Charting the proteome of Cryptosporidium parvum sporozoites using sequence similarity-based BLAST searching

Cryptosporidium (C.) spp. are important zoonotic parasites causing widespread diarrhoeal disease in man and animals. The recent release of the complete genome sequences for C. parvum and C. hominis has facilitated the comprehensive global proteome analysis of these opportunistic pathogens. The well-known approach for mass spectrometry (MS) based data analysis using the BLAST tool (MS BLAST) is a database search protocol for identifying unknown proteins by sequence similarity to homologous proteins using peptide sequences produced by mass spectrometry. We have used several complementary approaches to explore the global sporozoite proteome of C. parvum with available proteomic tools. To optimize the output of the MS data, a sequence similarity-based MS BLAST strategy was employed for bioinformatic analysis. Most significantly, almost all the constituents of glycolysis and several mitochondrion-related proteins were identified. In addition, many hypothetical Cryptosporidium proteins were validated by the identification of their constituent peptides. The MS BLAST approach was found to be useful during the study and could provide valuable information towards a complete understanding of the unique biology of Cryptosporidium.     

Molecular characterization of Cryptosporidium parvum detected in Japanese black and Holstein calves in Iwate Prefecture and Tanegashima Island,Kagoshima Prefecture,Japan

Cryptosporidium oocysts were found in 43 out of 77 calves from two farms in Iwate Prefecture and nine farms on Tanegashima Island, Kagoshima Prefecture, Japan. The DNA fragments of 18S ribosomal RNA (18S rRNA) gene were amplified by a nested PCR from 43 oocyst-positive as well as one oocyst-negative samples. All of them were precisely identified as C. parvum by analyzing the nucleotide sequences of the 18S rRNA gene. C. parvum oocyst-positive calves ranged in age from 6 to 13 days old and significantly have watery diarrhea (P<0.05). Sequences of the gene encoding the 60-kDa glycoprotein (GP60) in 43 Cryptosporidium oocyst-positive samples were identical to that of the zoonotic IIaA15G2R1 subtype. We therefore suggest that calves could be potential sources of C. parvum infections in humans.     

Occurrence of Giardia and Cryptosporidium in pigs on Prince Edward Island, Canada

In a cross-sectional study of 633 pigs from 21 herds on Prince Edward Island, Canada (PEI), the prevalence of infection with Cryptosporidium and Giardia, and the genotypes and species of isolates were determined in order to establish the zoonotic potential of pigs in this region. As determined by direct immunofluorescence microscopy (DFA), 18 herds (86%) and 163 animals (26%; 95% CI: 22-29%) tested positive for Cryptosporidium, while just 3 herds (14%) and 6 animals (1%; 95% CI: 0.4-2%) tested positive for Giardia. Cryptosporidium spp. isolates were detected in 39% (95% CI: 34-44%) of weanlings (1-3 months of age) and 9% (95% CI: 6-13) of sows (>8 months of age). Molecular characterization using the 18S rDNA and HSP70 gene fragments revealed the presence of Cryptosporidium sp. pig genotype II, C. suis, C. parvum, and Cryptosporidium sp. mouse genotype. Among the 113 isolates of Cryptosporidium spp. successfully genotyped, pig genotype II (61%) predominated, with C. suis (36%) being the next most prominant isolate. C. parvum (2%; two isolates) and Cryptosporidium sp. mouse genotype (0.9%; one isolate) were only occasionally isolated. The only two Cryptosporidium-positive genotyped isolates from sows included one each of C. suis and Cryptosporidium sp. pig genotype II.All but one of the six Giardia positive isolates were detected in weanling pigs. None of the Giardia-positive isolates was amenable to PCR. This study demonstrates that Cryptosporidium spp. are highly prevalent in pigs on PEI, Canada, are found mostly in weanlings (1-3 months of age). Furthermore, the pigs are primarily infected by the host-specific genotypes and species, Cryptosporidium sp. pig genotype II and C. suis, whereas the zoonotic C. parvum is rare. Giardia duodenalis is only occasionally found in pigs. These findings suggest that domestic pigs on PEI, Canada, likely do not pose a significant health risk to humans from these parasites.     

