Toxoplasma GRA15 and GRA24 are important activators of the host innate immune response in the absence of TLR11.

The murine innate immune response against Toxoplasma gondii is predominated by the interaction of TLR11/12 with Toxoplasma profilin. However, mice lacking Tlr11 or humans, who do not have functional TLR11 or TLR12, still elicit a strong innate immune response upon Toxoplasma infection. The parasite factors that determine this immune response are largely unknown. Herein, we investigated two dense granule proteins (GRAs) secreted by Toxoplasma, GRA15 and GRA24, for their role in stimulating the innate immune response in Tlr11-/- mice and in human cells, which naturally lack TLR11/TLR12. Our results show that GRA15 and GRA24 synergistically shape the early immune response and parasite virulence in Tlr11-/- mice, with GRA15 as the predominant effector. Nevertheless, acute virulence in Tlr11-/- mice is still dominated by allelic combinations of ROP18 and ROP5, which are effectors that determine evasion of the immunity-related GTPases. In human macrophages, GRA15 and GRA24 play a major role in the induction of IL12, IL18 and IL1ß secretion. We further show that GRA15/GRA24-mediated IL12, IL18 and IL1ß secretion activates IFNγ secretion by peripheral blood mononuclear cells (PBMCs), which controls Toxoplasma proliferation. Taken together, our study demonstrates the important role of GRA15 and GRA24 in activating the innate immune response in hosts lacking TLR11.

Systemic and local immune responses in sheep after Neospora caninum experimental infection at early, mid and late gestation.

Besides its importance in cattle, Neospora caninum may also pose a high risk as abortifacient for small ruminants. We have recently demonstrated that the outcome of experimental infection of pregnant sheep with 10(6) Nc-Spain7 tachyzoites is strongly dependent on the time of gestation. In the current study, we assessed peripheral and local immune response in those animals. Serological analysis revealed earlier and higher IFN-γ and IgG responses in ewes infected at early (G1) and mid (G2) gestation, when abortion occurred. IL-4 was not detected in sera from any sheep. Inflammatory infiltrates in the placenta mainly consisted of CD8+ and, to a lesser extent, CD4+ T cells and macrophages (CD163+). The infiltrate was more intense in sheep infected at mid-gestation. In the foetal mesenchyme, mostly free tachyzoites were found in animals infected at G1, while those infected in G2 displayed predominantly particulate antigen, and parasitophorous vacuoles were detected in sheep infected at G3. A similar pattern of placental cytokine mRNA expression was found in all groups, displaying a strengthened upregulation of IFN-γ and IL-4 and milder increases of TNF-α and IL-10, reminiscent of a mixed Th1 and Th2 response. IL-12 and IL-6 were only slightly upregulated in G2, and TGF-ß was downregulated in G1 and G2, suggestive of limited T regulatory (Treg) cell activity. No significant expression of TLR2 or TLR4 could be detected. In summary, this study confirms the pivotal role of systemic and local immune responses at different times of gestation during N. caninum infection in sheep.

Influence of the gestational stage on the clinical course, lesional development and parasite distribution in experimental ovine neosporosis.

Neospora caninum is considered one of the main causes of abortion in cattle, yet recent studies have also emphasised its relevance as an abortifacient in small ruminants. In order to gain deeper insight into the pathogenesis of ovine neosporosis, pregnant ewes were intravenously inoculated with 10(6) tachyzoites of the Nc-Spain7 isolate at days 40, 90 or 120 of gestation. Infection during the first term resulted in the death of all foetuses between days 19 and 21 post-infection, showing mainly necrotic lesions in foetal liver and the highest parasite DNA detection and burden in both placenta and foetal viscera. After infection at day 90, foetal death was also detected in all ewes, although later (34-48 days post-infection). In this group, lesions were mainly inflammatory. Foetal livers showed the lowest frequency of lesions, as well as the lowest parasite detection and burden. All ewes infected at day 120 delivered viable lambs, although 3 out of 9 showed weakness and recumbency. Neospora DNA was detected in all lambs but one, and parasite burden was similar to that observed in day 90 group. Lesions in this group showed more conspicuous infiltration of inflammatory cells and higher frequency in foetal brain and muscle when compared to both previous groups. These results highlight the crucial role that the stage of gestation plays on the course of ovine neosporosis, similar to that reported in bovine neosporosis, and open the doors to consider sheep as a valid model for exogenous transplacental transmission for ruminant neosporosis.

