scholarly journals Genetic Diversity of Cryptosporidium spp. within a Remote Population of Soay Sheep on St. Kilda Islands, Scotland

2013 ◽  
Vol 79 (7) ◽  
pp. 2240-2246 ◽  
Author(s):  
L. Connelly ◽  
B. H. Craig ◽  
B. Jones ◽  
C. L. Alexander
Keyword(s):  
Cryptosporidium Parvum ◽  
Nested Pcr ◽  
Fragment Length Polymorphism ◽  
Ovis Aries ◽  
Isolated Population ◽  
Content Type ◽  
Soay Sheep ◽  
Population Crash ◽  
Nested Pcr Assays ◽  
High Diversity

ABSTRACTThis is the first report to characterize the genotypes and subtypes ofCryptosporidiumspecies infecting a geographically isolated population of feral Soay sheep (Ovis aries) on Hirta, St. Kilda, Scotland, during two distinct periods: (i) prior to a population crash and (ii) as host numbers increased.CryptosporidiumDNA was extracted by freeze-thawing of immunomagnetically separated (IMS) bead-oocyst complexes, and species were identified following nested-PCR-restriction fragment length polymorphism (RFLP)/PCR sequencing at twoCryptosporidium18S rRNA loci. Two hundred fifty-five samples were analyzed, and the prevalentCryptosporidiumspecies in single infections were identified asC. hominis(11.4% of all samples tested),C. parvum(9%),C. xiaoi(12.5%), andC. ubiquitum(6.7%).Cryptosporidium parvumwas also present with otherCryptosporidiumspecies in 27.1% of all samples tested.Cryptosporidium parvum- andC. hominis-positive isolates were genotyped using two nested-PCR assays that amplify theCryptosporidiumglycoprotein 60 gene (GP60).GP60gene analysis showed the presence of twoCryptosporidiumgenotypes, namely,C. parvumIIaA19G1R1 andC. hominisIbA10G2. This study reveals a higher diversity ofCryptosporidiumspecies/genotypes than was previously expected. We suggest reasons for the high diversity ofCryptosporidiumparasites within this isolated population and discuss the implications for our understanding of cryptosporidiosis.

scholarly journals Detection and Discrimination of Cryptosporidium parvum and C. hominis in Water Samples by Immunomagnetic Separation-PCR

2005 ◽  
Vol 71 (2) ◽  
pp. 898-903 ◽  
Author(s):  
Yoshitsugu Ochiai ◽  
Chieko Takada ◽  
Mitsugu Hosaka
Keyword(s):  
Cryptosporidium Parvum ◽  
Water Samples ◽  
Nested Pcr ◽  
Fragment Length Polymorphism ◽  
Detection Method ◽  
Fluorescent Antibody ◽  
Immunomagnetic Separation ◽  
Antibody Assay ◽  
Pcr Products ◽  
Genetic Detection

ABSTRACT Cryptosporidium parvum and C. hominis have been the cause of large and serious outbreaks of waterborne cryptosporidiosis. A specific and sensitive recovery-detection method is required for control of this pathogen in drinking water. In the present study, nested PCR-restriction fragment length polymorphism (RFLP), which targets the divergent Cpgp40/15 gene, was developed. This nested PCR detected only the gene derived from C. parvum and C. hominis strains, and RFLP was able to discriminate between the PCR products from C. parvum and C. hominis. To evaluate the sensitivity of nested PCR, C. parvum oocysts inoculated in water samples of two different turbidities were recovered by immunomagnetic separation (IMS) and detected by nested PCR and fluorescent antibody assay (FA). Genetic detection by nested PCR and oocyst number confirmed by FA were compared, and the results suggested that detection by nested PCR depends on the confirmed oocyst number and that nested PCR in combination with IMS has the ability to detect a single oocyst in a water sample. We applied an agitation procedure with river water solids to which oocysts were added to evaluate the recovery and detection by the procedure in environmental samples and found some decrease in the rate of detection by IMS.

