Global prevalence of Cryptosporidium andersoni in dairy cattle: A systematic review and meta-analysis.

Cryptosporidium spp. are apicomplexan parasites commonly found in the gastrointestinal tracts of humans and in a wide range of animals. Infection is prevalent in dairy cattle and results in diarrhea and increased mortality with significant production losses. Cryptosporidium andersoni is commonly seen in asymptomatic adult cattle and has been associated with gastritis, reduced milk yield, and poor weight gain. However, a meta-analysis of C. andersoni infection in dairy cattle globally has not yet been published. We searched databases for studies on the global prevalence of C. andersoni infection in dairy cattle published from January 1, 2000, to December 31, 2022. The prevalence of C. andersoni infection in dairy cattle was estimated using a random effects model. In total, 86 publications from 30 countries were included in the final quantitative analysis. The global prevalence of C. andersoni in dairy cattle was 4.7 % (95 % confidence interval [CI]: 4.5-4.9 %, 2,554/54,627). European dairy cattle had the highest rate of C. andersoni infection at 8.8 % (961/10,944). A univariate meta-regression analysis indicated that the age of cattle (P = 0.002) and sample collection year (P = 0.025) might be sources of heterogeneity. This systematic review suggests that globally, dairy cattle exhibit a low level of C. andersoni infection; however, the geographical distribution of infection is extensive. C. andersoni mainly infects the stomach of cattle and causes no obvious clinical symptoms after infection but is thought to be responsible for reduced milk production. Therefore, subclinical Cryptosporidium infection in dairy cattle is easily overlooked. Cattle with subclinical infections can produce feces containing oocysts that are inadvertently not safely handled, which can then infect healthy dairy cattle and even cause Cryptosporidium infection in dairy cattle breeders. Therefore, prevention of C. andersoni transmission in asymptomatic cattle is an important issue that should not be neglected.

Cryptosporidium andersoni-associated proliferative abomasitis in a roan antelope.

A 2-y-old, intact male roan antelope (Hippotragus equinus) was submitted for routine postmortem investigation after a prolonged history of diarrhea and weight loss. The abomasal mucosa was diffusely thickened and corrugated. Abomasal gland hyperplasia was associated with abundant apical organisms consistent with Cryptosporidium spp. Genomic DNA was extracted from abomasal and intestinal contents and subjected to PCR using primers specific for the 18S rRNA gene of Cryptosporidium spp., followed by Sanger sequencing. The sequence was >99% homologous to Cryptosporidium andersoni. C. andersoni-associated proliferative abomasitis has not been reported previously in a captive hippotraginid, to our knowledge.

Molecular and phylogenetic characterization of Cryptosporidium species in the saffron finch Sicalis flaveola.

BACKGROUND: Cryptosporidium is the most common protozoan that can infect a wide variety of animals, including mammals and birds. Fecal samples of six saffron finches, Sicalis flaveola, from a commercial establishment were screened for the presence of Cryptosporidium by the modified Ziehl-Neelsen technique and nested PCR of the 18S rRNA gene followed by sequencing of the amplified fragments. RESULTS: The species Cryptosporidium galli was identified in all six saffron fiches, in addition to Cryptosporidium andersoni in one of the birds, indicating a mixed infection. Only two birds had feathers that were ruffled and dirty with feces. Concomitant infection with Isospora spp. was observed in all birds. CONCLUSIONS: Saffron finches are a possible host of C. andersoni and this is the first report of this species in a captive bird and the third report of parasitism by C. galli in Sicalis flaveola.

Tandem mass tag (TMT)-based proteomic analysis of Cryptosporidium andersoni oocysts before and after excystation.

BACKGROUND: Cryptosporidium andersoni initiates infection by releasing sporozoites from oocysts through excystation. However, the proteins involved in excystation are unknown. Determining the proteins that participate in the excystation of C. andersoni oocysts will increase our understanding of the excystation process. METHODS: Cryptosporidium andersoni oocysts were collected and purified from the feces of naturally infected adult cows. Tandem mass tags (TMT), coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic analysis, were used to investigate the proteomic expression profiles of C. andersoni oocysts before and after excystation. RESULTS: Proteomic analysis identified a total of 1586 proteins, of which 17 were differentially expressed proteins (DEPs) upon excystation. These included 10 upregulated and seven downregulated proteins. The 17 proteins had multiple biological functions associated with control of gene expression at the level of transcription and biosynthetic and metabolic processes. Quantitative real-time RT-PCR of eight selected genes validated the proteomic data. CONCLUSIONS: This study provides information on the protein composition of C. andersoni oocysts as well as possible excystation factors. The data may be useful in identifying genes for diagnosis, vaccine development, and immunotherapy for Cryptosporidium.