Prevalence and molecular characterization of Cryptosporidium spp. in dairy cattle from farms in China

Fecal samples of 2,056 dairy cattle from 14 farms were collected in three geographical regions of China and stained using a modified acid-fast staining technique to identify Cryptosporidium oocysts. A total of 387 (18.82%) positive samples were identified and further analyzed by polymerase chain reaction (PCR) using primers designed to amplify DNA fragments from the small subunit ribosomal RNA. The PCR products were sequenced and the sequences were deposited in the GenBank database under accession numbers EU369377-84 and GU070730-33. Phylogenetic analysis was performed and a distances matrix generated from these sequences confirmed the existence of Cryptosporidium (C.) parvum ''mouse'' genotype, C. bovis, C. andersoni, C. hominis, and C. serpentis in cattle. These results represent the first report on the prevalence and genetic identification of Cryptosporidium species, and may contribute to a better understanding of the epidemiology of Cryptosporidium in cattle in China.     

Occurrence of novel and rare subtype families of Cryptosporidium in bamboo rats (Rhizomys sinensis) in China

This report is the first to describe Cryptosporidium infection in bamboo rats (Rhizomys sinensis). Ninety-two fresh fecal specimens were collected from a pet market in Ya’an City, China. One Cryptosporidium isolate from an asymptomatic host and two isolates from separate hosts with diarrhea were obtained by using Sheather's sucrose flotation technique and modified acid-fast staining. The Cryptosporidium spp. were genotyped by nested PCR and nucleotide sequencing of the small subunit rRNA (SSU rRNA), 70-kDa heat shock protein (HSP70), oocyst wall protein (COWP), and actin genes: isolates were identified as Cryptosporidium parvum with minor nucleotide differences at all four loci. Further subtyping was performed by PCR amplification and DNA sequence analysis of the 60-kDa glycoprotein (gp60) gene: two subtype families were detected, including a novel C. parvum subtype IIpA9 and a rare subtype IIoA13G1 (only reported in diarrheal patients of Sweden). Our results suggest that the bamboo rat is a reservoir host of C. parvum. Significantly, we discovered that the rare C. parvum subtype family IIo is also a zoonotic subtype and confirmed C. parvum subtype IIpA9 as a novel subtype family.     

Occurrence of Cryptosporidium and Giardia on beef farms and water sources within the vicinity of the farms on Prince Edward Island, Canada

The objectives of this study were to determine the prevalence and assemblages of Giardia and species of Cryptosporidium on beef farms in Prince Edward Island (PEI), Canada, including the water sources associated with the farms, and to determine risk factors for infection of cattle with these parasites. Twenty beef farms were selected based on the presence of surface water < 500 m from the barn. Prevalence was determined by direct immunofluorescence microscopy, while genotyping and species determination were performed by nested-PCR and DNA sequencing. Giardia was detected in 42% (95% CI: 38-46%) of fecal samples from 100% farms while Cryptosporidium was detected in 17% (95% CI: 14-19%) of fecal samples from 80% of farms. The most predominant Giardia assemblage isolated was the livestock specific assemblage E (89%). The zoonotic assemblages A and B were found in 4 and 7% of the Giardia isolates that were genotyped, respectively. The Giardia assemblages were detected equally between the cows and calves examined. Overall, the most common Cryptosporidium species detected in this study was Cryptosporidium andersoni (49%), predominantly found in cattle >6 mo of age, while most Cryptosporidium bovis and Cryptosporidium pestis (previously Cryptosporidium parvum ‘bovine genotype’) isolates were detected in calves ≤ 6 mo of age. All Cryptosporidium ryanae isolates (four) were found in calves. Giardia cysts and Cryptosporidium oocysts were detected in 14 and 93% of surface water samples of 14 farms, respectively. Cryptosporidium oocysts were detected in three (15%) ground water samples of 20 farms. One Cryptosporidium-positive water sample, which was the only surface water sample amenable to genotyping, contained C. parvum. The farm-level risk factors investigated in this study, age of animals and location of the farm, were not associated with the risk of infection in cattle with either Cryptosporidium spp. or Giardia duodenalis.We conclude that beef cattle are a potential reservoir of Cryptosporidium spp. and G. duodenalis that could contaminate source water. There is the possibility of further transmission to humans on PEI if the source water is not properly treated prior to consumption.     

