Toxoplasma gondii: Infection natural congenital in cattle and an experimental inoculation of gestating cows with oocysts

Two studies, of a natural infection and an experimental infection, were performed in order to study congenital transmission of Toxoplasma gondii in cattle. In the first study, 50 fetuses were harvested from gestating cows that were eutanasied at a municipal slaughterhouse in Jaboticabal, São Paulo state, Brazil. In the second study, 11 gestating cows were divided into four groups for inoculation with T. gondii: GI consisted of three cows inoculated with 1.0 × 10⁵ oocysts during their first trimester of gestation; GII consisted of three cows inoculated with 1.0 × 10⁵ oocysts during their second trimester of gestation; GIII consisted of three cows inoculated with 1.0 × 10⁵ oocysts during their last trimester of gestation; and GIV consisted of two control cows, one during its first and the other during its second trimester of gestation. In both studies, the presence of T. gondii was confirmed both indirectly by immunofluorescence assay (IFAT). In the natural infection experiment, 18% (9/50) of the gestating cows were confirmed to have specific antibodies (IFAT – 1:64) against T. gondii. The bioassay was able to diagnose the presence of T. gondii in the tissue samples from three calves. In the second experiment, the nine cows from groups I, II and III presented with specific antibodies (IFAT) against T. gondii. In contrast, T. gondii could not be detected by IFAT, histopathological examination or the bioassay in any of the nine calves born to cows experimentally infected with T. gondii oocysts. Based on the results from both studies, we conclude that congenital infection of T. gondii in cattle, while infrequent, does occur naturally. The pathogenicity of the strain of T. gondii may influence the likelihood of this route of transmission.     

Trace elements (Al,Cd, Cr,Cu, Fe,Mn, Ni,Pb and Zn) in Mytilus galloprovincialis and Tapes decussatus from Faro and Ganzirri Lakes (Sicily,Italy): Flow cytometry applied for hemocytes analysis

BackgroundTrace elements present in sessile molluscs, are important because they are used in human consumption and it has significantly increased in recent years. While their filtering of the water can lead to their build-up of organic and inorganic materials that can be sampled and analyzed, this can also lead to bioaccumulation of harmful substances, such as essential and non-essential elements, that can harm the human health if in taken in high concentrations or for a long period of time.MethodsIn the present study, the trace metal content (Al, Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn) of two sessile crustaceans, 20 Mytilus galloprovincialis (mussel) and 20 Tapes decussatus (clam) in Faro and Ganzirri Lakes (Messina, Sicily, Italy) were analyzed. Haemolymph samples were taken on both molluscs in order to analyze the haemocyte population by flow cytometric analysis. Unpaired t-tests were used to determine significant differences for the essential and non-essential metallic elements concentrations in the lake waters and in the tissues of M. galloprovincialis and T. decussatus and for hemocyte populations R1 (halinocytes) and R2 (granulocytes).ResultsThe results suggested that that in Faro Lake, the tissue Al, Cr and Pb levels in M. galloprovincialis were higher than those for T. decussatus, in contrast to Mn, Fe, Ni, Cu, Zn and Cd, which were higher in T. decussatus. Unpaired t-tests showed that there were significantly higher proportions of halinocytes in M. galloprovincialis versus T. decussatus for both Faro Lake (41.8 % vs. 24.3 %; P < 0.001) and Ganzirri Lake (43.0 % vs. 22.4 %; P < 0.001). In contrast, while there were significantly higher proportions of granulocytes in Faro Lake (21.2 % vs. 9.1 %; P < 0.001), this difference was not seen for the granulocytes of M. galloprovincialis versus T. decussatus in Ganzirri Lake (9.6 % vs. 13.0 %).ConclusionThis study shows that M. galloprovincialis and T. decussatus can indeed bioaccumulate some of these metal, such that activation of the immune responses is specific to certain cell types. Future research must focus on the balance of trace elements in the consumption of these shellfish, and analyzes with more sophisticated tools can be used to diagnose the increased concentration of trace elements and the quantification of trace metals from shellfish to clams.     

