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.     

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.     

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.     

Participation of myosin in gliding motility and host cell invasion by Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that actively invades mammalian cells using a unique form of gliding motility that critically depends on actin filaments in the parasite. To determine if parasite motility is driven by a myosin motor, we examined the distribution of myosin and tested the effects of specific inhibitors on gliding and host cell invasion. A single 90 kDa isoform of myosin was detected in parasite lysates using an antisera that recognizes a highly conserved myosin peptide. Myosin was localized in T. gondii beneath the plasma membrane in a circumferential pattern that overlapped with the distribution of actin. The myosin ATPase inhibitor, butanedione monoxime (BDM), reversibly inhibited gliding motility across serum-coated slides. The myosin light-chain kinase inhibitor, KT5926, also blocked parasite motility and greatly reduced host cell attachment; however, these effects were primarily caused by its ability to block the secretion of microneme proteins, which are involved in cell attachment. In contrast, while BDM partially reduced cell attachment, it prevented invasion even under conditions in which microneme secretion was not affected, indicating a potential role for myosin in cell entry. Collectively, these results indicate that myosin(s) probably participate(s) in powering gliding motility, a process that is essential for cell invasion by T. gondii .     

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.     

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).     

Concerted action of two formins in gliding motility and host cell invasion by Toxoplasma gondii

The invasive forms of apicomplexan parasites share a conserved form of gliding motility that powers parasite migration across biological barriers, host cell invasion and egress from infected cells. Previous studies have established that the duration and direction of gliding motility are determined by actin polymerization; however, regulators of actin dynamics in apicomplexans remain poorly characterized. In the absence of a complete ARP2/3 complex, the formin homology 2 domain containing proteins and the accessory protein profilin are presumed to orchestrate actin polymerization during host cell invasion. Here, we have undertaken the biochemical and functional characterization of two Toxoplasma gondii formins and established that they act in concert as actin nucleators during invasion. The importance of TgFRM1 for parasite motility has been assessed by conditional gene disruption. The contribution of each formin individually and jointly was revealed by an approach based upon the expression of dominant mutants with modified FH2 domains impaired in actin binding but still able to dimerize with their respective endogenous formin. These mutated FH2 domains were fused to the ligand-controlled destabilization domain (DD-FKBP) to achieve conditional expression. This strategy proved unique in identifying the non-redundant and critical roles of both formins in invasion. These findings provide new insights into how controlled actin polymerization drives the directional movement required for productive penetration of parasites into host cells.     

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.     

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.     

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.     

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.     

Prevalence of antibodies to Neospora caninum, Sarcocystis neurona, and Toxoplasma gondii in wild horses from central Wyoming

Sarcocystis neurona, Neospora caninum, N. hughesi, and Toxoplasma gondii are 4 related coccidians considered to be associated with encephalomyelitis in horses. The source of infection for N. hughesi is unknown, whereas opossums, dogs, and cats are the definitive hosts for S. neurona, N. caninum, and T. gondii, respectively. Seroprevalence of these coccidians in 276 wild horses from central Wyoming outside the known range of the opossum (Didelphis virginiana) was determined. Antibodies to T. gondii were found only in 1 of 276 horses tested with the modified agglutination test using 1:25, 1:50, and 1:500 dilutions. Antibodies to N. caninum were found in 86 (31.1%) of the 276 horses tested with the Neospora agglutination test--the titers were 1:25 in 38 horses, 1:50 in 15, 1:100 in 9, 1:200 in 8, 1:400 in 4, 1:800 in 2, 1:1,600 in 2, 1:3,200 in 2, and 1:12,800 in 1. Antibodies to S. neurona were assessed with the serum immunoblot; of 276 horses tested, 18 had antibodies considered specific for S. neurona. Antibodies to S. neurona also were assessed with the S. neurona direct agglutination test (SAT). Thirty-nine of 265 horses tested had SAT antibodies--in titers of 1:50 in 26 horses and 1:100 in 13. The presence of S. neurona antibodies in horses in central Wyoming suggests that either there is cross-reactivity between S. neurona and some other infection or a definitive host other than opossum is the source of infection. In a retrospective study, S. neurona antibodies were not found by immunoblot in the sera of 243 horses from western Canada outside the range of D. virginiana.     