Identity and public health potential of Cryptosporidium spp. in water buffalo calves in Egypt

Little is known about the diversity and public health significance of Cryptosporidium species in water buffaloes. In this study, we examined the distribution of Cryptosporidium spp. in water buffalo calves in Egypt. Rectal fecal specimens from 179 calves and 359 adults were screened microscopically for Cryptosporidium oocysts using modified Ziehl–Neelsen stain. Cryptosporidium spp. in 17 microscopy-positive specimens from calves were genotyped by DNA sequence analysis of the small-subunit rRNA gene, and Cryptosporidium parvum was subtyped by sequence analysis of the 60 kDa glycoprotein gene. Cryptosporidium ryanae was found in 10 specimens and C. parvum in 7 specimens, with the former belonging to the newly identified C. ryanae buffalo variant and the latter belonging to the subtypes IIdA20G1 (in 5 specimens) and IIaA15G1R1 (in 2 specimens). The prevailing occurrence of C. ryanae and the subtype family IId of C. parvum and the absence of C. bovis and C. andersoni represent some features of Cryptosporidium transmission in water buffaloes in Egypt.     

Concurrent response to challenge infection with Cryptosporidium parvum in immunosuppressed C57BL/6N mice

We investigated the response to challenge infection with Cryptosporidium parvum oocysts in immunosuppressed C57BL/6N mice. In the primary infection, fecal oocyst shedding and parasite colonization were greater in immunosuppressed mice than in nonimmunosuppressed mice. Compared with primary infection, challenge infection with C. parvum didn''t show any oocyst shedding and parasite colonization. Especially, oocyst shedding and parasite colonization from the mice infected with heat-killed oocysts were not detected. After challenge infection with C. parvum oocysts, however, these mice were shedding small numbers of oocysts and parasite colonization. Except normal control and uninfected groups, the antibody titers of other groups appear similar. Based on the fecal oocyst shedding, parasite colonization of ilea, and antibody titers in the mice, these results suggest that the resistance to challenge infection with C. parvum in immunosuppressed C57BL/6N mice has increased.     

Cryptosporidiosis – an occupational risk and a disregarded disease in Estonia

Background

Cases of cryptosporidiosis have not been officially reported in Estonia after the year 2000, and the disease appears to be either under-diagnosed or under-reported.

Findings

Based on a human case of cryptosporidiosis contracted during faecal sampling in dairy farms, cattle considered to be sources of infection were analysed for Cryptosporidium spp. by a modified Ziehl Neelsen technique and molecular typing. C. parvum subtype IIaA16G1R1 was detected from the human case and from calves from one of nine farms enrolled in the study providing strong circumstantial evidence of zoonotic transmission from calves to humans.

Conclusion

Cryptosporidiosis presents an occupational risk to people with cattle contact, and may also be a risk to the human population in general. Thus increased public and medical awareness is warranted.     

Zoonotic Cryptosporidium parvum in Romanian newborn lambs (Ovis aries)

This study was undertaken to investigate the occurrence and public health significance of Cryptosporidium species/genotypes and subtypes in a newborn lambs. A total of 175 diarrheic fecal samples from lambs (younger than 21 days) were collected in seven sheep flocks located in western Romania, and were microscopically examined for the presence of Cryptosporidium oocysts after staining with modified Ziehl–Neelsen technique. Twenty-four (13.7%) fecal samples were tested Cryptosporidium positive by microscopy and were subjected for molecular characterization. All positive samples were successfully amplified through a nested polymerase chain reaction (PCR) of the small subunit (SSU) rRNA gene (18S). Cryptosporidium species were determined by restriction fragment length polymorphism (RFLP) analysis of the secondary PCR products using the conventional SspI and VspI restriction enzymes. The identified species were: Cryptosporidium parvum (20/24), C. ubiquitum (2/24) and C. xiaoi (2/24), respectively. PCR-RFLP results for C. ubiquitum and C. xiaoi isolates were confirmed by DNA sequencing. Subsequently, subtyping of seven randomly selected C. parvum isolates, based on sequence analysis of the GP60 gene, revealed the presence of five different subtypes (IIaA17G1R1, IIaA16G1R1, IIdA20G1, IIdA24G1 and IIdA22G2R1) belonging in two zoonotic subtype families (IIa and IId). These findings may suggest the potential role of the newborn lambs as a source for human cryptosporidiosis. This is the first published report about the presence of C. ubiquitum and C. xiaoi in lambs from Romania.     