Characterization of secreted recombinant <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> surface antigen 2 (SAG2) heterologously expressed by the yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The surface antigen 2 (SAG2) gene of the protozoan parasite, Toxoplasma gondii, was cloned and extracellularly expressed in the yeast Pichia pastoris. The effectiveness of the secreted recombinant SAG2 (rSAG2-S) as a serodiagnosis reagent was assessed by western blots and ELISA. In the western blot assay, rSAG2-S reacted with all Toxoplasma-antibody positive human serum samples but not with Toxoplasma-negative samples. In the ELISA, rSAG2-S yielded sensitivity rates ranging from 80% (IgG negative, IgM positive) to 100% (IgG positive, IgM negative). In vivo experiments showed that serum from mice immunized with rSAG2-S reacted specifically with the native SAG2 of T. gondii. These mice were protected when challenged with live cells of T. gondii.     

<Emphasis Type="Italic">Toxoplasma gondii</Emphasis> glycosylphosphatidylinositols are not involved in <Emphasis Type="Italic">T. gondii</Emphasis>-induced host cell survival

Toxoplasma gondii is an intracellular parasite able to both promote and inhibit apoptosis. T. gondii renders infected cells resistant to programmed cell death induced by multiple apoptotic triggers. On the other hand, increased apoptosis of immune cells after in vivo infection with T. gondii may suppress the immune response to the parasite. Glycosylphosphatidylinositol (GPI)-anchored proteins dominate the surface of T. gondii tachyzoites and GPIs are involved in the pathogenicity of protozoan parasites. In this report, we determine if GPIs are responsible for inhibition or induction of host cell apoptosis. We show here that T. gondii GPIs fail to block apoptosis that was triggered in human-derived cells via extrinsic or intrinsic apoptotic pathways. Furthermore, characteristics of apoptosis, e.g. caspase-3/7 activity, phosphatidylserine exposition at the cell surface or DNA strand breaks, were not observed in the presence of T. gondii GPIs. These results indicate that T. gondii GPIs are not involved in survival or in apoptosis of host cells. This absence of effect on apoptosis could be a feature common to GPIs of other parasites.     

Development of a qualitative PCR method for the Alexandrium spp. (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis)

Paralytic shellfish poisoning (PSP) is a syndrome caused by the consumption of shellfish contaminated with neurotoxins produced by organisms of the marine dinoflagellate genus Alexandrium. A. minutum is the most widespread species responsible for PSP in the Western Mediterranean basin. The standard monitoring of shellfish farms for the presence of harmful algae and related toxins usually requires the microscopic examination of phytoplankton populations, bioassays and toxin determination by HPLC. These procedures are time-consuming and require remarkable experience, thus limiting the number of specimens that can be analyzed by a single laboratory unit. Molecular biology techniques may be helpful in the detection of target microorganisms in field samples. In this study, we developed a qualitative PCR assay for the rapid detection of all potentially toxic species belonging to the Alexandrium genus and specifically A. minutum, in contaminated mussels. Alexandrium genus-specific primers were designed to target the 5.8S rDNA region, while an A. minutum species-specific primer was designed to bind in the ITS1 region. The assay was validated using several fixed seawater samples from the Mediterranean basin, which were analyzed using PCR along with standard microscopy procedures. The assay provided a rapid method for monitoring the presence of Alexandrium spp. in mussel tissues, as well as in seawater samples. The results showed that PCR is a valid, rapid alternative procedure for the detection of target phytoplankton species either in seawater or directly in mussels, where microalgae can accumulate.     

