Mónica Santín

A longitudinal study of cryptosporidiosis in dairy cattle from birth to 2 years of age

Fecal specimens were collected from 30 calves from birth to 24 months of age at a dairy farm in Maryland to determine the prevalence and age distribution of Cryptosporidium species/genotypes. After centrifugation to remove debris and concentrate oocysts, specimens were examined by immunofluorescence microscopy and polymerase chain reaction (PCR). Fragments of the SSU-rDNA gene amplified by PCR were purified and PCR products were sequenced. All 30 calves shed Cryptosporidium oocysts at some time during the 24 months of the study. Of 990 specimens, 190 were Cryptosporidium-positive (19.2%). The highest prevalence of infection was at 2 weeks of age when 29 of the 30 calves were excreting oocysts. Prevalence was higher in pre-weaned calves (1-8 weeks of age) (45.8%) than in post-weaned calves (3-12 months of age) (18.5%) and heifers (12-24 months of age) (2.2%). Sequence data for 190 PCR-positive specimens identified: C. parvum, C. bovis, the Cryptosporidium deer-like genotype and C. andersoni, with cumulative prevalences of 100, 80, 60, and 3.3%, respectively. C. parvum constituted 97% of infections in pre-weaned calves but only 4% and 0% of infections in post-weaned calves and heifers, respectively. All C. parvum GP60 nucleotide sequences were subtype IIaA15G2R1.

Cryptosporidium bovis n. sp. (Apicomplexa: Cryptosporidiidae) in cattle (Bos taurus).

A new species of Cryptosporidium, C. bovis, is described. Oocysts of C. bovis, previously identified as Cryptosporidium genotype Bovine B (GenBank AY120911), are morphologically indistinguishable from those of C. parvum. They are excreted fully sporulated and contain 4 sporozoites, but lack sporocysts. Oocysts measure 4.76–5.35 μm (mean = 4.89 μm) × 4.17–4.76 μm (mean = 4.63 μm), with a length-to-width ratio of 1.06 (n = 50). Oocysts were not infectious for neonatal BALB/ c mice, but were infectious for 2 calves that were previously infected with C. parvum. Oocysts were not infectious for 2 experimentally exposed lambs less than 1 wk of age and were not detected in 42 lambs 2–3 mo of age, but were detected in a 2-wk-old lamb. In an earlier study, 79 of 840 calves on 14 dairy farms in 7 states were found infected with the new species. Most calves were 2–7 mo of age and none exhibited signs of diarrhea. This new species has been found in 10 of 162 calves aged 9 to 11 mo on a beef farm in Maryland. Fragments of the 18S rDNA, HSP-70, and actin genes were amplified by PCR, and purified PCR products were sequenced. Multilocus analysis of the 3 unlinked loci demonstrated the new species to be distinct from C. parvum and also demonstrated a lack of recombination, providing further evidence of species status. Based on these biological and molecular data, we consider this highly prevalent Cryptosporidium that infects primarily postweaned calves to be a new species and propose the name Cryptosporidium bovis n. sp. for this parasite.

Microsporidiosis: Enterocytozoon bieneusi in domesticated and wild animals

Microsporidia are a ubiquitous group of obligate intracellular parasites that infect all major animal groups. Enterocytozoon bieneusi is the most commonly identified Microsporidia in humans and has also been reported worldwide in animals with importance in veterinary medicine (e.g., cats, dogs, horses, cattle and pigs). The identification of E. bieneusi in animals has raised the question of the importance of animal reservoirs in the epidemiology of this pathogen, and the implications of the infection with this pathogen in infected animals. Considerable genetic diversity within E. bieneusi has been found with over 90 genotypes identified based on the ITS nucleotide sequence of E. bieneusi spores recovered from the feces of infected humans and animals. Both host-adapted E. bieneusi genotypes with narrow host ranges and potentially zoonotic genotypes with wide host specificity have been identified. The information presented in this review should be useful in understanding the taxonomy, epidemiology, zoonotic potential, and importance in public health of E. bieneusi.

Enterocytozoon bieneusi genotype nomenclature based on the internal transcribed spacer sequence: a consensus.

