The Ingestion of Cryptosporidium Parvum Oocysts by Ceriodaphnia

Elissette Cabello

TX A & M University Research Center

 

ABSTRACT

            Cryptosporidium parvum has become an extremely important pathogen in drinking water.  It produces a high risk of waterborne disease particularly for the immunocompromised.  Ceriodaphnia, Daphnia, and Ostracods are all micro-invertebrates, which are common in water sediments where C. parvum oocysts may also be present. The main focus of this study was to determine whether Ceriodaphnia possessed the capability of ingesting C. parvum oocysts without the excretion of any viable oocysts.  Experiments were performed using five Ceriodaphnias.  They were fed 1,250 live C. parvum oocysts for one hour.  After a thorough microscopic examination of the remaining water samples, the number of oocysts detected were very few.  Ceriodaphnias were able to ingest a large number of oocysts given only an hour feeding time.  Less than 1% of oocysts were reported to have been (possibly) excreted.   

 

INTRODUCTION

            The contamination of water supplies is a serious global issue.  The urgency to better understand  C. parvum has become quite significant given the fact that it forms highly infectious oocysts (3). This protozoan is a serious health threat to those individuals who are immunocompromised and immunosuppressed (3).  Ingestion of C.parvum oocysts can lead to severe diarrhea, vomiting, abdominal pains, and in some extreme cases even death (1).  Cryptosporidium spp.  has been detected in sewage, filtered secondary waste water, surface waters, groundwater, and most importantly treated drinking water (5).  Cryptosporidium parvum oocysts are most commonly found in sites where infected domestic and wild animals excrete live oocysts.  These highly infectious oocysts can be transported by runoff finding their way into municipal water sources.  Ceriodaphnia, a micro-invertebrate, commonly found in water sediments, has shown the capability of ingesting large amounts of C.parvum oocysts with little to no oocysts excretion.

            Studies were performed in Stephenville, Texas using a micro-invertebrate, Alona guttata to show their filter feeding ability using fluorescent microbeads.  The fluorescent microbeads were used to simulate the protozoan porpagules.  Results show how A.guttata is highly efficient in the ingestion and digestion of these fluorescent microbeads (4).  The purpose of this study was to determine if C.parvum oocysts can be ingested by Ceriodaphnia and whether they can be excreted intact.   

 

MATERIALS AND METHODS

Cryptosporidium parvum oocysts  (106), suspended in water, were purchased from Waterborne Inc. (New Orleans, LA.).

Ceriodaphnia sp. were obtained from Forrest L. Mitchell at Stephenville, TX.    They were suspended in synthetic fresh water (DDH2O, synthetic sea salt, CaCl2 (11760mg/ml), NaHCO3 (2520mg/ml) SeO2 (140mg/ml) and stored in the laboratory at room temperature in the dark by using aluminum foil to cover the container.     

 

Experimental Study 

Feeding  Five Ceriodaphnias were pipetted and placed in a plastic petri-dish (VWR, Inc., San Diego, CA.) containing 1ml of synthetic fresh water.  A 10 µl of sample containing 1,250 oocysts were pipetted and placed into the petri-dish containing the Ceriodaphnia.  The petri-dish was covered with aluminum foil and incubated at room temperature for one hour.  The water sample after the feeding phase was referred to as “leftover”.  

Washing   After the hour of incubation, the Ceriodaphnias were pipetted and transferred into three separate petri-dishes each containing 1ml of synthetic fresh water to try and remove any C.parvum oocysts that may have been attached onto the exterior surface of the Ceriodaphnias.  The remaining water was pooled together using a pipetter and placed into a 10ml centrifuge tube.  This water was referred to as “carryover”.

Excretion   The Ceriodaphnias were then transferred into a 1.5 microcentrifuge tube.  They were fed diluted yeast and allowed to excrete any oocysts for a period of 24 hours.  The microcentrifuge tube was covered with aluminum foil and incubated at room temperature.  Upon completion, the Ceriodaphnias were pipetted out and transferred to a 1.5 microcentrifuge tube containing 1ml of synthetic fresh water.  The remaining water was referred to as “excretion”. 

