Aspergillosis in a colony of Humboldt penguins (Spheniscus humboldti) in a french zoological park: evaluation of environmental exposure.

Aspergillosis is a major health problem in captive penguins due to the inhalation and the development of airborne spores of opportunistic environmental molds of the genus Aspergillus. Diagnosis is often delayed and treatments, based on the use of azole antifungals, are not fully effective. This study assesses the risk of exposure to Aspergillus sp. and determines the environmental reservoirs in the direct environment of a colony of Humboldt penguins (Spheniscus humboldti) in a zoological park in Paris, and the risk of contamination with resistant isolates. Every 15 days between February and May 2022, environmental samples (air and subtract from the nests, pond water, pigeon and penguin droppings) were carried out in the penguin enclosure as well as clinical samples (one-time non-invasive sampling on chicks), and screened for Aspergillus sp. conidia. From 191 environmental samples, 264 strains of Aspergillus including 221 strains of A. fumigatus were isolated, mostly from ambient air, in the nests, and pond water. No "at risk" areas in the penguin environment have been highlighted, nor an increased risk because of the proximity with urban wild birds. However, the load of airborne Aspergillus in the nests increased significantly with outdoor temperature. Of the 221 strains isolated, we identified only one azole-resistant strain, displaying the TR34/L98H mutation in the cyp51A gene. This low prevalence of resistant strains may probably be partly explained by the urban location of the zoological park, surrounded by kilometers of urban areas without agricultural activities.

A case of feline gastrointestinal eosinophilic sclerosing fibroplasia associated with fungal colonisation: endoscopic features, treatment and follow-up.

Case summary: A 5-year-old castrated male domestic shorthair cat presented with a 3-month history of weight loss, chronic diarrhoea and vomiting. Examination revealed a large proximal duodenal lesion eventually diagnosed as feline gastrointestinal eosinophilic sclerosing fibroplasia (FGESF) associated with fungal filaments. Histological examination was performed following endoscopic biopsy. Direct examination and mycological culture of the duodenal biopsies revealed the presence of a siphomycetous fungus, which was further identified as Rhizopus microsporus. Treatment with prednisolone and ciclosporin for 3 months led to complete resolution of the clinical signs and marked improvement of the endoscopic lesions. Specific fungal treatment with amphotericin B was poorly tolerated. Relevance and novel information: To the best of our knowledge, this is the first report of the characterisation of a siphomycetous fungus associated with FGESF lesions, and the first endoscopic description and diagnosis of FGESF without surgical biopsies. We hypothesise that the presence of R microsporus occurred because of disrupted mucosal integrity.

Assessment of the Safety and Efficacy of an Oral Probiotic-Based Vaccine Against Aspergillus Infection in Captive-Bred Humboldt Penguins (Spheniscus humboldti).

Aspergillosis is a fungal infection caused mainly by Aspergillus fumigatus that often results in respiratory disease in birds. Aspergillosis is a major cause of morbidity and mortality in captive-bred penguin species. Currently, there is no registered vaccine to prevent aspergillosis. Recent research demonstrated that oral administration of gram-negative bacteria expressing high levels of galactose-α-1,3-galactose (α-Gal) modulates anti-α-Gal immunity and protects turkeys from clinical aspergillosis caused by experimental A. fumigatus infection. The role of anti-α-Gal immunity in penguins has not been studied. Here, we tested the distribution of α-1,3-galactosyltransferase (α1,3GT) genes in the fecal microbiome of Humboldt penguins (Spheniscus humboldti). The occurrence of natural anti-α-Gal antibodies (Abs) in sera and eggs of healthy Humboldt penguins was also assessed. A trial was then conducted to test whether oral administration of Escherichia coli Nissle, expressing high α-Gal levels, modulates anti-α-Gal immunity in a colony of Humboldt penguins. Animals in the vaccination and placebo groups were evaluated before the trial and followed for one year for aspergillosis detection using a diagnostic panel including computed tomography scans, capillary zone electrophoresis, 3-hydroxybutyrate levels, and anti-A. fumigatus Abs. Anti-α-Gal Abs were detected in sera (IgM and IgY) and eggs (IgY) of healthy penguins. Microbiota analysis and functional predictions revealed the presence of α1,3GT genes in the microbiota of Humboldt penguins and other penguin species. A strong decrease in anti-α-Gal IgM levels was observed in all animals in the placebo group three months after vaccination protocol. This decrease was not observed in E. coli Nissle-treated penguins. After the vaccination protocol, we found a positive correlation between anti-E. coli IgY and anti-α-Gal IgY in the E. coli Nissle group, suggesting a correlation between the presence of the bacteria and these Abs. During the study period, three penguins exhibited respiratory signs consistent with aspergillosis. Two were from the placebo group whose symptoms resolved with specific treatments, while a single vaccinated individual developed fatal respiratory aspergillosis eight months after the trial. We conclude that E. coli Nissle represents a safe potential probiotic with a protective effect against aspergillosis in Humboldt penguins that deserves to be further explored for therapeutic uses in these animals.

