Destructive effect of heating against toxoplasma oocysts.

Heating was examined for destructive effect against Toxoplasma oocysts, both unsporulated and sporulated, of the O-1 and O-3 strains. Sporulation-inhibition rates were measured by counting sporulated and unsporulated oocysts in each examination. As a result, the sporulation of Toxoplasma oocysts was completely inhibited by exposure to 60 or 70 degrees C for 10 seconds, 55 degrees C for 30 seconds, 50 degrees C for 2.5 minutes. 45 degrees C for 1 hour, or 37 degrees C for 48 hours. When examined by the mouse inoculation method, the infectivity of sporulated oocysts became extinct after heating at 90 degrees C for 30 seconds, 80 degrees C for 1 minute, 70 degrees C for 2 minutes, 55 or 60 degrees C for 15 minutes, or 50 degrees C for 30 minutes. It was confirmed that heating was effective for the sterilization of Toxoplasma oocysts.     

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.     

Infectivity of Cryptosporidium parvum oocysts following cryopreservation.

This study was undertaken to investigate the cryopreservation of Cryptosporidium parvum oocysts. Oocysts purified from mouse feces were suspended in distilled water, 10% glycerin, and 2.5% potassium dichromate. They were stored at -20 C and -80 C for 2, 7, and 30 days, respectively. In addition to the purified oocysts, the feces of C. parvum-infected mice were preserved under the same conditions described above. Purified and fecal oocysts were thawed at 4 C, and their viability was assessed by a nucleic acid stain, excystation test, tissue culture infectivity test, and infectivity to immunosuppressed adult mice. Oocysts purified from fecal material prior to cryopreservation lost most of their viability and all of their infectivity for tissue culture and mice. However, when oocysts were cryopreserved in feces, between 11.7 and 34.0% were judged to be viable and retained their infectivity for mice when stored at -20 C (but not -80 C) for 2, 7, and 30 days. Clearly, fecal material provides a cryoprotective environment for C. parvum oocysts stored at -20 C for at least 30 days.     

Loss of infectivity of Neospora caninum oocysts maintained for a prolonged time

The purpose of this study was to investigate whether sporulated Neospora caninum oocysts, which had been stored for 46 mo in a 2% sulfuric acid solution at 4 degrees C, remain morphologically viable and infective to gerbils (Meriones unguiculatus). Six gerbils were orally inoculated with doses of 400 or 1,200 oocysts. Two mo after inoculation, the animals did not show any clinical signs, had no histological lesions, and were seronegative for N. caninum at 1: 50 in an immunofluorescent antibody test. PCR using the brain from each gerbil did not reveal N. caninum specific DNA. We conclude that oocysts preserved for 46 mo are not infective, despite being morphologically intact.     

The effects of time and temperature on flow cytometry enumerated live Cryptosporidium parvum oocysts

AIMS: Recoveries of spiked standard suspensions are used to evaluate method performance. For many applications, gamma-irradiated Cryptosporidium oocysts are appropriate. In contrast, methods that determine viability, such as Cryptosporidium cell culture, require the use of live oocysts. Oocyst standards are usually prepared at a flow cytometry laboratory for use at another laboratory, and thus the samples are shipped. The goal of this study was to evaluate the shipping and storage stability of flow cytometry enumerated live oocysts over time at three temperatures: 4 degrees C, room temperature and 37 degrees C. METHODS AND RESULTS: Replicate samples containing 100 live C. parvum oocysts were prepared by flow cytometry and stored at 4 degrees C, room temperature and 37 degrees C. These samples were counted at various time points. Significant oocyst losses were observed after storage for 1 day at 37 degrees C, 7 days at room temperature and 21 days at 4 degrees C. CONCLUSIONS: Live C. parvum oocysts internal standards should be used within 10 days of preparation, and stored and shipped at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: When evaluating method performance with live oocysts, both the storage temperature and time are critical factors for obtaining reliable and accurate results.     

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.     

Effects of low temperatures on viability of Cryptosporidium parvum oocysts.