Membrane filtration    All the remaining water samples were filtered through a cellulose acetate membrane (Sartorius, Edgewood, NY.) using the Hoefer ® FH 225V 10-place filter manifold (Pharmacia Biotech, San Fransisco, CA.).  A 20X concentrated solution of Aqua-Glo (Waterborne, Inc., New Orleans, LA.) was diluted down to a working dilution of 1X using a Dilution/Blocking  buffer (PBS 1%, BSA 10%, NGS 0.02%) as per manufacturer’s recommended protocol .  Propidium iodine (PI: membrane impermeant (1) was used in determining the viability of the oocysts (2). 

Microscopic analysis   Following the filtration assay, each membrane filter was placed onto a 25 X 75 mm slide (VWR, Inc., San Diego, CA.).  Dabco/ glycerol mounting medium (60% glycerol, 40% 150mM PBS, 2% DABCO) was placed onto the membrane filter and covered with a 25mm sq. glass cover slip (Corning).  The membranes were viewed using the Olympus BH2 Fluorescence Microscope/Mercury-100 (Olympus, Lake Success, NY.)  under 40X objective for detection of any remaining oocysts in the water samples. 

 

RESULTS AND DISCUSSION

A total of three experimental runs were performed.  Table 1 provides information as to the number of oocysts initially fed to the Ceriodaphnias and the number of oocysts remaining from each water sample filtered.  In the first run, 60.4% of oocysts were ingested and no oocysts were excreted.  As for the second run, 74.32% of the oocysts were ingested.  Results also show there was a total of nine oocysts found in the excretion water sample, seven nonviable and two viable oocysts.  There is a probability that the oocysts may have been carried over from the washing phase to the final excretion phase, without properly being removed.  In the third run, 91.12% of the oocysts were ingested.  An average of 941 oocysts were ingested.  In all cases over 60% of the oocysts were consumed and less than 1% were excreted.  The three individual runs were performed on different days using a new selection of Ceriodaphnias each time.  We received a total of two Ceriodaphnia shipments from the laboratory of Forrest L. Mitchell.  The shipments  consisted of a variety of Ceriodaphnias.  This may be a possible explanation as to the different numbers of oocysts ingested per run given an hour feeding time.  Theoretically, given a 24 hour feeding period, five Ceriodaphnias would be able to ingest a minimum of 2.2 x 104 oocysts.  Given their small size and filter feeding ability, Ceriodaphnias would aid in the decontamination of different water sources.  Large quantities of Ceriodaphnias could be grown in local laboratories and spread on water bodies surrounding dairy farms, etc.  Previous studies have been conducted using freshwater benthic clams (Corbicula fluminea) (3).  Studies performed demonstrated the effectiveness of these clams in removing C. parvum oocysts from water.  Two hundred clams removed 3.8 x 107 oocysts from 38 liters of water within 24 hours. Due to the fact that C. parvum raises such a health threat, finding an economical source for aiding in the purification of different water sources is crucial.  Ceriodaphnia has shown the efficiency in the consumption of viable oocysts.

 

Table 1

Experimental Runs

1st Run

2nd Run

3rd Run

Number of oocysts fed

1,250

1,250

1,250

Leftover

483

310

109

Carryover

12

2

0

Excretion

0

9

2

 

  
REFERENCES

 

1.      Clancy, J. and Fricker, C.  1998.  Crypto’s protocol prospects.  Water Quality International.  May/June 11-15.

2.      Dowd, S.E. and Pillai, S.D.  1997.  A rapid viability assay for Cryptosporidium oocysts and Giardia cysts for use in conjunction with indirect fluorescent antibody detection.  Can. J. Microbiol 43:  658-661.

3.      Graczyk, T.K., Fayer, R., Cranfield, M.R., and Conn, D.B.  1998.  Recovery of Waterborne Cryptosporidium parvum Oocysts by Freshwater Benthic Clams (Corbicula fluminea).  Applied and Environmental Microbiology 64:  427-430.

4.      Mitchell, F.L. 1996.  Micro and Macro-Invertebrates as Bioconcentrators of Protozoan Parasites in Surface Water Systems.  Unpublished.

5.      Rose, J.B.  1997.  Environmental Ecology of Cryptosporidium and Public Health Implications.  Annu. Rev. Public Health 18:  135-61.

 

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