Toxocara cati and Toxoplasma gondii in French Birds of Prey.

Parasites have developed many strategies to ensure their development, multiplication, and dissemination, including the use of reservoir hosts that are often nondomesticated species. Despite drastic reductions in their populations, wild birds remain widespread worldwide and could constitute some of these reservoirs. We focused on the identification of wild bird species harboring parasite stages in their muscles. Breast muscles of 327 birds of 27 different species were collected at three different sites in France. After artificial digestion, isolated nematode larvae were identified by PCR sequencing or restriction fragment length polymorphism (PCR-RFLP). Toxocara cati was identified mainly in birds of prey. The presence of anti-Toxoplasma antibodies was investigated by modified agglutination test on muscle fluids. Anti-Toxoplasma antibodies were detected in 65 out of 166 samples from various bird species. Avifauna, particularly birds of prey, could help on the surveillance of parasite circulation and play a role as sentinel species.

Investigations upon the Improvement of Dermatophyte Identification Using an Online Mass Spectrometry Application.

Online MALDI-TOF mass spectrometry applications, such as MSI-2, have been shown to help identify dermatophytes, but recurrent errors are still observed between phylogenetically close species. The objective of this study was to assess different approaches to reduce the occurrence of such errors by adding new reference spectra to the MSI-2 application. Nine libraries were set up, comprising an increasing number of spectra obtained from reference strains that were submitted to various culture durations on two distinct culture media: Sabouraud gentamicin chloramphenicol medium and IDFP Conidia medium. The final library included spectra from 111 strains of 20 species obtained from cultures on both media collected every three days after the appearance of the colony. The performance of each library was then analyzed using a cross-validation approach. The spectra acquisitions were carried out using a Microflex Bruker spectrometer. Diversifying the references and adding spectra from various culture media and culture durations improved identification performance. The percentage of correct identification at the species level rose from 63.4 to 91.7% when combining all approaches. Nevertheless, residual confusion between close species, such as Trichophyton rubrum, Trichophyton violaceum and Trichophyton soudanense, remained. To distinguish between these species, mass spectrometry identification should take into account basic morphological and/or clinico-epidemiological features.

The Host Protein Aquaporin-9 is Required for Efficient Plasmodium falciparum Sporozoite Entry into Human Hepatocytes.

Hepatocyte invasion by Plasmodium sporozoites represents a promising target for innovative antimalarial therapy, but the molecular events mediating this process are still largely uncharacterized. We previously showed that Plasmodium falciparum sporozoite entry into hepatocytes strictly requires CD81. However, CD81-overexpressing human hepatoma cells remain refractory to P. falciparum infection, suggesting the existence of additional host factors necessary for sporozoite entry. Here, through differential transcriptomic analysis of human hepatocytes and hepatoma HepG2-CD81 cells, the transmembrane protein Aquaporin-9 (AQP9) was found to be among the most downregulated genes in hepatoma cells. RNA silencing showed that sporozoite invasion of hepatocytes requires AQP9 expression. AQP9 overexpression in hepatocytes increased their permissiveness to P. falciparum. Moreover, chemical disruption with the AQP9 inhibitor phloretin markedly inhibited hepatocyte infection. Our findings identify AQP9 as a novel host factor required for P. falciparum sporozoite hepatocyte-entry and indicate that AQP9 could be a potential therapeutic target.

