosis can be studied. Molecular investigations have shown that the vast majority of human cases are caused by C. hominis and C. parvum. Interestingly, differences in geographical and temporal distribution, disease presentations and risk factors for infection have been identified for both C. hominis and C. parvum. Further, molecular analyses have revealed that other species, including C. meleagridis, C. felis, C. canis, C. suis, C. muris and two Cryptosporidium genotypes, can infect humans and may be linked to clinical disease, not only in immunocompromised but also in immunocompetent individuals.     

Escherichia coli from animal reservoirs as a potential source of human extraintestinal pathogenic E. coli

Extraintestinal pathogenic Escherichia coli (ExPEC) are an important cause of urinary tract infections, neonatal meningitis and septicaemia in humans. Animals are recognized as a reservoir for human intestinal pathogenic E. coli, but whether animals are a source for human ExPEC is still a matter of debate. Pathologies caused by ExPEC are reported for many farm animals, especially for poultry, in which colibacillosis is responsible for huge losses within broiler chickens. Cases are also reported for companion animals. Commensal E. coli strains potentially carrying virulence factors involved in the development of human pathologies also colonize the intestinal tract of animals. This review focuses on the recent evidence of the zoonotic potential of ExPEC from animal origin and their potential direct or indirect transmission from animals to humans. As antimicrobials are commonly used for livestock production, infections due to antimicrobial-resistant ExPEC transferred from animals to humans could be even more difficult to treat. These findings, combined with the economic impact of ExPEC in the animal production industry, demonstrate the need for adapted measures to limit the prevalence of ExPEC in animal reservoirs while reducing the use of antimicrobials as much as possible.     

Molecular epidemiology, spatiotemporal analysis, and ecology of sporadic human cryptosporidiosis in Australia

Parasites from the Cryptosporidium genus are the most common cause of waterborne disease around the world. Successful management and prevention of this emerging disease requires knowledge of the diversity of species causing human disease and their zoonotic sources. This study employed a spatiotemporal approach to investigate sporadic human cryptosporidiosis in New South Wales, Australia, between January 2008 and December 2010. Analysis of 261 human fecal samples showed that sporadic human cryptosporidiosis is caused by four species; C. hominis, C. parvum, C. andersoni, and C. fayeri. Sequence analysis of the gp60 gene identified 5 subtype families and 31 subtypes. Cryptosporidium hominis IbA10G2 and C. parvum IIaA18G3R1 were the most frequent causes of human cryptosporidiosis in New South Wales, with 59% and 16% of infections, respectively, attributed to them. The results showed that infections were most prevalent in 0- to 4-year-olds. No gender bias or regional segregation was observed between the distribution of C. hominis and C. parvum infections. To determine the role of cattle in sporadic human infections in New South Wales, 205 cattle fecal samples were analyzed. Four Cryptosporidium species were identified, C. hominis, C. parvum, C. bovis, and C. ryanae. C. parvum subtype IIaA18G3R1 was the most common cause of cryptosporidiosis in cattle, with 47% of infections attributed to it. C. hominis subtype IbA10G2 was also identified in cattle isolates.     

Outbreak of cryptosporidiosis due to Cryptosporidium parvum subtype IIdA19G1 in neonatal calves on a dairy farm in China