Molecular analyses of Toxoplasma gondii calmodulin-like domain protein kinase isoform 3

Ca²⁺ signaling is thought to play an important role in Toxoplasma gondii motility, including invasion of and egress from host cells. Recently, it has been reported that phosphorylation of the glideosome apparatus components of T. gondii occurs during invasion. To elucidate the role of T. gondii calmodulin-like domain protein kinase in the signaling pathway that bridges Ca²⁺ stimulation and motility, we characterized T. gondii calmodulin-like domain protein kinase isoform 3 (TgCDPKif3). TgCDPKif3 is homologous to Plasmodium falciparum calcium-dependent protein kinase 1, which has been reported to phosphorylate P. falciparum glideosome components. TgCDPKif3 was purified as a fusion protein that was labeled with [γ-³²P]ATP, and the label was subsequently removed by phosphatase treatment. Phosphorylation was eliminated when the putative catalytic lysine residue of TgCDPKif3 was replaced with alanine. TgCDPKif3 phosphorylated Histone II_AS as a representative substrate in a Ca²⁺-dependent manner at a high Ca²⁺ concentration. TgCDPKif3 was localized to the apical ends of tachyzoites. TgCDPKif3 showed the translocation between intra- and extracellular tachyzoites. TgCDPKif3 could phosphorylate T. gondii aldolase 1 (TgALD1) in vitro. The interaction between TgCDPKif3 and TgALD1 was confirmed by the co-immunoprecipitation assay in mammal cells. We suggested that TgCDPKif3 could participate in the motility of T. gondii through the phosphorylation of glideosome complex member.     

Biases in determining the diet of jumbo squid <Emphasis Type="Italic">Dosidicus gigas</Emphasis> (D’ Orbigny 1835) (Cephalopoda: Ommastrephidae) off southern-central Chile (34°S–40°S)

The diet of jumbo squid (Dosidicus gigas) off southern-central Chile is described to examine potential biases in the determination of their main prey. Specimens were collected from catches using different fishing gear (jigging, trawl and purse-seine), from July 2003 to January 2004, and from December 2005 to October 2006. The stomach contents were analyzed in terms of frequency of occurrence, number, and weight of prey items and the diet composition was analyzed using Detrended Correspondence Analysis. In the industrial purse-seine fleet for jack mackerel (Trachurus murphyi), the dominant prey of D. gigas was T. murphyi. In the industrial mid-trawl fishery for Patagonian grenadier (Macruronus magellanicus), the dominant species in the diet of D. gigas was M. magellanicus. Similarly, Chilean hake (Merluccius gayi) was the main prey in the diet of D. gigas obtained in the industrial trawl fishery for Chilean hake; and, in both artisanal fisheries (purse-seine for small pelagics and jigging), small pelagic fish and D. gigas were the main prey in the stomach contents of D. gigas. Cannibalism in D. gigas varied between different fleets and probably is related to stress behavior during fishing. The Detrended Correspondence Analysis ordination showed that the main prey in the diet of D. gigas is associated with the target species of the respective fishery. Consequently, biases are associated with fishing gear, leading to an overestimate in the occurrence of the target species in the diet. We recommend analyzing samples from jigging taken at the same time and place where the trawl and purse-seine fleets are operating to avoid this problem, and the application of new tools like stable isotope, heavy metal, and fatty acid signature analyses.     

Seroprevalence of Toxoplasma gondii in pigs in Southwestern China

The seroprevalence of toxoplasmosis in pigs was investigated in Yunnan province, Southwestern China between March 2008 and January 2009. A total of 831 serum samples were collected from 9 counties and assayed for Toxoplasma gondii antibodies by indirect haemagglutination (IHA) test. Antibodies to T. gondii were found in 16.97% (141/831) with slaughter pigs having the highest rate (22.28%), followed by breeding sows (16.59%). The results of the present survey indicated that infection with T. gondii in pigs is widely spread in China, including the Yunnan province, and is of public health concern.     

