Monica Santin

Contamination of Atlantic coast commercial shellfish with Cryptosporidium

Abstract. Shellfish (oysters and/or clams) were obtained from 37 commercial harvesting sites in 13 Atlantic coast states from Maine to Florida and one site in New Brunswick, Canada. Gill washings from each of 25 shellfish at each site were examined by immunofluorescence microscopy (IFA) for oocysts of Cryptosporidium. Gill washings from another 25 shellfish at each site were grouped into five pools of five shellfish each. DNA from each pool was utilized for PCR and genotyping. Oocysts were found in 3.7% of 925 oysters and clams examined by IFA in shellfish from New Brunswick and 11 of 13 states. Cryptosporidium DNA was detected by PCR in 35.2% of 185 pools. Cryptosporidium parvum genotypes 1 and 2, and Cryptosporidium meleagridis, all of which have been identified in infected humans, were identified at 37.8% of the sites. Gill washings from every site were tested for the presence of infectious oocysts by biological assay in neonatal BALB/c mice but no mice were found infected, suggesting that either the oocysts were no longer infectious or infections in mice were below the level of detection. Collectively, these findings indicate that Cryptosporidium species, indicative of pollution from human and animal feces and potentially infectious for humans, were found in commercial shellfish from 64.9% of sites examined along the Atlantic coast by either microscopy or molecular testing. Previous reports link periods of high rainfall with the elevated numbers of pathogen contaminated shellfish. Because shellfish in the present study were examined during a period of exceptionally low precipitation, the data are thought to underestimate the number of Cryptosporidium contaminated shellfish likely to be found during periods of normal or above normal precipitation.

Enterocytozoon bieneusi genotypes in dairy cattle in the eastern United States

Fecal specimens were obtained from 12–24-month-old dairy heifers on farms in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. PCR positive specimens for Enterocytozoon bieneusi were found in 131 of 571 heifers examined (23%) and on all the farms visited. The prevalence of E. bieneusi varied considerably across farms, with the lowest prevalence (4.7%) on MD-2 and the highest prevalence (37.8%) on NY-2. All PCR positive specimens that amplified the ITS region as well as a portion of the flanking large and small subunit ribosomal RNA genes were sequenced to determine the genotype(s) of the E. bieneusi present and six genotypes were identified. Most were identified as cattle-specific genotypes, previously reported from cattle as BEB1, BEB2, BEB3, and BEB4. Two isolates were genetically identical or similar to E. bienesusi reported as the human pathogens Peru 6 and Peru 9 (or D) genotypes. Although our data demonstrate the presence of zoonotic genotypes in cattle, most genotypes found in cattle were host specific.

Identification of assemblage A Giardia in white-tailed deer.

Fecal samples were collected from hunter-killed white-tailed deer (Odocoileus virginianus) during a managed hunt in a central Maryland county. Fecal samples were cleaned of debris and concentrated by CsCl density gradient centrifugation and stained with MerIFluor™ reagents. Stained samples were examined by fluorescent microscopy for the presence of Giardia sp. cysts. One of 26 samples was found to be positive for Giardia sp. Polymerase chain reaction amplification using primers directed to the β-giardin and TPI genes identified the same sample as the only positive one. Sequencing of the β-giardin and TPI genes revealed that the Giardia sp. belonged to assemblage A, a genotype infectious for humans and also reported in a small percentage of cattle. This is the first report of assemblage A Giardia sp. in deer and suggests that deer could be a potential source of infectious cysts for humans and cattle.

A ZOONOTIC GENOTYPE OF ENTEROCYTOZOON BIENEUSI IN HORSES

Abstract This is the first report of Enterocytozoon bieneusi in an equid species. Feces from 195 horses from 4 locations in Colombia were examined for E. bieneusi by polymerase chain reaction. Of these, 21 horses (10.8%) were found positive for E. bieneusi. The prevalence of E. bieneusi in horses <1 yr of age was significantly higher (23.7%) than in horses >1 yr of age (2.5%). No significant differences in prevalence were observed between male (13.7%) and female horses (9%). Sequencing of the internal transcribed spacer region of the SSUrRNA locus identified 3 genotypes. Two genotypes appear to be unique to horses and were named Horse 1 and Horse 2. A third genotype, identified as genotype D, was detected in 4 horses. This genotype, previously reported to infect humans, beaver, cattle, dogs, falcons, foxes, macaques, muskrats, pigs, and raccoons, is the most ubiquitous of the E. bieneusi zoonotic genotypes. Our findings indicate that E. bieneusi from horses can be a potential source of infection for humans.

Prevalence and genotypes of Enterocytozoon bieneusi in weaned beef calves on cow-calf operations in the USA

To determine the prevalence and genotype distribution of Enterocytozoon bieneusi in weaned beef calves in the USA, fecal samples were collected from 819 calves (6–18xa0months of age) from 49 operations. Feces were sieved and subjected to density gradient centrifugation to remove fecal debris and to concentrate spores. DNA extracted from each sample was subjected to the polymerase chain reaction (PCR) to amplify the complete internal transcriber spacer (ITS). All PCR-positive specimens were sequenced to determine the genotype(s) present. Overall, E. bieneusi was detected in 34.8% of the 819 fecal samples. The highest prevalence was found in the Midwest region (42.7%) followed by the South (35.8%) and the West (23.2%). The prevalence of E. bieneusi varied considerably from operation to operation (0–100%). A prevalence of 100% was observed in three operations, one in the Midwest and two in the South; E. bieneusi was not found in six operations, three in the South and three in the West. Sequence analysis revealed the presence of six genotypes, four previously reported (I, J, BEB4, and type IV) and two novel genotypes (BEB8 and BEB9). Mixed infections were identified in five specimens, three contained I and BEB4 and two contained J and BEB4. Most of the positive calves (238 of 285) harbored genotypes with zoonotic potential including I (59), J (108), BEB4 (65), type IV (1), mixed I/BEB4 (3), and mixed J/BEB4 (2).