Aspergillosis in Wild Birds.

The ubiquitous fungi belonging to the genus Aspergillus are able to proliferate in a large number of environments on organic substrates. The spores of these opportunistic pathogens, when inhaled, can cause serious and often fatal infections in a wide variety of captive and free-roaming wild birds. The relative importance of innate immunity and the level of exposure in the development of the disease can vary considerably between avian species and epidemiological situations. Given the low efficacy of therapeutic treatments, it is essential that breeders or avian practitioners know the conditions that favor the emergence of Aspergillosis in order to put adequate preventive measures in place.

Detection and Control of Dermatophytosis in Wild European Hedgehogs (Erinaceus europaeus) Admitted to a French Wildlife Rehabilitation Centre.

The rising number of European hedgehogs (Erinaceus europaeus) admitted every year to wildlife rehabilitation centres might be a source of concern to animal and public health since transmissible diseases, such as dermatophytosis, can be easily disseminated. This study seeks to evaluate the frequency of dermatophyte detection in hedgehogs admitted to a wildlife rehabilitation centre located near Paris, France, and to assess the risk of contamination in the centre in order to adapt prevention measures. A longitudinal cohort study was performed on 412 hedgehogs hosted at the Wildlife Animal Hospital of the Veterinary College of Alfort from January to December 2016. Animals were sampled once a month for fungal culture. Dermatophyte colonies were obtained from 174 out of 686 skin samples (25.4%). Besides Trichophyton erinacei, Trichophyton mentagrophytes and Nannizzia gypsea were also found. Dermatophyte detection seemed to be associated with the presence of skin lesions, while more than one-third of T. erinacei-positive animals were asymptomatic carriers. Healing required several months of treatment with topical and systemic azoles, but dermatophytosis did not seem to reduce the probability of release. Daily disinfection procedures and early detection and treatment of infected and asymptomatic carriers succeeded in limiting dermatophyte transmission between hedgehogs and humans.

Cellular and molecular insights on the regulation of innate immune responses to experimental aspergillosis in chicken and turkey poults.

Across the world, many commercial poultry flocks and captive birds are threatened by infection with Aspergillus fumigatus. Susceptibility to aspergillosis varies among birds; among galliform birds specifically, morbidity and mortality rates seem to be greater in turkeys than in chickens. Little is known regarding the features of avian immune responses after inhalation of Aspergillus conidia, and to date, scarce information on inflammatory responses during aspergillosis exists. Thus, in the present study, we aimed to improve our understanding of the interactions between A. fumigatus and economically relevant galliform birds in terms of local innate immune responses. Intra-tracheal aerosolization of A. fumigatus conidia in turkey and chicken poults led to more severe clinical signs and lung lesions in turkeys, but leukocyte recovery from lung lavages was higher in chickens at 1dpi only. Interestingly, only chicken CD8+ T lymphocyte proportions increased after infection. Furthermore, the lungs of infected chickens showed an early upregulation of pro-inflammatory cytokines, including IL-1ß, IFN-γ and IL-6, whereas in turkeys, most of these cytokines showed a downregulation or a delayed upregulation. These results confirmed the importance of an early pro-inflammatory response to ensure the development of an appropriate anti-fungal immunity to avoid Aspergillus dissemination in the respiratory tract. In conclusion, we show for the first time that differences in local innate immune responses between chickens and turkeys during aspergillosis may determine the outcome of the disease.

Aspergillus fumigatus infection may cause mortality in poultry, depending on species sensitivity. This study confirms the earlier activation of chickens' pro-inflammatory effectors to control Aspergillus dissemination, whereas turkeys' immune response enables the exacerbation of lung lesions.

