Oocyst Form Research Articles

Detection of IgG antibodies against T. gondii and associated risk factors in domestic and stray cats in Lusaka, Zambia

Toxoplasma gondii (T. gondii) is a nearly ever-present organism that infects humans, wildlife, birds, and domestic and food animals. T. gondii has been reported in pigs, chickens, sheep, goats, and cattle (1). However, only members of the cat family (Felidae) are the definitive host and shed the environmentally resistant oocyst form of the organism in their faeces. Transmission to humans usually occurs by ingesting cysts in undercooked meat and exposure to soil water which is contaminated by oocysts (2). Feline infections are typically subclinical; congenitally infected kittens are the ones that are most likely to have clinical signs of infection, but previously clinically healthy adult cats may also be affected (3). Despite the disease’s significance in public health, very few studies have been done on T. gondii in Zambia. This study was a cross-sectional survey to investigate the seroprevalence and risk factors of Toxoplasmosis. A survey was carried out using a questionnaire that was distributed to cat owners who attended the veterinary clinics. The questionnaire had demographic data for the cats, questions related to the risk factors of Toxoplasmosis in cats, and practices surrounding the cat owners, like how they handle cat litter, whether their cat was indoor or outdoor, contact of the cat with other cats, and straying behaviour of their cat. A total of 178 blood samples were collected from both stray and domestic cats in this study. Of the 178 samples tested for T. gondii IgG antibodies, 88 were from domestic cats and 90 from stray cats. 30 out of 178 samples were positive for antibodies, indicating an overall seroprevalence of 16.85%. Of the 30 positive seropositive samples, 60% (18) came from domestic cats and 40% (12) from stray cats. The seroprevalence for domestic cats was found to be 20.4% and that of strays 13.3%. The two risk factors found to be associated with toxoplasma Seropositivity were sex (p=0.007, 95%, CI 0.071,0.976) and diet (p=0.038, 95%, CI 1.395,95.418) It was concluded that Toxoplasma gondii was widely spread in the study population. The presence of the antibodies indicated that at one point in their lives, these cats were actively infected and shedding oocysts in the environment, thereby posing a risk to humans and other species like sheep and chickens.

PIMMS43 is required for malaria parasite immune evasion and sporogonic development in the mosquito vector

After being ingested by a female Anopheles mosquito during a bloodmeal on an infected host, and before they can reach the mosquito salivary glands to be transmitted to a new host, Plasmodium parasites must establish an infection of the mosquito midgut in the form of oocysts. To achieve this, they must first survive a series of robust innate immune responses that take place prior to, during, and immediately after ookinete traversal of the midgut epithelium. Understanding how parasites may evade these responses could highlight new ways to block malaria transmission. We show that an ookinete and sporozoite surface protein designated as PIMMS43 (Plasmodium Infection of the Mosquito Midgut Screen 43) is required for parasite evasion of the Anopheles coluzzii complement-like response. Disruption of PIMMS43 in the rodent malaria parasite Plasmodium berghei triggers robust complement activation and ookinete elimination upon mosquito midgut traversal. Silencing components of the complement-like system through RNAi largely restores ookinete-to-oocyst transition but oocysts remain small in size and produce a very small number of sporozoites that additionally are not infectious, indicating that PIMMS43 is also essential for sporogonic development in the oocyst. Antibodies that bind PIMMS43 interfere with parasite immune evasion when ingested with the infectious blood meal and significantly reduce the prevalence and intensity of infection. PIMMS43 genetic structure across African Plasmodium falciparum populations indicates allelic adaptation to sympatric vector populations. These data add to our understanding of mosquito-parasite interactions and identify PIMMS43 as a target of malaria transmission blocking.

Proteomic Array Analysis of an Epithelial Cell Model Infected with Cryptosporidium

Cryptosporidiosis is a human diarrheal disease caused by the etiological agent Cryptosporidium. This parasite abounds in the environment in resistant oocyst form and can cause life-threatening consequences in immunosuppressed individuals. By evaluating the fundamental mechanisms of pathogenesis in the host, it is helpful to deduce proteins involved in virulence. Parasite-induced host cell signaling events and subsequent cytoskeletal remodeling were investigated by using cultured human ileocecal carcinoma epithelial (HCT-8) cells inoculated with C. parvum sporozoites. Although many efforts have been made to characterize gene expression changes, these changes may not necessarily reflect changes in protein expression within an infected cell. New applications in proteomic arrays have the added benefit of studying changes in protein expression and functionality. In this study, we use a protein array platform to observe changes in protein expression and function when host cells are infected with C. parvum sporozoites. Previous studies have shown that the invasion of epithelial cells involves host-signaling whereby host cell actin reorganization allows for the enveloping of the parasite. A major signaling pathway implicated in actin polymerization involves the activation and deactivation of proteins containing SH2 domains, such as CRKL, PIK3, ABL, and the Src protein tyrosine kinase (PTK) family. Our studies show that a phospho-proteomic array of HCT-8 cells inoculated with the pathogen have a 0.87-fold increase in the phosphorylated form of c-Abl. Moreover, an SH2 proteomic array overlayed with protein lysates from uninfected and infected cells revealed that infected protein lysates had decreased binding (66%) to the SH2 domain of CRKL and increased binding (45%) to the SH2 domain of PIK3. These findings demonstrate a prominent role for proteins that may play a role in formation and stabilization of microvillar protrusions which envelope the pathogen.