Epidemiology of nematode infections of Soay sheep (Ovis aries L.) on St Kilda

Parasitology ◽  
1992 ◽  
Vol 105 (3) ◽  
pp. 481-492 ◽  
Author(s):  
F. M. D. Gulland ◽  
M. Fox
Keyword(s):  
Population Density ◽  
Infection Intensity ◽  
Host Population ◽  
Ovis Aries ◽  
Sheep Population ◽  
Soay Sheep ◽  
Infective Larvae ◽  
Population Crash ◽  
Faecal Egg Counts ◽  
Bad Weather

SUMMARYThe epidemiology of nematode infections of Soay sheep on the island of St Kilda over a period of 2 years (August 1988–August 1990) spanning a host population crash is described. Infective larvae (L3) levels on pasture were high (2422±365 L3/kg D.M. grass in midsummer 1988) when host population density was high, decreasing after the sheep population declined by 70% in early 1989 (601 ±14 L3/kg D.M. in midsummer 1989). The availability of infective larvae to sheep increased during the winter of 1988–1989, probably as a result of concentration of existing larvae on grass as vegetation was destroyed by bad weather and overgrazing. Increased availability of pre-parasitic stages was accompanied by a marked increased in faecal egg counts from sheep of all ages and both sexes. Prevalence and intensity of infection (faecal egg counts) were higher in males than females throughout the 2-year study (χ2 = 208.3, P < 0.005 and F1.2000 = 304, P < 0.001 respectively), except during the lambing periods, and decreased with age in both sexes. Changes in prevalence and intensity of strongyle infections were associated with changes in host population density. Prevalence and intensity of Dictyocaulus filaria larvae in faeces increased during the host population crash. Infection intensity decreased with age (F1.203 = 44.02, P < 0.001) and was higher in males than females (F1.203 = 13.45, P < 0.001).

scholarly journals Species-Specific, Nested PCR-Restriction Fragment Length Polymorphism Detection of Single Cryptosporidium parvum Oocysts

2001 ◽  
Vol 67 (6) ◽  
pp. 2665-2668 ◽  
Author(s):  
Gregory D. Sturbaum ◽  
Carrie Reed ◽  
Paul J. Hoover ◽  
B. Helen Jost ◽  
Marilyn M. Marshall ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Cryptosporidium Parvum ◽  
Restriction Enzyme ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Nested Pcr ◽  
Fragment Length Polymorphism ◽  
Enzyme Digestion ◽  
Restriction Enzyme Digestion ◽  
Restriction Fragment Length

ABSTRACT Concurrent with recent advances seen with Cryptosporidium parvum detection in both treated and untreated water is the need to properly evaluate these advances. A micromanipulation method by which known numbers of C. parvum oocysts, even a single oocyst, can be delivered to a test matrix for detection sensitivity is presented. Using newly developed nested PCR-restriction fragment length polymorphism primers, PCR sensitivity was evaluated with 1, 2, 3, 4, 5, 7, or 10 oocysts. PCR detection rates (50 samples for each number of oocysts) ranged from 38% for single oocysts to 92% for 5 oocysts, while 10 oocysts were needed to achieve 100% detection. The nested PCR conditions amplified products from C. parvum,Cryptosporidium baileyi, and Cryptosporidium serpentis but no other Cryptosporidium sp. or protozoan tested. Restriction enzyme digestion with VspI distinguished between C. parvum genotypes 1 and 2. Restriction enzyme digestion with DraII distinguishedC. parvum from C. baileyi and C. serpentis. Use of known numbers of whole oocysts encompasses the difficulty of liberating DNA from the oocyst and eliminates the standard deviation inherent within a dilution series. To our knowledge this is the first report in which singly isolatedC. parvum oocysts were used to evaluate PCR sensitivity. This achievement illustrates that PCR amplification of a single oocyst is feasible, yet sensitivity remains an issue, thereby illustrating the difficulty of dealing with low oocyst numbers when working with environmental water samples.