Specific Detection of Pacific Oyster (Crassostrea gigas) Larvae in Plankton Samples Using Nested Polymerase Chain Reaction

Management of sustainable Pacific oyster fisheries would be assisted by an early, rapid, and accurate means of detecting their planktonic larvae. Reported here is an approach, based on polymerase chain reaction (PCR), for the detection of Pacific oyster larvae in plankton samples. Species-specific primers were designed by comparing partial mitochondrial cytochrome oxidase subunit I (COI) sequences from Crassostrea gigas, with other members of the family Ostreidae including those of Crassostrea angulata. Assay specificity was empirically validated through screening DNA samples obtained from several species of oysters. The assay was specific as only C. gigas samples returned PCR-positive results. A nested PCR approach could consistently detect 5 or more D-hinge-stage larvae spiked into a background of about 146 mg of plankton. The assay does not require prior sorting of larvae. We conclude that the assay could be used to screen environmental and ballast water samples, although further specificity testing against local bivalve species is recommended in new locations.     

Genetic characterisation of Toxoplasma gondii in wildlife from North America revealed widespread and high prevalence of the fourth clonal type

Little is known of the genetic diversity of Toxoplasma gondii circulating in wildlife. In the present study wild animals, from the USA were examined for T. gondii infection. Tissues of naturally exposed animals were bioassayed in mice for isolation of viable parasites. Viable T. gondii was isolated from 31 animals including, to our knowledge for the first time, from a bald eagle (Haliaeetus leucocephalus), five gray wolves (Canis lupus), a woodrat (Neotoma micropus), and five Arctic foxes (Alopex lagopus). Additionally, 66 T. gondii isolates obtained previously, but not genetically characterised, were revived in mice. Toxoplasma gondii DNA isolated from these 97 samples (31 + 66) was characterised using 11 PCR-restriction fragment length polymorphism (RFLP) markers (SAG1, 5′- and 3′-SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22–8, c29–2, L358, PK1 and Apico). A total of 95 isolates were successfully genotyped. In addition to clonal Types II, and III, 12 different genotypes were found. These genotype data were combined with 74 T. gondii isolates previously characterised from wildlife from North America and a composite data set of 169 isolates comprised 22 genotypes, including clonal Types II, III and 20 atypical genotypes. Phylogenetic network analysis showed limited diversity with dominance of a recently designated fourth clonal type (Type 12) in North America, followed by the Type II and III lineages. These three major lineages together accounted for 85% of strains in North America. The Type 12 lineage includes previously identified Type A and X strains from sea otters. This study revealed that the Type 12 lineage accounts for 46.7% (79/169) of isolates and is dominant in wildlife of North America. No clonal Type I strain was identified among these wildlife isolates. These results suggest that T. gondii strains in wildlife from North America have limited diversity, with the occurrence of only a few major clonal types.     

Warm temperature acclimation impacts metabolism of paralytic shellfish toxins from Alexandrium minutum in commercial oysters

Species of Alexandrium produce potent neurotoxins termed paralytic shellfish toxins and are expanding their ranges worldwide, concurrent with increases in sea surface temperature. The metabolism of molluscs is temperature dependent, and increases in ocean temperature may influence both the abundance and distribution of Alexandrium and the dynamics of toxin uptake and depuration in shellfish. Here, we conducted a large‐scale study of the effect of temperature on the uptake and depuration of paralytic shellfish toxins in three commercial oysters (Saccostrea glomerata and diploid and triploid Crassostrea gigas, n = 252 per species/ploidy level). Oysters were acclimated to two constant temperatures, reflecting current and predicted climate scenarios (22 and 27 °C), and fed a diet including the paralytic shellfish toxin‐producing species Alexandrium minutum. While the oysters fed on A. minutum in similar quantities, concentrations of the toxin analogue GTX1,4 were significantly lower in warm‐acclimated S. glomerata and diploid C. gigas after 12 days. Following exposure to A. minutum, toxicity of triploid C. gigas was not affected by temperature. Generally, detoxification rates were reduced in warm‐acclimated oysters. The routine metabolism of the oysters was not affected by the toxins, but a significant effect was found at a cellular level in diploid C. gigas. The increasing incidences of Alexandrium blooms worldwide are a challenge for shellfish food safety regulation. Our findings indicate that rising ocean temperatures may reduce paralytic shellfish toxin accumulation in two of the three oyster types; however, they may persist for longer periods in oyster tissue.     

