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Biomedical subjects

J M Trout

Publications and source records attributed to J M Trout.

At least 19 recordsLinked to original sources
PubMed2005

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.

Animals↗
PubMed2004

Characterization and potential use of a Cryptosporidium parvum virus (CPV) antigen for detecting C. parvum oocysts.

The purpose of this study was to characterize the viral symbiont (CPV) of Cryptosporidium parvum sporozoites and evaluate the CPV capsid protein (CPV40) as a target for sensitive detection of the parasite. Recombinant CPV40 was produced in Escherichia coli, purified by affinity chromatography, and used to prepare polyclonal rabbit sera specific for the viral capsid protein. Anti-rCPV40 recognized a 40 kDa and a 30 kDa protein in C. parvum oocysts and appeared to localize to the apical end of the parasite. Anti-rCPV40 serum was capable of detecting as few as 1 C. parvum oocyst in a dot blot assay, the sensitivity being at least 1000-fold greater than sera reactive with total native C. parvum oocyst protein or specific for the 41 kDa oocyst surface antigen. Water samples were seeded with C. parvum oocysts and incubated at 4, 20, or 25 degrees C for greater than 3 months to determine if CPV levels were correlated with oocyst infectivity. Samples were removed monthly and subjected to mouse and cell culture infectivity, as well as PCR analysis for infectivity and viral particle presence. While sporozoite infectivity declined by more than 75% after 1 month at 25 degrees C, the CPV signal was similar to that of control samples at 4 degrees C. By 3 months at 20 degrees C, the C. parvum oocysts were found to be non-infectious, but retained a high CPV signal. This study indicates that CPV is an excellent target for sensitive detection of C. parvum oocysts in water, but may persist for an indefinite time after oocysts become non-infectious.

Animals↗
PubMed2003

First detection of microsporidia in dairy calves in North America.

Fecal specimens were obtained from a total of 413 dairy calves from farms in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. After removal of fecal debris by sieving and density gradient centrifugation, specimens were examined by fluorescence microscopy, polymerase chain reaction (PCR), and DNA sequencing analysis for the presence of microsporidia. Microscopic examination revealed no spores. PCR using generic primers for microsporidia revealed 70 positive calves. PCR was then conducted using specific primers for Enterocytozoon bieneusi, the most frequently found microsporidian in human infections. These primers revealed 13 positive calves from six farms in five states. DNA sequencing analysis of the 13 E. bieneusi-positive specimens confirmed the PCR results and indicated 96.8-99.8% similarity with E. bieneusi sequences in GenBank. This is the first report of E. bieneusi in cattle in North America.

Animals↗
PubMed2003

Infectivity of microsporidia spores stored in water at environmental temperatures.

To determine how long waterborne spores of Encephalitozoon cuniculi, E. hellem, and E. intestinalis could survive at environmental temperatures, culture-derived spores were stored in water at 10, 15, 20, 25, and 30 C and tested for infectivity in monolayer cultures of Madin Darby bovine kidney (MDBK) cells. At 10 C, spores of E. intestinalis were still infective after 12 mo, whereas those of E. hellem and E. cuniculi were infective for 9 and 3 mo, respectively. At 15 C, spores of the same species remained infective for 10, 6, and 2 mo, and at 20 C, for 7, 5, and 1 mo, respectively. At 25 C, spores of E. intestinalis and E. hellem were infective for 3 mo, but those of E. cuniculi were infective for only 3 wk. At 30 C, the former 2 species were infective for 3 wk and 1 mo, respectively, and the latter species for only 1 wk. These findings indicate that spores of different species of Encephalitozoon differ in their longevity and temperature tolerance, but at temperatures from 10 to 30 C, all 3 have the potential to remain infective in the environment long enough to become widely dispersed.

Animals↗
PubMed2003

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 beta-giardin and TPI genes identified the same sample as the only positive one. Sequencing of the beta-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.

Animals↗
PubMed2002

Contamination of Atlantic coast commercial shellfish with Cryptosporidium.

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.

Animals↗
PubMed2002

Comparison of tests for viable and infectious Cryptosporidium parvum oocysts.

