Ultrastructure of tachyzoites, bradyzoites and tissue cysts of Neospora caninum

Dividing tachyzoites of Neospora caninum were 4 x 3 microns and had ultrastructural characteristics typical for the cyst-forming coccidia. Unusual ultrastructural characteristics of fully-formed tachyzoites included no micropores, 8-12 anterior and 4-6 posterior rhoptries, and a few posterior micronemes. Most tachyzoites were located free in the host cell cytoplasm; only a few occurred within a parasitophorous vacuole. Parasite multiplication appeared to be rapid because most organisms were in various stages of endodyogeny. Neural tissue cysts of N. caninum were 24.3 x 19.2 microns and contained 50-200 bradyzoites (7.3 x 1.5 microns), which lacked micropores. The cyst wall was 0.74-1.12 microns thick and consisted of the primary cyst wall (the parasitophorous vacuole membrane) and a thick granular layer with electron-dense vesicles.     

Autofluorescence of Toxoplasma gondii and Neospora caninum cysts in vitro

Autofluorescence of Toxoplasma gondii and Neospora caninum was studied by fluorescence microscopy during their differentiation from tachyzoites to bradyzoites in vitro using Vero as host cells. Stage conversion into bradyzoites and cysts was confirmed by immunofluorescent microscopy and Western blot analysis using SAG1- and BAG1-specific antibody, respectively. From day 4 postinfection (PI), pale blue autofluorescence of the bradyzoites and tissue cysts was observed with UV light at 330-385 nm, which coincided with the onset of cyst development. This autofluorescence under UV light of bradyzoites and tissue cysts increased in intensity from days 8 to 10 PI. In contrast to the autofluorescence shown by bradyzoites and cysts, tachyzoites and parasitophorous vacuoles containing tachyzoites never autofluoresced at any time examined. Autofluorescence of the cystic stages was of sufficient intensity and duration to allow the detection of cysts and bradyzoites of T. gondii and N. caninum. In this study, we describe for the first time the autofluorescence properties of in vitro-induced bradyzoites and cysts of T. gondii and N. caninum.     

Developmental differentiation between tachyzoites and bradyzoites of Toxoplasma gondii

An important event in the pathogenesis of toxoplasmosis is the interconversion between the bradyzoite and the tachyzoite stage of Toxoplasma gondii within the intermediate host. The factors that influence either cyst formation (bradyzoites) or reactivation (tachyzoites) are unknown. Uwe Gross, Wolfgang Bohne, Martine Soête and Jean Fran?ois Dubremetz here describe current knowledge about the mechanisms that might lead to the induction of stage differentiation of this protozoan parasite.     

Application of proteomics for comparison of proteome of Neospora caninum and Toxoplasma gondii tachyzoites

Protein profiles of two isolates of Neospora caninum (KBA-2 and JPA1) and Toxoplasma gondii RH strain were investigated by proteomic approach. Approximately, 78% of protein spots on two-dimensional gel electrophoresis (2-DE) profiles and 80% of antigen spots on 2-DE immunoblotting profiles were exhibited to share the same pI and M(r) between KBA-2 and JPA1 of N. caninum. On the other hand, a total of 30 antigen spots of T. gondii were recognized on 2-DE immunoblotting profile using rabbit antiserum against N. caninum KBA-2. A number of homologue proteins, such as heat shock protein 70, tubulin alpha- and beta-chain, putative protein disulfide isomerase, actin, enolase and 14-3-3 protein homologue are believed as the conserved proteins in both N. caninum and T. gondii. On the contrary, NcSUB1, NcGRA2 and NCDG1 (NcGRA7) might be the species-specific proteins for N. caninum tachyzoites. The present study showed that the high degree of similarity between N. caninum isolates (KBA-2 and JPA1), whereas large differences between N. caninum and T. gondii were noticed by proteome comparisons.     