Phylogenetic analysis of Veneridae (Bivalvia): Comparison of molecular and palaeontological data

An approximately 400-by-long portion of the 16s rRNA gene sequence has been determined for the venerid clamsChamelea gallina (Chioninae),Dosinia lupinus (Dosiniinae),Pitar rudis,Callista chione (Pitarinae),Tapes decussatus,T. philippinarum,Venerupis (=Paphia)aurea (Tapetinae), andVenus verrucosa (Venerinae). Neighbor-joining and maximum parsimony trees support the results of traditional classification methods at the subfamily level but do not support the concept of a genusTapes. The transversion divergence rate estimated on the basis of the palaeontological record for theC. gallina/V. verrucosa separation and for the Pitarinae is very close (0.14–0.16% per Myr, respectively) to that of ungulates and cetaceans, while the Tapetinae exhibit a much higher (0.36% per Myr) rate. Correspondence to: E. Olmo     

Proteomic approach to probe for larval proteins of the mussel Mytilus galloprovincialis

A proteomic approach was used to search for larval proteins specific to the mussel Mytilus galloprovincialis from Galicia in northwest Spain. The study included both a comparative analysis, through two-dimensional electrophoresis, of protein expression maps of the larvae of the mussel and of 5 abundant and commercially important bivalve species from the region (Ostrea edulis, Cerastoderma edule, Pecten maximus, Tapes decussatus, and Venarupis pullastra) and subsequent mass spectrometric analysis of some of the protein spots. A total of 18 spots were selected and isolated from gels of M. galloprovincialis larvae. From their relative position on the electrophoresis gels, 6 of these were clearly exclusive to the mussel species. However, it was not clear whether the other spots were shared by other species. To overcome this ambiguity, first an analysis using matrix assisted laser desorption ionization with time-of-flight (MALDI-TOF) was conducted on the 6 spots of Mytilus that could possibly be shared with only one species. The peptide mass fingerprinting was completely different for the proteins compared. This result confirmed that the 6 proteins were exclusively mussel proteins, but demonstrated the utility of this approach when working with species that are poorly represented at the protein level in databases.     

Whole-cell antigens of members of the sulfate-reducing genus Desulfovibrio

Antisera have been developed against the wholecell antigens of Desulfovibrio africanus Benghazi and Walvis Bay, D. vulgaris Hildenborough, D. salexigens British Guiana, D. gigas, and D. desulfuricans Essex 6. An enzymelinked immunoadsorption assay (ELISA) was developed to measure the reaction of these antisera with the homologous and heterologous antigens. The ELISA method demonstrated a reaction between pre-immune sera and cells of D. africanus, D. gigas and D. desulfuricans, suggesting the presence of a lectin-like substance on these cell surfaces. Extensive cross-reactions were seen between the antisera and heterologous cells, suggesting the sharing of a number of surface antigens amongst the Desulfovibrio. However, the pattern of these cross-reactions was different from that observed for an ELISA reaction developed for the cytochrome c₃ from various Desulfovibrio.Abbreviation ELISA enzyme-linked immunoadsorption assay     

High levels of congenital transmission of Toxoplasma gondii in a commercial sheep flock