Neospora caninum infection during early pregnancy in cattle: how the isolate influences infection dynamics, clinical outcome and peripheral and local immune responses.

This work studies the influence of Neospora caninum intra-species diversity on abortion outcome, infection dynamics in terms of parasite dissemination and peripheral-local immune responses in pregnant cattle. Animals were intravenously inoculated at day 70 of pregnancy with 107 tachyzoites of two isolates showing marked differences in virulence in vitro and in pregnant mouse models: Nc-Spain7, a high virulence isolate, and Nc-Spain8, a low-to-moderate virulence isolate. After inoculation, pregnancy was monitored, and dams were culled when foetal death was detected. Foetal mortality occurred in all infected heifers between days 24 and 49 post-infection (pi), however, it was detected sooner in Nc-Spain7-infected animals (median day = 34) than those inoculated with Nc-Spain8 (median day = 41) with a trend towards significance (P < 0.11). Similar histological lesions were observed in placentomes and in most of the foetuses from the two infected groups. However, parasites were more frequently detected in the placenta and foetuses by PCR and in the foetal brain by immunohistochemistry in Nc-Spain7-infected animals. Specific antibodies were detected starting at day 13 post-infection in all infected cattle, with higher IgG levels in Nc-Spain7-infected group. IFN-γ and IL-4 profiles also varied between infected groups in PBMC stimulation assays. Infected animals showed significant increases in their cytokine mRNA levels (IFN-γ, IL-4, IL-10, IL-12p40 and TNF-α) in the caruncle at time of foetal death. Differences between the infected groups were also observed for cytokine profiles. These results demonstrate the influence of the N. caninum isolate on foetal death outcome, infection dynamics and immune responses in cattle.

A combination of GRA3, GRA6 and GRA7 peptides offer a useful tool for serotyping type II and III Toxoplasma gondii infections in sheep and pigs.

The clinical consequences of toxoplasmosis are greatly dependent on the Toxoplasma gondii strain causing the infection. To better understand its epidemiology and design appropriate control strategies, it is important to determine the strain present in infected animals. Serotyping methods are based on the detection of antibodies that react against segments of antigenic proteins presenting strain-specific polymorphic variations, offering a cost-effective, sensitive, and non-invasive alternative to genotyping techniques. Herein, we evaluated the applicability of a panel of peptides previously characterized in mice and humans to serotype sheep and pigs. To this end, we used 51 serum samples from experimentally infected ewes (32 type II and 19 type III), 20 sheep samples from naturally infected sheep where the causative strain was genotyped (18 type II and 2 type III), and 40 serum samples from experimentally infected pigs (22 type II and 18 type III). Our ELISA test results showed that a combination of GRA peptide homologous pairs can discriminate infections caused by type II and III strains of T. gondii in sheep and pigs. Namely, the GRA3-I/III-43 vs. GRA3-II-43, GRA6-I/III-213 vs. GRA6-II-214 and GRA6-III-44 vs. GRA6-II-44 ratios showed a statistically significant predominance of the respective strain-type peptide in sheep, while in pigs, in addition to these three peptide pairs, GRA7-II-224 vs. GRA7-III-224 also showed promising results. Notably, the GRA6-44 pair, which was previously deemed inefficient in mice and humans, showed a high prediction capacity, especially in sheep. By contrast, GRA5-38 peptides failed to correctly predict the strain type in most sheep and pig samples, underpinning the notion that individual standardization is needed for each animal species. Finally, we recommend analyzing for each animal at least 2 samples taken at different time points to confirm the obtained results.

Late Embryogenesis Abundant Proteins Contribute to the Resistance of Toxoplasma gondii Oocysts against Environmental Stresses.