scholarly journals Detection and Resolution of Cryptosporidium Species and Species Mixtures by Genus-Specific Nested PCR-Restriction Fragment Length Polymorphism Analysis, Direct Sequencing, and Cloning

2011 ◽  
Vol 77 (12) ◽  
pp. 3998-4007 ◽  
Author(s):  
Norma J. Ruecker ◽  
Rebecca M. Hoffman ◽  
Rachel M. Chalmers ◽  
Norman F. Neumann
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Nested Pcr ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Content Type ◽  
Pcr Rflp ◽  
Pcr Products ◽  
Restriction Fragment Length

ABSTRACTMolecular methods incorporating nested PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene ofCryptosporidiumspecies were validated to assess performance based on limit of detection (LoD) and for detecting and resolving mixtures of species and genotypes within a single sample. The 95% LoD was determined for seven species (Cryptosporidium hominis,C. parvum,C. felis,C. meleagridis,C. ubiquitum,C. muris, andC. andersoni) and ranged from 7 to 11 plasmid template copies with overlapping 95% confidence limits. The LoD values for genomic DNA from oocysts on microscope slides were 7 and 10 template copies forC. andersoniandC. parvum, respectively. The repetitive nested PCR-RFLP slide protocol had an LoD of 4 oocysts per slide. When templates of two species were mixed in equal ratios in the nested PCR-RFLP reaction mixture, there was no amplification bias toward one species over another. At high ratios of template mixtures (>1:10), there was a reduction or loss of detection of the less abundant species by RFLP analysis, most likely due to heteroduplex formation in the later cycles of the PCR. Replicate nested PCR was successful at resolving many mixtures ofCryptosporidiumat template concentrations near or below the LoD. The cloning of nested PCR products resulted in 17% of the cloned sequences being recombinants of the two original templates. Limiting-dilution nested PCR followed by the sequencing of PCR products resulted in no sequence anomalies, suggesting that this method is an effective and accurate way to study the species diversity ofCryptosporidium, particularly for environmental water samples, in which mixtures of parasites are common.

scholarly journals High Diversity of Cryptosporidium Species and Subtypes Identified in Cryptosporidiosis Acquired in Sweden and Abroad

Pathogens ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 523
Author(s):  
Marianne Lebbad ◽  
Jadwiga Winiecka-Krusnell ◽  
Christen Rune Stensvold ◽  
Jessica Beser
Keyword(s):  
Cryptosporidium Parvum ◽  
Ribosomal Rna ◽  
Diarrheal Disease ◽  
Protozoan Parasite ◽  
Small Subunit ◽  
Cryptosporidium Species ◽  
Intestinal Protozoan ◽  
Genotype I ◽  
Glycoprotein Gene ◽  
High Diversity

The intestinal protozoan parasite Cryptosporidium is an important cause of diarrheal disease worldwide. The aim of this study was to expand the knowledge on the molecular epidemiology of human cryptosporidiosis in Sweden to better understand transmission patterns and potential zoonotic sources. Cryptosporidium-positive fecal samples were collected between January 2013 and December 2014 from 12 regional clinical microbiology laboratories in Sweden. Species and subtype determination was achieved using small subunit ribosomal RNA and 60 kDa glycoprotein gene analysis. Samples were available for 398 patients, of whom 250 (63%) and 138 (35%) had acquired the infection in Sweden and abroad, respectively. Species identification was successful for 95% (379/398) of the samples, revealing 12 species/genotypes: Cryptosporidium parvum (n = 299), C. hominis (n = 49), C. meleagridis (n = 8), C. cuniculus (n = 5), Cryptosporidium chipmunk genotype I (n = 5), C. felis (n = 4), C. erinacei (n = 2), C. ubiquitum (n = 2), and one each of C. suis, C. viatorum, C. ditrichi, and Cryptosporidium horse genotype. One patient was co-infected with C. parvum and C. hominis. Subtyping was successful for all species/genotypes, except for C. ditrichi, and revealed large diversity, with 29 subtype families (including 4 novel ones: C. parvum IIr, IIs, IIt, and Cryptosporidium horse genotype VIc) and 81 different subtypes. The most common subtype families were IIa (n = 164) and IId (n = 118) for C. parvum and Ib (n = 26) and Ia (n = 12) for C. hominis. Infections caused by the zoonotic C. parvum subtype families IIa and IId dominated both in patients infected in Sweden and abroad, while most C. hominis cases were travel-related. Infections caused by non-hominis and non-parvum species were quite common (8%) and equally represented in cases infected in Sweden and abroad.