Parasite's Double Play Helps Evade Mouse Immune Response

Researchers estimate that about onethird of the world’s population has been infected by Toxplasma gondii. Most have silent infections; their immune system keeps the parasite in check and there are no clinical signs of disease. However, when T. gondii does cause illness, the resulting disease, toxoplamosis, can be unpleasant. Mild cases may feel like the flu, but more serious cases can result in eye infections and damage. Most importantly, pregnant women who become infected may miscarry, or the child may suffer neurological symptoms. Except for congenital transmission, the parasite does not appear to spread between people. Human infections generally result from handling or eating raw meat or from eating food or drinking water contaminated with the oocyst form of the parasite. Cats play a key role in T. gondii evolution. The parasite reproduces sexually when an infected animal is eaten by a cat, which then sheds oocysts in its feces; this is why pregnant women are often advised to have someone else change their cat’s litter box. Rodents, which cats eat, are therefore important intermediate hosts. New research by Martin Fleckenstein and colleagues examines the virulence of various T. gondii strains, and highlights the role mice play in the evolution of the parasite. The researchers demonstrate how some T. gondii strains deflect the mouse immune response and result in a symptomatic infection. They elucidate for the first time the role of a pseudokinase called ROP5, which is essential for virulence. Mice exposed to two of the three main strains of T. gondii develop a chronic infection with relatively mild symptoms. This is due to a class of proteins called interferon-gamma-inducible immunity-related GTPases (IRG proteins), which are key to the mouse immune response. Shortly after infection, IRGs attach to the vacuole surrounding the parasite and aid in its destruction, resulting in clearance of the parasite and thus restricting the spread of the infection. However, infection by one of the three strains (type I) can produce serious disease in mice. Researchers knew that this virulent T. gondii strain secretes a protein kinase called ROP18 that inactivates the mouse IRG proteins by phosphorylating specific sites. T. gondii also produces a pseudokinase called ROP5 when it invades mouse cells. Both these proteins are key to the progression of disease, but how ROP5 affected virulence was not understood. To understand ROP5’s role in virulence, the researchers began by demonstrating that ROP5 binds directly to IRG proteins in T. gondii–infected cells. Three ROP5 isoforms exist; all were identified in this assay. Then, researchers infected mouse cells with wild-type virulent parasites, and also parasites in which either ROP5 or ROP18 was deleted. The deletion strains did not phosphorylate IRG proteins, and as a result were not virulent. That the ROP5 deletion strain was avirulent suggested a necessary role in virulence for the protein. To demonstrate that the ROP5 loss was actually responsible for the loss of virulence in ROP5 deletion strains, the researchers took deletion strains and restored the expression of two isoforms, ROP5AIII and ROP5BIII. These restored parasites were as virulent as the wild-type strains. However, as ROP5 had previously been shown to not have an inherent catalytic activity, this suggests that it acts as a co-factor for ROP18. Finally, researchers identified where ROP5 binds to IRG proteins, and what conformational changes that binding produces. They found that ROP5 remained relatively rigid upon binding to IRG proteins—that is, they saw little change in ROP5’s structure in its bound and unbound states. The IRG proteins, however, do undergo a conformational change on binding, which could explain how ROP5 facilitates binding of ROP18 to these proteins. The scientists identified a likely binding site and inactive conformation of the IRG protein. Further work using mutagenesis showed that while the ROP5 binding site is near the IRG activation site, it is clearly distinct—and that the ROP5 binding site is also clearly distinct from ROP18 targets. That is, ROP5 binding may prepare the IRG

Obtaining highly purified Toxoplasma gondii oocysts by a discontinuous cesium chloride gradient.

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii cysts in meat1, 2. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk3-5. To date, research on understanding the prevalence of T. gondii oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment 5, 6. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation7.

Obtaining Highly Purified <em>Toxoplasma gondii</em> Oocysts by a Discontinuous Cesium Chloride Gradient

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii cysts in meat1, 2. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk3-5. To date, research on understanding the prevalence of T. gondii oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment 5, 6. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation7.

