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Therapy based on the protective passive immunity of hyperimmune bovine colostrum (HBC) (raised against Cryptosporidium parvum in cows) was applied to 4 Savanna monitors (Varanus exanthematicus) with gastric Cryptosporidium sp. infections. All lizards were moderately emaciated, and their fecal and gastric lavage samples contained moderate numbers of Cryptosporidium sp. oocysts. The first 3 of 7 gastric HBC treatments at 1-wk interval each decreased the numbers of oocysts in the fecal and gastric samples to undetectable levels. Neither feces nor lavages of the HBC-treated lizards contained Cryptosporidium sp. oocysts after the HBC therapy, whereas such samples of a single control lizard remained positive for oocysts. Two of the HBC-treated lizards died spontaneously due to metastasized carcinoma and septicemia of unknown etiology, respectively, and 2 lizards treated and killed during the experiment were histologically negative for developmental stages of Cryptosporidium sp. The control lizard died spontaneously of septicemia of unknown etiology and contained developmental stages of Cryptosporidium sp. in the gastric region. The HBC therapy was efficacious in V. exanthematicus and is recommended for lizards with gastric cryptosporidiosis.

For conservation purposes and because of growing ecotourism, some mountain gorilla (Gorilla gorilla beringei) populations have been habituated to humans. Fecal specimens (n = 100) of nonhabituated and human-habituated gorillas (5 populations; 6 age classes) were tested for Cryptosporidium sp. oocysts and Giardia sp. cysts by conventional staining and immunofluorescent antibody (IFA). Cryptosporidium sp. infections (prevalence 11%) were not restricted to very young gorillas but were observed in 3-yr-old to12-yr-old gorillas; most of the infections (73%) occurred in human-habituated gorillas. The prevalence of Giardia sp. infections was 2%; 1 nonhabituated gorilla was concomitantly infected. Oocysts of Cryptosporidium sp. in the gorilla stools were morphologically, morphometrically, and immunologically undistinguishable from a bovine isolate of Cryptosporidium parvum oocysts. Mean concentration of Cryptosporidium sp. oocysts and Giardia sp. cysts in gorilla stools was 9.39x10(4)/g, and 2.49x10(4)/g, respectively. There was no apparent relationship between oocyst concentration and gorilla age, sex, or habituation status. Most Cryptosporidium sp. infections found in gorillas with closest proximity to people may be a result of the habituation process and ecotourism. This study constitutes the first report of Cryptosporidium sp. infections in the family Pongidae, in the free-ranging great apes, and in the species of gorilla.

Habituation to humans of free-ranging populations of endangered mountain gorillas (Gorilla gorilla beringei) raised concern of anthropozoonotic transmission of parasitic helminths and protozoans. Examinations of liver tissue of 19 gorillas found dead in the Parc National de Volcans, Rwanda, revealed 10 cases of hepatic nematodiasis due to Capillaria hepatica. Identifiable C. hepatica eggs were present in the liver of 4 gorillas (3 juveniles, 1 adult), and nematode cross-sections were found in 1 juvenile gorilla. Six other adult gorillas had areas of periportal and subcapsular fibrosis with calcified eggs. Histologically, the lesions surrounded by the areas of mild inflammatory reaction were characterized by subcapsular, periportal foci of fibrosis in which were embedded numerous C. hepatica eggs. Control of hepatic capillariasis in the remaining populations of mountain gorillas should be focused on eradication or control of populations of rodent pests (i.e., mice and rats) that sustain the reservoir of C. hepatica in habitats shared by gorillas and humans.

Sequence alignment of a polymerase chain reaction-amplified 713-base pair region of the Cryptosporidium 18S rDNA gene was carried out on 15 captive reptile isolates from different geographic locations and compared to both Cryptosporidium parvum and Cryptosporidium muris isolates. Random amplified polymorphic DNA (RAPD) analysis was also performed on a smaller number of these samples. The data generated by both techniques were significantly correlated (P0.002), providing additional evidence to support the clonal population structure hypothesis for Cryptosporidium. Phylogenetic analysis of both 18S sequence information and RAPD analysis grouped the majority of reptile isolates together into 1 main group attributed to Cryptosporidium serpentis, which was genetically distinct but closely related to C. muris. A second genotype exhibited by 1 reptile isolate (S6) appeared to be intermediate between C. serpentis and C. muris but grouped most closely with C. muris, as it exhibited 99.15% similarity with C. muris and only 97.13% similarity with C. serpentis. The third genotype identified in 2 reptile isolates was a previously characterized 'mouse' genotype that grouped closely with bovine and human C. parvum isolates.