Toxoplasma gondii oocysts, which are shed in large quantities in the feces from infected felines, are very stable in the environment, resistant to most inactivation procedures, and highly infectious. The oocyst wall provides an important physical barrier for sporozoites contained inside oocysts, protecting them from many chemical and physical stressors, including most inactivation procedures. Furthermore, sporozoites can withstand large temperature changes, even freeze-thawing, as well as desiccation, high salinity, and other environmental insults; however, the genetic basis for this environmental resistance is unknown. Here, we show that a cluster of four genes encoding Late Embryogenesis Abundant (LEA)-related proteins are required to provide Toxoplasma sporozoites resistance to environmental stresses. Toxoplasma LEA-like genes (TgLEAs) exhibit the characteristic features of intrinsically disordered proteins, explaining some of their properties. Our in vitro biochemical experiments using recombinant TgLEA proteins show that they have cryoprotective effects on the oocyst-resident lactate dehydrogenase enzyme and that induced expression in E. coli of two of them leads to better survival after cold stress. Oocysts from a strain in which the four LEA genes were knocked out en bloc were significantly more susceptible to high salinity, freezing, and desiccation compared to wild-type oocysts. We discuss the evolutionary acquisition of LEA-like genes in Toxoplasma and other oocyst-producing apicomplexan parasites of the Sarcocystidae family and discuss how this has likely contributed to the ability of sporozoites within oocysts to survive outside the host for extended periods. Collectively, our data provide a first molecular detailed view on a mechanism that contributes to the remarkable resilience of oocysts against environmental stresses. IMPORTANCE Toxoplasma gondii oocysts are highly infectious and may survive in the environment for years. Their resistance against disinfectants and irradiation has been attributed to the oocyst and sporocyst walls by acting as physical and permeability barriers. However, the genetic basis for their resistance against stressors like changes in temperature, salinity, or humidity, is unknown. We show that a cluster of four genes encoding Toxoplasma Late Embryogenesis Abundant (TgLEA)-related proteins are important for this resistance to environmental stresses. TgLEAs have features of intrinsically disordered proteins, explaining some of their properties. Recombinant TgLEA proteins show cryoprotective effects on the parasite's lactate dehydrogenase, an abundant enzyme in oocysts, and expression in E. coli of two TgLEAs has a beneficial effect on growth after cold stress. Moreover, oocysts from a strain lacking all four TgLEA genes were more susceptible to high salinity, freezing, and desiccation compared to wild-type oocysts, highlighting the importance of the four TgLEAs for oocyst resilience.

Fatal Sarcocystis calchasi hepatitis in a captive Indian ringneck parakeet (Psittacula krameri manillensis).

An adult Indian ringneck parakeet (Psittacula krameri manillensis) from an outdoor aviary in Sacramento, California was found dead on the nest box. Postmortem examination showed firm, enlarged, yellow­tinged liver and splenomegaly. Multifocal to coalescing, acute necrosis with macrophages, lymphocytes, plasma cells, and periportal ductular reaction were seen on liver histology with extra- and intracellular schizonts and merozoites. A few schizonts and lymphohistiocytic inflammation were seen in the spleen. Toxoplasma gondii, Sarcocystis neurona, S. falcatula and Neospora caninum were ruled out by immunohistochemistry. PCR of the liver for Sarcocystis spp. Based on the positive amplification/PCR of ITS1 segment and sequencing of 28S rRNA fragment, S. calchasi was confirmed. The splanchnic presentation of S. calchasi in this parakeet resembles the acute infection described experimentally in domestic pigeons (Columba livia f. domestica) and cockatiels (Nymphicus hollandicus). Since large populations of red-tailed hawks (Buteo jamaicensis) and Cooper's hawk (Accipiter cooperi), the likely definitive hosts of S. calchasi in North America, inhabit the Sacramento area, their presence near outdoor aviaries may account for the source of S. calchasi infective sporocysts.

New Avenues to Design Toxoplasma Vaccines Based on Oocysts and Cysts.