Our current understanding of congenital transmission of Toxoplasma gondii from ewe to lamb dictates that infection frequently results in abortion and the death of the developing foetus, that the birth of live infected lambs occurs rarely and that the cat is the predominant source of infection in ewes. Using direct polymerase chain reaction detection of T. gondii, we report high levels of congenital transmission occurring in a commercially managed sheep flock. We sampled foetal-derived placental tissue and tissues from aborted lambs and showed that congenital transmission was detected in these tissues from 61% of all pregnancies. Where pregnancies resulted in the death of one or more lambs, T. gondii was detected in the lamb tissue for all but one of 18 (94%) pregnancies. Of the successful pregnancies resulting in the birth of live lambs we were able to detect T. gondii in foetal-derived placental tissue from 37 of 70 (42%) pregnancies. These results show that congenital transmission is occurring in a high percentage of lambings including normal healthy lambings, at this farm, suggesting that this route of transmission from generation to generation may be much more significant than that reported previously. These results may have implications for sheep husbandry and future epidemiological studies of T. gondii.     

Toxoplasma gondii: protective immunity against experimental toxoplasmosis induced by a DNA vaccine encoding the perforin-like protein 1

Toxoplasma gondii is an important zoonotic parasite infecting about one third of the world population, causing congenital infections and eye disease. T. gondii perforin-like protein 1 (TgPLP1) is believed to be involved in the acute virulence of T. gondii in mice, and is therefore of interest as a vaccine candidate. In this study, we constructed a DNA vaccine expressing TgPLP1, and evaluated the immune response in Kunming mice. The gene sequence encoding TgPLP1 was inserted into the eukaryotic expression vector pVAX I, and Kunming mice were immunized intramuscularly with the plasmid. After immunization, we evaluated the immune response using lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally with 1 × 10³ tachyzoites of the virulent T. gondii RH strain. The results showed that pVAX/TgPLP1 alone or with pVAX/IL-18 developed specific anti-TLA (T. gondii lysate antigen) antibodies and specific lymphocyte proliferative responses. Co-injection of pVAX/IL-18 significantly increased the production of IFN-γ and IL-2. Further, challenge experiments showed that co-immunization of pVAX/TgPLP1 with pVAX/IL-18 significantly (P < 0.05) increased survival time (12.7 ± 1.2 days) of immunized mice, compared with pVAX/TgPLP1 alone (11.3 ± 0.9 days). These results demonstrate that TgPLP1 is a potential vaccine candidate against toxoplasmosis, worth further evaluation in other animal hosts. IL-18 could enhance the immune effect of TgPLP1, prolonging the survival time of immunized mice.     

The ectoparasitic mite Tropilaelaps mercedesae (Acari, Laelapidae) as a vector of honeybee viruses

The ectoparasitic mites Varroa destructor and Tropilaelaps mercedesae share life history traits and both infect honeybee colonies, Apis mellifera. Since V. destructor is a biological vector of several honeybee viruses, we here test whether T. mercedesae can also be infected and enable virus replication. In Kunming (China), workers and T. mercedesae mites were sampled from three A. mellifera colonies, where workers were exhibiting clinical symptoms of deformed wing virus (DWV). We analysed a pooled bee sample (15 workers) and 29 mites for the presence of Deformed wing virus (DWV), Black queen cell virus (BQCV), Sacbrood virus (SBV), Kashmir bee virus (KBV), Acute bee paralysis virus (ABPV), and Chronic bee paralysis virus (CBPV). Virus positive samples were analysed with a qPCR. Only DWV +RNA was found but with a high titre of up to 10⁸ equivalent virus copies per mite and 10⁶ per bee. Moreover, in all DWV positive mites (N= 12) and in the bee sample virus–RNA was also detected using RT-PCR and tagged RT-PCR, strongly suggesting virus replication. Our data show for the first time that T. mercedesae may be a biological vector of DWV, which would open a novel route of virus spread in A. mellifera. Received 6 June 2008; revised 14 August 2008; accepted 10 September 2008.     