Viable Cryptosporidium serpentis oocysts originating from 6 captive snakes were gastrically delivered to 12 Cryptosporidium-free African clawed frogs and 9 tadpoles and 3 recently metamorphosed adults of Cryptosporidium-free wood frogs. On days 7 and 14 postinoculation, no life-cycle stage of Cryptosporidium was observed in any of the histological sections of stomach, jejunum, ileum, cloaca, and cecum. However, viable inoculum-derived C. serpentis oocysts were recovered from the water in which the amphibians were kept. Amphibians may disseminate C. serpentis oocysts in the natural habitat.

The potential cross-reactivity of the combined Cryptosporidium/Giardia direct immunofluorescence antibodies (IFA) of MERIFLUOR and HYDROFLUOR-COMBO tests was examined against tissues containing known developmental stages of 12 pathogens causing the principal infectious diseases in oysters. Spores of Haplosporidium nelsoni and Haplosporidium costale produced positive acid-fast stain (AFS) reactions similar in intensity to Cryptosporidium parvum oocysts. Hexamia nelsoni trophozoites produced positive IFA reactions in both IFA tests; however, the intensity of fluorescence was considerably lower and the fluorescein-staining pattern different than those of Giardia cysts. The applicability of AFS for screening oysters for Cryptosporidium oocysts is low, and positive identification of Cryptosporidium oocysts cannot be accomplished based on the AFS. Presumptive IFA identification of the Cryptosporidium oocysts or Giardia cysts in the oyster tissue should fulfill 3 criteria, i.e., bright-green fluorescence of the same intensity as C. parvum oocysts and Giardia cysts in the positive control, correct size and shape of the fluorescein-stained objects, and oocyst or cyst shell clearly visible.

It was demonstrated by an in vitro slide phagocytosis assay that hemocytes of the Eastern oyster, Crassostrea virginica Gmelin, 1791 are capable of rapid recognition and internalization of infectious Cryptosporidium parvum (AUCP-1 strain) oocysts. The incubation of hemocyte monolayers (8.5 x 10(4) cells) that had received 6.8 x 10(5) or 3.4 x 10(5) oocysts was arrested at 5, 15, 30, 60, 90, and 120 min and the oocytes detected by acid-fast stain and immunofluorescent antibody (IFAT). An average of 20.5, 38.3, 50.2, 58.9, 69.0, and 75.0% oocysts were phagocytosed after 5, 15, 30, 60, 90, and 120 min, respectively. The intensity of fluorescence of phagocytosed oocysts significantly decreased over time (P0.01), and their round shape was altered. The number of cells containing oocysts and the mean number of ingested oocysts (range: 1.2-4.5 per cell) increased significantly over time (P0.01), whereas the numbers of nonphagocytosed oocysts that were adherent to the glass slides significantly decreased (P0.05). By extrapolation, the results indicate that Cr. virginica is capable of internalizing up to 6.4 x 10(6) Cryptosporidium oocysts per ml of its hemolymph.

Cryptosporidium parvum oocysts infectious to neonatal BALB/c mice were processed by the cellulose-acetate membrane (CAM) filter dissolution method to determine if the procedure that utilizes acetone incubation and alcohol centrifugations alters their viability (determined by in vitro excystation) or infectivity (determined by infectivity bioassay). In addition, most oocysts with altered viability by desiccation, heat inactivation, and snap freezing that were processed by the CAM filter dissolution method were nonrefractile, unstained oocyst ghosts. The remaining organisms, oocyst shells, were lightly stained with the acid-fast stain. Infectious oocysts retained their infectivity and nonviable oocysts (oocyst shells) retained their morphology when processed by the CAM dissolution method. Infectious oocysts, oocyst shells, and oocyst ghosts produced positive reactions of similar intensity in direct immunofluorescence antibody staining, utilizing the MERIFLUOR Cryptosporidium/Giardia test kit. Cryptosporidium oocysts recovered from finished drinking water by the CAM dissolution method can be subjected to testing for their viability and infectivity.