Direct Detection of Cryptosporidium parvum Oocysts by Immunomagnetic Separation–Polymerase Chain Reaction in Raw Milk

Cryptosporidium parvum is an emerging protozoan parasite responsible for several serious outbreaks of cryptosporidiosis, an enteric infection characterized by severe intestinal distress. This parasite can be transmitted through contaminated water and raw food in the oocyst form, which is resistant to many environmental stresses and food processes. C. parvum is also commonly found on dairy farms and could be transmitted to humans through contaminated raw milk and dairy products. Thus, an immunomagnetic separation–polymerase chain reaction assay for direct detection of C. parvum oocysts in milk was developed. The procedure was able to detect <10 C. parvum oocysts. Thus, it could be used for monitoring milk samples.

Regulation and trafficking of three distinct 18 S ribosomal RNAs during development of the malaria parasite

The human malaria parasite Plasmodium vivax has been shown to regulate the transcription of two distinct 18 RNAs during development. Here we show a third and distinctive type of ribosome that is present shortly after zygote formation, a transcriptional pattern of ribosome types that relates closely to the developmental state of the parasite and a phenomenon that separates ribosomal types at a critical phase of maturation. The A-type ribosome is predominantly found in infected erythrocytes of the vertebrate and the mosquito blood meal. Transcripts from the A gene are replaced by transcripts from another locus, the O gene, shortly after fertilization and increase in number as the parasite develops on the mosquito midgut. Transcripts from another locus, the S gene, begins as the oocyst form of the parasite matures. RNA transcripts from the S gene are preferentially included in sporozoites that bud off from the oocyst and migrate to the salivary gland while the O gene transcripts are left within the oocyst. Although all three genes are typically eukaryotic in structure, the O gene transcript, described here, varies from the other two in core regions of the rRNA that are involved in mRNA decoding and translational termination. We now can correlate developmental progression of the parasite with changes in regions of rRNA sequence that are broadly conserved, where sequence alterations have been related to function in other systems and whose effects can be studied outside of Plasmodium. This should allow assessment of the role of translational control in parasite development.

Gamma-Irradiated and Nonirradiated Eimeria tenella Sporozoites Exhibit Differential Uracil Uptake and Expression of a 7- to 10-kDa Metabolic Antigen

Eimeria tenella sporozoites were exposed in the oocyst form to either an optimum (15 kRad) or a high (25 kRad) dose of gamma irradiation and used to infect cultured chicken embryo fibroblasts (CEF). The sporozoite-infected CEF monolayer was pulsed at time of infection or 24 hr postinfection with [ 3H]uracil and harvested 24 hr later to measure sporozoite metabolic activity. Sporozoites exposed to either 0 or 15 kRad gamma irradiation incorporated similar ( P > 0.05) amounts of [ 3H]uracil during the first and second 24-hr periods after infection. However, there was a significant decrease ( P < 0.05) in [ 3H]uracil uptake by 25 kRad-exposed sporozoites compared to nonirradiated and 15 kRad-irradiated sporozoites. Indirect immunofluorescence (IFA) staining of E. tenella sporozoite-infected CEFs using monoclonal antibodies (MAb) specific for somatic or "metabolic" antigens showed that gamma irradiation also affected the release of intracellular metabolites. Regardless of irradiation dose, extracellular sporozoites exhibited similar intensity of immunofluorescence when stained with either somatic antigen- or metabolic antigen-reactive MAb. Also, somatic antigen expression was similar for intracellular parasites irrespective of radiation dose. However, metabolic 7- to 10-kDa antigen expression by 25 kRad-irradiated sporozoites was markedly reduced compared to nonirradiated or 15 kRad-irradiated intracellular sporozoites. These results were corroborated by immunostaining sporozoite/CEF protein impregnated Immobilon membrane with somatic or metabolic 7- to 10-kDa antigen-reactive MAb. These findings may indicate that the metabolic 7- to 10-kDa antigen is involved in protective immunity elicited by nonirradiated and/or 15 kRad-irradiated E. tenella sporozoites.

Identification of Cryptosporidium in Paraffin-Embedded Tissue Sections with the Use of a Monoclonal Antibody

Recently a monoclonal antibody has been developed against the oocyst form of Cryptosporidium species for use in detecting the organism in fecal material. The authors undertook this study to determine if this antibody could be used in identifying Cryptosporidium species in paraffin-embedded tissue sections. Three biopsies showing Cryptosporidiosis as diagnosed by characteristic appearance by light and electron microscopy were selected from the surgical pathology files at the authors' institution. Paraffin sections were examined with the use of both an indirect immunofluorescence and an avidin-biotin-peroxidase technique. In all the cases, prominent staining of oocysts was seen; however, intracellular trophozoites did not react with the antibody. The authors conclude that this antibody can be used to identify Cryptosporidium species oocysts in tissue sections and that the antibody appears to be specific for the oocyst form only.