Toxoplasmosis is a worldwide disease affecting all warm-blooded animals, including humans. Vaccination strategies aimed at inducing an efficient immune response while preventing transmission have been attempted in the past. While many different approaches can partially protect immunized animals against subsequent infections, full and lasting protection is rarely attained and only with live-attenuated vaccines. In addition, vaccines based on mutant strains that are deficient in forming the chronic phase of the parasite (such as Toxovax™) cannot be extensively used due to their zoonotic potential and the possibility of reversion to virulent phenotypes. An increasing number of studies using emerging genetic-engineering tools have been conducted to design novel vaccines based on recombinant proteins, DNA or delivery systems such as nanoparticles. However, these are usually less efficient due to their antigenic simplicity. In this perspective article we discuss potential target genes and novel strategies to generate live-attenuated long-lasting vaccines based on tissue cysts and oocysts, which are the environmentally resistant chronic forms of Toxoplasma. By selectively disrupting genes important for parasite dissemination, cyst formation and/or sporozoite invasion, alone or in combination, a vaccine based on a live-attenuated strain that elicits a protective immune response while preventing the transmission of Toxoplasma could be created. Finally, further improvements of protocols to generate Toxoplasma sexual stages in vitro might lead to the production of oocysts from such a strain without the need for using mice or cats.

Sarcocystis calchasi and other Sarcocystidae detected in predatory birds in California, USA.

Outbreaks of neurological disease associated with Sarcocystis calchasi have been observed in captive and free-ranging rock pigeons (Columba livia) in Europe and the United States as well as in wild Brandt's cormorants (Phalacrocorax penicillatus) and captive psittacines in California, USA. Experimental and field studies have identified northern goshawks (Accipiter gentilis) and European sparrowhawks (A. nisus) as definitive hosts in Europe while the definitive hosts elsewhere remain unknown. In this study, we aimed to identify the potential definitive host(s) of S. calchasi through molecular analysis of intestinal samples from seven predatory (n = 85) and one omnivorous (n = 11) bird species in California. In total, apicomplexan-generic 28S rRNA PCR products were obtained and sequenced for 42 raptors. Three of 16 (18.8%) Cooper's hawks (A. cooperii) and two of 26 (5.6%) red-tailed hawks (Buteo jamaicensis) also tested positive for the S. calchasi-specific ITS1 PCR and sequencing of the 28S rRNA PCR product was 100% homologous to S. calchasi. In addition to S. calchasi (5.9%; 5/85), other Sarcocystis spp. detected in raptors included: S. jamaicensis (21.2%; 18/85), S. columbae (8.2%; 7/85), S. turdusi (7.1%; 6/85), and S. halieti (4.7; 4/85%). Infections with closely related S. jamaicensis and S. (Frenkelia) microti (9.4%; 8/85) could not be distinguished for eight raptors. Eumonospora henryae (1.2%; 1/85) was detected in one raptor. Our results indicate for the first time that S. calchasi may have a definitive host range in North America that includes at least two raptors, Cooper's hawks and red-tailed hawks, within the family Accipitridae.

Pampas fox (Lycalopex gymnocercus) of the Argentine Pampas as intermediate host for Neospora caninum.

The Pampas fox (Lycalopex gymnocercus) is the most abundant wild canid from South America. This wild canid inhabits grasslands, open woodlands, and areas highly modified by extensive ranching and agricultural activities. We aimed to evaluate Neospora caninum infection in tissues from the Pampas fox from Argentina. A total of 41 free-living Pampas foxes were sampled in rural areas located in the Humid Pampas region, Argentina. Brain tissue and different muscles were assessed by histologic and molecular methods. No N. caninum cysts were observed in brain and muscle tissue samples analyzed by histology and immunohistochemistry. Molecular N. caninum identification from brain tissue was based on amplification by PCR of Nc-5 gene and ITS1 rRNA fragments and subsequent sequencing. The presence of N. caninum DNA was 74% (23/31) for the Nc-5 gene and was confirmed by a second ITS1 PCR in 55% (17/31) of the brain tested. Thirteen ITS1 consensus sequences were obtained, and all have a 99.58-100% similarity with N. caninum reference sequences. Only 4% (1/23) of muscles samples analyzed were positive for the Nc-5 gene of N. caninum. This study demonstrated a high prevalence of N. caninum DNA in brain from free-ranging Pampas fox of the Pampa Argentine, thus confirming that this wild canid is a wide distributed intermediate host.

First description of naturally acquired Tritrichomonas foetus infection in a Persian cattery in Spain.

