Assessment of a dye permeability assay for determination of inactivation rates of Cryptosporidium parvum oocysts

Jenkins, Michael B.; Anguish, Lynne J.; Bowman, Dwight D.; Walker, Mark J.; Ghiorse, William C.

doi:10.1128/aem.63.10.3844-3850.1997

Cited by 129 publications

(75 citation statements)

References 20 publications

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“…Oocysts used in the oral challenge were purified as previously described. 11 Briefly, feces were collected from naturally infected 6-to 14-day-old calves from a commercial dairy operation. Fecal matter was processed by continuous-flow differential density flotation and stored at 4 C until needed.…”

Section: Methodsmentioning

confidence: 99%

Correlation Between Diarrhea Severity and Oocyst Count via Quantitative PCR or Fluorescence Microscopy in Experimental Cryptosporidiosis in Calves

Operario¹,

Bristol²,

Liotta³

et al. 2015

The American Society of Tropical Medicine and Hygiene

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Abstract. Cryptosporidium is an important diarrhea-associated pathogen, however the correlation between parasite burden and diarrhea severity remains unclear. We studied this relationship in 10 experimentally infected calves using immunofluorescence microscopy and real-time polymerase chain reaction (qPCR) (N = 124 fecal samples). The qPCR data were corrected for extraction/amplification efficiency and gene copy number to generate parasite counts. The qPCR and microscopic oocyst quantities exhibited significant correlation (R 2 = 0.33, P 0.05), however qPCR had increased sensitivity. Upon comparison with diarrhea severity scores (from 0 to 3), a PCR-based count of 2.6

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Section: Methodsmentioning

confidence: 99%

Correlation Between Diarrhea Severity and Oocyst Count via Quantitative PCR or Fluorescence Microscopy in Experimental Cryptosporidiosis in Calves

Operario¹,

Bristol²,

Liotta³

et al. 2015

The American Society of Tropical Medicine and Hygiene

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show abstract

“…The oocysts used to dose the calves were purified using a procedure previously described (Jenkins et al, 1997). In brief, feces were collected from naturally infected 6-to 14day-old calves from a separate commercial dairy operation and processed by continuous-flow differential density flotation.…”

Section: Calf Management Sampling and Inoculationmentioning

confidence: 99%

“…Before inoculation, oocysts were first cleaned for one minute in 0.6% sodium hypochlorite to inactivate viruses and bacteria co-purified with the oocysts, then washed four times with phosphate buffered saline to remove the sodium hypochlorite, quantified using a hemocytometer and finally viability determined using a dye permeability assay as described previously (Campbell et al, 1992;Anguish and Ghiorse, 1997;Jenkins et al, 1997). Viable oocysts were the sum of 4,6-diamidino-2-phenylindolenegative (DAPI−) propidium iodide-negative (PI−) oocysts and DAPI-positive (DAPI+) PI− oocysts; DAPI+ PI+ oocysts were considered inactivated (Jenkins et al, 1999).…”

Section: Preparation Of Inoculummentioning

confidence: 99%

Cryptosporidium parvum: Determination of ID50 and the dose–response relationship in experimentally challenged dairy calves