ABSTRACT. The standard method for determining the genotypes of Enterocytozoon bieneusi is based on the DNA sequence of the internal transcribed spacer (ITS) region of the rRNA gene. There are 81 genotypes with 111 genotype names: 26 genotypes have been identified exclusively in humans, eight have been identified in humans and in other hosts, 27 have been identified exclusively in cattle and pigs, six have been identified exclusively in cats and dogs, and 14 have been identified in miscellaneous hosts. Because none of these genotypes has taxonomic status and therefore do not adhere to the International Code of Zoological Nomenclature regarding naming, some genotypes have received multiple names, each different and in separate publications by different authors. Because of the proliferation of genotypes with overlapping names and multiple hosts the scientific literature has become confusing and difficult to efficiently utilize. To reduce confusion and provide guidance for future publications we tabulated all names, GenBank accession numbers, and author citations and propose that the first published name has precedence and should become the primary name used in all subsequent publications in which genotyping is based on ITS sequencing. In those publications the names and GenBank numbers that were submitted at later dates should also be provided by the authors as synonyms to aid readers and reviewers.

Cryptosporidium ubiquitum n. sp. in animals and humans

A new species, Cryptosporidium ubiquitum, previously identified as the Cryptosporidium cervine genotype and infrequently as the cervid, W4 or genotype 3 genotype, is described. In published studies this genotype was reported in wild and domesticated ruminants, rodents, carnivores and primates including humans. In the present study oocysts were found in feces from a captive prehensile-tailed porcupine and her infant. Oocysts from the porcupine were transmitted to 4 boer goats. Oocysts from the goats were transmitted to a calf (calf 1) and oocysts from calf 1 were transmitted to gerbils and BALB/c mouse pups. Calf 2 housed near calf 1 became contaminated and excreted oocysts of C. ubiquitum. Oocysts collected from calf 2 were transmitted to a calf 3. When calf 2 stopped excreting C. ubiquitum oocysts it was challenged with oocysts of C. parvum and became infected, indicating a lack of cross-species immunity. Oocysts of C. ubiquitum from calf 1 measured 4.71-5.32 microm x 4.33-4.98 microm (mean=5.04 microm x4.66 microm) with a length/width shape index of 1.08 (n=50). Purified PCR products of the SSU rRNA, actin and COWP genes were sequenced and analysis of the 3 unlinked loci demonstrated the new species to be distinct from all other species and also demonstrated a lack of recombination, providing further evidence of species status. Based on morphological, molecular and biological data, this geographically widespread parasite infectious for a wide range of mammalian hosts is recognized as a new species and is named C. ubiquitum.

Cryptosporidium ryanae n. sp. (Apicomplexa: Cryptosporidiidae) in cattle (Bos taurus)

A new species, Cryptosporidium ryanae, is described from cattle. Oocysts of C. ryanae, previously identified as the Cryptosporidium deer-like genotype and recorded as such in GenBank (AY587166, EU203216, DQ182597, AY741309, and DQ871345), are similar to those of Cryptosporidium parvum and Cryptosporidium bovis but smaller. This genotype has been reported to be prevalent in cattle worldwide. Oocysts obtained from a calf for the present study are the smallest Cryptosporidium oocysts reported in mammals, measuring 2.94-4.41micromx2.94-3.68microm (mean=3.16micromx3.73microm) with a length/width shape index of 1.18 (n=40). The pre-patent period for two Cryptosporidium-naïve calves fed C. ryanae oocysts was 11 days and the patent period was 15-17 days. Oocysts were not infectious for BALB/c mice or lambs. Fragments of the SSU-rDNA, HSP-70, and actin genes amplified by PCR were purified and PCR products were sequenced. Multi-locus analysis of the three unlinked loci demonstrated the new species to be distinct from all other species and also demonstrated a lack of recombination, providing further evidence of species status. Based on morphological, molecular and biological data, this geographically widespread parasite found only in Bos taurus calves is recognized as a new species and is named C. ryanae.

