Development and application of different methods for the detection of Toxoplasma gondii in water

Two methods, centrifugation and flocculation, were evaluated to determine their efficiencies of recovery of Toxoplasma gondii oocysts from contaminated water samples. Demineralized and tap water replicates were inoculated with high numbers of sporulated or unsporulated T. gondii oocysts (1 x 10(5) and 1 x 10(4) oocysts). The strain, age, and concentration of the seeded oocysts were recorded. Oocysts were recovered either by centrifugation of the contaminated samples at various g values or by flocculation with two coagulants, Fe(2)(SO(4))(3) and Al(2)(SO(4))(3). The recovery rates were determined with the final pellets by phase-contrast microscopy. Sporulated oocysts were recovered more effectively by flocculation with Al(2)(SO(4))(3) (96.5% +/- 21.7%) than by flocculation with Fe(2)(SO(4))(3) (93.1% +/- 8.1%) or by centrifugation at 2,073 x g (82.5% +/- 6.8%). For the unsporulated oocysts, flocculation with Fe(2)(SO(4))(3) was more successful (100.3% +/- 26.9%) than flocculation with Al(2)(SO(4))(3) (90.4% +/- 19.1%) or centrifugation at 2,565 x g (97.2% +/- 12.5%). The infectivity of the sporulated oocysts recovered by centrifugation was confirmed by seroconversion of all inoculated mice 77 days postinfection. These data suggest that sporulated Toxoplasma oocysts purified by methods commonly used for waterborne pathogens retain their infectivity after mechanical treatment and are able to induce infections in mammals. This is the first step in developing a systematic approach for the detection of Toxoplasma oocysts in water.     

Failure of Development of the Sexual Phase of Eimeria intricata in Heavily Inoculated Sheep*

SYNOPSIS Each of 4 bottle-fed lambs received totals of 3500-8100 sporulated oocysts of Eimeria intricata in series of 3-6 inoculations during 4- to 14-day periods; all produced oocysts of the parasite. The periods of patency in some infections indicated that only the earliest inoculations of a series may have produced oocysts. Fifteen other bottle-fed lambs were inoculated, each with a single dose of 0.01-2 million sporulated oocysts, and necropsied at intervals of 1-2 days over 29 days. Parasites apparently resulting from the inoculations were found in only the lambs killed up to and including 17 days postinoculation; the most advanced stages were 2nd generation schizonts. It was concluded that a reaction occurred against the large numbers of asexual stages of E. intricata, which were evidently present, and the infections were terminated before formation of sexual stages.     

Sporulation and Survival of Toxoplasma gondii Oocysts in Seawater

ABSTRACT. We have been collaborating since 1992 in studies on southern sea otters ( Enhdyra lutris nereis ) as part of a program to define factors, which may be responsible for limiting the growth of the southern sea otter population. We previously demonstrated Toxoplasma gondii in sea otiers. We postulated that cat feces containing oocysts could be entering the marine environment through storm run-off or through municipal sewage since cat feces are often disposed down toilets by cat owners. The present study examined the sporulation of T. gondii oocysts in seawater and the survival of sporulated oocysts in seawater. Unsporulated oocysts were placed in 1.5 ppt artificial seawater, 32 ppt artificial seawater or 2% sulfuric acid (positive control) at 24 C in an incubator. Samples were examined daily for 3 days and development monitored by counting 100 oocysts from each sample. From 75 to 80% of the oocysts were sporulated by 3 days post-inoculation under all treatment conditions. Groups of 2 mice were fed 10,000 oocysts each from each of the 3 treatment groups. All inoculated mice developed toxoplasmosis indicating that oocysts were capable of sporulating in seawater. Survival of sporulated oocysts was examined by placing sporulated T. gondii oocysts in 15 ppt seawater at room temperature 22–24 C (RT) or in a refrigerator kept at 4 C. Mice fed oocysts that had been stored at 4C or RT for 6 months became infected. These results indicate that T. gondii oocysts can sporulate and remain viable in seawater for several months.     