Development and ultrastructure of Besnoitia oryctofelisi tachyzoites,tissue cysts,bradyzoites, schizonts and merozoites

Development and structure of different life cycle stages of Besnoitia oryctofelisi which has a rabbit-cat life cycle was studied by light and transmission electron microscopy. For light microscopy, Besnoitia oryctofelisi-infected tissues were stained with haematoxylin-eosin, periodic acid Schiff (PAS) reagent, and immunohistochemically with rabbit anti-B. oryctofelisi polyclonal antibodies and anti-BAG-1 antibodies. In vitro and in vivo-derived tachyzoites were 5-6 microm long and they were found to divide by endodyogeny. In tachyzoites, the nucleus was often central, and micronemes were few and located anterior to the nucleus. Earliest tissue cysts were seen in gerbils starting 12 days p.i. Early tissue cysts had an outer PAS-positive cyst wall, a middle PAS-negative host cell layer, and an inner PAS-negative parasitophorous vacuolar membrane. Organisms in early tissue cysts were PAS-negative, did not stain with anti-BAG-1 antibodies, and amylopectin granules and enigmatic bodies were absent. Tissue cysts beginning 17 days p.i. contained organisms that became PAS-positive and reacted with anti-BAG-1 antibodies, indicating they were bradyzoites. Immunoreactivity with polyclonal anti-B. oryctofelisi antibodies suggested that Besnoitia species bradyzoites are encapsulated by the host cell. Bradyzoites (10 microm) were about twice the length of tachyzoites and contained enigmatic bodies characteristic of Besnoitia bradyzoites. Unlike tachyzoites and tissue cysts, schizonts were located intravascularly in the lamina propria of the small intestine of cats. Merozoites were 5-6 microm long, had few rhoptries and amylopectin granules, had numerous micronemes and had a terminal nucleus.     

Examination of tissue cyst formation by Toxoplasma gondii in cell cultures using bradyzoites, tachyzoites, and sporozoites

Tissue cyst formation by a goat isolate (GT-1) of Toxoplasma gondii was examined in bovine monocyte, human fetal lung, and Madin-Darby bovine kidney cell cultures. Transmission electron microscopy (TEM) and cat feeding studies indicated that tissue cysts were present in all 3 cell lines examined. Tissue cysts were first seen 3 days postinoculation (PI) using TEM. Standard cell culture procedures were used and no additional condition was needed to induce tissue cyst formation. Cats fed cell cultures excreted T. gondii oocysts in their feces 5-7 days PI. These oocysts caused lethal infections in mice. Tissue cysts were produced in cell cultures regardless if the initiating inoculum consisted of bradyzoites, sporozoites, or a mixture of bradyzoites and tachyzoites. Tissue cyst formation has been followed through 40 subpassages of infected cells. By TEM tissue cysts still were present after 40 passages, but when 40th-passaged cultures were fed to cats, oocytsts were not excreted. This indicates that the parasite had become oocystless after repeated passage in vitro.     

Infections in mice with tachyzoites and bradyzoites of Neospora caninum (Protozoa: Apicomplexa)

Tachyzoites of 2 isolates of Neospora caninum (NC-1 and NC-2) were inoculated subcutaneously (s.c.), intraperitoneally (i.p.), or orally into mice to compare the effects of route of inoculation on pathogenicity. Mice developed more severe disease, and disease occurred sooner when inoculated with the NC-1 isolate compared to the NC-2 isolate. Deaths occurred earlier in mice inoculated i.p. with either isolate. Mice inoculated orally or s.c. with tachyzoites responded similarly to infection. Tissue cysts of the NC-2 isolate produced infections in mice following oral or s.c. inoculation. Lesions seen in mice inoculated with tachyzoites or bradyzoites were primarily acute pneumonia, myositis, encephalitis, ganglioradiculoneuritis, and pancreatitis. In vitro studies demonstrated that tachyzoites of both isolates were killed by incubation in pepsin-HCl solution but not 1% trypsin solution. Bradyzoites of the NC-2 isolate were able to withstand treatment with pepsin-HCl solution.     

Neospora caninum emerges from the shadow of Toxoplasma gondii

It is sometimes easy to make the mistake of assuming that everything that holds true for Toxoplasma gondii is also true for its relative Neospora caninum. However, a recurring theme in the recent review by Hemphill et al. is not the similarities but the striking differences between the two parasites.     