Neonatal diarrhea is one of the most important syndromes in dairy cattle. Among enteropathogens, Cryptosporidium spp. are primary causes of diarrhea, but outbreaks due to cryptosporidiosis are rarely reported in cattle. From January to April in 2016, severe diarrhea was observed in over 400 neonatal dairy calves on a large dairy farm in Jiangsu Province of East China. Approximately 360 calves died due to watery diarrhea despite antibiotic therapy. In this study, 18 fecal specimens were collected from seriously ill calves on this farm during the diarrhea outbreak, and analysed for common enteropathogens by enzymatic immunoassay (EIA). In a post-outbreak investigation, 418 and 1372 specimens collected from animals of various age groups were further analysed for rotavirus and Cryptosporidium spp. by EIA and PCR, respectively, to assess their roles in the occurrence of diarrhea on the farm. Cryptosporidium spp. were genotyped using established techniques. Initial EIA tests showed that 15/18 seriously ill calves during the outbreak were positive for Cryptosporidium parvum, while 8/18 were positive for rotavirus. The overall infection rate of Cryptosporidium in pre-weaned calves on the farm was 22.7%, with odds of the Cryptosporidium infection during the outbreak 4.4–23.5 times higher than after the outbreak. Four Cryptosporidium spp. were identified after the outbreak including C. parvum (n = 79), Cryptosporidium ryanae (n = 48), Cryptosporidium bovis (n = 31), and Cryptosporidium andersoni (n = 3), with co-infections of multiple species being detected in 34 animals. Infection with C. parvum (73/79) was found in the majority of calves aged ≤3 weeks, consistent with the age of ill calves during the outbreak. All C. parvum isolates were identified as subtype IIdA19G1. In the post-outbreak investigation, C. parvum infection was associated with the occurrence of watery diarrhea in pre-weaned calves, C. ryanae infection was associated with moderate diarrhea in both pre- and post-weaned calves, while no association was identified between rotavirus infection and the occurrence of diarrhea. Results of logistic regression analysis further suggested that C. bovis infection might also be a risk factor for moderate diarrhea in calves. Thus, we believe this is the first report of a major outbreak of severe diarrhea caused by C. parvum IIdA19G1 in dairy calves. More attention should be directed toward preventing the dissemination of this virulent subtype in China.     

Substance P receptor antagonist reverses intestinal pathophysiological alterations occurring in a novel ex-vivo model of Cryptosporidium parvum infection of intestinal tissues derived from SIV-infected macaques

BACKGROUND: Cryptosporidium infection leads to life-threatening diarrhea in AIDS patients. Pathogenesis of cryptosporidiosis is due to intestinal physiological alterations. We devised an ex-vivo model using ex-vivo Cryptosporidium parvum infection of jejunal tissues derived from SIV-infected macaques and studied the role of substance P (SP) in the pathogenesis of cryptosporidiosis. METHODS: We measured jejunal SP protein levels using ELISA, and electrophysiological alterations using the Ussing chamber technique in an ex vivo model of Cryptosporidium infection. Paraformaldehyde-fixed jejunum from SIV-infected macaques with and without naturally occurring cryptosporidiosis was studied for SP protein expression by immunohistochemistry and fluorescence deconvolution microscopy. RESULTS: Ex-vivo Cryptosporidium-infected tissues and tissues from SIV-infected macaques with naturally occurring cryptosporidiosis demonstrated elevated SP protein levels compared with tissues from SIV-infected animals without ex-vivo C. parvum infection or tissues from SIV-infected animals that have no evidence of cryptosporidiosis. In our ex-vivo model of Cryptosporidium infection, we demonstrated pathophysiological alterations that were blocked by SP-receptor antagonist treatment. CONCLUSIONS: These studies suggest that SP-receptor antagonists could prove useful for treatment of AIDS-related cryptosporidiosis.     

Cryptosporidium in farmed animals: the detection of a novel isolate in sheep.

We describe the discovery of polymorphisms in the Cryptosporidium oocyst wall protein (COWP) gene conferring a novel restriction fragment length polymorphism (RFLP) pattern in 26/60 (43%) isolates from a flock of sheep sampled following a waterborne outbreak of human cryptosporidiosis. The sheep isolates showed identical PCR-RFLP patterns to each other by COWP genotyping but different from those of most currently recognised genotypes, including the major Cryptosporidium parvum genotypes 1 and 2. Sequence analysis of the 550bp amplicon from the COWP gene was compared with a DNA coding region employed in previous studies and showed the novel isolate to differ from other Cryptosporidium species and C. parvum isolates by 7-21%. The sheep-derived isolates were compared at this and further three Cryptosporidium gene loci with isolates from other farmed animals. The loci employed were one in the thrombospondin related adhesive protein (TRAP-C2) gene and two in the 70kDa heat shock protein (HSP70) gene (CPHSP1 and 2). Other animal samples tested in our laboratory were from clinically ill animals and all contained C. parvum genotype 2. The sheep in which the novel isolate was identified were healthy and showed no symptoms of cryptosporidiosis, and the novel sheep isolate could represent a non-pathogenic strain. Our studies suggest that a previously undetected Cryptosporidium sub-type may exist in sheep populations, reflecting the increasingly recognised diversity within the parasite genus.     

Cryptosporidium and wildlife: a risk for humans?