Tritrichomonas foetus has been identified as the causative agent of feline intestinal trichomonosis, characterized by clinical signs of chronic large bowel diarrhoea. This disease has been reported in cats from the USA, Europe and Australia. However, its epidemiology is still unclear. The aim of the present study was to describe T. foetus infection in a Persian cattery in Spain. T. foetus infection was sequentially diagnosed in 20 cats by direct faecal smear examined under the microscope, specific culture (In Pouch TF medium) and PCR. A standard coprological sedimentation method was also performed in order to screen for other intestinal parasites in all the cats included. In addition, sera were tested for IgG antibodies against Leishmania infantum, Toxoplasma gondii, and for the detection of feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV). Five out of 20 cats were positive for T. foetus (25%), two of them by microscopy, culture and PCR and three by culture and PCR. No association was found between T. foetus infection and age or sex. L. infantum and T. gondii seroprevalence rates were 15% and 10%, respectively. The prevalence of FeLV p27 antigen and of FIV antibodies in the study population was zero. Cystoisospora spp. oocysts were detected in one cat. These preliminary results show that the transmission of T. foetus infection in cluster conditions may occur between asymptomatic cats and young or immunocompromised animals.

Toxoplasma Effectors that Affect Pregnancy Outcome.

As an immune-privileged organ, the placenta can tolerate the introduction of antigens without inducing a strong inflammatory response that would lead to abortion. However, for the control of intracellular pathogens, a strong Th1 response characterized by the production of interferon-γ is needed. Thus, invasion of the placenta by intracellular parasites puts the maternal immune system in a quandary: The proinflammatory response needed to eliminate the pathogen can also lead to abortion. Toxoplasma is a highly successful parasite that causes lifelong chronic infections and is a major cause of abortions in humans and livestock. Here, we discuss how Toxoplasma strain type and parasite effectors influence host cell signaling pathways, and we speculate about how this might affect the outcome of gestation.

CRISPR screen to determine the in vivo fitness of Toxoplasma genes.

The virulence of eukaryotic parasites like Toxoplasma gondii depends on their capacity to escape from the host immune response and disseminate throughout the host organism. However, Toxoplasma gene products essential for its in vivo pathogenesis remain uncharacterized. Here, we present the complete workflow of a CRISPR-Cas9 in vivo loss-of-function screen to identify Toxoplasma fitness-conferring genes. This protocol can be used to uncover gene products that play a role in Toxoplasma immune evasion, nutrient acquisition, dissemination, and tissue colonization. For complete details on the use and execution of this protocol, please refer to Sangaré et al. (2019).

Toxoplasma GRA Peptide-Specific Serologic Fingerprints Discriminate Among Major Strains Causing Toxoplasmosis.

The severity of toxoplasmosis depends on a combination of host and parasite factors. Among them, the Toxoplasma strain causing the infection is an important determinant of the disease outcome. Type 2 strains dominate in Europe, whereas in North America type 2, followed by type 3 and 12 strains are commonly isolated from wildlife and patients. To identify the strain type a person is infected with, serological typing provides a promising alternative to the often risky and not always possible biopsy-based DNA methods of genotyping. However, despite recent advances in serotyping, improvements in the sensitivity and specificity are still needed, and it does not yet discriminate among the major Toxoplasma lineages infecting people. Moreover, since infections caused by non-1/2/3 strains have been associated with more severe disease, the ability to identify these is critical. In the present study we investigated the diagnostic potential of an ELISA-based assay using 28 immunogenic Toxoplasma peptides derived from a recent large-scale peptide array screen. Our results show that a discrete number of peptides, derived from Toxoplasma dense granule proteins (GRA3, GRA5, GRA6, and GRA7) was sufficient to discriminate among archetypal strains that infect mice and humans. The assay specifically relies on ratios that compare individual serum reactivities against GRA-specific polymorphic peptide variants in order to determine a "reactivity fingerprint" for each of the major strains. Importantly, nonarchetypal strains that possess a unique combination of alleles, different from types 1/2/3, showed either a non-reactive, or different combinatorial, mixed serum reactivity signature that was diagnostic in its own right, and that can be used to identify these strains. Of note, we identified a distinct "HG11/12" reactivity pattern using the GRA6 peptides that is able to distinguish HG11/12 from archetypal North American/European strain infections.

Influence of the Host and Parasite Strain on the Immune Response During Toxoplasma Infection.