Recombinant Expression And Indirect ELISA For COWP And HSP70 Proteins From Cryptosporidium andersoni.

Cryptosporidium spp. infect cattle at a high rates, and reduce milk production. Cryptosporidiosis has caused economic losses for the dairy industry. Studies in Western countries have shown that Cryptosporidium can also infect humans. Therefore, the development of methods for the early detection of Cryptosporidium is an important public health objective. Total RNA isolated from C. andersoni was used as template for generating cDNA encoding the COWP and HSP70 proteins. The recombinant plasmid, pET-32a(+)-COWP-HSP70, was constructed by double digestion and subcloning. The expression of the three recombinant proteins was induced in Escherichia coli BL21 using isopropyl-ß-D-thiogalactopyranoside. The antigenicity of the recombinant proteins was examined using western blotting and indirect ELISA. The identities of the COWP fusion protein (CFP), HSP70 fusion protein (HFP), and COWP-HSP70 fusion protein (CHFP) were confirmed by BLAST searches of known sequences in GenBank respectively. The ELISA and western blot analyses indicated that all three of the proteins were highly immunogenic and antigenic. An indirect ELISA method was developed using the three recombinant proteins as coating antigens for the analysis of 40 clinical samples. The results showed that CHFP was the best candidate antigen for clinical testing, with a detection rate of 100%, compared with general parasitological screening. Above of all, the recombinant CHFP protein represents the best candidate antigen among three ones for detecting anti-Cryptosporidium antibodies in clinical samples. The development of the indirect ELISA lays the foundation for further research in immunodiagnosis and disease prevention of cryptosporidiosis.

The molecular characterisation of Cryptosporidium species in relinquished dogs in Great Britain: a novel zoonotic risk?

Surveillance was conducted to investigate the occurrence of protozoan parasites of the genus Cryptosporidium in dogs newly admitted to a dog rehoming charity in London, Great Britain. Voided faecal samples were collected from all new admissions between 2011 and 2012 during six separate 4-week sampling periods. Information on host signalment, including age, breed and reason for submission and faecal consistency, was collected. Polymerase Chain Reaction (PCR) targeting the 18S ribosomal RNA gene, confirmed by sequencing, was conducted on the faecal samples to detect Cryptosporidium genomic DNA and determine Cryptosporidium identity. In total, 677 dogs were included in the study. The prevalence of Cryptosporidium-positive faecal samples was 4.6% (31/676). There were positive samples in all of the six sampling periods. Cryptosporidium canis (n = 28), C. parvum (n = 2) and C. andersoni (n = 1) were identified. Sixty KDa glycoprotein (gp60) gene amplicon sequencing of the C. parvum samples identified genotypes IIaA17G1R1 and IIaA15G2R1 for the first time from a dog. There were no significant associations between signalment data and Cryptosporidium status. While this was a study of one rehoming shelter, the presence of the potentially zoonotic C. parvum and C. canis in dogs highlights a public health concern. Further research is needed to better understand the epidemiology and potential impacts of Cryptosporidium infection in dogs.

Prevalence and multilocus genotyping of Cryptosporidium andersoni in dairy cattle and He cattle in Xinjiang, China.