Toxoplasma gondii: effects of neuwiedase, a metalloproteinase from Bothrops neuwiedi snake venom, on the invasion and replication of human fibroblasts in vitro

The major aim to the present study was to determine the effects of neuwiedase, a metalloproteinase isolated from Bothrops neuwiedi snake venom, on invasion and replication of Toxoplasma gondii in human fibroblasts in vitro. Neuwiedase treatment was done on host cells previously infected with T. gondii or on parasite before fibroblast infection. When treatments were done after or before infection, infection rates were inhibited in 71% and 61%, respectively. Considering that therapy protocols currently used in T. gondii infection cause considerable side effects, particularly in immunocompromised individuals and pregnant women, the results of neuwiedase treatment described herein could be taken into account for the development of new synthetic therapeutic agents, mainly due to the capacity of this enzyme to degrade extracellular matrix components, such as laminin, fibronectin and type I collagen, which is important to interfere in T. gondii host cell invasion.     

Isolation and Genetic Characterization of Toxoplasma gondii from Black Bears (Ursus americanus), Bobcats (Lynx rufus), and Feral Cats (Felis catus) from Pennsylvania

Toxoplasma gondii infects virtually all warm‐blooded hosts worldwide. Recently, attention has been focused on the genetic diversity of the parasite to explain its pathogenicity in different hosts. It has been hypothesized that interaction between feral and domestic cycles of T. gondii may increase unusual genotypes in domestic cats and facilitate transmission of potentially more pathogenic genotypes to humans, domestic animals, and wildlife. In the present study, we tested black bear (Ursus americanus), bobcat (Lynx rufus), and feral cat (Felis catus) from the state of Pennsylvania for T. gondii infection. Antibodies to T. gondii were found in 32 (84.2%) of 38 bears, both bobcats, and 2 of 3 feral cats tested by the modified agglutination test (cut off titer 1:25). Hearts from seropositive animals were bioassayed in mice, and viable T. gondii was isolated from 3 of 32 bears, 2 of 2 bobcats, and 2 of 3 feral cats. DNA isolated from culture‐derived tachyzoites of these isolates was characterized using multilocus PCR‐RFLP markers. Three genotypes were revealed, including ToxoDB PCR‐RFLP genotype #1 or #3 (Type II, 1 isolate), #5 (Type 12, 3 isolates), and #216 (3 isolates), adding to the evidence of genetic diversity of T. gondii in wildlife in Pennsylvania. Pathogenicity of 3 T. gondii isolates (all #216, 1 from bear, and 2 from feral cat) was determined in outbred Swiss Webster mice; all three were virulent causing 100% mortality. Results indicated that highly mouse pathogenic strains of T. gondii are circulating in wildlife, and these strains may pose risk to infect human through consuming of game meat.     

Immunofluorescent Localization of Myosin at the Anterior Pole of the Coccidian,Toxoplasma gondi1

Indirect immunofluorescence using anti-myosin rabbit sera showed myosin localized in a characteristic pattern at the anterior pole of Toxoplasma gondii. This polar fluorescent staining was abolished by pre-absorption of the anti-sera with myosin extracted from avian muscle. Both intracellular and extracellular T. gondii showed similar patterns when formaldehyde-fixed, but neither showed polar fluorescence when acetone was used as the sole fixative. Immunofluorescent staining of live T. gondii revealed no polar fluorescence, suggesting that myosin is not present on the outer parasite membrane. Anti-myosin serum did not prevent host cell invasion and plaque formation in the presence of human complement. Inhibition of contractile proteins with cytochalasin D inhibited T. gondii motility and infectivity in a plaque formation assay. The pattern of polar fluorescence described here resembles the IgM-associated polar staining frequently detected in human sera, but we believe it is a different phenomenon because human sera that showed such staining retained their activity after pre-absorption with avian myosin. The unusual localization of myosin at the anterior pole of T. gondii tachyzoites may play a role in the function of anterior organelles, which are thought to facilitate the invasion of host cells.