Comparison of the major antigens of Neospora caninum and Toxoplasma gondii

The Apicomplexa are a diverse group of parasitic protozoa with very ancient phylogenetic roots. Consistent with their phylogeny, the extant species share conserved proteins and traits that were found in their apicomplexan progenitor, but at the same time they have diverged to occupy different biological niches (e.g. host-range and cell type). Characterisation of gene and protein diversity is important for distinguishing between related parasites, for determining their phylogeny, and for providing insight into factors that determine host restriction, cell preference, and virulence. The value of molecular characterisations and comparisons between species is well illustrated by the close phylogenetic relationship between Neospora caninum and Toxoplasma gondii. These two organisms have nearly identical morphology and can cause similar pathology and disease. Consequently, N. caninum has often been incorrectly identified as T. gondii, thus demonstrating the need for studies addressing the molecular and antigenic composition of Neospora. In this review, we describe the major antigenic proteins that have been characterised in N. caninum. These show homology to T. gondii proteins, yet possess unique antigenic characteristics that distinguish them from their homologues and enable their use for specific serological diagnoses and parasite identification.     

Endogenous and exogenous transplacental infection in Neospora caninum and Toxoplasma gondii

It is clear from researching the vertical transmission of Neospora caninum in cattle that the terms 'vertical', 'congenital' and, indeed, 'transplacental' are inadequate for describing two extremely different situations that have fundamentally different immunological, epidemiological and control implications. A similar situation pertains to Toxoplasma gondii in different hosts. We advocate the use of the terms 'endogenous transplacental infection (TPI)' to define foetal infection from a recrudescent maternal infection acquired before pregnancy (and probably prenatally) and 'exogenous TPI' to define foetal infection that occurs as a result of an infection of the dam during pregnancy.     

Neospora caninum and Toxoplasma gondii antibodies in dogs from Durango City, Mexico

Toxoplasma gondii and Neospora caninum are structurally similar parasites, with many hosts in common. The prevalence of antibodies to T. gondii and N. caninum was determined in sera from dogs from Durango City, Mexico. Using a modified agglutination test, antibodies to T. gondii were found in 52 (51.5%) of the 101 dogs with titers of 1:25 in 27, 1:50 in 11, 1:100 in 5, 1:200 in 4, 1:400 in 2, 1:800 in 2, and 1:3,200 or higher in 1. Antibodies to N. caninum were determined by the indirect immunofluorescent antibody test (IFAT) and the Neospora sp. agglutination test (NAT). Two of the 101 dogs had N. caninum antibodies; these dogs did not have T. gondii antibodies, supporting the specificity of the tests used. The N. caninum antibody titers of the 2 dogs were: 1:400 by IFAT and 1:200 by NAT in 1, and 1:25 by NAT and IFAT in the other. Results indicate that these 2 structurally similar protozoans are antigenically different.     

Growth of and competition between Neospora caninum and Toxoplasma gondii in vitro

Competitive interactions between Neospora caninum and Toxoplasma gondii were studied because both species appear to have identical ecological niches in vitro. Tachyzoites of N. caninum (NC-1 isolate) and T. gondii (RH isolate) were compared in three in vitro studies: (1) rate of penetration of host cells; (2) generation time; and (3) competition between the two species when grown together in the same flask and allowed to compete for space. When tachyzoites of the two species were inoculated onto human foreskin fibroblasts, 3.24-times more N. caninum tachyzoites penetrated cells by 1 h p.i. At 3 h p.i., there were 2.87-times more N. caninum intracellular tachyzoites than T. gondii tachyzoites. The generation times for N. caninum (NC-1 isolate) and T. gondii (RH isolate) were approximately 14-15 h and 8-10 h, respectively. Before exponential growth occurred, both species displayed a lag period, which was 10-12 h for N. caninum and 8-10 h for T. gondii. To observe competition, equal numbers of tachyzoites of each species were mixed and inoculated into flasks of host cells, and the monolayers were allowed to proceed to >90% lysis before the next transfer. Competition was analysed for 31 days by labelling samples of each flask with a species-specific monoclonal antibody and determining the ratio of each species. In all trials, T. gondii outcompeted N. caninum. By 4 days p.i., 70% of the tachyzoites were T. gondii; this percentage increased to 97% by 23 days p.i. When the starting inoculum contained 75% N. caninum and 25% T. gondii tachyzoites, T. gondii was still competitively superior. When infected monolayers that were labelled with T. gondii-specific antibodies were examined, it was noted that both species can occupy and undergo endodyogeny in the same host simultaneously.     