A recent report suggested that an isolate of Cryptosporidium parvum had established infections in fish, amphibians, and reptiles and raises concern that animals other than mammals might be a potential source of waterborne Cryptosporidium oocysts. To test this possibility, viable C. parvum oocysts, infectious for neonatal BALB/c mice, were delivered by gastric intubation to bluegill sunfish, poison-dart frogs, African clawed frogs, bearded dragon lizards, and corn snakes. Histological sections of the stomach, jejunum, ileum, and cloaca prepared from tissues collected on days 7 and 14 postinoculation (PI) were negative for Cryptosporidium developmental stages. However, inoculum-derived oocysts were detectable by fluorescein-labeled monoclonal antibody in feces of inoculated animals from day 1 to day 12 PI in fish and frogs, and up to day 14 PI in lizards. Snakes did not defecate for 14 days PI. Impression smears taken at necropsy on days 7 and 14 PI revealed C. parvum oocysts in the lumen of the cloaca of 2 fish and 1 lizard on day 7 PI only. Because tissue stages of the pathogen were not found, it appears that C. parvum was not heterologously transmitted to lower vertebrates. Under certain circumstances, however, such as after the ingestion of C. parvum-infected prey, lower vertebrates may disseminate C. parvum oocysts in the environment.

Indigenous to Hawaii, the Hawaiian crow (Corvus hawaiiensis) is the world's most severely endangered species with only 3 reproductively active pairs remaining in the wild. Seven captive-reared, avian malaria-naive C. hawaiiensis were exposed in an outdoor aviary and hematologically and serologically monitored for 9 wk. Three birds showed Plasmodium relictum capistranoae parasitemia (6.35%, 2.15%, and 0.60%). All birds were seroconverted for malaria on week 7 as determined by enzyme-linked immunosorbent assay (ELISA). Malaria IgG levels of exposed parasitemic birds did not differ from those of exposed nonparasitemic C. hawaiiensis and were not significantly correlated with the level of parasitemia. Four of 9 hematological parameters, e.g., white blood cell count (WBC), relative and absolute lymphocytosis, and total solids (TS), showed significant increases related to ELISA-determined malarial infection. The sensitivity, specificity, and the positive predictive values of these 4 parameters for malarial infections in C. hawaiiensis were higher than 66%, with the WBC and TS sensitivity reaching 100%. The reference range of 9 hematological parameters was established based on uninfected, clinically healthy C. hawaiiensis. Seven birds were successfully treated and released, increasing the total wild C. hawaiiensis world population by approximately 50%.

Blood samples of 191 adult Jackass penguins (Spheniscus demersus) from South Africa were tested by indirect enzyme-linked immunosorbent assays (ELISA) with Plasmodium falciparum antigen (R32tet32) for avian malaria antibodies (Ab). The samples originated from free-ranging penguins from offshore islands and southern coast colonies (3 groups, n = 110), from 2 penguin groups (n = 66) rescued after offshore oil-spill contamination and rehabilitated at the Rescue Station of the South African National Foundation for the Conservation of Coastal Birds (SANCCOB) in Cape Town, and from SANCCOB-resident penguins (n = 15). The total average malaria Ab seroprevalence was 39%, and the mean malaria seropositivity ranged from 20% to 62% among the 6 S. demersus groups, with a mean of 55% for oiled penguins, and 31% for the remaining birds. The total mean absorbance value was 0.57 for ELISA-positive penguins, 0.43 for birds kept at SANCCOB facilities, and 0.70 for the penguins from wild colonies. The 2 groups of oiled penguins exhibited higher malaria Ab seroprevalence (38% and 62%) than the 3 groups of non-oiled birds (29%, 33% and 35%). Malaria Ab seroprevalence of free-ranging penguins was significantly lower (P0.01) than in the 3 groups of birds at SANCCOB facilities and did not differ significantly among 3 wild penguin colonies.

An enzyme-linked immunosorbent assay (ELISA) is described utilizing the surface glycocalyx crude antigen of adult blood trematodes Learedius learedi, Hapalotrema dorsopora, and Carettacola hawaiiensis, for the detection of circulating antibodies (Ab) in Hawaiian green turtles (Chelonia mydas) naturally infected with the parasites and with or without green turtle fibropapillomatosis (GTFP). A concentration of 10.0 micrograms/ml of antigen was optimal in terms of test specificity and sensitivity. A direct ELISA with anti-reptilian/amphibian phosphatase-labeled IgG identified C. mydas Ab at a dilution of 1/12,800. Utilizing indirect ELISA, it was possible to detect Ab to blood flukes at a dilution of 1/3,200. The gross lesions and histopathology were typical for cardiovascular spirorchidiasis in C. mydas. No significant relationship was found between the size of turtles and the degree of GTFP severity. Forty-seven of 59 (80%) samples, from 5 sites, gave a positive ELISA reaction; 6 of the 47 (13%) specimens gave significantly (P0.001) higher absorbance values, with 5 from the same location. All 12 (20%) ELISA-negative turtles originated from 1 site, and the absorbance values of the 41 animals from this location were significantly lower (P0.015) when compared with the other 4 sites. The proposed assay is fast, has the feature of visual scoring, and can be used for determination of exposure of C. mydas to the spirorchid trematodes.