Gut Microbiota Abrogates Anti-α-Gal IgA Response in Lungs and Protects against Experimental Aspergillus Infection in Poultry.

Naturally occurring human antibodies (Abs) of the isotypes IgM and IgG and reactive to the galactose-α-1,3-galactose (α-Gal) epitope are associated with protection against infectious diseases, caused by pathogens expressing the glycan. Gut microbiota bacteria expressing α-Gal regulate the immune response to this glycan in animals lacking endogenous α-Gal. Here, we asked whether the production of anti-α-Gal Abs in response to microbiota stimulation in birds, confers protection against infection by Aspergillus fumigatus, a major fungal pathogen that expresses α-Gal in its surface. We demonstrated that the oral administration of Escherichia coli O86:B7 strain, a bacterium with high α-Gal content, reduces the occurrence of granulomas in lungs and protects turkeys from developing acute aspergillosis. Surprisingly, the protective effect of E. coli O86:B7 was not associated with an increase in circulating anti-α-Gal IgY levels, but with a striking reduction of anti-α-Gal IgA in the lungs of infected turkeys. Subcutaneous immunization against α-Gal did not induce a significant reduction of lung anti-α-Gal IgA and failed to protect against an infectious challenge with A. fumigatus. Oral administration of E. coli O86:B7 was not associated with the upregulation of lung cytokines upon A. fumigatus infection. We concluded that the oral administration of bacteria expressing high levels of α-Gal decreases the levels of lung anti-α-Gal IgA, which are mediators of inflammation and lung damage during acute aspergillosis.

Infection with Toxocara canis Inhibits the Production of IgE Antibodies to α-Gal in Humans: Towards a Conceptual Framework of the Hygiene Hypothesis?

α-Gal syndrome (AGS) is a type of anaphylactic reaction to mammalian meat characterized by an immunoglobulin (Ig)E immune response to the oligosaccharide α-Gal (Galα1-3Galß1-4GlcNAc-R). Tick bites seems to be a prerequisite for the onset of the allergic disease in humans, but the implication of non-tick parasites in α-Gal sensitization has also been deliberated. In the present study, we therefore evaluated the capacity of helminths (Toxocara canis, Ascaris suum, Schistosoma mansoni), protozoa (Toxoplasma gondii), and parasitic fungi (Aspergillus fumigatus) to induce an immune response to α-Gal. For this, different developmental stages of the infectious agents were tested for the presence of α-Gal. Next, the potential correlation between immune responses to α-Gal and the parasite infections was investigated by testing sera collected from patients with AGS and those infected with the parasites. Our results showed that S. mansoni and A. fumigatus produce the terminal α-Gal moieties, but they were not able to induce the production of specific antibodies. By contrast, T. canis, A. suum and T. gondii lack the α-Gal epitope. Furthermore, the patients with T. canis infection had significantly decreased anti-α-Gal IgE levels when compared to the healthy controls, suggesting the potential role of this nematode parasite in suppressing the allergic response to the glycan molecule. This rather intriguing observation is discussed in the context of the 'hygiene hypothesis'. Taken together, our study provides new insights into the relationships between immune responses to α-Gal and parasitic infections. However, further investigations should be undertaken to identify T. canis components with potent immunomodulatory properties and to assess their potential to be used in immunotherapy and control of AGS.

Malaria Sporozoites Traverse Host Cells within Transient Vacuoles.

Plasmodium sporozoites are deposited in the host skin by Anopheles mosquitoes. The parasites migrate from the dermis to the liver, where they invade hepatocytes through a moving junction (MJ) to form a replicative parasitophorous vacuole (PV). Malaria sporozoites need to traverse cells during progression through host tissues, a process requiring parasite perforin-like protein 1 (PLP1). We find that sporozoites traverse cells inside transient vacuoles that precede PV formation. Sporozoites initially invade cells inside transient vacuoles by an active MJ-independent process that does not require vacuole membrane remodeling or release of parasite secretory organelles typically involved in invasion. Sporozoites use pH sensing and PLP1 to exit these vacuoles and avoid degradation by host lysosomes. Next, parasites enter the MJ-dependent PV, which has a different membrane composition, precluding lysosome fusion. The malaria parasite has thus evolved different strategies to evade host cell defense and establish an intracellular niche for replication.

