Pathogen screening in adult frozen feeder mice commonly reveals zoonotic rodent-adapted Cryptosporidium spp.

Advances in molecular epidemiology of cryptosporidiosis in dogs and cats

Humans and animals who have an acute case of diarrhea can be infected with Cryptosporidium spp. Within the category of water-borne disease, it is a zoonotic disease. The zoonotic disease Cryptosporidium is among the several pathogens carried by wild rats (Rattus spp.). The risk of spreading this disease is rather significant in urban environments because rats are often close to people. This study aims to detect Cryptosporidium spp. infection in wild rats in Surabaya, East Java, Indonesia. Through necropsy, a total of 100 wild rats' intestines were sampled for feces. Microscopic observation of the presence of Cryptosporidium was carried out using the float test with a combination of Ziehl Neelsen (ZN) staining. Molecular detection of Cryptosporidium spp. positive results used the Cryptosporidium oocyst wall protein (COWP) gene with polimerase chain reaction method. The results showed that 69 samples were positive for containing Cryptosporidium spp. oocysts and with ZN staining to confirm the diagnosis, the staining results showed Cryptosporidium spp. oocysts dark pink with a clear cavity inside with a percentage of 95.65% in Rattus norvegicus and 61.03% in Rattus tanezumi. In residential and densely populated environments the percentage of Cryptosporidium spp. amounted to 66.66% and in the market environment amounted to 74.19%. The percentage of Cryptosporidium spp. in the North Surabaya region was 42.85%, South Surabaya 100%, West Surabaya 37.5%, East Surabaya 81.39%, and Central Surabaya 65.38%. Molecular detection of Cryptosporidium spp. positive results were obtained using the COWP gene 550 bp. This study aims to detect Cryptosporidium spp. infection in wild rats in Surabaya, East Java, Indonesia. The high number of cases of cryptosporidiosis in wild rats has the potential to be a reservoir for the spread of the disease. The Cryptosporidium spp can detected with COWP in 550 bp in wild rats in Surabaya, East Java, Indonesia.

Detection and genotyping of Cryptosporidium spp. in large Japanese field mice, Apodemus speciosus

BackgroundSynanthropic filth flies thrive in human and animal habitats, posing health risks through the transmission of infectious agents. They breed on organic waste, including animal feces, making them carriers of various pathogens. In Egypt, where livestock farming is common and poor sanitation, these flies may contribute to zoonotic disease transmission. The current study investigates parasitic infections in filth flies from three livestock farms in Assiut Governorate, Upper Egypt, highlighting their role as vectors for zoonotic infections, particularly Cryptosporidium, via morphological and molecular tools.MethodsA total of 12,749 flies were collected from the study sites via sweep nets. After taxonomic identification, the flies were examined microscopically for parasites using various concentration and staining techniques. Positive samples were further confirmed for infections, particularly for Cryptosporidium parasites, via nested PCR and sequence analysis targeting the COWP and SSU rRNA genes.ResultsThis study revealed the presence of several fly species from seven dipteran families, particularly the family Muscidae, primarily Musca domestica, which presented a high parasite infestation rate of 96.6%. This study revealed a high prevalence of various protozoans and helminths in the collected flies. Cryptosporidium was the most prevalent parasite (64.4–100%), infecting all fly species. Entamoeba and Balantidium were also significant, especially in M. domestica (22.6–90.1%, 8.9–100%), Fannia canicularis (10.5–74.4%, 44.2–88.2%), and Borborillus vitripennis (11.1–50%, 37.2–91.4%). Giardia, Trichuris, and Trichostrongylidae had low to moderate prevalence in multiple fly species. Mites are commonly detected on fly exoskeletons, with high infestation rates observed in Musca domestica (77–100%) and Physiphora alceae (66.7–100%). The present study also reported sporadic infections with Trichomonas, Toxocara vitulorum, and pseudoscorpions, along with notable midge larval infestations (52.1%), mainly at site B. Parasitic infections were highest in autumn and spring, with the lowest rates in winter. Molecular identification confirmed the presence of the zoonotic species Cryptosporidium parvum and Cladotanytarsus gedanicus.ConclusionThis study revealed that zoonotic parasites exist in flies and pose potential risks when they are found near humans. Cryptosporidium parvum is the prevalent parasite causing diarrhea outbreaks in animals. This is the first genetic evidence of Cladotanytarsus gedanicus midge from Upper Egypt.