Toxoplasma gondii and Neospora caninum infections in goat abortions from Argentina

The aims of this study were to identify the occurrence of Toxoplasma gondii and Neospora caninum abortions in goats from Argentina by serological, macroscopical and microscopical examination and bioassay, and to characterize the obtained isolates by molecular techniques. For this purpose, 25 caprine fetal fluids, 18 caprine fetal brains and 10 caprine placentas from 8 dairy/meat goat farms from Argentina were analyzed. Gestational age of the aborted fetuses was determined in 18 cases. Protozoal infections were detected by at least one of the applied diagnostic techniques in 44% (11/25) of examined fetuses; specifically, 24% (6/25) were positive to T. gondii, 8% (2/25) were positive to N. caninum and 12% (3/25) were positive to both parasites. In this study IFAT titers were similarly distributed in younger and older fetuses. Macroscopical and microscopical examination of one placenta revealed chalky nodules in the fetal cotyledons and normal intercotyledonary areas, as well as necrosis and calcification of mesenchymal cells in villi. Tachyzoites were observed in peritoneal wash from 2 mice inoculated with brain and a pool of brain and placenta of two fetuses. Cell culture growth of tachyzoites was achieved from one inoculated mouse, and confirmed as T. gondii by PCR. The T. gondii isolate was identified as atypical or non-canonical by nested-PCR-RFLP. This is the first study that investigated the involvement of N. caninum and T. gondii in cases of goat abortion in Argentina.     

Toxoplasma gondii and Neospora caninum antibodies in dogs from Grenada, West Indies

Toxoplasma gondii and Neospora caninum are structurally similar parasites with many common hosts. The prevalence of antibodies to T. gondii and N. caninum was determined in sera from dogs in Grenada, West Indies. Using a modified agglutination test, antibodies to T. gondii were found in 52 (48.5%) of the 107 dogs, with titers of 1:25 in 17, 1:50 in 19, 1:100 in 7, 1:1,600 in 5, and 1:3,200 or higher in 4. Seroprevalence increased with age from 2.2% in dogs <6 mo old to 18.9% in dogs older than 2 yr, indicating postnatal transmission of T. gondii in this population of canines. There was no correlation between the health of the dogs and the seroprevalence or magnitude of the T. gondii titer. Antibodies to N. caninum were determined by the indirect immunofluorescent antibody test (IFAT). Two of the 107 dogs had N. caninum antibodies (IFAT titers 1:100 and 1:400); these dogs had T. gondii titers of 1:1,600 and 1:50, respectively. Results indicate that these 2 structurally similar protozoa are antigenically different.     

The in vitro development of Neospora caninum bradyzoites

Neospora caninum is a recently identified apicomplexan protozoan parasite that is closely related to Toxoplasma gondii. Neospora caninum is of significant economic importance as it causes neurological disease and abortion in numerous animals. Antibodies to BAG1/hsp30 (also known as BAG5), a T. gondii bradyzoite-specific protein, have been demonstrated to react with N. caninum tissue cysts in vivo. Bradyzoite differentiation of N. caninum in vitro was investigated using culture conditions previously utilised for T. gondii in vitro bradyzoite development. Utilising the NC-Liverpool isolate of N. caninum, cyst-like structures developed within 3-4 days of culture of this parasite in human fibroblasts. In addition, an antigen reacting with mAb 74.1.8 (anti-BAG1) and rabbit anti-recombinant BAGI was demonstrable by immunofluorescence, fluorescence-activated cell sorter, and immunoblot analyses. Expression of this antigen was increased by stress conditions, similar to that which has been described for T. gondii bradyzoite induction. Cyst-wall formation in vitro, as assayed by lectin binding, did not occur as readily for N. caninum as it does for T. gondii.     

Prevalence of Antibodies to Neospora caninum and Toxoplasma gondii in Swedish Dogs

Neospora caninum is a newly described coccidian parasite which has been found in various species such as the dog, cattle, horse, sheep and goat. Morphologically it resembles Toxoplasma gondii with which it is related (Holmdahl et al. 1994), and with which it has earlier been confused. The life cycle of N caninum is only partially known. Tachyzoites and tissue cysts are the only known stages of the parasite, and transplacental transmission is the only known route of infection. Subclini-cally infected dams can transmit the parasite to their fetuses and successive offspring from the same mother might be born infected (Dubey et al. 1990b). Clinical neosporosis is mostly seen in pups or young dogs, and the majority or all pups in a litter are often affected. The disease is characterized by ascending paralysis of the legs, with the hind legs more severely affected than the front legs, paralysis of the jaw, difficulty in swallowing and muscle flaccidity and atrophy (Dubey 1992, Dubey & Lindsay 1993). Fatal infections with N caninum in dogs have been reported from many countries, e.g. Norway (Bjerkäs & Presthus 1988), USA (Dubey et al. 1988), Sweden (Uggla et al. 1989a,b) and the United Kingdom (Dubey et al. 1990a). Serological surveys for antibodies to N. caninum in dogs from Kansas, USA and England have shown a prevalence of 2 and 13%, respectively (Lindsay et al. 1990, Trees et al. 1993).     