Coccidian parasites (Apicomplexa: Eimeriidae) from insectivores. IV. Four new species in Talpa europaea from England

Moles from England were examined for coccidian oocysts and all 64 Talpa europaea were infected; of 64 infected hosts, 56 (88%) had multiple infections representing two to six coccidian species when examined. Oocysts in 31 of the 64 samples remainedunsporulated. Three eimerians and one isosporan were studied from the 33 fecal samples that had sporulated oocysts and these are described as new species; Cyclospora talpae Pellérdy & Tanyi, 1968, and Isospora sofiae (Golemansky, 1978) Levine & Ivens, 1979, are redescribed; and Cyclospora sp., similar to C. talpae, is discussed. Sporulated oocysts of C. talpae are ellipsoidal, 14.3 X 9.6 (12-19 X 6-13) microns with sporocysts ovoid, 9.4 X 5.7 (6-13 X 4-8) microns; it was found in 21 of the 33 (63.6%) sporulated samples. Sporulated oocysts of Cyclospora sp. are subspheroidal to ellipsoidal, 12.5 X 8.9 (10-14 X 6-12) microns with sporocysts ovoid, 8.6 X 5.3 (6-10 X 4-6) microns; it was found in 21 of the 33 (63.6%) sporulated samples. Sporulated oocysts of Eimeria avonensis n. sp. are elongate-ellipsoidal, 15.0 X 9.6 (13-20 X 7-12) microns with sporocysts ovoid, 6.6 X 3.6 (5-9 X 3-7) microns; it was found in 15 of the 33 (45.5%) sporulated samples. Sporulated oocysts of Eimeria berea n. sp. are subspheroidal, 12.1 X 10.5 (10-15 X 8-14) microns with sporocysts ovoid, 6.3 X 3.9 (5-10 X 2-5) microns; it was found in 8 of the 33 (24.2%) sporulated samples.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of the anticoccidial arprinocid on production, sporulation, and infectivity of Eimeria oocysts

Medication of broilers with arprinocid [MK-302, 9-(2-chloro-6-fluorbenzyl adenine)] had 3 distinct effects on oocysts; (1) the number of oocysts produced was decreased, (2) fewer of the oocysts sporulated, and (3) those oocysts which did sporulate were less infective than those from unmedicated birds. The drug level necessary to prevent passage of oocysts depended on the species and strain of coccidia. To essentially eliminate oocyst production (less than 5% of controls) required medication with the following levels of arprinocid: 70 ppm with Eimeria maxima; 60 ppm with E. mivati, E. E. necatrix, and E. brunetti; and 50 ppm with E. tenella. With E. acervulina, oocysts were completely eliminated by 60 ppm of arprinocid with one field strain but were still numerous at 70 ppm with a second field strain. Oocysts recovered from birds on medication often failed to sporulate. No sporulation was seen at drug levels of 30 ppm or above with E. maxima and E. mivati. The level of arpinocid required to prevent sporulation with other species depended on the strain being studied, but varied from 30 ppm to 70 ppm. The oocysts of E. acervulina, E. mivati, E. tenella, and E. brunetti recovered from medicated birds that subsequently sporulated, were less infective when inoculated into susceptible birds, than oocysts from unmedicated birds. Oocysts from low medication level with E. necatrix (30 ppm) and E. maxima (10 ppm), once sporulated, were as infective as oocysts from unmedicated control birds, even though the numbers produced were less. No differences were detected in the time oocysts were produced between medicated and unmedicated birds infected with E. acervulina, E. maxima, E. brunetti, and E. tenella.     

Five new species of Coccidia (Apicomplexa: Eimeriidae) from colubrid snakes of Ecuador.

Fecal samples from 11 colubrid snakes, representing 10 species, collected in Ecuador during October 1994 were examined for coccidian parasites. Feces of 4 individuals, representing 4 host species, contained coccidian oocysts. Three species of Eimeria and 2 species of Isospora were observed and are described here as new. Oocysts of both Eimeria and Isospora were found in the feces of a slug-eating snake, Dipsas vermiculata. Sporulated oocysts of the Eimeria sp. are spheroid to subspheroid, 16.7 by 16.6 microm (14-18 by 14-18 microm) and those of the Isospora sp. are spheroid and 15.0 microm (13-18 microm) in diameter. Imantodes cenchoa, the common bluntheaded treesnake, was infected with a species of Eimeria. These sporulated oocysts are ellipsoid, 23.3 by 16.2 microm (25-21 by 15-17 microm). Sporulated eimerian oocysts from Leptodeira annulata, the southern cat-eyed snake, are subspheroid, 22.5 by 18.8 microm (19-26 by 17-21 microm). Feces of a juvenile Imantodes lentiferus, the bluntheaded vine snake, contained ovoid to ellipsoid isosporan oocysts, which measured 21.6 by 15.0 microm (20-23 by 14-16 microm) when sporulated.     