Absorption of amino acids from the human buccal cavity.

appropriate controls the concentration of each amino acid could be established. The absence of glycylproline ( < 0 . 5 m ~ ) from the samples were established by t.1.c. From each of two to six animals a minimum of four sacs were prepared from the ileum. Examination of sacs after incubation by lightmicroscopy failed to show any disruption caused by the experimental procedure. Fig. ] ( a ) shows the glycine and proline concentrations of sac contents (serosal fluid) expressed as percentages of those in sacs from uninfected control animals ( n = 15). Control values were 4.17 A 0.13 mM for glycine and 3.55 f 0. l O m M for proline. Less amino acid appeared in sacs as the infection progressed, with only a slight return towards normal within 21 days. Weight gain in infected animals was less than in controls, with a maximum difference on day 8. This correlates with the life-cycle of the parasite, which, after invading epithelial cells of the small intestine within hours of inoculation, leaves the animal around day 9 in the form of oocysts that can be detected in faeces. The extent to which the epithelial layer had recovered by day 21 is not yet known, but reports from chickens suggest that it is exceptional for macroscopic lesions to persist beyond day 12 (Turk, 1978).

Prevalence of Naturally Occurring Toxoplasma gondii Infections in Cats from U. S. Military Installations in Japan

To assess the possible role of the domestic cat as a source of human infection with Toxoplasma gondii, cats belonging to U. S. Forces families were obtained from installations in the Kanto Plain (Tokyo) area. A total of 90 cats of all ages were examined for serum antibodies by the indirect fluorescent antibody test (IFAT), for tissue cysts by mouse inoculation, and for fecal oocysts by microscopic examination and mouse inoculation. Forty (44%) had antibodies to toxoplasma, and the organism was isolated from tissues of 11%. An isolation was made from feces of 1 adult cat. This indicates that domestic cats could be a significant source of soil contamination with infectious oocysts in U. S. military housing areas. For many years the mechanism of transmission of human-acquired toxoplasmosis was unknown. Other than the implication of raw or undercooked meat in a few situations (Desmonts et al., 1965; Kean et al., 1969; Magaldi et al., 1969) no sources of natural human infections have been identified. The recent discovery of a resistant oocyst form of Toxoplasma gondii, which is shed in the feces of the cat (Frenkel et al., 1970; Hutchison et al., 1970; Sheffield and Melton, 1970), has offered a simple and apparently logical explanation for the means of transmission of this widespread infection. However likely a source of human infection the oocyst might appear to be, the true role of this stage in the epidemiology of human infection has yet to be determined. The intestinal cycle producing the infectious oocyst is known to occur only in the domestic cat and in two species of wild felines (Frenkel, 1970). Experimental feedings of tissue cysts to ferrets and dogs (Witte and Piekarski, 1970), mice, rats, hamsters, guinea pigs, dogs, domestic rabbits, opossums, Japanese quail, chickens, and pigeons (Frenkel et al., 1970) have all failed to yield oocysts. Although these studies do not rule out the possibility of other species serving as reservoir Received for publication 17 March 1972. * Present address: Department of Biology, Northern Michigan University, Marquette, Michigan 49855. tPresent address: U. S. Army Medical Research Unit, Panama, Box 2011, Balboa Heights, Canal Zone. ho ts, they do leave the domestic cat as the only current likely suspect. The house cat has been shown to be frequently infected in many parts of the world (Dubey, 1968; Work, 1969). In Japan, only small numbers have been examined, but high infection rates, over 60%, were reported (Katsube et al., 1967). The domestic cat is frequently maintained as a household pet by U. S. military families, but the high cost of shipping usually precludes taking them back to the U. S. This, combined with the close official control over pets within U. S. Forces installati ns, provided an unusual opportunity to gain additional information regarding the prevalence of infection in cats in Japan, and specifically those of U. S. families, to assess their role as a potential reservoir of Toxoplasma gondii. MATERIALS AND METHODS Cats were obtained from donors and from pounds at triservice military installations throughout the Kanto Plain area of Japan. Immediately after acquisition, 1 to 3 stools were collected whenever possible from each cat and processed by sucrose (sp gr 1.18) flotation. Microscopic examination was made, and following incubation at room temperature for 6 to 10 days in 2% potassium dichromate, 0.5 ml of washed fecal suspension was injected ip into each of 5 mice. The cats were bled by heart puncture, killed by sodium phenobarbital injection, and examined as follows: (1) In the largest group consisting of 72 cats, approximately 3 g of brain tissue was homogenized in a tissue grinder in 10 ml of physiologic saline with 0.05 ml of penicillin-streptomycin solution, and 0.5 ml was injected ip into each of 6 mice. (2) In a second group of 9 cats, in addition to