CD81 is required for rhoptry discharge during host cell invasion by Plasmodium yoelii sporozoites.

Plasmodium sporozoites are transmitted by Anopheles mosquitoes and first infect the liver of their mammalian host, where they develop as liver stages before the onset of erythrocytic infection and malaria symptoms. Sporozoite entry into hepatocytes is an attractive target for anti-malarial prophylactic strategies but remains poorly understood at the molecular level. Apicomplexan parasites invade host cells by forming a parasitophorous vacuole that is essential for parasite development, a process that involves secretion of apical organelles called rhoptries. We previously reported that the host membrane protein CD81 is required for infection by Plasmodium falciparum and Plasmodium yoelii sporozoites. CD81 acts at an early stage of infection, possibly at the entry step, but the mechanisms involved are still unknown. To investigate the role of CD81 during sporozoite entry, we generated transgenic P. yoelii parasites expressing fluorescent versions of three known rhoptry proteins, RON2, RON4 and RAP2/3. We observed that RON2 and RON4 are lost following rhoptry discharge during merozoite and sporozoite entry. In contrast, our data indicate that RAP2/3 is secreted into the parasitophorous vacuole during infection. We further show that sporozoite rhoptry discharge occurs only in the presence of CD81, providing the first direct evidence for a role of CD81 during sporozoite productive invasion.

A rapid and robust selection procedure for generating drug-selectable marker-free recombinant malaria parasites.

Experimental genetics have been widely used to explore the biology of the malaria parasites. The rodent parasites Plasmodium berghei and less frequently P. yoelii are commonly utilised, as their complete life cycle can be reproduced in the laboratory and because they are genetically tractable via homologous recombination. However, due to the limited number of drug-selectable markers, multiple modifications of the parasite genome are difficult to achieve and require large numbers of mice. Here we describe a novel strategy that combines positive-negative drug selection and flow cytometry-assisted sorting of fluorescent parasites for the rapid generation of drug-selectable marker-free P. berghei and P. yoelii mutant parasites expressing a GFP or a GFP-luciferase cassette, using minimal numbers of mice. We further illustrate how this new strategy facilitates phenotypic analysis of genetically modified parasites by fluorescence and bioluminescence imaging of P. berghei mutants arrested during liver stage development.

Health impact evaluation of alternative management systems in vicuña (Vicugna vicugna mensalis) populations in Peru.

To determine the impact of farming over vicuña population in Peru, serum samples were collected from 207 vicuñas (126 captive vicuñas and 81 free-ranging vicuñas) and 614 domestic South American camelids (571 alpacas and 43 llamas), in ten Andean communities at the Salinas y Aguada Blanca reserve, province of Arequipa, southern Peru. Samples were tested for the presence of leptospirosis, foot and mouth disease (FMD), bovine viral diarrhea (BVD), bovine herpesvirus type 1 (BHV-1), brucellosis, bluetongue disease (BT), paratuberculosis, and neosporosis. Serological results showed that 1.9% (4/207) of vicuñas, 18.6% (106/571) of alpacas, and 23.3% (10/43) of llamas were positive to one or more Leptospira serovars. One percent of vicuñas (2/207) and 2.4% of domestic camelids (15/614) had Neospora caninum antibodies tested by ELISA, but only two vicuñas and two alpacas were confirmed by Western blot. Epidemiological evaluation found an association of leptospirosis to sex and age (p < 0.001), with female subjects older than 2.5 years at higher risk of infection. Interestingly, antibodies against Leptospira serovars were only found in captive vicuñas. This is the first study where health status of free-ranging and captive vicuñas has been compared. Results indicate minimal to nil presence of FMD, BVD, BHV-1, brucellosis, BT, paratuberculosis, and neosporosis allied to health disorders in our sample. The detection of seropositive animals against Leptospira, however, unveils the likely significance of leptospirosis in wild and domestic South American camelids, the impact of mixed husbandry over vicuña population and the risk to human health.