Neospora caninum: antibodies directed against tachyzoite surface protein NcSRS2 inhibit parasite attachment and invasion of placental trophoblasts in vitro

Polyclonal and monoclonal antibodies to native Neospora caninum tachyzoite surface protein NcSRS2 were generated and tested in vitro for their ability to neutralize tachyzoite attachment to and invasion of host cells. Host cells included Vero cells and a newly cloned, immortalized ovine trophoblast cell line obtained from primary cultures of ovine placenta. The ovine trophoblasts had morphology consistent with fetal trophoblasts and expressed mRNA for interferon-tau, a marker for trophoblasts. Native NcSRS2 was used to immunize mice to obtain monospecific anti-NcSRS2 polyclonal serum and anti-NcSRS2 monoclonal antibodies. Compared to irrelevant antibodies, monospecific anti-NcSRS2 serum and two anti-NcSRS2 monoclonal antibodies, 100.2.4.4 and 119.4.9.10, significantly blocked invasion of tachyzoites into both trophoblasts and Vero cells. Parasite attachment, assessed by IFA, was significantly reduced by anti-NcSRS2 mAb 100.2.4.4 and monospecific serum. The findings provide rationale to investigate a role for antibodies to NcSRS2 in prevention of N. caninum transplacental transmission in vivo.     

Neospora caninum and Toxoplasma gondii: a novel adhesion/invasion assay reveals distinct differences in tachyzoite-host cell interactions

This paper describes an adhesion/invasion assay, based on combined pyrrolidine dithiocarbamate (PDTC) and antibody treatment of parasites followed by quantitative real-time PCR. This PDTC-PCR assay can be used to comparatively assess the participation of host cell- and parasite-associated components during host cell adhesion and entry by Neospora caninum and Toxoplasma gondii tachyzoites, respectively, and is potentially applicable to any other apicomplexan parasite. The assay allows to determine the parasite invasion rate in relation to the overall number of parasites which interact with host cells in any given experiment, and thus represents a significant improvement to conventional microscopic assays in terms of accuracy and reproducibility. Using this assay it was possible to show that adhesion and invasion of N. caninum tachyzoites are two distinct and separated events, in that N. caninum tachyzoites preferentially utilise host cell surface chondroitin sulphates for adhesion, but not for the host cell invasion process. Application of the PDTC-PCR assay also demonstrated that N. caninum and T. gondii tachyzoites differ largely with regard to the functional involvement of proteases in adhesion and invasion of host cells. Thus, although phylogenetically closely related, N. caninum and T. gondii are biologically quite different and exhibit distinct dissimilarities with regard to host cell interactions.     

Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii

Apicomplexan parasites rely on actin-based gliding motility to move across the substratum, cross biological barriers, and invade their host cells. Gliding motility depends on polymerization of parasite actin filaments, yet ～98% of actin is nonfilamentous in resting parasites. Previous studies suggest that the lack of actin filaments in the parasite is due to inherent instability, leaving uncertain the role of actin-binding proteins in controlling dynamics. We have previously shown that the single allele of Toxoplasma gondii actin depolymerizing factor (TgADF) has strong actin monomer-sequestering and weak filament-severing activities in vitro. Here we used a conditional knockout strategy to investigate the role of TgADF in vivo. Suppression of TgADF led to accumulation of actin-rich filaments that were detected by immunofluorescence and electron microscopy. Parasites deficient in TgADF showed reduced speed of motility, increased aberrant patterns of motion, and inhibition of sustained helical gliding. Lack of TgADF also led to severe defects in entry and egress from host cells, thus blocking infection in vitro. These studies establish that the absence of stable actin structures in the parasite are not simply the result of intrinsic instability, but that TgADF is required for the rapid turnover of parasite actin filaments, gliding motility, and cell invasion.     