Cryptosporidium parvum infection and management-based risk factors of dairy calves in Taiwan

Cryptosporidiosis is one of the major causes of diarrhea in calves. Cryptosporidium parvum is considered the most important calf diarrhea pathogen in the Cryptosporidium species. Not only could infected calves spread C. parvum, but infected adult cattle could also shed oocysts. The objectives of this study were (1) to investigate the prevalence of C. parvum in dairy herds in Taiwan, including calves, the dams in delivery enclosure, the floor, and the drinking water; (2) to clarify the relationship of diarrhea, management, and C. parvum infection. Twenty dairy herds in Taiwan were selected by random sampling, including 226 calves and 198 dams, and other environmental samples were collected. A questionnaire was filled out by the farm owners to collect information regarding the management of calves and the delivery enclosure. Hierarchical logistic regression was used to analyze the risk factors for C. parvum infection. The prevalence of C. parvum infection in calves was 26.5% (60/226), while in dams, it was 19.7% (39/198). The C. parvum infection in calves increased with environmental contamination of C. parvum and clinical signs of diarrhea, while it decreased with a yard provided in the delivery enclosure. In conclusion, the management of the delivery enclosure appears to be more important for preventing cryptosporidiosis in calves in Taiwan.     

Prevalence and genotyping of bovine Cryptosporidium species in the Mediterranean and Central Anatolia Region of Turkey

The prevalence of Cryptosporidium species in calves and heifers with relation to diarrhea from several herds was investigated in this study. Fecal samples were collected from 135 and 120 pre-weaned calves and 79 and 130 heifers raised in the Central Anatolia (CAR) and Mediterranean Regions (MR) of Turkey, respectively. A total of 86 post-weaned calves in CAR were also included in the study. For diagnostic comparison, all samples were examined by microscopic examination, SSU rRNA nested PCR and TaqMan real-time PCR for the presence of oocyst and Cryptosporidium DNA. In total, 102 (34.0 %) and 93 (37.2 %) of the examined samples from CAR and MR were found positive for Cryptosporidium DNA with both nested PCR and real-time PCR analyses, respectively with an overall prevalence of 35.5 %. The diagnostic sensitivity and specificity of microscopic examination were determined as 68.7 % and 100.0 % compared to molecular tools, respectively. RFLP and sequence analyses of the SSU rRNA from the PCR products revealed that 138 (70.8 %) out of 195 positive isolates were C. parvum further confirming the species-specific real-time PCR results. Among the remaining 57 (29.2 %) positive isolates, 30 (15.4 %) and 27 (13.8 %) were characterized as C. ryanae and C. bovis, respectively. C. parvum was the dominant species in pre-weaned calves especially with diarrhea while C. bovis and C. ryanae were mostly found in post-weaned calves and heifers. The sequence analyses of the gp60 gene of C. parvum isolates revealed two subtypes (IIaA13G2R1, IIaA14G1R1) belonging to zoonotic family IIa, with IIaA13G2R1 being the most common in diarrheic calves.     

Molecular characterization of Giardia intestinalis and Cryptosporidium parvum from calves with diarrhoea in Austria and evaluation of point-of-care tests