The present review underlines the knowledge of Cryptosporidium, especially its biodiversity and transmission. The presence of the parasite in different mammal host species is discussed with real, potential risk of transmission to humans. The potential role of insects in mechanical transmission of the parasite is evaluated by experimental protocols. The cost of cryptosporidiosis at health and economic levels are mentioned, which emphasises the importance of detection and identification of the parasite in the environment and in wild mammal species, using specific molecular tools. Potential measures to be accomplished in order to fight off cryptosporidiosis are also noted.     

Zoonotic cryptosporidiosis

The widespread usages of molecular epidemiological tools have improved the understanding of cryptosporidiosis transmission. Much attention on zoonotic cryptosporidiosis is centered on Cryptosporidium parvum. Results of genotype surveys indicate that calves are the only major reservoir for C. parvum infections in humans. The widespread presence of human-adapted C. parvum, especially in developing countries, is revealed by recent subtyping and multilocus typing studies, which have also demonstrated the anthroponotic transmission of C. parvum subtypes shared by humans and cattle. Developing and industrialized countries differ significantly in disease burdens caused by zoonotic species and in the source of these parasites, with the former having far fewer human infections caused by C. parvum and little zoonotic transmission of this species. Exclusive anthroponotic transmission of seemingly zoonotic C. parvum subtypes was seen in Mid-Eastern countries. Other zoonotic Cryptosporidium spp. are also responsible for substantial numbers of human infections in developing countries, many of which are probably transmitted by anthroponotic pathways. The lower pathogenicity of some zoonotic species in some populations supports the occurrence of different clinical spectra of Cryptosporidium spp. in humans. The use of a new generation of molecular diagnostic tools is likely to produce a more complete picture of zoonotic cryptosporidiosis.     

Genotype analysis of Cryptosporidium spp. prevalent in a rural village in Hwasun-gun, Republic of Korea

Two species of Cryptosporidium are known to infect man; C. hominis which shows anthroponotic transmission between humans, and C. parvum which shows zoonotic transmission between animals or between animals and man. In this study, we focused on identifying genotypes of Cryptosporidium prevalent among inhabitants and domestic animals (cattle and goats), to elucidate transmittal routes in a known endemic area in Hwasun-gun, Jeollanam-do, Republic of Korea. The existence of Cryptosporidium oocysts was confirmed using a modified Ziehl-Neelsen stain. Human infections were found in 7 (25.9%) of 27 people examined. Cattle cryptosporidiosis cases constituted 7 (41.2%) of 17 examined, and goat cases 3 (42.9%) of 7 examined. Species characterizations were performed on the small subunit of the rRNA gene using both PCR-RFLP and sequence analysis. Most of the human isolates were mixtures of C. hominis and C. parvum genotypes and similar PCR-RFLP patterns were observed in cattle and goat isolates. However, sequence analyses identified only C. hominis in all isolates examined. The natural infection of cattle and goats with C. hominis is a new and unique finding in the present study. It is suggested that human cryptosporidiosis in the studied area is caused by mixtures of C. hominis and C. parvum oocysts originating from both inhabitants and domestic animals.     

Randomly Amplified Polymorphic DNA PCR Analysis of Bovine Cryptosporidium parvum Strains Isolated from the Watershed of the Red River of the North

Cryptosporidium parvum is a protozoan parasite that causes the disease cryptosporidiosis in a variety of mammals, including neonatal calves and humans. Millions of oocysts are shed during acute cryptosporidiosis, and zoonotic transmission is inferred, though not proven, to be a general phenomenon. Very little is known about the degree of strain variation exhibited by bovine and human isolates, though such knowledge would enable the amount of bovine-to-human transmission to be more precisely analyzed. This research was initiated to determine whether variations exist among bovine strains isolated from a localized geographic area, the watershed of the Red River of the North. Sixteen strains were isolated and compared to each other and to two human and two calf strains from Australia by randomly amplified polymorphic DNA PCR. A statistical analysis of the data indicated that the isolates belonged to four different groups of strains.     

