Vol. 57 (2)
2002
Kanyari, P.W.N.
1
, Oyejide, A.O.
2
, Alak, J.I.B.
2
, Anderson, D.L.
2
,
Wilson, S.T.
2
and Srivastava, K.
2
1. Department of Pathology, Microbiology and Parasitology, Faculty of
Veterinary Medicine, University of Nairobi, Kenya.
2. Department of Pathobiology, College of Veterinary Medicine, Nursing and Allied Health,Tuskegee University, Tuskegee, Alabama, USA
The progression of Cryptosporidium parvum infection in pregnant C57BL/6J mice with and without immunosuppression was studied. Dexamethasone was given daily to one group intraperitoneally at the dose rate of 125 ug/mouse/day. Infection was initiated orally with two million oocysts per mouse on day six of immunosuppression (day 13 of pregnancy). Faecal oocyst output was monitored on alternate days starting at day three post-infection (PI) the presence of cryptosporidial antigenemia determined by antigen-ELISA test using rat anti-cryptosporidium polyvalent sera as the primary antibody. Establishment of patent infection in the adult mice and subsequent fetal infection was evaluated by histopathological examination of intestinal tissues and transmission electron microscopy (TEM). Immunosuppressed mice shed more oocysts than their controls (P<0.01). In both groups peak oocyst shedding was on day 5 PI. Antigenemia developed faster in immunosuppressed mice, but once infection was fully established, no difference was noted between the two infected groups. On day 5 PI, fetal infection with Cryptosporidium was observed among immunosuppressed mice, which were at day 18 of pregnancy. The parasite stages were uninucleate, occurring characteristically at the intracellular extracytoplasmic position of the intestinal epithelial brush border. By H&E staining, they showed prominent basophilic nuclei and eosinophilic cytoplasm. By TEM, merozoites, meronts and oocysts were distinctly identifiable.
Oocysts were frequently encountered in the intestinal lumen in various stages of maturation. This could be the first report of transplacental cryptosporidial infection in any host. These findings could have serious transmission implications in relation to pregnant human AIDS patients and other vertebrate hosts of Cryptosporidium parvum .
Cryptosporidiosis is a zoonosis caused by a protozoan parasite of the genus
Cryptosporidium . Although the disease affects mainly the gastro-intestinal tract of a wide range of vertebrate hosts, significant effects of infections have been noted mainly in humans and calves. The condition occurs in immunocompetent hosts including humans
(1,2,3) and mice (4). The organism is primarily an opportunist but in immuncompromised individuals, the infection fulminates and might be life-threatening. Death as a direct result of cryptosporidiosis has been reported among patients with Acquired Immunodeficiency
Syndrome (AIDS)[5]. The vertical route of transmission is the major avenue of infection with Human Immunodeficiency Virus (HIV) in infants and children (6). Cryptosporidiosis is one of several opportunistic complications of HIV infections and is reported as a significant cause of disease in neonates (7).
Cryptosporidiosis in cattle and other hosts appears to parallel the disease in humans, for example, clinical cryptosporidiosis has been observed in calves as young as 4 days (8). In

the gazella, the condition was observed among day-old neonates (9). Such early occurrence of patent infections calls for investigations on the parasite at critical times such as during pregnancy. A murine cryptosporidiosis model is used here to study the effects of drug-induced immunosuppression and pregnancy on parasite development in the gut and subsequent invasion of the circulatory system and foetal intestinal tissues.
Mice
An inbred strain of mice C57BL/6J from the Jackson Laboratories (Bar Harbor, ME) was used in this study. Thirty-four female mice, at five-day-old pregnancy, as determined by vaginal plug, were treated according to the recommendations of The American
Association for the Laboratory Animal Science (10). They were put in individual cages in an animal facility in a micro-isolator unit maintained at 20-22 0 C, 60 - 80% relative humidity and a 12-hour dark-light cycle. Food (mouse chow- Purina Company, St. Louis, MO) and water were freely provided. They were allowed two days to acclimatize. On the third day, they were randomly divided into three groups, A=13; B=13 and C=8. Starting from the third day and each day during the study period, group A were injected intraperitoneally with 0.2 ml of physiological saline containing 125 ug dexamethasone (Sigma Chemical
Company, St. Louis, MO). Following a similar regimen, an equal volume of physiological saline was administered to mice in groups B and C.