Identification of Besnoitia besnoiti proteins that showed differences in abundance between tachyzoite and bradyzoite stages by difference gel electrophoresis.

Bovine besnoitiosis is a chronic and debilitating disease, caused by the apicomplexan parasite Besnoitia besnoiti. Infection of cattle by B. besnoiti is governed by the tachyzoite stage, which is related to acute infection, and the bradyzoite stage gathered into macroscopic cysts located in subcutaneous tissue in the skin, mucosal membranes and sclera conjunctiva and related to persistence and chronic infection. However, the entire life cycle of this parasite and the molecular mechanisms underlying tachyzoite-to-bradyzoite conversion remain unknown. In this context, a different antigenic pattern has been observed between tachyzoite and bradyzoite extracts. Thus, to identify stage-specific proteins, a difference gel electrophoresis (DIGE) approach was used on tachyzoite and bradyzoite extracts followed by mass spectrometry (MS) analysis. A total of 130 and 132 spots were differentially expressed in bradyzoites and tachyzoites, respectively (average ratio ± 1.5, P<0.05 in t-test). Furthermore, 25 differentially expressed spots were selected and analysed by MALDI-TOF/MS. As a result, 5 up-regulated bradyzoite proteins (GAPDH, ENO1, LDH, SOD and RNA polymerase) and 5 up-regulated tachyzoite proteins (ENO2; LDH; ATP synthase; HSP70 and PDI) were identified. The present results set the basis for the identification of new proteins as drug targets. Moreover, the role of these proteins in tachyzoite-to-bradyzoite conversion and the role of the host cell environment should be a subject of further research.

[Plasmodium sporozoite entry pathways during malaria liver infection]. / Phase hépatique du paludisme : voies d'entrée des sporozoïtes de Plasmodium.

Plasmodium parasites, the causative agents of malaria, are transmitted by female Anopheles mosquitoes, which inject sporozoites into the skin of the host. The motile sporozoites enter the blood stream and, upon reaching the liver, transform into liver stages inside hepatocytes. The parasites enter host cells actively, using their actomyosin motor machinery to propel themselves through a specialized structure called junction. Penetration inside an invagination of the host cell plasma membrane results in the formation of the parasitophorous vacuole, which is essential for parasite further development. The mechanisms of sporozoite entry into host cells remain poorly understood at the molecular level. We reported for the first time a host factor required for infection of hepatocytes by Plasmodium sporozoites, the tetraspanin CD81, which also serves as a receptor for the hepatitis C virus. CD81 is involved at an early step of the infection, however no evidence for a direct interaction between CD81 and the parasite could be found. Although sporozoites can use several independent pathways to enter hepatocytes, depending on the parasite species and the host cell type, we showed that P. falciparum, the deadliest human malaria parasite, depends on CD81 to infect hepatocytes. We identified structural determinants in the CD81 large extracellular domain, and demonstrated that CD81 function is regulated by its molecular environment in specialized tetraspanin-enriched membrane microdomains. Based on these data we propose that CD81 acts indirectly during malaria infection, by interacting with other essential but still unidentified factor(s), possibly a receptor for the sporozoites, within specific microdomains of the hepatocyte plasma membrane.

Evaluation of the protection conferred by a naturally attenuated Neospora caninum isolate against congenital and cerebral neosporosis in mice.