Enzyme variation of Eimeria arizonensis from Peromyscus truei and P. boylii

Cricetid rodents, Peromyscus truei and P. boylii, were inoculated with sporulated oocysts of Eimeria arizonensis collected from wild P. truei maintained in the lab. In P. truei the prepatent period was 4-5 days, the patent period was 9-11 days, and sporulated oocysts were 21.5 x 25.0 (20-23 x 24-26) microns with sporocysts 7.7 x 12.0 (6-8 x 10-13) microns. In P. boylii the prepatent period was 6-7 days, the patent period was 8-9 days, and sporulated oocysts were 20.1 x 23.2 (18-22 x 21-24) microns with sporocysts 6.8 x 10.0 (5-8 x 9-12) microns. Sporulated oocysts from both host species were used in direct side-by-side comparison of isozyme banding patterns using protein electrophoresis. The parasite has polytypic loci for leucine aminopeptidase (LAP), lactate dehydrogenase (LDH), and 6-phosphogluconate dehydrogenase (6-PGD). In oocysts from P. truei, LAP showed one band with fast migration and LDH and 6-PGD each showed two bands, one with fast and one with slow migration. In oocysts from P. boylii, LAP and LDH each had one band with slow migration and 6-PGD had one band with moderate migration. Oocysts of E. arizonensis collected from P. boylii were used to inoculate P. truei. The prepatent and patent periods, structural measurements, and isozyme banding patterns of the resultant oocysts were the same as those from P. truei when inoculated with oocysts from P. truei.     

Fixing coccidian oocysts is not an adequate solution to the problem of preserving protozoan type material

Fresh (36 days old) sporulated oocysts of Eimeria nieschulzi were divided into 7 groups. Control oocysts were maintained at 23 C in 2% aqueous (w/v) K2Cr2O7. The 6 experimental groups were mixed with either Bouin's solution, 10% aqueous (v/v) buffered formalin, Karnovsky's solution, glutaraldehyde, paraformaldehyde, or 70% aqueous (v/v) ethanol (EtOH). After 115 days, oocysts from all 7 groups were examined under oil immersion to determine the effect of fixation on their structural integrity. The parameters examined were lengths and widths of oocysts and sporocysts, percent sporulation (%S), and percent crenation (%C) of oocysts and sporocysts. The highest destruction (%S and %C) occurred in oocysts exposed to glutaraldehyde and Karnovsky's fixatives where 100% of both oocysts and sporocysts crenated and only 8% and 48%, respectively, remained sporulated. Of the oocysts in paraformaldehyde, 93% remained sporulated, but 95%of these oocysts and 100% of the sporocyst crenated. In Bouin's solution, 75% of the oocysts were intact structurally, but of these, only 60% were still sporulated with 70% of their sporocysts crenated. Oocysts preserved in 70% EtOH were 80% intact and 70% remained sporulated, but nearly 60% of their sporocysts collapsed even though the oocyst walls were intact. Oocysts preserved in 10% buffered formalin maintained structural integrity but had lower numbers of sporulated oocysts (84%) and greater numbers of crenated oocysts (18%) than control oocysts maintained in the dichromate solution (95% and 0%, respectively).     

Experimental Transmission of Caryospora simplex (Apicomplexa: Eimeriidae) to Palestine Vipers, Vipera xanthina palestinae (Serpentes: Viperidae)

ABSTRACT. Four littermate, laboratory-reared Palestine vipers, Vipera xanthina palestinae (#149, #150, #151, #152) (Serpentes: Viperidae) were used to determine modes by which Caryospora simplex (Apicomplexa: Eimeriidae) could be transmitted to snakes. Viper #149 was inoculated orally by stomach tube with 5.0 x 10⁴ sporulated oocysts of C. simplex obtained from the feces of an Ottoman viper, V. x. xanthina and began passing unsporulated oocysts of C. simplex 121 days post-inoculation (DPI). Viper #150 was fed five mice that had been inoculated orally £25 days previously with 5.0 x 10⁴ sporulated oocysts of C. simplex and it began passing unsporulated oocysts of C. simplex 33 days after being fed the first two of five mice. Viper #151 was inoculated orally with sporulated oocysts of C. simplex obtained from viper #150 and began passing oocysts 52 DPI. Viper #152 served as an uninoculated control and did not pass oocysts of any species of coccidian. This study demonstrates that snake/snake and mouse/snake transmission of C. simplex readily occurs.     