Cryptosporidium xiaoi n. sp. (Apicomplexa: Cryptosporidiidae) in sheep (Ovis aries).

A new species, Cryptosporidium xiaoi, is described from sheep. Oocysts of C. xiaoi, previously identified as the Cryptosporidium bovis-like genotype or as C. bovis from sheep in Spain, Tunisia, United Kingdom, and the United States are recorded as such in GenBank (EU408314-EU408317, EU327318-EU327320, EF362478, EF514234, DQ991389, and EF158461). Oocysts obtained from naturally infected sheep were infectious for a lamb and oocysts from that lamb were infectious for three other lambs. The prepatent period for C. xiaoi in these four Cryptosporidium-naive lambs was 7-8 days and the patent period was 13-15 days. Oocysts are similar to those of C. bovis but slightly smaller, measuring 2.94-4.41 microm x 2.94-4.41 microm (mean=3.94 microm x 3.44 microm) with a length/width shape index of 1.15 (n=25). Oocysts of C. xioai were not infectious for BALB/c mice, Bos taurus calves, or Capra aegagrus hircus kids. Fragments of the SSU-rDNA, HSP-70, and actin genes were amplified by PCR, purified, and PCR products were sequenced. The new species was distinct from all other Cryptosporidium species as demonstrated by multi-locus analysis of the 3 unlinked loci. Based on morphological, molecular and biological data, this geographically widespread parasite found in Ovis aries is recognized as a new species and is named C. xiaoi.

Distribution of Cryptosporidium parvum subtypes in calves in eastern United States

Cryptosporidium parvum DNA from 175 neonatal calves on 16 farms in eight eastern states in the United States was subtyped by sequence analysis of the 60-kDa glycoprotein gene to determinate the parasite genetic diversity. Six subtypes of the IIa subtype family were found. Subtype IIaA15G2R1, which is the predominant C. parvum subtype in calves in many parts of the world, was identified in 77% of the C. parvum DNA from calves. Several farms had more than one C. parvum subtype and a few calves had infections with mixed subtypes. Distribution of subtypes differed geographically. Diversity of C. parvum in calves in eastern United States was lower than that previously seen in Michigan and southern Ontario. The high prevalence of one subtype in calves worldwide and frequent detection of this subtype in humans suggests that parasite fitness probably plays an important role in transmission of cryptosporidiosis among cattle and in zoonotic infections.

Subtyping Cryptosporidium ubiquitum, a Zoonotic Pathogen Emerging in Humans

Cryptosporidium ubiquitum is an emerging zoonotic pathogen. In the past, it was not possible to identify an association between cases of human and animal infection. We conducted a genomic survey of the species, developed a subtyping tool targeting the 60-kDa glycoprotein (gp60) gene, and identified 6 subtype families (XIIa-XIIf) of C. ubiquitum. Host adaptation was apparent at the gp60 locus; subtype XIIa was found in ruminants worldwide, subtype families XIIb-XIId were found in rodents in the United States, and XIIe and XIIf were found in rodents in the Slovak Republic. Humans in the United States were infected with isolates of subtypes XIIb-XIId, whereas those in other areas were infected primarily with subtype XIIa isolates. In addition, subtype families XIIb and XIId were detected in drinking source water in the United States. Contact with C. ubiquitum-infected sheep and drinking water contaminated by infected wildlife could be sources of human infections.

Detection of concurrent infection of dairy cattle with Blastocystis, Cryptosporidium, Giardia, and Enterocytozoon by molecular and microscopic methods

Of fecal specimens examined from 47 dairy cattle ranging in age from neonates to multiparous cows, 9, 10, 24, and 17 were positive for Blastocystis spp., Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi, respectively, as determined by PCR. Eight 3- to 5-month-old cattle were concurrently infected with three or four of these parasites. This is the first report to identify multiple concurrent infections with these four potentially zoonotic protist pathogens in cattle. None of the cattle exhibited signs of illness or effects of infection on growth and are regarded as healthy carriers. A commercially available immunofluorescence (IFA) microscopic test confirmed six of seven available PCR-positive Blastocystis specimens and identified one IFA-positive cow that was PCR negative.