Antibodies to Toxoplasma gondii and Neospora caninum in captive neotropical and exotic wild canids and felids

This study was designed to detect antibodies to Toxoplasma gondii and Neospora caninum in wild captive carnivores maintained in Brazilian zoos. Blood samples were collected from 142 Brazilian wild felids and 19 exotic felids in zoos, and 3 European wolves (Canis lupus) and 94 Brazilian wild canids maintained in captivity in Brazilian zoos of S?o Paulo, Mato Grosso states and Federal District. One hundred and two (63.4%) and 70 (50.3%) of the 161 wild felids tested were seropositive for T. gondii and N. caninum by indirect immunofluorescent assay test (IFAT), respectively. Among sampled wild canids, 49 (50.5%) and 40 (41.2%) animals were seropositive for T. gondii and N. caninum antigens by IFAT, respectively. To our knowledge, this is the first serological detection of antibodies to N. caninum in Brazilian wild captive felids and bush dogs (Speothos venaticus (Lund)).     

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.     

Seroprevalence of Toxoplasma gondii and Neospora caninum in slaughtered pigs in the Czech Republic

In the Czech Republic, sera from 551 clinically healthy adult slaughtered pigs (females, 6-8 months old) were collected during the first half of June in 2010. Sera were tested for Toxoplasma gondii-specific IgG antibodies by an enzyme-linked immunosorbent assay; samples with more than 50% S/P were considered as positive. The same samples were also analysed for Neospora caninum antibodies using a commercial competitive-inhibition enzyme-linked immunosorbent assay; samples with more than 30% inhibition were considered as positive. Antibodies against T. gondii were found in 198 pigs (36%) in all districts with prevalences ranging from 18% to 75%. Antibodies against N. caninum were found in 16 pigs (3%); positive animals were found in 4 districts with prevalences ranging from 1% to 20%. Indication of mixed infections (concurrent presence of both N. caninum and T. gondii antibodies) was found in 8 (1·5%) pigs. The results of our study indicate that pigs in the Czech Republic have a relatively high seroprevalence for T. gondii, while they have only a low seroprevalence for N. caninum. Therefore, natural infection with T. gondii seems to be very common in Czech pigs. It is the first evidence of N. caninum antibodies in pigs in the Czech Republic. These results complete data about N. caninum infection in pigs in Europe.     

Prevalence of Neospora caninum and Toxoplasma gondii antibodies in wild ruminants from the countryside or captivity in the Czech Republic

In the Czech Republic, sera from 720 wild ruminants were examined for antibodies to Neospora caninum by screening competitive-inhibition enzyme-linked immunosorbent assay and confirmed by indirect fluorescence antibody test (IFAT); the same sera were also examined for antibodies to Toxoplasma gondii by IFAT. Neospora caninum antibodies were found in 14% (11 positive/79 tested) roe deer (Capreolus capreolus), 14% (2/14) sika deer (Cervus nippon), 6% (24/ 377) red deer (Cervus elaphus), 1% (2/143) fallow deer (Dama dama), 3% (3/105) mouflon (Ovis musimon), and none of 2 reindeer (Rangifer tarandus). Toxoplasma gondii antibodies were found in 50% (7/14) sika deer, 45% (169/377) red deer, 24% (19/79) roe deer, 17% (24/143) fallow deer, 9% (9/105) mouflon, and 1 of 2 reindeer. In 42 samples of wild ruminants that tested positive for N. caninum antibodies, 28 (67% of the positive N. caninum samples) reacted solely to N. caninum. This is the first evidence of N. caninum infection in mouflon, the first N. caninum seroprevalence study in farmed red deer, and the first survey of N. caninum in wild ruminants from the Czech Republic.