Little information is available on the occurrence of the zoonotic protists Cryptosporidium spp. and none on Enterocytozoon bieneusi in camels. This preliminary study was conducted to examine the identity of Cryptosporidium subtypes and E. bieneusi genotypes in dromedary camels in Algeria. A total of 39 fecal specimens were collected from young camels. PCR-sequence analysis of the small subunit rRNA was used to detect and genotype Cryptosporidium spp. Cryptosporidium parvum present was further subtyped by sequence analysis of the 60kDa glycoprotein gene. PCR-sequence analysis of the ribosomal internal transcribed spacer gene was used to detect and genotype E. bieneusi. Altogether, two and eight of the specimens analyzed were positive for C. parvum and E. bieneusi, respectively. The former was identified as a new subtype that is genetically related to the C. hominis If subtype family, whereas the latter was identified as two related genotypes (Macaque1 and a novel genotype) in the newly assigned E. bieneusi genotype group 8. Although they are not known hosts for C. parvum and E. bieneusi, camels are apparently infected with genetically distinct variants of these pathogens.

Common occurrence of zoonotic pathogen Cryptosporidium meleagridis in broiler chickens and turkeys in Algeria

Longitudinal monitoring of Cryptosporidium species in pre-weaned dairy calves on five farms in Shanghai, China

Enteric protozoan parasites Giardia duodenalis, Cryptosporidium spp., and, to a lesser extent, the ciliate Balantioides coli are responsible for severe human and animal intestinal disorders globally. However, limited information is available on the occurrence and epidemiology of these parasites in domestic, but especially wild species in Portugal. To fill this gap of knowledge, we have investigated G. duodenalis, Cryptosporidium spp., and B. coli occurrence, distribution, genetic diversity, and zoonotic potential by analyzing 756 fecal samples from several wild carnivores (n = 288), wild ungulates (n = 242), and domestic species (n = 226) collected across different areas of mainland Portugal. Overall, infection rates were 16.1% (122/756; 95% CI: 13.59–18.96) for G. duodenalis and 2.7% (20/756; CI: 1.62–4.06) for Cryptosporidium spp., while no ungulate sample analyzed yielded positive results for B. coli. Giardia duodenalis was found across a wide range of hosts and sampling areas, being most prevalent in the Iberian lynx (26.7%), the Iberian wolf (24.0%), and the domestic dog (23.9%). Cryptosporidium spp. was only identified in wild boar (8.4%), red fox (3.4%), Iberian lynx (3.3%), red deer (3.1%), and Iberian wolf (2.5%). Sequence analysis of G. duodenalis determined zoonotic assemblage A (subassemblage AI) in one roe deer sample, canine-specific assemblages C and D in Iberian wolf, red fox, and domestic dog, and ungulate-specific assemblage E in wild boar, sheep, cattle, and horse. Six Cryptosporidium species were identified: C. scrofarum in wild boar, C. canis in the Iberian wolf and red fox, C. ubiquitum in red deer and wild boar, C. felis in the Iberian lynx, and both C. ryanae and C. occultus in red deer. Giardia duodenalis and Cryptosporidium spp. coinfections were observed in 0.7% (5/756) of the samples. This is the first, most comprehensive, and largest molecular-based epidemiology study of its kind carried out in Portugal, covering a wide range of wild and domestic hosts and sampling areas. The detection of zoonotic Cryptosporidium spp. and G. duodenalis subassemblage AI demonstrates the role of wild and domestic host species in the transmission of these agents while representing a potential source of environmental contamination for other animals and humans.