The parasite Neospora caninum is an important abortifacient agent in cattle worldwide. At present, the development of an effective and safe vaccine against bovine neosporosis is of great relevance. Recently, a new isolate of N. caninum (Nc-Spain 1 H) which was obtained from the brain of an asymptomatic congenitally infected calf, exhibited non-virulent behaviour in mouse and bovine infection models. The aim of this study was to determine the safety and efficacy of Nc-Spain 1 H when used as a vaccinal isolate in well-established BALB/c models of congenital and cerebral neosporosis. Mice were subcutaneously immunised twice at 3-week intervals and were challenged with 2 × 106 tachyzoites of the virulent Nc-Liv isolate. After immunisation with live Nc-Spain 1 H tachyzoites, no parasitic DNA was detected in the dams' brains before challenge and microsatellite analysis performed in PCR-positive mice showed that the profiles corresponded to the challenge isolate Nc-Liv, indicating the Nc-Spain 1 H isolate to be a safe vaccine candidate. The efficacy of the live vaccine was evaluated in the first experiment after the immunisation of mice with 5 × 105 live Nc-Spain 1 H tachyzoites. This immunisation protocol significantly reduced the neonatal mortality to 2.4%, reduced the vertical transmission from 89.1% to 2.3% and completely limited the cerebral infection. These results were associated with a Th1-type immune response. In the second experiment, the effect of various immunising doses was established using ten-fold dilutions of the tachyzoites (from 5 × 105 to 5 × 10). In all the cases, congenital protection rates above 60% were observed, and the mice that were immunised with the lowest dose (5 × 10) presented the highest protection rate (86%). Moreover, low immunising doses of Nc-Spain 1 H induced an IgG2a response, and high parasitic doses induced an IgG1 response. These results evidence the safety and the efficient protection that was conferred by Nc-Spain 1 H against congenital neosporosis, even when the mice were immunised with low parasitic doses.

Identification of Neospora caninum proteins regulated during the differentiation process from tachyzoite to bradyzoite stage by DIGE.

Identification of differentially expressed proteins during Neospora caninum tachyzoite-bradyzoite conversion processes may lead to a better knowledge of the pathogenic mechanisms developed by this important parasite of cattle. In the present work, a differential expression proteomic study of tachyzoite and bradyzoite stages was accomplished for the first time by applying DIGE technology coupled with MS analysis. Up to 72 differentially expressed spots were visualized (1.5-fold in relative abundance, p<0.05, t-test). A total of 53 spots were more abundant in bradyzoites and 19 spots in tachyzoites. MS analysis identified 26 proteins; 20 of them overexpressed in the bradyzoite stage and 6 in the tachyzoite stage. Among the novel proteins, enolase and glyceraldehyde-3-phosphate dehydrogenase (involved in glycolysis), HSP70 and HSP90 (related to stress response) as well as the dense granule protein GRA9, which showed higher abundance in the bradyzoite stage, might be highlighted. On the other hand, isocitrate dehydrogenase 2, involved in the Krebs cycle, was found to be more abundant in tachyzoites extract. Biological functions from most novel proteins were correlated with previously reported processes during the differentiation process in Toxoplasma gondii. Thus, DIGE technology arises as a suitable tool to study mechanisms involved in the N. caninum tachyzoite to bradyzoite conversion.

Failure of a vaccine using immunogenic recombinant proteins rNcSAG4 and rNcGRA7 against neosporosis in mice.

The development of an effective vaccine against Neospora caninum infection in cattle is an important issue due to the significant economic impact of this parasitic disease worldwide. In this work, the immune response, safety and efficacy of different vaccine formulations using the N. caninum recombinant proteins rNcSAG4 (the first bradyzoite-specific protein assayed as a vaccine) and rNcGRA7 were evaluated in mouse models. The survival curves of pups from all vaccinated groups showed a slight delay in time to death compared to control groups; this difference was statistically significant for rNcSAG4+adjuvant group. Immune response of mice vaccinated with rNcSAG4 was characterized by reduced specific IgG and cytokine levels with an equilibrated IFN-gamma/IL-10 balance. Regarding mice vaccinated with rNcGRA7, a very strong humoral and cellular immune response was generated characterized by a hyper-production of IFN-gamma. This response was not accompanied by significant protection. Vaccination with a mixture of both recombinant proteins reduced infection in lung and brain during acute and chronic infection, respectively, although it was not statistically significant. In summary, no significant protection was obtained with these vaccine formulations in the present mouse models. However, the study reveals some positive results on immune response and efficacy for both recombinant proteins; these results are being discussed in order to suggest new approaches with new chronic infection mouse models and adjuvants.