Cryptosporidium andersoni is the predominant species in post-weaned and adult cattle in China. The aim of this study was to determine the prevalence and understand the transmission of cattle cryptosporidiosis in the Xinjiang Uyghur Autonomous Region, China, a total of 1827 fecal samples (436 from He cattle and 1391 from dairy cattle) were examined for the presence of C. andersoni-like oocysts by microscopy after Sheather's sugar flotation technique. The overall prevalence of C. andersoni-like was 3.8% (70/1827) and all the C. andersoni-like isolates were identified as C. andersoni at the SSU rRNA locus. Among the C. andersoni isolates, a total of 60 isolates were successfully characterized into eight multilocus sequence typing (MLST) subtypes using MLST analysis at the four microsatellite/minisatellite loci (MS1, MS2, MS3 and MS16), and three new subtypes were identified. The MLST subtype A4,A4,A4,A1 showed a predominance and a wide distribution among the eight MLST subtypes obtained in the investigated areas. The MLST subtypes A2,A4,A2,A1 and A4,A5,A2,A1 showed a unique distribution in the investigated areas. A linkage disequilibrium analysis showed the presence of an epidemic population genetic structure of C. andersoni isolated from dairy and He cattle in Xinjiang. These findings provide new insights into the genetic structure of C. andersoni isolates and are also helpful to explore the infection source of C. andersoni in cattle in Xinjiang, China.

Microscopic and Molecular Detection of Cryptosporidium andersoni and Cryptosporidium xiaoi in Wastewater Samples of Tehran Province, Iran.

BACKGROUND: As a waterborne pathogen, Cryptosporidium is one of the most common causes of gastroenteritis in human and hoofed livestock animals. This study aimed to investigate the distribution of Cryptosporidium spp. in human and livestock wastewaters in Iran, by the 18S rRNA sequence analysis. METHODS: A total of 54 raw wastewater samples collected from three urban treatment plants and two slaughterhouses during 2014-2015 in Tehran, Iran. The presence of the Cryptosporidium oocysts was assessed by immunofluorescence with monoclonal antibodies. To characterize the oocysts at the molecular level, the 18S rRNA gene of Cryptosporidium was PCR amplified and sequenced. RESULTS: Of the 54 wastewater samples examined, 34 (62.9%) were positive for Cryptosporidium oocysts using the IFA. Of these, 70.5% (24/34) were positive by PCR, that 91.6% (22/24) were successfully sequenced. The species of C. andersoni (95.4%) and C. xiaoi (4.6%) were detected in livestock wastewater samples. CONCLUSION: C. andersoni was the major Cryptosporidium sp. found in the aquatic environmental wastewater samples. The high rate of detection of C. andersoni in domestic wastewater was probably the result of the predominancy of this species in cattle herds in Iran. The current study is the first report of C. xiaoi in Iran.

Cloning and Iron Transportation of Nucleotide Binding Domain of Cryptosporidium andersoni ATP-Binding Cassette (CaABC) Gene.

Cryptosporidium andersoni ATP-binding cassette (CaABC) is an important membrane protein involved in substrate transport across the membrane. In this research, the nucleotide binding domain (NBD) of CaABC gene was amplified by PCR, and the eukaryotic expression vector of pEGFP-C1-CaNBD was reconstructed. Then, the recombinant plasmid of pEGFP-C1-CaNBD was transformed into the mouse intestinal epithelial cells (IECs) to study the iron transportation function of CaABC. The results indicated that NBD region of CaABC gene can significantly elevate the transport efficiency of Ca(2+), Mg(2+), K(+), and HCO3 (-) in IECs (P<0.05). The significance of this study is to find the ATPase inhibitors for NBD region of CaABC gene and to inhibit ATP binding and nutrient transport of CaABC transporter. Thus, C. andersoni will be killed by inhibition of nutrient uptake. This will open up a new way for treatment of cryptosporidiosis.

Survey on gastrointestinal parasites and detection of Cryptosporidium spp. on cattle in West Java, Indonesia.

OBJECTIVE: To evaluate the presence of gastrointestinal parasites on cattle in Indonesia because the prevalence of parasites varies between countries depending on the terrain surrounding livestock farms and investigations in Indonesia have never been performed. METHODS: Fecal samples from cattle at 35 farms in 7 districts in West Java, Indonesia, has been examined using the floatation or sedimentation methods, and a immunofluorescence assay and experimentally inoculation to mice for Cryptosporidium or Giardia.spp. RESULTS: 153 of 394 examined cattle (38.8%) were infected with gastrointestinal parasites. The prevalence of Eimeria spp., Nematoda spp. (including Oesophagustomum and Bunostomum-like), Fasciola gigantica and Paramphistomum spp. was 22.4%, 11.2%, 12.5% and 3.8%, respectively. Cryptosporidium andersoni (C. andersoni) was also found in two samples. One isolate of this parasite was confirmed to be transmitted to mice, in contrast to the isolates from other countries. CONCLUSIONS: although this survey is preliminary, the results shows that the infection of gastrointestinal parasites in Indonesia was not high, but these infected cattle could be as a potential source leading to economic losses in livestock production.