Simple SummaryWild small mammals can be a veterinary and public health concern, because they can act as reservoir hosts for numerous pathogens and potentially transmit them to humans, domestic animals and other wildlife species. This study represents the first investigation of the diarrhea-causing parasites Cryptosporidium spp. and Giardia spp. in wild rodents and shrews from Portugal. Cryptosporidium spp. was rarely and Giardia was frequently detected in the feces of the analyzed species, with the southwestern water voles (Arvicola sapidus) and Lusitanian pine voles (Microtus lusitanicus) showing the highest infection rates of Giardia spp. Genetic characterization based on common genomic marker sequences revealed the rodent-adapted Giardia microti and potentially zoonotic Cryptosporidium muris as the only circulating species. These findings suggest the limited role of wild rodents and shrews as natural sources of human infections in Portugal regarding the investigated parasites. Moreover, the host ranges of Giardia and Cryptosporidium spp. were extended and the obtained genetic sequences of Giardia microti are useful for future comparative studies. From the One-Heath perspective, this study helps to understand the epidemiology of Giardia spp. and Cryptosporidium spp. in wildlife.Cryptosporidium spp. and Giardia spp. are important diarrhea-causing protozoan parasites worldwide that exhibit broad host ranges. Wild small mammals can harbor host-adapted and potentially zoonotic species of both parasites. The aim of this study was to investigate Cryptosporidium spp. and Giardia spp. in wild rodents and shrews in Portugal, focusing on the protist’s occurrence and genetic diversity. Molecular screening by PCR at the small subunit (SSU) rRNA gene locus of 290 fecal samples from wood mice (Apodemus sylvaticus), southwestern water voles (Arvicola sapidus), Cabrera’s voles (Microtus cabrerae), Lusitanian pine voles (Microtus lusitanicus), Algerian mice (Mus spretus) and greater white-toothed shrews (Crocidura russula) in Northeast Portugal revealed the low occurrence of Cryptosporidium spp. (1%) and high occurrence of Giardia spp. (32.8%). The analysis revealed that “species” was the only significant factor associated with the increasing probability of Giardia spp. infection, with the highest prevalence reported in southwestern water voles and Lusitanian pine voles. Cryptosporidium and Giardia species determination at the SSU rRNA gene locus revealed C. muris and G. microti as the only circulating species, respectively. Subtyping of the glutamate dehydrogenase (gdh) and beta-giardin (bg) genes provided evidence of the high genetic diversity within the G. microti clade. This study suggests that rodent-adapted G. microti occurs to a large extent in cricetid hosts and supports the limited role of wild rodents and shrews as natural sources of human infections in Northeast Portugal regarding the investigated parasites. Moreover, this is the first record of G. microti in southwestern water voles, Lusitanian pine voles, Algerian mice, wood mice and Cabrera’s voles and C. muris in Cabrera’s voles. Finally, this study improves the database of sequences relevant for the sequence typing of G. microti strains and provides new insights about the epidemiology of Giardia spp. and Cryptosporidium spp. in wild rodents and shrews, two parasite genera of high importance for public and animal health.