Serum antibodies against Toxoplasma gondii and Neospora caninum in southeast Queensland dugongs

The dugong (Dugong dugon) is an herbivorous marine mammal that inhabits tropical inshore waters and thus may be vulnerable to pollutants and terrestrial pathogens as a result of coastal runoff. In this study, serum samples collected from live, wild dugongs (n = 114) in an embayment located on the urbanized southeast Queensland coast of Australia during 2008–2014, were measured for IgG antibody levels specific to Toxoplasma gondii and Neospora caninum. An ELISA used to measure T. gondii tachyzoite antibodies indicated a non-Gaussian distribution of antibody level, with five dugongs identified as high outliers. Mean levels of antibodies specific for T. gondii in dugongs sampled in 2014 were significantly higher than in 2010 (p = .006) and 2011 (p = .009) with an elevation in mean antibody levels after a major 2011 flood event relative to antibody levels prior to the flood (p < .0001). A competitive ELISA to detect N. caninum antibody indicated a normal distribution of antibody with no high outliers. Mean antibody level for N. caninum was highest in 2012 and declined significantly in 2014 (p = .004). This is the first survey of antibodies directed against T. gondii and N. caninum in dugongs and suggests future health monitoring of this species.     

Ingestion of Neospora caninum tissue cysts by Mustela species

Dogs are a definitive host of Neospora caninum, a protozoal parasite that causes abortion in cattle. Mustelids were tested to determine if they could also be definitive hosts. The procedures used were the same as those previously used to test dogs. Ermine (Mustela erminea), weasels (Mustela frenata) and ferrets (Mustela putorius) were fed N. caninum-infected mice. Neospora caninum oocysts were not observed. Mustelid faeces were fed to mice. The mice did not seroconvert and N. caninum was not detected in murine brains using tissue culture and PCR. The hypothesis that Mustela spp. are definitive hosts of N. caninum is not supported.     

Prevalence of Toxoplasma gondii and Neospora caninum in sika deer from eastern Hokkaido, Japan

Brain and serum were collected from 120 and 12 free-ranging sika deer (Cervus nippon yesoensis), respectively, from six regions in eastern Hokkaido during controlled hunts in the autumn of 2003. Brains were tested for Neospora caninum and Toxoplasma gondii DNA by polymerase chain reaction (PCR) assays. Antibodies to Toxoplasma gondii were measured by means of a latex agglutination test. No brain tested positive for either type of DNA, and no antibody to Toxoplasma gondii was detected in serum, suggesting a low prevalence of infection with these organisms in free-ranging sika deer from eastern Hokkaido. Further examination of multiple tissues by PCR and serologic surveys will be necessary to confirm this.     

A survey of Neospora caninum and Toxoplasma gondii infection in urban rodents from Brazil

Toxoplasma gondii is a protozoan parasite that infects humans and other warm-blooded animals; it uses feral and domestic cats as the definitive hosts. Neospora caninum is a protozoan parasite of animals whose life cycle is very similar to T. gondii but uses canids as definitive hosts. Small rodents play an important role in the life cycle of T. gondii , and a few findings indicated that they may be natural intermediate hosts for N. caninum . The present study was aimed at identifying infections by T. gondii and N. caninum in urban rodents. Infections by T. gondii were quantified using isolation of the parasite by bioassay in mice; molecular methods were also used for both parasites. Overall, 217 rodents were captured. Brain and heart tissues of all rodents were bioassayed in mice for the detection of T. gondii infection. Brain and heart tissues of 121 rodents had the DNA extracted for molecular analysis. Toxoplasma gondii was isolated by bioassay from a single rodent. From the 121 rodents tested for the presence of T. gondii DNA, 2 animals were positive. In contrast, DNA of N. caninum was not detected in any of the samples. In conclusion, the surveys of N. caninum and T. gondii infection in Rattus rattus , Rattus norvegicus , and Mus musculus captured in urban areas of S?o Paulo reveal a striking low frequency of occurrence of these infections.