Cryptosporidium parvum and Cryptosporidium andersoni infection in naturally infected cattle of northwest Iran.

The protozoan intestinal parasite Cryptosporidium commonly infects cattle throughout the world and Iran. The present study was undertaken to determine the abundance and associated risk factors of Cryptosporidium infection in cattle herds of northwestern Iran. A total number of 246 fecal samples from 138 (56.1%) diarrheic (D) and 108 (43.9%) non-diarrheic (ND) cattle were randomly collected and examined by fecal smears stained with Ziehl-Neelsen. For molecular specification, DNA was extracted from collected Cryptosporidium oocysts and a fragment of 1325 bp in size from 18S rRNA gene was amplified. The overall prevalence of Cryptosporidium infection was 22.3% (55/246). The prevalence of Cryptosporidium infection in examined calves less than 6 month-old was significantly higher than adult cattle. C. parvum and C. andersoni were identified in 20.3% (50/246) and 2.03% (5/246) of examined cattle, respectively. The highest prevalence of C. parvum infection was found in D calves < 6 month-old (13.4%, 33/246), while C. andersoni was only detected in ND cattle (8.9%, 22/246). There was significant difference in the prevalence between male than female cattle. There was no significant difference between prevalence and seasons of investigation. It was concluded that C. parvum was the prevalent species in younger animals compared to older ones as a potentially zoonotic agent in the region.

The Lesser Egyptian Gerbil (Gerbillus gerbillus) is a suitable host for the long-term propagation of Cryptosporidium andersoni.

We describe the course of infection of Cryptosporidium andersoni LI03, originally isolated from cattle, in outbred Gerbillus gerbillus (Lesser Egyptian Gerbil), Meriones unguiculatus (Mongolian gerbil), and Meriones tristrami (Tristram's jird). While both Meriones spp. partially cleared the infection and shed a low number of oocysts (less than 15,000 oocysts per gram (OPG)), chronic infection with a mean infection intensity reaching 200,000 OPG was observed in G. gerbillus. These data suggest that G. gerbillus can be used as a laboratory model for the long-term maintenance and study of C. andersoni without the need for host immunosuppression.

First description of heterogeneity in 18S rRNA genes in the haploid genome of Cryptosporidium andersoni Kawatabi type.

The Apicomplexan Cryptosporidium andersoni, is a species of gastric Cryptosporidium, is frequently detected in older calves and adult cattle. Genotyping analyses based on 18S ribosomal RNA gene sequences have been performed on a novel C. andersoni genotype, namely the Kawatabi type, and the oocysts were classified into two distinct groups genotypically: Type A (the sequence in GenBank) and Type B (with a thymine nucleotide insertion not in Type A). This study analyzed 3775 cattle at a slaughterhouse and 310 cattle at a farm using microscopy and found 175 Cryptosporidium-positive animals: 171 from the slaughterhouse and four from the farm, and all infecting parasites were determined to be C. andersoni from 18S rRNA gene sequences determined from fecal DNA. In genotyping analyses with single isolated oocysts, about a half of analyzed ones were clearly classified into well known two genotypes (Type A and B). In addition to these two known genotypes, we have detected some oocysts showing mixed signals of Types A and B in the electropherogram from the automated sequencer (the Type C genotype). To determine the genotypic composition of sporozoites carried by the Type C oocysts, we analyzed their 18S rRNA gene sequences using a single sporozoite isolation procedure. Some sporozoites were classified as either Type A or Type B. However, more than half of the analyzed isolated sporozoites showed a mixed signal identical to that of Type C oocysts, and both the Type A and B signals were surely detectable from such sporozoites after a cloning procedure. In conclusion, C. andersoni carries two different genotypes heterogeneously in its haploid genome.