Schistosoma mansoni cercariae have been shown to aggregate in the presence of glass slides treated with clear nail varnish and linoleic acid. In choice chambers cercariae move toward the stimulant, but this behavior is not seen when linoleic acid is omitted. After 30-45 min, the cercariae were concentrated near the end of the choice-chamber containing the linoleic acid slide. When the cercariae were added in the center of the choice chamber, they formed a diffuse cloud that dispersed slowly in both directions in the absence of linoleic acid. Cercariae aggregating in the vicinity of a stimulant surface are not immediately stimulated to commence penetration; this appears to be time and dose related.

Erythrocytic stages of Haemoproteus columbae were extracted from the cytoplasm of nucleated red blood cells (RBC) of Rock dove pigeons (Columba livia) using cationic detergent (N,N',N'-polyoxyethylene(10)-N-tallow-1,3-diaminopropane [EDTA-20]) and discontinuous Percoll gradient density. Crude RBC extract (CRBCE) antigen was prepared. Parasitized RBCs were more resistant to EDTA-20 action than unparasitized cells. An enzyme-linked immunosorbent assay (ELISA) was developed for detection of anti-H. columbae immunoglobulins in 30 wild-captured C. livia. Whole blood, serum, and dried blood on filter paper gave similar results; the latter was selected for sampling convenience. Optimal antigen concentration was 5 micrograms/ml, and anti-H. columbae immunoglobulins were detectable at a 10(-4.11) dilutions. The binding efficacy of anti-chicken IgG to the pigeon immunoglobulins was significantly higher than anti-duck IgG or anti-turkey IgG. Parasitemia by Giemsa-stained thin blood smears ranged from 20.0 to 47.5%, mean = 32.4 +/- 8.3%; 17 of 30 birds had multiply infected RBCs with a mean parasitemia of 2.4 +/- 1.1%, range 0.7-4.9%; 27 birds were positive by the ELISA. No clinical signs of infection were observed. ELISA absorbance values were not correlated with the level of parasitemia in individual birds. All pigeons were negative for anti-Plasmodium relictum and anti-P. elongatum immunoglobulins as determined by ELISA. The pigeons were not subclinically infected with Plasmodium spp. as determined by inoculation of domestic ducklings with blood from dexamethasone-immunosuppressed pigeons.

The shedding pattern of fecal Cryptosporidium serpentis oocysts, histopathologic changes in the gastric region, and the effect of spiramycin treatment were investigated in 6 experimentally infected, captive black rat (Elaphe obsoleta obsoleta), 4 yellow rat (Elaphe obsoleta quadrivittata), and 2 corn snakes (Elaphe guttata guttata). Feces were monitored for up to 2 years postinfection (PI). No significant (P0.07) differences were observed between expected and observed numbers of PI oocyst-positive feces. Two of 5 control animals acquired natural infections of C. serpentis over the period of study. No morphological differences were observed between oocysts from experimental and natural infections. Clinical signs included postprandial regurgitation in 5 of 13 (38%) snakes, not coinciding with the shedding of fecal oocysts. Midbody swelling and self-cure were not observed. Spiramycin treatment of 4 of 12 experimentally infected animals resulted in negative fecal examinations in 2 snakes and reduced the percentage of oocyst-positive feces in 2 other snakes from 75.5% to 24.5% and from 83.9% to 33.6%. Biopsies and necropsies revealed stages of Cryptosporidium in the gastric mucosa of all spiramycin-treated animals. The gastric mucosa was thickened and edematous, with focal necrosis, mucosal petechiae, and brush hemorrhages. Fibroplasia of lamina propria associated with chronic mucosal inflammation were common. Examination of direct fecal smears was found not to be a reliable technique for diagnosis of cryptosporidial infections in snakes.