Despite the clinical and public health importance of Cryptosporidium parvum, little is known about its transmission dynamics in cattle and other farm animals, especially in Iran and other Mideast countries. Currently, the maintenance of the parasites on cattle farms and the role of herd-to-herd transmission in cryptosporidiosis epidemiology are not clear (1). Recent molecular epidemiologic studies of cryptosporidiosis have helped researchers to better understand the transmission of cryptosporidiosis in humans and the public health significance of Cryptosporidium spp. in animals and the environment (2, 3). Because of the ability of Cryptosporidium spp. to infect humans and a wide variety of animals, and because of the ubiquitous presence of Cryptosporidium oocysts in the environment, humans can acquire Cryptosporidium infections through several transmission routes, such as direct contact with infected persons (person-to-person transmission) or animals (zoonotic transmission), and ingestion of contaminated food (foodborne transmission) and water (waterborne transmission). The relative importance of these transmission routes in the epidemiology of cryptosporidiosis is not entirely clear, largely due to the fact that traditional diagnostic tools do not have the ability to differentiate sources of parasites (3). The use of molecular tools has been helpful in assessing the zoonotic potential of various Cryptosporidium species and the sources of human infection; it has begun to play a significant role in the characterization of transmission dynamics in different areas and in the determination of host specificity of various Cryptosporidium spp. The 60 kDa glycoprotein (gp60, also known as Cpgp15/45) gene encodes a precursor protein that is proteolytically cleaved to yield mature surface glycoproteins gp45 and gp15 (also known as Cp17), both of which are implicated in the attachment and invasion of enterocytes by sporozoites and merozoites. An important feature of this gene is its high degree of sequence polymorphism in C. hominis, C. parvum, and C. meleagridis isolates. Several subtype families have been identified in these species: 7 subtype families in C. hominis (Ia–Ig), 2 zoonotic (IIa, IId) and 10 non-zoonotic (IIb, IIc, IIe–IIl) subtype families in C. parvum, and 6 subtype families in C. meleagridis (4). Within each subtype family, there are multiple subtypes based primarily on the number of tri-nucleotide repeats coding for the amino acid serine, as suggested by Sulaiman et al. (2005) (5). The use of gp60 subtyping has allowed the identification of geographic and temporal differences in the transmission dynamics of cryptosporidiosis, the role of zoonotic infections in epidemiology, better appreciation of the public health significance of Cryptosporidium species/genotypes in ruminants and significance of parasite subtypes/strains in clinical manifestations and outbreak potentials, and the tracking of infection and contamination sources during outbreak and endemic investigations (1–5). To our knowledge, there are several molecular epidemiological studies that have documented the presence of C. parvum and C. hominis In Iran (Table 1) (6–12). However, the distribution of subtypes of the two species in humans, animals and environmental is unclear. In the first characterization of Cryptosporidium subtypes in humans and cattle in Iran by sequence analysis of the gp60 gene, 47 samples of C. parvum (22 from children and 25 from cattle) and three of C. hominis (all from children) were characterized. Nine subtypes (two of C. hominis and seven of C. parvum) belonging to four subtype families were found. Cattle were mainly infected with C. parvum IIa subtypes and humans mostly with C. parvum IIa and IId subtypes (Table 2). The predominance of IIa and IId subtypes underlines the importance of zoonotic Cryptosporidium transmission in Iran. Thus, cattle could be a source of human infection with C. parvum IIa in Iran (13–15). Although the source of IId subtypes in humans is not yet clear, IId subtypes are known to be common in sheep and goats in some countries such as Spain (16) and in dairy cattle in some other countries such as Egypt (17) and China (18). Further molecular study in humans and animals is needed in order to determine the extent and animal source of zoonotic transmission of cryptosporidiosis in Iran. Table 1 Distribution of Cryptosporidium spp. in humans in Mideast countries Table 2 Distribution of C. parvum subtypes in humans and cattle in Iran. The dominance of C. parvum and wide occurrence of IId C. parvum subtypes in humans in Iran is similar to the situation seen in other Mideast countries (9–12, 19–25) (Tables 1 and ​and3).3). Children in the Kuwait City are almost exclusively infected with IIa and IId subtypes, although they have little contact with farm animals. As the city uses desalinized sea water as drinking water, the C. parvum transmission appears to be anthroponotic in origin (5, 20). IId subtypes are also common in children in Saudi Arabia and Jordan (Table 3). In many industrialized nations in other areas, C. parvum infections are much less common in humans than C. hominis infections, with the exception of European countries and New Zealand, where both C. parvum and C. hominis are commonly seen in humans. In these industrialized nations, most C. parvum infections are caused by the IIa subtypes commonly found in cattle, indicating zoonotic transmission plays a significant role in cryptosporidiosis epidemiology. In contrast, humans in developing countries are much less commonly infected with C. parvum and most of the few C. parvum infections are caused by the anthroponotic IIc subtype family (2). Table 3 Distribution of C. parvum subtype families in humans in Mideast countries In conclusion, preliminary molecular epidemiological studies have revealed some unique features of cryptosporidiosis transmission in humans in Iran and other Mideast countries. As the C. parvum subtype family IId was the dominant family causing cryptosporidiosis in humans in Iran (13–15), zoonotic transmission could possibly be involved. However, more extensive sampling of both humans and farm animals, especially sheep and goats, and collection of epidemiological data in case-control and longitudinal studies are needed for a better understanding of the sources of C. parvum infections in humans in Iran and other Mideast countries.

BackgroundHorses interact with humans in a wide variety of sport competitions and non-competitive recreational pursuits as well as in working activities. Cryptosporidium spp are one of the most important zoonotic pathogens causing diarrhea of humans and animals. The reports of Cryptosporidium in horses and the findings of zoonotic Cryptosporidium species/genotypes show a necessity to carry out molecular identification of Cryptosporidium in horses, especially in diarrheic ones. The aim of the present study was to understand Cryptosporidium infection and species/genotypes in diarrheic horses, and to trace the source of infection of horse-derived Cryptosporidium isolates at a subtype level.FindingsFecal specimens of 29 diarrheic adult horses were collected in Taikang County in northeastern China’s Heilongjiang Province. Cryptosporidium oocysts were concentrated by Sheather’s sugar flotation technique, and then examined by a bright-field microscope. Meanwhile, all the specimens were subjected to PCR amplification of the small subunit (SSU) rRNA gene of Cryptosporidium. C. andersoni isolates were further subtyped by multilocus sequence typing (MLST) at the four microsatellite/minisatellite loci (MS1, MS2, MS3 and MS16).One and two Cryptosporidium-positive isolates were obtained in horses by microscopy and by PCR, respectively. The two C. andersoni isolates were identified by sequencing of the SSU rRNA gene of Cryptosporidium. Both of them were identical to each other at the MS1, MS2, MS3 and MS16 loci, and MLST subtype A4,A4,A4,A1 was found here.ConclusionsThis is the first report of C. andersoni in horses. The fact that the MLST subtype A4,A4,A4,A1 was reported in cattle suggests a large possibility of transmission of C. andersoni between cattle and horses.