Infection of Mice
After six days of dexamethasone administration, mice in groups A and B were each given 2 million purified oocysts of sporulated Cryptosporidium parvum , enumerated using improved Neubauer Chambers ( Hawksley and Son, London). Group C were left as infection controls. Oocysts were administered by the oral-gastric route using a curved blunt needle. The oocysts were of calf origin and were obtained from The National Animal
Research Centre (Ames, IA), courtesy of Dr. John Alak of Carver Research Foundation,
Tuskegee School of Veterinary Medicine.
Fecal and Blood Sample collection
Fecal pellets were collected directly from the rectum of the mice on alternate days starting on day 3 PI and continued till day 11. Pellets were put into 1.5 ml Eppendorf safelock tubes (Sigma Chemical Company, St. Louis, MO) and stored at room temperature. At day 18 of pregnancy, blood was collected individually from 4 mice of groups A and B and one mouse from group C, then frozen untill testing. They were then euthanised with ether.
Similarly at day 19 of pregnancy, blood was obtained from each mouse followed by euthanasia.
Histopathology
At post-mortem, adult ileal intestinal portions were sectioned and preserved in 10 % buffered formalin while those from fetuses were preserved whole as they were too delicate to disect. They were then processed routinely for staining with H & E and observed microscopically.
Transmission Electron Microscopy
Intestinal fetal tissues were cut into 1-2 mm pieces and fixed as recommended (9) in glutaraldehyde (1.25%) / paraformaldehyde (2%) in cacodylate buffer (pH 7.2, 0.067 M) for 5 minutes. They were transferred to a second mixture of 4 % glutaraldehyde and 2% paraformaldehyde in the same buffer as above for a minimum of 2 hours. They were postfixed in 1% osmium tetroxide and then stained 'en block' with 5% uranyl acetate. The tissues were then dehydrated through graded ethanol concentrations and then embedded in epon/araldite. Sections were cut with glass knives made with a KLB knife-maker. Semithin sections (1-2 um thick) were stained with 1% toluidine blue on a hot plate (60 o C) and mounted in Depex (Hopkins and Williams). These sections were used to determine the area suitable for thin sectioning. Thin sections were mounted on copper grids (22-300 mesh) and then stained with uranyl acetate or lead citrate. Section were examined with a
Zeiss 10 electron microscope or a Corinth (A.E.I.) and photographed with a 35 mm camera.

All measurements were made from developed prints of known original magnification. All prints enlarged from the original negatives had their magnification factors calculated accordingly.
Fecal Oocyst Enumeration
Fecal samples were weighed individually and then soaked in a known volume of phosphate buffered saline (PBS). The sample was then homogenized and the resultant mixture divided into two. One half was used for confirmatory immunoflourescent staining of oocysts using the Meriflour direct assay kit
(Meridian Diagnostics Inc., Cincinnati, Ohio). The other half was used to enumerate the oocysts using the improved Neubauer chambers following a previously described method
(7). Using the original fecal weight, the oocyst counts were expressed per gram (OPG) of feces. The oocyst counts were compared by Student’s t-test.
Oocyst Antigen
Antigen was prepared from oocysts of the same source as those used for infection. This antigen was prepared by suspending 252 m oocysts in physiological saline, which was filtered through 0.2 micron disposable sterile filter (Corning). This was followed by sonication in an Ultra Cell Disruptor (Micron) for 20 min. in 2 min. cycles at 70% power setting. The protein concentration was then determined as 42 ug/ml using the Micro-BCA
Protein Assay Reagent kit (Pierce, Rockford, Il). This preparation was used as positive control in the antigen-ELISA test and to immunize the rat for producing hyperimmune anticryptosporidium serum.
Immunization
A male adult rat was inoculated with the antigen preparation subcutaneously after swabbing the injection site with ethyl alcohol. On day 1, 20 ug of oocyst protein were injected in 0.5 ml complete Freunds adjuvant (FCA). The second and third boosters were administered after 10 and 25 days respectively and consisted of 10 ug oocyst protein in incomplete Freunds adjuvant.