Microcentrifuge tubes containing 8 x 10(6) purified oocysts of Cryptosporidium parvum suspended in 400 microliters of deionized water were stored at 5 degrees C for 168 h or frozen at -10, -15, -20, and -70 degrees C for 1 h to 168 h and then thawed at room temperature (21 degrees C). Fifty microliters containing 10(6) oocysts was administered to each of five to seven neonatal BALB/c mice by gastric intubation. Segments of ileum, cecum, and colon were taken for histology from each mouse 72 or 96 h later. Freeze-thawed oocysts were considered viable and infectious only when developmental-stage C. parvum organisms were found microscopically in the tissue sections. Developmental-stage parasites were not found in tissues from any mice that received oocysts frozen at -70 degrees C for 1, 8, or 24 h. All mice that received oocysts frozen at -20 degrees C for 1, 3, and 5 h had developmental-stage C. parvum; one of 6 mice that received oocysts frozen at -20 degrees C for 8 h had a few developmental-stage parasites; mice that received oocysts frozen at -20 degrees C for 24 and 168 h had no parasites. All mice that received oocysts frozen at -15 degrees C for 8 and 24 h had developmental-stage parasites; mice that received oocysts frozen at -15 degrees C for 168 h had no parasites. All mice that received oocysts frozen at -10 degrees C for 8, 24, and 168 h and those that received oocysts stored at 5 degrees C for 168 h had developmental-stage parasites. These findings demonstrate for the first time that oocysts of C. parvum in water can retain viability and infectivity after freezing and that oocysts survive longer at higher freezing temperatures.     

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.     

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)

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.     

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.     

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.     

Methods in coccidiosis research: separation of oocysts from faeces.

Factors which may be important in the large-scale extraction of coccidial oocysts from faeces ha.ve been investigated with Eimeria tenella. Age of bird, inoculum, feeding status at the time of inoculation, period of collection, feeding status during collection, collection medium, homogenization and sieving, flotation, washing, sporulation and further purification have all been considered. The aim has been to establish a method to produce the maximum number of oocysts of a required degree of purity and viability, with the expenditure of the minimum amount of physical effort, time, animals and chemicals. In our method, groups of chickens 3-4 weeks of age are inoculated with 5000 oocysts of E. tenella and food is supplied ad lib. Over the period 5-8 days after inoculation, faeces are collected in trays containing 2% (w/v) potassium dichromate solution, while food intake is restricted. The faecal material is homogenized, passed once through 40 and 100 mesh sieves, centrifuged and the oocysts recovered from the sediment by 3 flotations in saturated salt solution. Following washing, oocysts are sporulated by forced aeration at 30 degrees C and may be further purified by hypochlorite treatment, or passage in 5% Tween 80 solution through a glass bead column followed by sucrose density gradient centrifugation. Routine passages along these lines over a 5 year period have given a recovery of 46% of the oocysts excreted by over 7000 birds.     

Life Cycle of Eimeria christenseni Levine,Ivens & Fritz, 1962 from the Domestic Goat,Capra hircus L.1

The endogenous development of Eimeria christenseni was studied in 10 two- to four-week-old kids inoculated with 10⁶-10⁷ sporulated oocysts. They were killed at intervals from two to 26 days after inoculation, and their tissues were examined for endogenous stages of the coccidian by light microscopy. Such stages were found in the small intestine and mesenteric lymph nodes. In the sexual cycle, two generations of meronts were found. The first generation developed in endothelial cells of lacteals in the jejunun and ileum and mesenteric lymph nodes, and mature meronts were first seen 14 days after inoculation. The second generation developed in epithelial cells of the glands of Lieberkuehn in the jejunum and ileum and in mesenteric lymph nodes, and its mature meronts were first seen by 16 days. Sexual stages were present mostly in epithelial cells of the tips and sides of the villi and less frequently in crypt cells of the jejunum and ileum. Mature macrogametes and microgamonts and oocysts were also first seen by 16 days. The prepatent period was 17 (14-23) days; the patent ranged from 8 to more than 30 days. Sporulation time was 3-4 days at 30°C. E. christenseni was found to be pathogenic, kids inoculated with 1-5 × 10⁵ sporulated oocysts exhibited the following signs: severe diarrhea, anorexia, polydipsia, poor hair coat, and extreme weakness. They recovered about a month later, but their growth rates appeared to be lower than those of uninoculated animals kept under the same conditions. One kid died 20 days after inoculation with 10⁷ oocysts.     