Comparison of Microscopy and PCR for Detection of Three Species of Encephalitozoon in Feces

As the name implies Microsporidia are very small. Spores rarely exceed 3 lm in size, making them difficult to see, difficult to differentiate from other tiny particulate matter in clinical and environmental specimens, and difficult or impossible to differentiate some species of Microsporidia even with excellent microscopic equipment. Therefore, several investigators have developed methods to detect spores in biological specimens or water using the polymerase chain reaction (PCR) [1–3,5,7,8]. Because the small size of spores and non-specific staining characteristics make direct detection of spores in feces by light microscopy difficult and because little is known of the sensitivity, or limits of detection, for either microscopic or molecular methods the present study was undertaken. In this study microscopic versus molecular methods of detection were compared when known numbers of spores from three microsporidian species were seeded into animal feces. Additional attempts were made to determine if detection varied with feces from different animal sources.

Molecular identification of Enterocytozoon bieneusi, Cryptosporidium, and Giardia in Brazilian captive birds

A total of 85 fecal samples from captive birds collected from October 2013 to September 2014 in Uberlândia and Belo Horizonte in the state of Minas Gerais (Brazil) were evaluated for the presence of Enterocytozoon bieneusi, Cryptosporidium, and Giardia by PCR. Of these, three birds were found positive for E. bieneusi (3.5%), two for Cryptosporidium (2.3%), and one for Giardia (1.2%). Two genotypes of E. bieneusi were detected by nucleotide sequence analysis of the ITS region, genotypes D and Peru 6 in a swan goose and in two rock pigeons, respectively. For Cryptosporidium and Giardia, nucleotide sequence analysis of the SSU rRNA identified Cryptosporidium baileyi and Duck genotype in a swan goose and a mandarin duck, respectively, and Giardia duodenalis assemblage A in a toco toucon. Our results demonstrate that human-pathogenic E. bieneusi genotypes D and Peru6 and G. duodenalis assemblage A are present in captive birds in Brazil, corroborating their potential role as a source of human infection and environmental contamination.

Detection of Encephalitozoon hellem in feces of experimentally infected chickens

Encephalitozoon hellem, initially isolated from an AIDS patient, was found infectious for athymic mice [5] and has been reported in natural infections of psittacine birds, an ostrich and hummingbirds [4,6]. The specific routes of transmission have not been well documented. However, like other fecal borne pathogens, the infectious spore stage is probably transmitted by direct contact or by ingestion of contaminated food and water. Because the range of avian hosts has been found to be so great the likelihood of there being additional avian species susceptible to infection with E. hellem seems a reasonable expectation. Therefore, the present study was conducted to determine if chickens and turkeys, easily accessible laboratory species, species of economic importance, and species in close contact with humans, were also susceptible to infection with E. hellem.

Detection and Comparison of Giardia Virus (GLV) from Different Assemblages of Giardia duodenalis

Abstract Five assemblages of Giardia duodenalis were identified from cysts in cattle, dog, cat, sheep, and reindeer feces using ribosomal DNA (rDNA) sequencing. Assemblage A was present in cattle and reindeer feces, Assemblages C and D were present in dog feces, Assemblage E was present in cattle and sheep feces, and Assemblage F was present in cat feces. Giardia virus, originally referred to as Giardia lamblia virus (GLV), is a double-stranded RNA virus. Primers designed for the GLV capsid protein gene identified GLV sequences in G. lamblia from a reindeer (Assemblage A) and from a dog (Assemblage C). Two distinct GLV sequences were identified in the dog specimen and 1 sequence was identified in the reindeer specimen. None of these GLV sequences was identical with previously published GLV sequences. It appears that GLVs are genetically diverse and that more than 1 virion can be present in a single sample. Because many of the specimens that contained cysts were found to be negative for GLV, it appears that this test for capsid protein is of limited value for the purposes of detecting G. lamblia.

First Report of Giardia in Coyotes (Canis latrans)

Giardia is a ubiquitous enteric parasite that affects humans and domestic and wild mammals worldwide 161. The genus Giardia currently comprises 6 species: G. intestinalis, G. muris, G. agilis, G. psittuci, G. ardeae, and G. microti. Out of these 6 species of Giardia, G. intestinalis is the only species recognized as causing disease in humans, but it is also found in other mammals. The similar morphology within G. intestinalis masks genetic and biotypic differences that are large enough for G. intestinalis now to be considered a species complex. Giardia isolated from humans falls exclusively into Assemblages A and B. Some animal-derived isolates appear to be similar or identical to human-derived genotypes while others represent unique genotypes that seem to be host-specific [6-71. The role of animals in transmission of Giardia to humans is unclear. Wetlands mammals such as beaver have been implicated as sources of contamination in waterborne outbreaks (41. But the potential role of other animals, such as the ubiquitous coyote, in the epidemiology of Giardia is unknown. To determine if coyotes serve as hosts for Giardiu a survey was conducted on coyotes trapped in Pennsylvania. Microscopy and molecular analysis were perfonned on Giardia isolated from these coyotes.