The rat was bled one week after the last injection using general anaesthesia by inhalation of isoflurane, USP (Rhone-Poulenc Chemicals Ltd. Avonmouth, Bristol, BS11
9YF, UK). The area to the left of the xiphoid cartilage was swabbed with ethyl alcohol and
5-7 ml blood was drawn from the heart with a 21G needle and a 10 ml syringe. This blood sample yielded about 1.5 ml serum.
Antigen-ELISA Test
A chequer-board titration was conducted to determine the appropriate dilutions for the antigen, primary antibody and the conjugated secondary antibody with slight modifications
(11, 12). Briefly, microtitre plates were coated with 100 µl of mice sera (suspected to contain the circulating parasite antigen) diluted in an equal volume of coating carbonate buffer (pH 9.6). Negative control sera from uninfected mice (group C) and a positive control (sonicated oocyst preparation diluted 1:20) were included in the assay.
Plates were incubated at 37
0
C for 3 hours, excess antigen discarded and 100 µl per well of blocking solution consisting of 150 ul rabbit serum stock in 10 ml buffer (Vector
Laboratories, Burlingame, CA.) was added and plates incubated for 30 min. at 37 0 C. The plates were then washed three times in PBSTween 20 (3 min. /wash) and 100 µl of the hyperimmunised rat serum diluted 1:20 in PBS-Tween 20 in buffer were then added.
Incubation at 37
0
C was followed by removal of excess antibody and washing 3 times in
PBS-Tween 20. T o each well was then added 100 µl of biotinylated rabbit anti-rat IgG
(Vector Laboratories) diluted 1:200 in PBS buffer. The plates were then incubated for 30 min., washed in buffer and 100 µl Vectastain Elite ABC Reagent mixed and added as recommended. Incubation for 30 min. and washing (x 3) in buffer was followed by addition of 150200 µl fresh peroxidase substrate solution. This solution was made by mixing 100
µl of buffer with 5.0 ml glass distilled water, two drops of ABST stock solution and 100 µl of hydrogen peroxide solution.
The plate was let to stand in the dark at room temperature for 30 min. and the optical density (O.D) values read using a Microplate Autoreader (Bio-Tek Instruments) at 405 nm.

Antigen ELISA - test
Positive control samples had an average OD of 1.31, which represented a protein concentration of 60 ng/ml. At 5 day PI, half the mice sacrificed from group A had antigenemia with OD values above the cut-off point (which was the reading of the sera from the uninfected mice) while all mice in group B had values lower than this with a mean of 0.67. However, at day 11 PI cryptosporidium antigenemia was detected by OD values above 0.96 in sera of all mice in both infected groups. At this time, sera from group B mice had a mean OD value of 1.3 compared to 1.2 of group A (Fig. 2).

Fecal Oocyst Output
From day 3 to day 11 of fecal sampling, group A (immunosuppressed) mice had higher
OPG values than group B. Oocyst output in both groups of mice peaked at day 5 PI, subsequently the output declined steadily among the immunosuppressed group. Group B mice had the lowest values at day 7 PI after which a gradual increase was noted during the next two days of sampling (Fig. 1). A statistical comparison of the means of OPG between the groups revealed a significant difference, P< 0.01.
Light and Electron Microscopy of Fetal Endogenous Parasite Stages
All intestinal sections examined from fetuses of non-immunosuppressed (group B) and the control mice (group C) did not show any parasitic endogenous stages. However, sections from fetuses of immunosuppressed dams examined microscopically revealed that
60% had Cryptosporidium developmental stages. These stages were located at the epithelial brush border. They were stained distinctively from the host epithelial cells by having a basophilic nucleus surrounded by an equally well-defined eosinophilic cytoplasm
(Fig. 3). They measured 9.8 - 6.9 µm. Samples positive in light microscopy were then further processed for transmission electron microscopy.