scholarly journals High Diversity of Magnetotactic Deltaproteobacteria in a Freshwater Niche

2013 ◽  
Vol 79 (8) ◽  
pp. 2813-2817 ◽  
Author(s):  
Yinzhao Wang ◽  
Wei Lin ◽  
Jinhua Li ◽  
Yongxin Pan
Keyword(s):  
Magnetotactic Bacteria ◽  
Natural Environments ◽  
Content Type ◽  
Operational Taxonomic Units ◽  
Geological Processes ◽  
Freshwater Site ◽  
High Diversity

ABSTRACTKnowledge of the diversity of magnetotactic bacteria in natural environments is crucial for understanding their contribution to various biological and geological processes. Here we report a high diversity of magnetotactic bacteria in a freshwater site. Ten out of 18 operational taxonomic units (OTUs) were affiliated with theDeltaproteobacteria. Some rod-shaped bacteria simultaneously synthesized greigite and magnetite magnetosomes.

Sex Differences in Feeding Behaviour at Feeding Station Scale in Soay Sheep (Ovis Aries)

Behaviour ◽  
2004 ◽  
Vol 141 (8) ◽  
pp. 999-1020 ◽  
Author(s):  
◽  
◽  
Keyword(s):  
Feeding Behaviour ◽  
Ovis Aries ◽  
Intake Rate ◽  
Same Sex ◽  
Soay Sheep ◽  
Food Items ◽  
Feeding Station ◽  
Foraging Decisions ◽  
Mouth Size ◽  
Mechanistic Response

AbstractHerbivorous ungulates live in a spatially heterogeneous environment making foraging decisions at a range of hierarchical scales, from bite size to landscape. We investigated the factors that control intake rate in Soay sheep (Ovis aries) when discrete items of food were sparsely distributed at the feeding station scale. Within the feeding station we varied the difficulty of accessing food, distance between items of food, difficulty of finding the food and complexity of the feeding station and recorded how intake rate responded in relation to body size, mouth size and the sex of the animal. Our findings demonstrated how increasing difficulty of accessing food, and increasing complexity of the feeding station negatively affected intake rate. The expected mechanistic response that smaller animals or animals with smaller mouth size were better at handling discrete small items of food, was overridden by individual and sexual differences in behaviour. We also considered that intake rate within a feeding station could be maximised by optimising the spatial pattern of offtake, and the results clearly indicated that both sexes tended to show clustered patterns of offtake. Animals of the same sex responded in a similar way to the difficulty in handling food items; males persevered more than females and consequently were less handicaped by having larger mouths. We discussed these results in relation to behavioural and body mass differences between the sexes and animals.

scholarly journals Comparison of Passively Transferred Antibodies in Bighorn and Domestic Lambs Reveals One Factor in Differential Susceptibility of These Species to Mannheimia haemolytica-Induced Pneumonia

2011 ◽  
Vol 18 (7) ◽  
pp. 1133-1138 ◽  
Author(s):  
Caroline N. Herndon ◽  
Sudarvili Shanthalingam ◽  
Donald P. Knowles ◽  
Douglas R. Call ◽  
Subramaniam Srikumaran
Keyword(s):  
Differential Susceptibility ◽  
Critical Factor ◽  
Surface Antigens ◽  
Ovis Canadensis ◽  
Ovis Aries ◽  
Mannheimia Haemolytica ◽  
Domestic Sheep ◽  
Experimental Conditions ◽  
Content Type ◽  
Impaired Ability