Experimental transmission of Caryospora simplex (Apicomplexa: Eimeriidae) to Palestine vipers, Vipera xanthina palestinae (Serpentes: Viperidae)

Four littermate, laboratory-reared Palestine vipers, Vipera xanthina palestinae (#149, #150, #151, #152) (Serpentes: Viperidae) were used to determine modes by which Caryospora simplex (Apicomplexa: Eimeriidae) could be transmitted to snakes. Viper #149 was inoculated orally by stomach tube with 5.0 X 10(4) sporulated oocysts of C. simplex obtained from the feces of an Ottoman viper, V. x. xanthina and began passing unsporulated oocysts of C. simplex 121 days post-inoculation (DPI). Viper #150 was fed five mice that had been inoculated orally greater than or equal to 25 days previously with 5.0 X 10(4) sporulated oocysts of C. simplex and it began passing unsporulated oocysts of C. simplex 33 days after being fed the first two of five mice. Viper #151 was inoculated orally with sporulated oocysts of C. simplex obtained from viper #150 and began passing oocysts 52 DPI. Viper #152 served as an uninoculated control and did not pass oocysts of any species of coccidian. This study demonstrates that snake/snake and mouse/snake transmission of C. simplex readily occurs.     

Sporogony of the Oocysts of Isospora felis Wenyon, 1923 from the Cat*

SYNOPSIS Sporogony of oocysts of Isospora felis from the cat was studied by observing the individual oocysts. Unsporulated oocysts were passed with the fresh feces. The sporont divided into 2 ball-like sporoblasts which elongated and changed into sporocysts each of which 4 sporozoites then formed. All of the sporulating oocysts completed sporulation at 20 C in 40 hr, at 25 C in 24 hr, at 30 C in 12 hr, and at 38 C in 8 hr. The percentages of oocysts which sporulated at 20, 25, 30 and 38 C were 96, 95, 95 and 95 respectively. No sporulation occurred at 45 C and 50 C when oocysts were incubated for 4 hr. These oocysts evidently died because, on reincubation at 30 C for 4 hr, they failed to develop.     

Infection of llamas with stored Eimeria macusaniensis oocysts obtained from guanaco and alpaca feces

Oocysts obtained from a guanaco and an alpaca with natural infections were identified as Eimeria macusaniensis and evaluated for host specificity and infectivity over time. In 3 separate trials conducted over 4 yr, 4 adult llamas were fed 500-5,000 sporulated oocysts obtained from guanaco feces stored under laboratory conditions for 41-84 mo. Infections with prepatent periods of 36-41 days and patent periods of 38-55 days developed in 4/4 llamas. In a fourth trial, 3 adult llamas and 1 alpaca were each fed 1,000 sporulated E. macusaniensis oocysts obtained from alpaca feces stored in the laboratory for 3 mo. Infections with prepatent periods of 33-34 days and patent periods of 14-20 days developed in 3/3 llamas. Infection in the alpaca had a prepatent period of 58 days and a patent period of 1 day. Clinical signs associated with infection, if any, were minimal and included increased fecal mucus and occasional soft feces. These results provide evidence that E. macusaniensis is a single species transmissible amongst alpacas, llamas, and guanacos and that oocysts of this species can remain infective for many years.     

Sporulation of Cyclospora sp. oocysts.

Cyclospora sp. oocysts sporulated maximally at 22 and 30 degrees C for 14 days retarded sporulation. Up to 12% of human- and baboon-derived oocysts previously stored at 4 degrees C for 1 to 2 months sporulated when stored for 6 to 7 days at 30 degrees C.     

柔嫩艾美耳球虫孢子发育阶段虫体抑制性消减文库的构建

为了分离鉴定柔嫩艾美耳球虫(Eimeria tenella)孢子发育阶段虫体的差异表达基因,分别以柔嫩艾美耳球虫未孢子化卵囊和孢子化卵囊为驱动组、子孢子为实验组,或未孢子化卵囊为驱动组、孢子化卵囊为实验组,利用抑制性消减杂交(SSH)技术,构建了2个子孢子cDNA消减文库和1个孢子化卵囊cDNA消减文库。随机从3个cDNA消减文库中分别挑取50个克隆,经PCR鉴定2个子孢子cDNA消减文库的重组率都为96%,孢子化卵囊cDNA消减文库的重组率为98%。从每个文库中随机挑取50个克隆测序,并进行同源性比较分析,结果显示:从孢子化卵囊cDNA消减文库中获得了13个单一有效序列,其中8个EST与已知蛋白同源性很高;从2个子孢子cDNA消减文库中共获得了40个单一有效序列,其中9个EST与已知蛋白同源,其余可能为柔嫩艾美耳球虫的新基因。这些结果为分离柔嫩艾美耳球虫新功能基因和进一步探索防治球虫病的方法提供了理论基础。     