Various endogenous stages of the parasite at various levels of development were recognized. They were observed both at the intracellular extracytoplasmic (within the outer cell membrane but outside the cytoplasm) position of the intestinal epithelial
Section of fetal intestine at day 18 of
TEM section of a merozoite of pregnancy (5 day PI) showing many
Cryptosporidium parvum developmental
Cryptosporidium parvum in the intestinal lumen tapering anteriorly at the region of the stages. The arrow depicts one of them at their apical complex (AP) with the blunt posterior typical site of development at the epithelial brush border. (H & E, x 600) end having the feeding organelle or the intravascular tubes ( IT). x 24,000

TEM fetal intestinal section showing the presence of Cryptosporidium parvum maturing oocyst [O] with a prominent nucleus
(N) and amylopectien granules (A). It is located within the intestinal lumen, which is richly lined with microvilli (v). x 12,000
The pattern of oocyst shedding among both infected groups (A and B) of adult mice compared closely with observations among calves infected with Cryptosporidium parvum .
The peak oocyst output at day 5 PI agrees with previous observations and also the subsequent sharp drop was recorded in calves 5 to 7 days post-infection. The role played by dexamethasone in enhancing infection is clearly evident here. This justifies its use to establish cryptosporidial infections in various hosts (12,13,14,15,16).
The development of antigenemia was accelerated by dexamethasone as seen in group A mice at day 5 PI in comparison to group B. However once infection was established, antigenemia developed fully in both groups (A and B) as seen on day 11 PI. The antigen detection assay developed in this study was able to pick up antigen concentrations of 60 ng/ml compared to that reported earlier (11) where detection levels were 100 ng/ml of calf sera.This assay could be developed further as there are advantages of detecting circulating cryptosporidial antigens such as in sub-clinical infections in immunosuppressed patients (11). Such antigens are possibly merozoites that later lead to transuterine fetal infections encountered in this study.
The use of rat anti-cryptosporidium polyvalent sera directed against purified parasite membranes followed by goat anti-rat IgG conjugated to horseradish peroxidase and
3,3',5,5'-tetramethyl-benzidine as substrate has been reported (12). These workers were able to assess the antimicrobial activity of various compounds using an in vitro grown C. parvum . The polyvalent serum used here was also developed in the rat while the secondary conjugated antibody was from rabbits and worked quite well at dilutions of

1:200 to 1:400. It is significant that rats can be successfully immunized with protein concentrations of cryptosporidial oocyst antigen that are much lower than that reported previously considering that several million oocysts are required to yield any appreciable amounts of protein (11,12).
The detection of C. parvum developmental stages in the foetal intestinal epithelium among immunosuppressed mice is of great significance. At light microscopy theses stages were mainly oval to round in shape and had a distinct nucleus with eosinophilic cytoplasm.
The observations were made in foetuses from 60% of drug-immunosuppressed dams. No attempts were made to examine exhaustively the whole length of the fetal intestinal tract, thus this percentage could be higher. If by extension this situation is compared to that of human female patients with HIV infections, or are on immunosuppressive therapy, one can postulate that infections may be transmitted to babies in utero. This finding suggests an urgent need to evaluate neonates of immunocompromised as well as immunocompetent mothers for the presence of vertically transmitted cryptosporidial infections.
Our results suggest that observations of neonatal cryptosporidiosis in various hosts including a one-day-old gazella (9) can be interpreted to mean that infections may have been acquired transplacentally. The infective stages in this case would have to be the merozoites which systemically invade the maternal tissues including the circulatory system resulting in the fetal infection. The exact route of fetal infection may be oral by ingestion of merozoites possibly contained in amniotic fluids or via the blood. The infection establishes itself in the fetal gut where the life cycle is completed successfully. This parallels the parasite behaviour described in mice where patent gut infections resulted following intravenous inoculation of Cryptosporidium parvum oocysts (17).
This is possibly the first record of transplacental transmission of C. parvum in any host. It is quite different from the intrauterine infection of adult mice reported earlier (18). The ability of C. parvum to infect unborn fetuses requires more investigation considering the importance of this disease even among human AIDS patients and the need to control vertical transmission.
Acknowledgement
This work was financially supported by a Fulbright Senior Scholar Fellowship Program through the Council for International Exchange of Scholars (C.I.E.S.). We express our thanks to Tuskegee University through the Dean, School of Veterinary Medicine for availing the facilities and the University of Nairobi, Kenya for giving the senior author leave of absence. We would further like thank Mr. Clive Wells of ILRI, Kenya and his assistant
Chris for their technical support in the TEM procedures.
1.