Breeding for immune responsiveness and disease resistance

Animal production efficiency, and product volume and quality can be greatly increased by reducing disease losses. Genetic variation, a prerequisite for successful selection, has been found in animals and poultry exposed to a variety of viral, bacterial and parasitic infections. Breeding for disease resistance can play a significant role alone or in combination with other control measures including disease eradication, vaccination and medication. Feasibility of simultaneously improving resistance to specific diseases and production traits has been demonstrated. However, selection for specific resistance to all diseases of animals and poultry is impossible. Development of general disease resistance through indirect selection primarily on immune response traits may be the best long-term strategy but its applicability is presently limited by insufficient understanding of resistance mechanisms. Another hindrance may be negative genetic correlations among various immune response functions: phagocytosis, cell mediated and humoral immunity. To better assess the feasibility of increasing general disease resistance by indirect selection we must obtain estimates of heritability for immune response, disease resistance, and economic production traits, as well as genetic correlations among these traits. The present level of disease resistance in farm animals resulted from natural selection and from correlated responses to selection for production traits while the influence of artificial selection for resistance was minimal. Future research should be directed towards developing and applying breeding techniques that will increase resistance to diseases without compromising production efficiency and product quality. This will require cooperation of immunogeneticists, veterinarians and animal and poultry breeders. Significant progress in the improvement of resistance to diseases may result from the application of new techniques of molecular genetics and cell manipulation.     

Breeding for immune responsiveness and disease resistance1

Animal production efficiency, and product volume and quality can be greatly increased by reducing disease losses. Genetic variation, a prerequisite for successful selection, has been found in animals and poultry exposed to a variety of viral, bacterial and parasitic infections. Breeding for disease resistance can play a significant role alone or in combination with other control measures including disease eradication, vaccination and medication. Feasibility of simultaneously improving resistance to specific diseases and production traits has been demonstrated. However, selection for specific resistance to all diseases of animals and poultry is impossible. Development of general disease resistance through indirect selection primarily on immune response traits may be the best long-term strategy but its applicability is presently limited by insufficient understanding of resistance mechanisms. Another hindrance may be negative genetic correlations among various immune response functions: phagocytosis, cell mediated and humoral immunity. To better assess the feasibility of increasing general disease resistance by indirect selection we must obtain estimates of heritability for immune response, disease resistance, and economic production traits, as well as genetic correlations among these traits. The present level of disease resistance in farm animals resulted from natural selection and from correlated responses to selection for production traits while the influence of artificial selection for resistance was minimal. Future research should be directed towards developing and applying breeding techniques that will increase resistance to diseases without compromising production efficiency and product quailty. This will require cooperation of immunogeneticists, veterinarians and animal and poultry breeders. Significant progress in the improvement of resistance to diseases may result from the application of new techniques of molecular genetics and cell manipulation.     

Genetic diversity of Campylobacter jejuni isolates from farm animals and the farm environment

The genetic diversity of Campylobacter jejuni isolates from farm animals and their environment was investigated by multilocus sequence typing (MLST). A total of 30 genotypes, defined by allelic profiles (assigned to sequence types [STs]), were found in 112 C. jejuni isolates originating in poultry, cattle, sheep, starlings, and slurry. All but two of these genotypes belonged to one of nine C. jejuni clonal complexes previously identified in isolates from human disease and retail food samples and one clonal complex previously associated with an environmental source. There was some evidence for the association of certain clonal complexes with particular farm animals: isolates belonging to the ST-45 complex predominated among poultry isolates but were absent among sheep isolates, while isolates belonging to the ST-61 and ST-42 complexes were predominant among sheep isolates but were absent from the poultry isolates. In contrast, ST-21 complex isolates were distributed among the different isolation sources. Comparison with MLST data from 91 human disease isolates showed small but significant genetic differentiation between the farm and human isolates; however, representatives of six clonal complexes were found in both samples. These data demonstrate that MLST and the clonal complex model can be used to identify and compare the genotypes of C. jejuni isolates from farm animals and the environment with those from retail food and human disease.     

Modification of a rapid method for the identification of gene-specific polymorphisms in Cryptosporidium parvum and its application to clinical and epidemiological investigations.

The application of genotyping to clinical isolates of Cryptosporidium has increased significantly our knowledge and understanding of the distribution and epidemiology of this parasite. However, some methods can be laborious and demand specialist technical expertise. PCR-restriction fragment length polymorphism (RFLP) techniques represent a more rapid and simple method of genotyping to support epidemiological and clinical investigations than conventional DNA analytical techniques. We describe a nested PCR-RFLP technique that identifies polymorphisms in the C. parvum thrombospondin-related adhesive protein gene locus; this method offers a sensitive and specific tool for the confirmation and investigation of disease associated with C. parvum. The potential of this enhanced method is demonstrated by its application to the confirmation and epidemiological investigation of an outbreak of cryptosporidiosis associated with a school visit to an open farm.