Survival of Toxoplasma gondii oocysts in Eastern oysters (Crassostrea virginica)

Toxoplasma gondii has recently been recognized to be widely prevalent in the marine environment. It has previously been determined that Eastern oysters (Crassostrea virginica) can remove sporulated T. gondii oocysts from seawater and that oocysts retain their infectivity for mice. This study examined the long-term survival of T. gondii oocysts in oysters and examined how efficient oysters were at removing oocysts from seawater. Oysters in 76-L aquaria (15 oysters per aquarium) were exposed to 1 x 10(6) oocysts for 24 hr and examined at intervals up to 85 days postexposure (PE). Ninety percent (9 of 10) of these oysters were positive on day 1 PE using mouse bioassay. Tissue cysts were observed in 1 of 2 mice fed tissue from oysters exposed 21 days previously. Toxoplasma gondii antibodies were found in 2 of 3 mice fed oysters that had been exposed 85 days previously. In another study, groups of 10 oysters in 76-L aquaria were exposed to 1 x 10(5), 5 x 10(4), or 1 x 10(4) sporulated T. gondii oocysts for 24 hr and then processed for bioassay in mice. All oysters exposed to 1 x 10(5) oocysts were infected, and 60% of oysters exposed to 5 x 10(4) oocysts were positive when fed to mice. The studies with exposure to 1 x 10(4) oocysts were repeated twice, and 10 and 25% of oysters were positive when fed to mice. These studies indicate that T. gondii can survive for several months in oysters and that oysters can readily remove T. gondii oocysts from seawater. Infected filter feeders may serve as a source of T. gondii for marine mammals and possibly humans.     

Further studies on the development of gamonts and oocysts of Eimeria magna in cultured cells

Monolayer, cell-line cultures of embryonic bovine trachea, Madin-Darby bovine kidney (MDBK), and monolayers (RK-1) or aggregates of primary rabbit kidney cells were inoculated with merozoites obtained from rabbits that had been inoculated 3 to 5 1/2 days earlier with Eimeria magna. Merozoites obtained from from rabbits 3 days entered cells and underwent only merogony, whereas 3 1/2-5 1/2-day-old merozoites formed gamonts as well as meronts. Merozoites arising from the first or second meront generation in culture formed another meront generation or gamonts. Third-generation merozoites formed only gamonts. Most merozoites remained within the parasitophorous vacuole of the original host cell and transformed into macro- or microgamonts or meronts. Some such macro- and microgamonts then fused with each other to form larger multinucleated bodies. Such microgamonts formed microgametes, but multinucleate macrogamonts did not form oocysts. Mature microgamonts were 34 microns in diameter, and contained several hundred biflagellate microgametes. Mature macrogamonts measured 29.1 x 21.5 microns, unsporulated oocysts were 31.2 x 22 microns, and sporulated oocysts were 32 x 23.1 microns. Oocysts obtained from cell cultures were sporulated and then inoculated by gavage into rabbits, which passed E. magna oocysts 6--10 days later. Sporozoites, obtained from oocysts produced in culture or from rabbits that had been inoculated with the vitro-produced oocysts, developed to first- and second-generation meronts in MDBK or RK-1 cultures.     

Effects of temperature and different food matrices on Cyclospora cayetanensis oocyst sporulation

Effects of temperature on the sporulation of the parasite Cyclospora cayetanensis were studied in 2 food substrates, dairy and basil. Unsporulated Cyclospora oocysts were subjected to freezing and heating conditions for time periods ranging from 15 min to 1 wk. Oocysts were then removed from the food substrates and placed in 2.5% potassium dichromate for 2 wk to allow viable unsporulated oocysts to differentiate and fully sporulate, and to determine the percentage sporulation as an indicator of viability. Sporulation occurred when oocysts resuspended in dairy substrates were stored within 24 hr at -15 C. When oocysts were placed in water or basil, sporulation occurred after incubation for up to 2 days at -20 C, and up to 4 days at 37 C. Few oocysts sporulated when incubated for 1 hr at 50 C. Sporulation was not observed in basil leaves or water at -70 C, 70 C, and 100 C. Sporulation was not affected when incubated at 4 C and 23 C for up to 1 wk, which was the duration of the experiment in both of the tested substrates.