Examination of attachment and survival of Toxoplasma gondii oocysts on raspberries and blueberries

The consumption of Toxoplasma gondii oocysts on fresh produce may be a means of its transmission to humans. Cats shed T. gondii oocysts, which contaminate produce directly or contaminate water sources for agricultural irrigation and pesticide and fertilizer applications. Cyclospora cayetanensis is a related coccidial parasite, and outbreaks of diarrhea caused by C. cayetanensis have been associated with the ingestion of contaminated raspberries. The oocysts of these coccidians are similar in size and shape, indicating that they may attach to and be retained on produce in a similar manner. In the present study the attachment and survival of T. gondii oocysts on 2 structurally different types of berries were examined. Raspberries and blueberries were inoculated individually with 1.0 x 10(1) to 2.0 x 10(4) oocysts of sporulated T. gondii. Berries inoculated with 2.0 x 10(4) oocysts were stored at 4 C for up to 8 wk. Oocyst viability and recovery were analyzed by feeding processed material to mice. Mice fed T. gondii-inoculated berries stored at 4 C for 8 wk developed acute infections. In other experiments mice fed raspberries inoculated with > or = 1.0 x 10(1) oocysts became infected, whereas only mice fed blueberries inoculated with > or = 1.0 x 10(3) oocysts became infected. This study demonstrates that T. gondii oocysts can adhere to berries and can be recovered by bioassays in mice and that raspberries retain more inoculated oocysts than do blueberries. The results suggest that T. gondii may serve as a model for C. cayetanensis in food safety studies.     

Eimeria scholtysecki n. sp. from Ord's Kangaroo Rat Dipodomys ordii*

SYNOPSIS. Eimeria scholtysecki n. sp. is described from Ord's kangaroo rat Dipodomys ordii. The sporulated oocysts are broadly ovoid to ellipsoid, averaging 24.6 by 19.6 μ. A polar granule is present. A micropyle and oocyst residuum are absent. The ovoid sporocysts average 12.1 by 8.0 μ, and have small, flattened Stieda bodies. The distinctive sporocyst residuum is composed of coarse granules. The mean prepatent period was 8.2 days. Five inoculated rats apparently became reinfected and discharged oocysts for 30 days or more.     

Viability of preserved Cryptosporidium baileyi oocysts

The present study was undertaken to determine the viability and infectivity of oocysts of Cryptosporidium baileyi that had been stored from 1 to 40 months at 4 degrees C preserved in 2.5% potassium dichromate solution. Oocysts of C. baileyi were purified from the feces of experimentally infected chickens using discontinuous sucrose gradients. Subsequently, the purified oocysts were suspended in 2.5% potassium dichromate solution at a concentration of 1 x 10(7) organism/ml, and their viabilities were assessed by nucleic acid staining, histologic examination, and infectivity to 2-day-old chickens. All chickens inoculated with oocysts that had been stored for 1-18 months developed patent infections, while chickens infected with older oocysts remained uninfected. Between 5.8% and 82.2% of the oocysts, stored at 4 degrees C in 2.5% potassium dichromate solution, were found to be viable, as determined by nucleic acid staining. Parasite colonization in the bursa of Fabricius was detected in the microvillus border of bursal epithelium. The finding that C. baileyi oocysts remain infective to chickens for at least 18 months offers important time-saving advantages to investigators who frequently require large numbers of oocysts.     

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.     

Extraintestinal development of Caryospora simplex (Apicomplexa: Eimeriidae) in experimentally infected mice, Mus musculus

Developmental stages of Caryospora simplex were found in connective tissue of the cheek, tongue, and nose of Swiss-Webster and C57 BL/6 mice (Mus musculus) from 8 through 70 days after oral inoculation with 50,000 or 250,000 oocysts, or 60,000 free sporocysts of the same species obtained from an Ottoman viper, Vipera xanthina xanthina. The earliest developmental stages were seen on day 8 post-inoculation (PI) and consisted of two types of meronts and gamonts (undifferentiated sexual stages). Gamonts, microgametocytes, macrogametes, and unsporulated oocysts were found on days 10 and 12 PI. Fully sporulated, thin-walled oocysts containing eight sporozoites surrounded by a thin sporocyst membrane were first seen 12 days PI. Monozoic cysts (caryocysts) were first seen 12 days PI and appeared fully viable throughout the duration of the study, 70 days PI. Four mice injected intra-peritoneally with 150,000 free sporozoites and killed 12 days PI contained unsporulated and sporulated oocysts in connective tissues of the cheek, tongue, and nose, suggesting that sporozoites may be carried to the site of infection via the lymphatic/circulatory system. Four cotton rats, Sigmodon hispidus, inoculated orally with 250,000 oocysts all had unsporulated and sporulated oocysts of C. simplex in connective tissue of the cheek, tongue, and nose when killed on day 12 PI, indicating extraintestinal development in the secondary host is not species specific. This is the first report of a heteroxenous coccidium with both asexual and sexual development in the primary (predator) and secondary (prey) hosts.