The purpose of this study was to compare different assays for viable Cryptosporidium parvum incubated in water at a temperature commonly found in the environment. C. parvum oocysts were stored in sterile water for 9 months at 15 degrees C. A sample was removed monthly and analyzed by five different assays to determine oocyst viability. Mouse infection and cell culture showed that C. parvum oocysts remained viable and infectious when stored for 7 months at this temperature. Fluorescence in situ hybridization (FISH) using probes directed to ribosomal RNA was also applied to these oocysts. The proportion of FISH-positive oocysts was 70-80% for the first 2 months of storage, decreased and remained nearly constant at 40-50% for 3-7 months, then decreased to 20% by 8 months, and to 0% by 9 months. Amylopectin content and mRNA for amyloglucosidase (CPAG), as measured by RT-PCR, decreased much more rapidly. By 3 months and for the remainder of the incubation period, amylopectin content was 20% of the original amount present in the oocysts. The CPAG RT-PCR signal at 3 months was 50% of that observed after 1 month storage, 20% at 4 months, and was not detected thereafter. Thus, results from cell culture and mouse infection assay exhibited the best agreement, the FISH assay showed modest agreement with these assays, and CPAG RT-PCR and the amylopectin assay displayed marginal agreement with the other three assays.

Amylopectin↗
PubMed2002

Temporal variability of Cryptosporidium in the Chesapeake Bay.

Although Cryptosporidium has been found worldwide in molluscan shellfish from waters contaminated with human and animal feces, little or no related environmental data have been obtained. In the present study, oysters ( Crassostrea virginica) were collected eight times over 3 years from seven sites in the Chesapeake Bay or its tributaries, with accompanying data on water temperature, salinity, rainfall, and streamflow. Oyster gill washings were examined by immunofluorescence microscopy for Cryptosporidium oocysts. Of 1,590 oysters collected, 19.6% had detectable oocysts. Of 53 collections, oocysts were detected 81% of the time. The time when the greatest percentage of oysters at most sites had detectable oocysts coincided with the time of greatest weekly and monthly rainfall, greatest streamflow into the Bay, and lowest water temperatures. In 28% of 53 collections, C. parvum genotypes 1 and 2 and C. baileyi were identified by PCR and gene sequencing. Oocyst infectivity was confirmed from 37.5% of 40 collections by initiating C. parvum genotype 2 infections in mice.

Animals↗
PubMed2002

Effects of gamma radiation on viability of Encephalitozoon spores.

Spores of Encephalitozoon cuniculi, E. hellem, and E. intestinalis harvested from cultured mammalian cells were suspended in deionized water, exposed to gamma irradiation at doses of 0-3.0 kGy, and then tested for infectivity by inoculating spores into monolayer cultures of Madin-Darby bovine kidney cells. The cultures were examined for developing microsporidia 4 days later. As the dosage level of radiation increased, corresponding decreases were observed in the number of developing microsporidia for all 3 species. For E. cuniculi and E. intestinalis, 100% inhibition of development was observed after exposure to 1.5 and 2.0 kGy, respectively. Although development of E. hellem was greatly inhibited (97.6% inhibition) after exposure to 3.0 kGy, complete inhibition was not obtained. These findings provide a baseline for investigating the dose levels required to render food products safe when kept under varying temperature, moisture, and other storage conditions.

Animals↗
PubMed2002

Rotifers ingest Giardia cysts.

Seven species of rotifers representing 6 genera, Epiphanes, Plationus, Asplanchna, Philodina species A, Philodina species B. Platyias, and Brachionus, were exposed to Giardia cysts isolated from the feces of experimentally infected holstein calves. Giardia cysts were prestained with a fluorescein isothiocyanate-conjugated monoclonal antibody and mixed with viable rotifers on 3-well Teflon-coated microscope slides. Organisms were observed with phase-contrast, differential interference contrast, and fluorescence microscopy. Five rotifer species, Epiphanes brachionus, Plationus patulus, Philodina (both A and B), and Platyias quadricornis, ingested varying numbers of cysts, which were retained within the rotifers' bodies throughout the observation period. Rotifer ingestion of Giardia cysts may represent a means of reducing water contamination.

Animals↗
PubMed2001

Real-time PCR for the detection of Cryptosporidium parvum.