An enzyme-linked immunosorbent assay (ELISA) utilizing surface glycocalyx membrane crude antigen of adult Echinostoma caproni was developed for the detection of circulating anti-E. caproni immunoglobulins in sera from experimentally infected ICR mice. Whole blood, serum, and dried blood on filter paper gave similar results. The latter was selected for convenience. A concentration of 10.0 micrograms/ml of antigen was optimal in terms of specificity, sensitivity, and test speed. It was possible to detect anti-E. caproni immunoglobulins at a dilution of 10(-4.11). Low absorbance values (0.050) of nonspecific background were observed. In a blind trial the described ELISA accurately differentiated sera taken from infected and uninfected mice. All experimentally infected mice had ELISA-detectable anti-E. caproni immunoglobulins reactive on day 8 postinfection (PI) with the surface glycocalyx antigen. The antibody level varied widely over a 40-day period showing a characteristic and consistent pattern. Mice developed antibodies to E. caproni rapidly, with antibody levels rising by day 8 PI and peaking 6-10 days later. Experimentally infected mice showed time-dependent changes in antibody levels. The proposed ELISA is fast, easy to perform, reproducible, and requires a minimal amount of equipment to collect blood samples. The assay can be used for the detection of anti-E. caproni immunoglobulins in experimentally infected mice, along with monitoring antibody levels in selected groups of mice or for surveys of laboratory experiments where evidence of infection is required.

Twenty-nine juvenile, captive-reared African black-footed penguins (Spheniscus demersus) were hematologically monitored every 2 wk over the period of 24 wk during their first outdoor exposure. Blood samples taken from the penguins were screened for 12 blood evaluation parameters. Parasitemic penguins were medically treated. Eighteen birds (62.1%) experienced naturally acquired malaria and 11 birds (37.9%) remained nonparasitemic. A total of 32 avian malaria episodes were noted; 25 (78.1%) were identified as Plasmodium elongatum, 5 (15.6%) as Plasmodium relictum, and 2 (6.3%) as Plasmodium spp. One P. elongatum (3.4%) and 3 P. relictum (10.3%) infections were fatal. All deaths occurred during the first episode of parasitemia. Gross lesions of the birds that died included hepatomegaly and splenomegaly. Interstitial pneumonia with schizonts was observed on histological examinations. The range, mean, and SD of 12 hematological parameters were determined for nonparasitemic and parasitemic penguins. Differences between these groups in total white blood cell (WBC) counts and relative lymphocytosis (LYMPHS) were not significant. The combined classes of total WBC counts (20.0 x 10(3)/microliters) and LYMPHS (60.0%) are not indicative of avian malaria infection in African penguins. No correlations were found between changes in the values of blood parameters with season or age of penguins. Treatment of parasitemic birds significantly reduced expected mortality from 50.0% to 13.8%.

An enzyme-linked immunosorbent assay (ELISA) with 3 Plasmodium falciparum NF-54 antigens, R32tet32, P.F.R27, and crude red blood cell extract (CRBCE), was tested for detection of anti-Plasmodium relictum and anti-Plasmodium elongatum antibodies in sera from experimentally infected ducklings. Whole blood, serum, and dried blood on filter paper gave similar results. The latter was selected for convenience. All birds infected by experimental blood challenge, but not exposed to sporozoites, had detectable antibody (up to 1.0 x 10(-3.8) dilution) reactive with R32tet32, P.F.R27, and CRBCE antigens. Ducklings infected with P. elongatum had higher antibody levels than those infected with P. relictum. In a blind trial, the described ELISA accurately distinguished sera taken from infected and uninfected ducklings. This study provides the first evidence on cross reactivity in the ELISA format between P. falciparum antigens and antibodies induced by P. relictum and P. elongatum in experimentally infected ducklings. The proposed ELISA is fast, easy to perform, reproducible, and requires a minimal amount of equipment. The assay can be used for the detection of P. relictum and P. elongatum antibodies in captive or wild ducks, along with monitoring the level of antibody in selected groups of birds or for surveys of laboratory experiments where evidence of infection is required.

An acid-medium pretreatment appeared to be essential for in vitro excystation of Cotylurus cornutus metacercarial cysts. Both acid-pepsin or acid-saline were effective but optimum excystation occurred after 15 min acid-pepsin pretreatment and subsequent 2 hr treatment at 41.5 C in an alkaline bile salt-trypsin medium. The rate of excystation was rapid, reaching 78.1% by 1 hr and increasing to 84.3% after 2 hr. Storage of metacercarial cysts up to 16 wk at 4 C in sterile Locke's 1:1 solution did not alter cyst viability.

A simple trap for schistosome cercariae has been developed using clear nail varnish as a matrix and linoleic acid as stimulant affixed to a 75 x 25-mm glass slide. Schistosoma mansoni cercariae will attach to the surface and easily can be visualized either stained or unstained. The trap works well under laboratory and field conditions where the retrieval rate varies from 30% to 100%. Under laboratory conditions there appears to be some form of affinity for cercariae to the surface.