ABSTRACTMannheimia haemolyticaconsistently causes fatal bronchopneumonia in bighorn sheep (BHS;Ovis canadensis) under natural and experimental conditions. Leukotoxin is the primary virulence factor of this organism. BHS are more susceptible to developing fatal pneumonia than the related speciesOvis aries(domestic sheep [DS]). In BHS herds affected by pneumonia, lamb recruitment is severely impaired for years subsequent to an outbreak. We hypothesized that a lack of maternally derived antibodies (Abs) againstM. haemolyticaprovides an immunologic basis for enhanced susceptibility of BH lambs to population-limiting pneumonia. Therefore, the objective of this study was to determine the titers of Abs directed againstM. haemolyticain the sera of BH and domestic lambs at birth through 12 weeks of age. Results revealed that BH lambs had approximately 18-fold lower titers of Ab against surface antigens ofM. haemolyticaand approximately 20-fold lower titers of leukotoxin-neutralizing Abs than domestic lambs. The titers of leukotoxin-neutralizing Abs in the serum and colostrum samples of BH ewes were approximately 157- and 50-fold lower than those for domestic ewes, respectively. Comparatively, the higher titers of parainfluenza 3 virus-neutralizing Abs in the BH lambs ruled out the possibility that these BHS had an impaired ability to passively transfer Abs to their lambs. These results suggest that lower levels of leukotoxin-neutralizing Abs in the sera of BH ewes, and resultant low Ab titers in their lambs, may be a critical factor in the poor lamb recruitment in herds affected by pneumonia.

scholarly journals Detection of Viable Cryptosporidium parvum in Soil by Reverse Transcription–Real-Time PCR Targetinghsp70mRNA

2011 ◽  
Vol 77 (18) ◽  
pp. 6476-6485 ◽  
Author(s):  
Zhanbei Liang ◽  
Ann Keeley
Keyword(s):  
Cryptosporidium Parvum ◽  
Reverse Transcription ◽  
Rna Binding ◽  
Direct Detection ◽  
Rna Extraction ◽  
Milk Powder ◽  
Extraction Methods ◽  
Content Type ◽  
Total Rna ◽  
Rna Extraction Methods

ABSTRACTExtraction of high-quality mRNA fromCryptosporidium parvumis a key step in PCR detection of viable oocysts in environmental samples. Current methods for monitoring oocysts are limited to water samples; therefore, the goal of this study was to develop a rapid and sensitive procedure forCryptosporidiumdetection in soil samples. The efficiencies of five RNA extraction methods were compared (mRNA extraction with the Dynabeads mRNA Direct kit after chemical and physical sample treatments, and total RNA extraction methods using the FastRNA Pro Soil-Direct, PowerSoil Total RNA, E.Z.N.A. soil RNA, and Norgen soil RNA purification kits) for the direct detection ofCryptosporidiumwith oocyst-spiked sandy, loamy, and clay soils by using TaqMan reverse transcription-PCR. The study also evaluated the presence of inhibitors by synthesis and incorporation of an internal positive control (IPC) RNA into reverse transcription amplifications, used different facilitators (bovine serum albumin, yeast RNA, salmon DNA, skim milk powder, casein, polyvinylpyrrolidone, sodium hexametaphosphate, andSalmonella entericaserovar Typhi) to mitigate RNA binding on soil components, and applied various treatments (β-mercaptoethanol and bead beating) to inactivate RNase and ensure the complete lysis of oocysts. The results of spiking studies showed thatSalmonellacells most efficiently relieved binding of RNA. With the inclusion ofSalmonelladuring extraction, the most efficient mRNA method was Dynabeads, with a detection limit of 6 × 102oocysts g−1of sandy soil. The most efficient total RNA method was PowerSoil, with detection limits of 1.5 × 102, 1.5 × 103, and 1.5 × 104C. parvumoocysts g−1soil for sandy, loamy, and clay samples, respectively.

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