Real time, TaqMan PCR assays were developed for the Cp11 and 18S rRNA genes of the protozoan parasite Cryptosporidium parvum. The TaqMan probes were specific for the genus Cryptosporidium, but could not hybridize exclusively with human-infectious C. parvum species and genotypes. In conjunction with development of the TaqMan assays, two commercial kits, the Mo Bio UltraClean Soil DNA kit, and the Qiagen QIAamp DNA Stool kit, were evaluated for DNA extraction from calf diarrhea and manure, and potassium dichromate and formalin preserved human feces. Real-time quantitation was achieved with the diarrhea samples, but nested PCR was necessary to detect C. parvum DNA in manure and human feces. Ileal tissues were obtained from calves at 3, 7, and 14 days post-infection, and DNA extracted and assayed. Nested PCR detected C. parvum DNA in the 7-day post-infection sample, but neither of the other time point samples were positive. These results indicate that real-time quantitation of C. parvum DNA, extracted using the commercial kits, is feasible on diarrheic feces, with large numbers of oocysts and small concentrations of PCR inhibitor(s). For samples with few oocysts and high concentrations of PCR inhibitor(s), such as manure, nested PCR is necessary for detection.

Animals↗
PubMed2001

Cryptosporidium canis n. sp. from domestic dogs.

Oocysts of Cryptosporidium, from the feces of a naturally infected dog and from an HIV-infected human, were identified as the previously reported canine genotype of Cryptosporidium parvum, hereafter referred to as Cryptosporidium canis n. sp. Also among the oocysts from the dog, a trace amount of C. parvum bovine genotype was detected. Cryptosporidium canis oocysts from both the dog and human were infectious for calves. Oocysts excreted by calf 1 (dog source) were approximately 90% C. canis and 10% C. parvum, whereas those excreted by calf 3 (human source) were 100% C. canis. Oocysts from calf 1 infected calf 2 resulting in excretion by calf 2 of oocysts approximately 90% C. parvum and 10% C. canis. Oocysts of C. canis were not infectious for BALB/c neonatal mice or immunosuppressed C57 juvenile mice, although all control mice became infected with the C. parvum Beltsville isolate. Oocysts of C. canis from calf 1 and the human were structurally indistinguishable from oocysts of the C. parvum Beltsville isolate (bovine). However, C. canis oocysts differed markedly at the molecular level from all known species of Cryptosporidium based on sequence data for the 18S rDNA and the HSP 70 gene. The differences in genetics and host specificity clearly differentiate C. canis as a new species.

Animals↗
PubMed2000

Prevalence of Cryptosporidium, Giardia and Eimeria infections in post-weaned and adult cattle on three Maryland farms.

The prevalence of Cryptosporidium, Giardia and Eimeria, in healthy, asymptomatic, post-weaned and mature cattle was investigated on three Maryland farms. One farm, a dairy research facility, had 150 multiparous Holstein milking cows; 24 were examined and Cryptosporidium andersoni was detected in three (12.5%) but neither Giardia nor Eimeria was detected. The second farm, a commercial dairy, had 57 multiparous Holstein milking cows and an equal number of heifers. Of 19 cows examined, C. parvum, Giardia duodenalis, and Eimeria bovis and/or E. ellipsoidalis were detected in two (10.5%), two (10.5%) and one (5.26%) cow, respectively. Of 23 heifers examined, C. parvum, Giardia, and E. bovis and E. ellipsoidalis, was detected in two (8.7%), four (17.4%), and five (21.7%), heifers, respectively. The third farm, a beef cattle breeding and genetics research facility, had 180 7- to 9-month old purebred black Angus. Of 118 examined for C. parvum and Giardia, 34 (28.8%) and 44 (37.3%) were positive, respectively, of 97 examined for E. bovis and/or E. ellipsoidalis 32 (33.0%) were positive. These findings, based on a method with a minimum detection level of 100 oocysts of C. parvum/g of feces, which underestimates the number of infected cattle, clearly demonstrate the presence of low level, asymptomatic infections in post-weaned and adult cattle in the United States and indicate the potential role of such cattle as reservoirs of infectious parasites.

Animals↗
PubMed1999

Cryptosporidium oocysts in Bent mussels (Ischadium recurvum) in the Chesapeake Bay.