To obtain information about the occurrence and genotype distribution of G. intestinalis and C. parvum in Austrian cattle, faecal samples from diarrhoeic calves younger than 180 days of age originating from 70 farms were examined. Of the 177 faecal samples, 27.1% were positive for Giardia cysts (immunofluorescence microscopy) and 55.4% for Cryptosporidium oocysts (phase-contrast microscopy). Positive samples were characterized by nested PCR for Giardia, 83.3% (triosephosphate isomerase; tpi) and 89.6% (β-giardin; bg) were positive, while the Cryptosporidium nested PCR returned 92.5% (60-kDa glycoprotein) positive results. Sequence analysis revealed one assemblage A-positive sample and 30 (bg) respectively 29 (tpi) assemblage E-positive samples for G. intestinalis. For C. parvum four subtypes within the IIa family (IIaA15G2R1, n = 29; IIaA19G2R2, n = 3; IIaA21G2R1, n = 2; IIaA14G1R1, n = 1) could be differentiated. Validation of two immunochromatographic point-of-care tests resulted in a sensitivity of 29.2% and 77.6%; a specificity of 98.4% and 91.1% for the detection of Giardia intestinalis and Cryptosporidium parvum, respectively. Results confirm the widespread occurrence of both protozoa in diarrhoeic calves in Austria.     

Molecular epidemiology of Cryptosporidium subtypes in cattle in England

Samples of Cryptosporidium spp., collected in a cross-sectional study of calves (median age 26 days) from 41 farms in Cheshire, UK, underwent molecular typing. The aim was to determine naturally occurring species/genotypes and to investigate transmission pathways within a local area. Of 60 positive samples, 54 were sequenced at an 18S rRNA locus and 51 were typed at a GP60 locus. C. parvum was identified in 50 samples, three cases were C. bovis and one was Cryptosporidium deer-like genotype. Six GP60 subgenotypes were identified. One subgenotype (IIaA15G2R1) was highly prevalent throughout the study area. A single subgenotype was identified on 20/22 farms. Two subgenotypes were found on 2/22 farms. The spatial scan statistic detected a cluster of subgenotype IIaA15G2R1 comprising seven farms. This suggests local transmission of the parasite between farms. As most of the isolates detected were the potentially zoonotic C. parvum allele IIa, intervention strategies should be considered to reduce the threat to public health. Biosecurity measures may reduce transmission between farms and result in lower levels of environmental contamination.     

Molecular characterisation of Cryptosporidium isolates from rivers,water treatment plants and abattoirs in Ibadan,Nigeria

To understand the molecular characteristics of Cryptosporidium species contaminating rivers, water treatment plants and abattoirs in Ibadan Nigeria, water samples were obtained from ten rivers used for household and agricultural purposes, three major functional water treatment plants and three major abattoirs located within Ibadan metropolis during dry and rainy seasons between November, 2016 to October, 2017. Obtained samples were examined for Cryptosporidium oocysts using microscopy after using modified formalin–ether concentration method and modified acid-fast staining. Cryptosporidium oocysts were detected in samples from five rivers with mean oocyst count/field ranging from 7.70 ± 0.57–1.34 ± 0.57, oocysts were also detected in samples from two abattoirs with mean oocyst count/field ranging from 4.60 ± 0.33–2.50 ± 0.33. Genomic DNA were extracted from microscopy positive river and abattoir samples using sucrose gradient purification method and genotypes and subtypes of parasites were detected by nested PCR amplification and nucleotide sequence analysis of both 18S rRNA and 60-kDa glycoprotein (gp60) genes. Cryptosporidium parvum, C. muris and C. fragile were the only genotypes detected in some river samples, while gp60 gene sequence analysis showed that the C. parvum strain detected was subtype IIa. This study provides evidence that rivers used for household and agricultural purposes in studied area may be potential reservoirs and infection sources for Cryptosporidium species and zoonotic subtypes of public health importance.     

Comparative evaluation of Cryptosporidium infection in malnourished and well-nourished children: Parasitic infections are affected by the interaction of nutritional status and socio-demographic characteristics