Protozoan parasites of the Cryptosporidium genus cause severe cryptosporidiosis in newborn lambs. However, asymptomatic infections also occur frequently in lambs and ewes. In sheep, the most commonly detected Cryptosporidium species are C. ubiquitum, C. xiaoi and C. parvum. Due to a lack of relevant information about such infections in France, we investigated the situation on five dairy sheep farms in the Pyrénées-Atlantiques Department in south-western France in December 2017. Individual fecal samples were collected from 79 female lambs (5–17 days old) and their mothers (72 ewes). Oocysts were screened using Heine staining before and after Bailenger concentrations. Cryptosporidium species identification and genotyping were performed using real-time PCR and gp60 gene sequencing. No cases of clinical cryptosporidiosis were observed in the 79 lambs. Microscopically, Cryptosporidium spp. oocysts were observed in only one lamb on one farm (prevalence 1.3%) and one ewe on another farm (prevalence 1.4%). By contrast, Cryptosporidium spp. DNA was detected in 17 ewes (prevalence ranging from 10.5% to 50% depending on the farm) and in 36 lambs (prevalence ranging from 0% to 77.8% depending on the farm). Only zoonotic Cryptosporidium parvum IId and IIa genotypes were identified when genotyping was possible. Cryptosporidium ubiquitum and C. xiaoi were detected on one and three farms, respectively. We conclude that healthy young lambs and their mothers during the peripartum period could be a source of environmental contamination with oocysts.

Cryptosporidium spp. cause cryptosporidiosis in humans through zoonotic and anthroponotic transmission. Previous studies illustrated the significance of domestic animals as reservoirs of this parasite. Cryptosporidium occurs in Njoro River; a main source of water to humans and animals. However, there is no information on the Cryptosporidium spp. and genotypes circulating in Njoro Sub County. A total of 2174 samples from humans, cattle, chickens, sheep and goats were assessed for the presence of Cryptosporidium spp. Thirty-three positive samples were subsequently successfully sequenced and compared to Cryptosporidium sequences in the GenBank repository using NCBI's (National Center for Biotechnology Information) online BLAST (Basic Local Alignment Search Tool) algorithmic program. Sequence alignment was done using the Clustal W program and phylogenetic analysis was executed in MEGA X (Molecular Evolutionary Genetics Analysis version X). The prevalence of cryptosporidiosis in Njoro Sub County is 6.99%. Cryptosporidium spp. present in the watershed showed great genetic diversity and nine Cryptosporidium species were recorded: Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium ubiquitum, Cryptosporidium meleagridis, Cryptosporidium andersoni, Cryptosporidium baileyi, Cryptosporidium muris, Cryptosporidium xiaoi and Cryptosporidium viatorum. This is the first study to report the presence of C. viatorum in Kenya. Cattle is the major reservoir of zoonotic Cryptosporidium spp. while goats harbored the lowest number of species. Humans and domestic animals drink the contaminated water from Njoro River, humans are therefore, exposed to a high cryptosporidiosis risk.

Genetic characterizations of Cryptosporidium spp. from pet rodents indicate high zoonotic potential of pathogens from chinchillas

The objective of this study was to assess the potential role that wildlife plays in environmental degradation of watersheds through the contamination of the water supply with zoonotic genotypes of Cryptosporidium. Cryptosporidium isolates recovered from wildlife in the New York City (NYC) watershed were examined to determine genotype using a polymerase chain reaction protocol targeting the 18-Small Subunit (SSU) rRNA locus. Seventy-seven DNA samples recovered from 12 wildlife host species captured in the NYC watershed were amplified and sequenced. Data on risk factors associated with the perpetuation of these genotypes also were collected and analyzed. Although many genotypes appeared to be host-specific, 38% of the samples examined were identified as Cryptosporidium parvum, indicating the presence of zoonotic Cryptosporidium. Adult animals were more likely to shed the zoonotic strains of Cryptosporidium spp. Animals captured in the fall and winter were more likely to be infected with C. parvum than those captured in spring and summer.