Toxoplasma gondii is an exceptionally successful parasite that infects a very broad host range, including humans, across the globe. The outcome of infection differs remarkably between hosts, ranging from acute death to sterile infection. These differential disease patterns are strongly influenced by both host- and parasite-specific genetic factors. In this review, we discuss how the clinical outcome of toxoplasmosis varies between hosts and the role of different immune genes and parasite virulence factors, with a special emphasis on Toxoplasma-induced ileitis and encephalitis.

Prevalence of intestinal parasite infections in stray and farm dogs from Spain.

Dogs play a potential role as reservoirs for zoonotic parasites, being especially problematic uncontrolled dog populations such as stray and farm dogs with access to populated areas. In order to investigate the prevalence of canine intestinal parasites in at-risk dog populations, we tested a total of 233 faecal samples shed by stray and dairy farm dogs from northern Spain. Telemann method was used to detect the presence of eggs and (oo)cysts of common dog intestinal parasites and Cryptosporidium was detected by PCR. One hundred and forty eight out of 233 samples (63.5%) were positive for at least one intestinal parasite, being Ancylostomidae (35.6%; 83/233) and Trichuris (35.2%; 82/233) the parasites most frequently identified. Cryptosporidium DNA was not detected in any of the faecal samples analysed. The overall prevalence was significantly higher in stray dogs than in farm dogs (72.5% vs 58.8%). Specifically, stray dogs had a significantly higher prevalence of Ancylostomatidae, Toxocara, Toxascaris and Taenidae. These dog populations are an important source of environmental contamination with intestinal parasite forms, which could be of significance to animal and human health.

Protozoal encephalitis associated with Sarcocystis calchasi and S. falcatula during an epizootic involving Brandt's cormorants (Phalacrocorax penicillatus) in coastal Southern California, USA.

Between March and May 2019, wildlife rehabilitation centers along coastal southern California admitted increased numbers of Brandt's cormorants (Phalacrocorax penicillatus) with neurological disease including head tilt, nystagmus, torticollis, tremors, paresis, paralysis, and ataxia. Seven cormorants from Los Angeles County and one cormorant from Orange County were submitted for postmortem examination. Gross findings included thin to fair body condition, generalized congestion/hyperemia, nematode parasites in the ventriculus, and diarrhea in the seven birds from Los Angeles County while the one bird from Orange County had icterus. Histologic examination revealed sarcocysts in the adductor muscles and meningoencephalitis characterized by coalescing infiltrations of macrophages, lymphocytes and plasma cells with severe perivascular cuffing and gliosis in all eight cormorants. Rare to few numbers of schizonts were seen in the cerebrum of the seven cormorants from Los Angeles County whereas the cormorant from Orange County had numerous schizonts in various stages of development in the cerebrum, cerebellum, and brainstem. All eight birds were positive for the generic Sarcocystis spp. 28S PCR. The seven cormorants from Los Angeles County tested positive for the S. calchasi-specific ITS1 and confirmed by sequencing, while the analysis of the 28S sequence in the cormorant from Orange County showed a 100% homology to S. falcatula. This bird also was positive by immunohistochemistry for Sarcocystis spp. using a polyclonal antibody that detects S. falcatula and S. neurona. This report demonstrates for the first time that seabirds such as Brandt's cormorants may be intermediate or dead-end hosts for S. calchasi and/or S. falcatula, and that S. calchasi can cause epizootic infection in a seabird.

Multilocus analysis reveals further genetic differences between Tritrichomonas foetus from cats and cattle.