Filter-feeding molluscan shellfish can concentrate environmentally derived waterborne pathogens of humans, which can be utilized in the sanitary assessment of water quality. In the present study, oocysts of Cryptosporidium were detected in Bent mussels (Ischadium recurvum) at two Chesapeake Bay sites from which C. parvum-contaminated oysters had previously been collected. Spiking of Cryptosporidium-free blue mussel (Mytilus edulis) tissue with C. parvum oocysts showed a 51.1% recovery rate of oocysts, giving an oocyst detection limit of 19 oocysts/0.7 ml of mussel tissue homogenate. The results indicate that Bent mussels, which are common throughout the Chesapeake Bay region, may prove to be useful as biological indicators of water contamination with Cryptosporidium oocysts.

Animals↗
PubMed1998

The role of neuroendocrine immune interactions in the initiation of humoral immunity in chickens.

The presence of neuroendocrine immune interaction in mammalian species has been studied extensively and has been established. However, such an interaction is not as well established in avian species. Furthermore, the role of such an interaction in the initiation of humoral immunity is not well understood. Therefore, the present studies were conducted to determine mechanisms involved in the initiation of humoral immunity in chickens. Cornell K-strain White Leghorn immature male chickens were used for all the experiments. Changes in hormonal and leukocyte profiles after antigen stimulation were studied. The ability of different leukocytes to produce ACTH was also investigated. It was concluded that the first step in the initiation of humoral immunity after antigen exposure is the release of interleukin-1 by macrophages, which in turn stimulates the production of CRF by hypothalamus and/or leukocytes. It is important to mention that CRF production could also be a direct effect of antigen stimulation. The CRF will then stimulate ACTH production by anterior pituitary and/or leukocytes. In addition, CRF will directly enhance lymphocyte activities in the spleen. Corticosteroid production will be stimulated by ACTH and will cause redistribution of lymphocytes from circulation to secondary lymphoid organs such as the spleen for antigen processing and eventual production of antibodies against the invading antigens. Finally, both ACTH and corticosteroids will later act in a negative feedback manner to regulate and control the process of antibody production by inhibiting lymphocyte activities and/or reducing the responsiveness to different stimuli.

Adrenocorticotropic Hormone↗
PubMed1998

Survival of infectious Cryptosporidium parvum oocysts in seawater and eastern oysters (Crassostrea virginica) in the Chesapeake Bay.

Oocysts of Cryptosporidium parvum placed in artificial seawater at salinities of 10, 20, and 30 ppt at 10 degrees C and at 10 ppt at 20 degrees C were infectious after 12 weeks. Those placed in seawater at 20 ppt and 30 ppt at 20 degrees C were infectious for 8 and 4 weeks, respectively. These findings suggested that oocysts could survive in estuarine waters long enough to be removed by filter feeders such as oysters. Thereafter, 30 Eastern oysters, Crassostrea virginica, were collected with a dredge or with hand tongs at each of six sites within Maryland tributaries of the Chesapeake Bay in May and June and in August and September of 1997. Hemocytes and gill washings from all oysters were examined for the presence of Cryptosporidium oocysts and Giardia cysts by immunofluorescence microscopy utilizing a commercially available kit containing fluorescein isothiocyanate-conjugated monoclonal antibodies. Giardia was not detected by this method from any of the 360 oysters examined. Presumptive identification of Cryptosporidium oocysts was made in either hemocytes or gill washings of oysters from all six sites both times that surveys were conducted. In addition, during August and September, for each of the six sites, hemocytes from the 30 oysters were pooled and gill washings from the oysters were pooled. Each pool was delivered by gastric intubation to a litter of neonatal mice to produce a bioassay for oocyst infectivity. Intestinal tissue from two of three mice that received gill washings from oysters collected at a site near a large cattle farm and shoreline homes with septic tanks was positive for developmental stages of C. parvum. These findings demonstrate for the first time that oysters in natural waters harbor infectious C. parvum oocysts and can serve as mechanical vectors of this pathogen.

Animals↗
PubMed1998

Giardia sp. cysts and infectious Cryptosporidium parvum oocysts in the feces of migratory Canada geese (Branta canadensis).

Fecal droppings of migratory Canada geese, Branta canadensis, collected from nine sites near the Chesapeake Bay (Maryland), were examined for the presence of Cryptosporidium parvum and Giardia spp. Cryptosporidium sp. oocysts were found in feces at seven of nine sites, and Giardia cysts were found at all nine sites. The oocysts from three sites were infectious for mice and molecularly identified as the zoonotic genotype of Cryptosporidium parvum. Waterfowl can disseminate infectious C. parvum oocysts in the environment.

Animals↗