Cryptosporidium, as a small protozoan parasite, is a leading cause of persistent diarrhea in children in developing countries and has both a short and long-term impact on the growth of children. In the present study, Cryptosporidium infection was compared in malnourished and well-nourished children by modified acid-fast staining, nested-polymerase chain reaction (nested-PCR) and loop-mediated isothermal amplification (LAMP) methods. As a case-control study, Cryptosporidium infection in 94 malnourished children was evaluated and compared with those of 188 age and gender-matched well-nourished children. Oocysts of Cryptosporidium were detected by modified acid-fast staining method. The extracted DNA was amplified by nested-PCR and LAMP techniques. In addition, positive amplicons were directly sequenced for phylogenetic analysis. Cryptosporidium oocysts were found in the stools of two (2.12 %) children who were hospitalized and had diarrhea by nested-PCR while three isolates (3.2 %) were found by LAMP. Cryptosporidium-positive children were more malnourished compared to those who were negative for Cryptosporidium infection but this important finding was not statistically significant. C. parvum was the main species of Cryptosporidium detected in malnourished children in northwest Iran. LAMP can be considered as a sensitive field monitoring assay in patients with low parasite burden. Nutritional status and socio-demographic factors may have interactive effects on the incidence and severity of parasitic diseases.     

Comparative proteomic analysis of proteins expression changes in the mammary tissue of cows infected with Escherichia coli mastitis

Cows infected with Escherichia (E.) coli usually experience severe clinical symptoms, including damage to mammary tissues, reduced milk yield, and altered milk composition. In order to investigate the host response to E. coli infection and discover novel markers for mastitis treatment, mammary tissue samples were collected from healthy cows and bovines with naturally occurring severe E. coli mastitis. Changes of mammary tissue proteins were examined using two-dimensional gel electrophoresis and label-free proteomic approaches. A total of 95 differentially expressed proteins were identified. Of these, 56 proteins were categorized according to molecular function, cellular component, and biological processes. The most frequent biological processes influenced by the proteins were response to stress, transport, and establishment of localization. Furthermore, a network analysis of the proteins with altered expression in mammary tissues demonstrated that these factors are predominantly involved with binding and structural molecule activities. Vimentin and α-enolase were central "functional hubs" in the network. Based on results from the present study, disease-induced alterations of protein expression in mammary glands and potential markers for the effective treatment of E. coli mastitis were identified. These data have also helped elucidate defense mechanisms that protect the mammary glands and promote the pathogenesis of E. coli mastitis.     

Molecular detection of Cryptosporidium spp. infections in water buffaloes from northeast Thailand

The objectives of this study were to determine the individual and herd-level prevalence and genotype of Cryptosporidium and to identify putative risk factors associated with Cryptosporidium spp. infections in water buffaloes in northeast Thailand. Fecal samples from 600 water buffaloes of 287 farms in six provinces were collected and tested using DMSO-modified acid-fast staining and polymerase chain reaction. The overall prevalence of Cryptosporidium infections in buffaloes was 5.7 and 8.7 % among individual animals and herds, respectively. The provinces with highest infected Cryptosporidium were located in the Sakon Nakhon Basin in the northern part of the region. In addition, higher herd prevalence was observed among farms with more than five buffaloes (30 %) than those with five or less animals (16.2 %). Thirty (88.2 %) of the 34 Cryptosporidium-positive samples were Cryptosporidium parvum and four (11.8 %) were Cryptosporidium ryanae.     

Prevalence of Cryptosporidium species isolated from HIV/AIDS patients in southwest of Iran

This study aimed to determine the prevalence and species of Cryptosporidium among HIV/AIDS patients in southwest of Iran. Two hundred fifty faecal samples from HIV patients were examined for the presence of Cryptosporidium oocysts using a conventional coproscopic approach. Such oocysts were detected in 18 (7.2%) out of 250 faecal samples. Genomic DNAs from 250 samples were then subjected to a nested-PCR-RFLP technique targeting different loci of 18S rRNA gene for species identification. Out of 250 samples, 27 (10.8%) were positive for different Cryptosporidium spp; Restriction patterns resulting from the digestion of the nested amplicon with restriction endonucleases VspI and SspI showed that C. parvum (70.38%) was the most prevalent species, followed by C. hominis (25.92%) and C. meleagridis (3.7%), respectively. The mean CD4+ T-cell count was 215 cells/μL. There was a strong association between cryptosporidiosis and CD4+ T-cell count (P = 0.000) with the highest prevalence recorded among patients with CD4+ T-cell count < 200 cells/μL. This confirms that there is a low opportunity for this parasite to get established as the patients CD4+ T-cell count increases. Also HIV infection increased the risk of having Cryptosporidium. Our epidemiological findings are useful for any preventive intervention to control disease diffusion.