Tritrichomonas foetus isolates from feline and bovine origin has been previously shown to carry a certain degree of genetic heterogeneity. Here, novel candidate molecular markers were developed by means of multilocus sequence typing of the gap2 gene (encoding for T. foetus glyceraldehyde-3-phosphate dehydrogenase), ITS region, the TR7/TR8 variable-length repeat and microsatellite genotyping. These markers were used to characterize T. foetus field isolates from bulls and domestic cats and to compare phylogenetically with the following ATCC isolates: T. foetus isolated from cattle and pig (syn. Tritrichomonas suis), Tritrichomonas mobilensis, Tetratrichomonas gallinarum and Pentatrichomonas hominis. Among them, TFMS10 and TFMS7 were found to be the most polymorphic markers. Moreover, an 809 bp fragment of the gap2 gene was successfully amplified from all the trichomonads included in this study and the sequence analysis revealed differences between T. foetus porcine and feline genotypes and T. mobilensis in comparison to the bovine T. foetus ATCC isolate. The TR7/TR8 repeat pattern was not reproducible, being only consistent the fragments of approximately 110 and 217 bp. Sequence analysis of the latter revealed the existence of 3 SNPs resulting in 98.6 % homology between bovine and feline isolates. A search for similar sequences was carried out to develop a Restriction Length Fragment Polymorphism analysis. A 503 bp region, named TF1, revealed the existence of two BbvI restriction enzyme sites that were able to generate different length fragments for T. foetus feline and bovine isolates. Finally, the neighbour-joining analyses showed that T. foetus porcine genotype clusters together with bovine genotype, whereas T. mobilensis and the feline genotype form a separate cluster.

Serotyping of Toxoplasma gondii Infection Using Peptide Membrane Arrays.

The intracellular parasite Toxoplasma gondii can cause chronic infections in most warm-blooded animals, including humans. In the USA, strains belonging to four different Toxoplasma clonal lineages (types 1, 2, 3, and 12) are commonly isolated, whereas strains not belonging to these lineages are predominant in other continents such as South America. Strain type plays a pivotal role in determining the severity of Toxoplasma infection. Therefore, it is epidemiologically relevant to develop a non-invasive and inexpensive method for determining the strain type in Toxoplasma infections and to correlate the genotype with disease outcome. Serological typing is based on the fact that many host antibodies are raised against immunodominant parasite proteins that are highly polymorphic between strains. However, current serological assays can only reliably distinguish type 2 from non-type 2 infections. To improve these assays, mouse, rabbit, and human infection serum were reacted against 950 peptides from 62 different polymorphic Toxoplasma proteins by using cellulose membrane peptide arrays. This allowed us to identify the most antigenic peptides and to pinpoint the most relevant polymorphisms that determine strain specificity. Our results confirm the utility of previously described peptides and identify novel peptides that improve and increase the specificity of the assay. In addition, a large number of novel proteins showed potential to be used for Toxoplasma diagnosis. Among these, peptides derived from several rhoptry, dense granule, and surface proteins represented promising candidates that may be used in future experiments to improve Toxoplasma serotyping. Moreover, a redesigned version of the published GRA7 typing peptide performed better and specifically distinguished type 3 from non-type 3 infections in sera from mice, rabbits, and humans.

Immunization with a cocktail of antigens fused with OprI reduces Neospora caninum vertical transmission and postnatal mortality in mice.

OprI is an outer membrane lipoprotein from Pseudomonas aeruginosa, and when fused to a recombinant antigen, will exert adjuvant properties by engaging Toll-like receptor 2, leading to dendritic cell activation. Previous studies have shown that the Neospora caninum (Nc) antigens NcPDI, NcROP2 and NcROP40 are implicated in host cell interactions and are promising vaccine candidates. In two independent experiments, the efficacy of a polyvalent vaccine formulation composed of OprI-NcPDI, OprI-NcROP2 and OprI-NcROP40 (collectively named O-Ags) was assessed in non-pregnant and pregnant Balb/c mouse models challenged with tachyzoites of the high-virulence isolate Nc-Spain7. Parameters that were investigated were clinical signs, fertility, parasite burden in adult mice, humoral and cellular immune responses at different time-points prior to and after challenge infection, vertical transmission and post-natal survival of offspring mice, all to explore potential correlations with efficacy. Vaccination of mice with O-Ags induced a mixed Th1/Th2 immune response in adult mice and led to significantly increased protection against cerebral infection. Vaccination with O-Ags also resulted in reduced vertical transmission, and postnatal disease in offspring was significantly inhibited at a rate not observed in mice infected with a high-virulence isolate to date. However, O-Ags mixed with TLR ligands targeting TLR3 and TLR7, which are known to induce clear Th1-biased responses, or vaccination with OprI fused to the non-N. caninum antigen ovalbumin (OprI-OVA) did not confer protection.