Zambriski

Nydam

Wilcox

et al. 2013

Veterinary Parasitology

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The objectives were to determine the median infective dose (ID₅₀) of Cryptosporidium parvum and to describe the dose-response relationship including associated clinical illness in experimentally challenged dairy calves. Within the first 24h of life, 27 test calves were experimentally challenged with C. parvum oocysts and 3 control calves were sham dosed. Test calves received 1 of 8 possible doses (25, 50, 100, 500, 1 × 10(3), 1 × 10(4), 1 × 10(5), and 1 × 10(6) oocysts). All 27 test calves developed diarrhea. Fecal oocyst shedding occurred in 25 (92.6%) test calves and in 0 control calves. The 2 non-shedding test calves both received 25 oocysts. There was an inverse relationship between dose and time to onset of fecal oocyst shedding (P=0.005). There was no relationship found between dose and duration (P=0.2) or cessation (P=0.3) of fecal oocyst shedding. In addition, there was not a significant relationship between log-dose and the log-peak oocysts (P=0.2) or log-total oocysts (P=0.5) counted/g of feces across the dose groups. There was a positive dose-response relationship between log-dose and diarrhea (P=0.01). However, when controlling for other factors, such as onset and cessation of fecal oocyst shedding, dose was not a significant predictor of diarrhea (P=0.5). Onset and cessation of fecal oocyst shedding were found to be the best predictors of diarrhea (P=0.0006 and P=0.04, respectively). The ID₅₀ for fecal oocyst shedding was 5.8 oocysts, for diarrhea was 9.7 oocysts, and for fecal oocyst shedding with diarrhea was 16.6 oocysts. Given that the ID₅₀ of C. parvum is far less than would be excreted into the environment by a naturally infected calf, prevention and control of cryptosporidiosis is a formidable challenge.

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“…The oocysts used to dose the calves were purified using a procedure previously described (Jenkins et al 1997). In brief, feces were collected from naturally infected 6-to 14-day-old calves from a separate commercial dairy operation and processed by continuous-flow differential density flotation.…”

Section: Challenge Modelmentioning

confidence: 99%

“…Before inoculation, oocysts were first cleaned for 1 min in 0.6 % sodium hypochlorite to inactivate viruses and bacteria co-purified with the oocysts, then washed four times with phosphate-buffered saline to remove the sodium hypochlorite, quantified using a hemocytometer, and finally, viability determined using a dye permeability assay as described previously (Campbell et al 1992;Anguish and Ghiorse 1997;Jenkins et al 1997). Viable oocysts were the sum of 49,6-diamidino-2-phenylindole-negative (DAPI−), propidium iodide-negative (PI−) oocysts, and DAPI-positive (DAPI+) PI-oocysts; DAPI+PI+oocysts were considered inactivated (Jenkins et al 1999).…”

Section: Challenge Modelmentioning

confidence: 99%

Description of fecal shedding of Cryptosporidium parvum oocysts in experimentally challenged dairy calves

et al. 2013

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The objective was to describe the probability of Cryptosporidium parvum fecal oocyst shedding at different magnitudes of exposure, the pattern of fecal shedding over time, and factors affecting fecal shedding in dairy calves. Within the first 24 h of life, 36 calves were experimentally challenged with C. parvum oocysts at one of four possible magnitudes of oral exposure (1 × 10(3), 1 × 10(4), 1 × 10(5), and 1 × 10(6) oocysts), and 7 control calves were sham dosed. Fecal shedding occurred in 33 (91.7 %) experimentally challenged calves and in none of the control calves. There was a difference in the log-total number of oocysts counted per gram of feces dry weight among the four exposure groups; calves with the lowest magnitude of exposure (1 × 10(3) oocysts) shed less than the other three groups. At higher magnitudes of exposure, there was more variability in the range of fecal oocyst shedding. There was an inverse relationship between the log-total amount of oocysts counted per gram of feces dry weight and the number of days to the onset of fecal shedding per calf, i.e., the more time that elapsed to the onset of fecal shedding, the fewer oocysts that were shed. The pattern of fecal shedding over time for all calves shedding oocysts was curvilinear; the number of oocysts increased with time, reached a peak, and declined. Therefore, the dynamics of oocyst shedding can be influenced in part by limiting exposure among calves and delaying the onset of fecal oocyst shedding.

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Assessment of a dye permeability assay for determination of inactivation rates of Cryptosporidium parvum oocysts

Cited by 129 publications

References 20 publications

Correlation Between Diarrhea Severity and Oocyst Count via Quantitative PCR or Fluorescence Microscopy in Experimental Cryptosporidiosis in Calves

Correlation Between Diarrhea Severity and Oocyst Count via Quantitative PCR or Fluorescence Microscopy in Experimental Cryptosporidiosis in Calves

Cryptosporidium parvum: Determination of ID50 and the dose–response relationship in experimentally challenged dairy calves

Description of fecal shedding of Cryptosporidium parvum oocysts in experimentally challenged dairy calves

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