Effects of triploid induction on innate immunity and hematology in Astyanax altiparanae.

Triploid induction is a promising biotechnique that could be used to enhance aquaculture yields in the near future. However, studies conducted with several fish species have demonstrated that the presence of an extra set of chromosomes may result in deleterious health effects. Furthermore, studies of fish immune responses still need to be conducted before these specimens can be readily commercialized. In the study presented herein, we evaluated the effects of triploid induction on hematology, erythrocyte morphometry and morphology, phagocytosis, and the expression levels of IL-1ß and TGF-ß using specimens of the Neotropical species, Astyanax altiparanae. In general, the cell counts of erythrocytes, leukocytes, and neutrophils in triploid fish were lower than those in diploid fish. The erythrocytes of triploid fish were larger than those found in diploid fish, but also demonstrated considerably higher frequencies of cellular and nuclear abnormalities. Although not statistically significant, triploid induction resulted in a phagocytic capacity (PC) 20% lower than that found with diploid fish. No notable differences were observed in phagocytic index (PI). Gene expression levels for the cytokine IL-1 were lower in tissues from the head kidney, liver, and spleen of triploid fish with respect to diploid fish. Gene expression levels of TGF-ß were lower only in the spleen of triploids compared to diploids. In conclusion, triploid induction resulted in A. altiparanae specimens with immune impairments and potentially lower resistances to disease and low-quality environments.

Molecular characterization and gene expression analysis of the pro-inflammatory cytokines IL-1ß and IL-8 in the South American fish Piaractus mesopotamicus challenged with Aeromonas dhakensis.

In the present study, the complete characterization of cDNA and genomic sequences of IL-1ß and IL-8, as well as the expression profile of these genes in the South American fish pacu (Piaractus mesopotamicus) is provided. The full-length pmIL-1ß cDNA was composed of 1208 nucleotides that would produce a precursor peptide with 273 amino acid residues. A putative caspase-1 cleavage site, similar to what is found in mammalian IL-1ß, was identified producing a mature peptide with a theoretical molecular weight of 17.21 kDa. The pmIL-8 cDNA sequence consisted of 1019 nucleotides which encoded a 95-amino acid protein with a theoretical molecular weight of 10.43 kDa that showed all typical CXC chemokine features, including a 20-residue signal peptide and four conserved cysteine residues. Constitutive mRNA expression was detected for both genes in the liver, head kidney, gill, intestine, skin and spleen. After a bacterial challenge, up-regulation was detected for both pmIL-1ß and pmIL-8 in the spleen and head kidney at 12 h post-infection. At 24 h post-infection there was a decrease in the expression of both genes, with pmIL-8 showing a significant down-regulation in the liver and head kidney when compared to the control groups.

Immune recognition of salivary proteins from the cattle tick Rhipicephalus microplus differs according to the genotype of the bovine host.

BACKGROUND: Males of the cattle tick Rhipicephalus microplus produce salivary immunoglobulin-binding proteins and allotypic variations in IgG are associated with tick loads in bovines. These findings indicate that antibody responses may be essential to control tick infestations. Infestation loads with cattle ticks are heritable: some breeds carry high loads of reproductively successful ticks, in others, few ticks feed and they reproduce inefficiently. Different patterns of humoral immunity against tick salivary proteins may explain these phenotypes. METHODS: We describe the profiles of humoral responses against tick salivary proteins elicited during repeated artificial infestations of bovines of a tick-resistant (Nelore) and a tick-susceptible (Holstein) breed. We measured serum levels of total IgG1, IgG2 and IgE immunoglobulins and of IgG1 and IgG2 antibodies specific for tick salivary proteins. With liquid chromatography followed by mass spectrometry we identified tick salivary proteins that were differentially recognized by serum antibodies from tick-resistant and tick-susceptible bovines in immunoblots of tick salivary proteins separated by two-dimensional electrophoresis. RESULTS: Baseline levels of total IgG1 and IgG2 were significantly higher in tick-susceptible Holsteins compared with resistant Nelores. Significant increases in levels of total IgG1, but not of IgG2 accompanied successive infestations in both breeds. Resistant Nelores presented with significantly higher levels of salivary-specific antibodies before and at the first challenge with tick larvae; however, by the third challenge, tick-susceptible Holsteins presented with significantly higher levels of IgG1 and IgG2 tick salivary protein-specific antibodies. Importantly, sera from tick-resistant Nelores reacted with 39 tick salivary proteins in immunoblots of salivary proteins separated in two dimensions by electrophoresis versus only 21 spots reacting with sera from tick-susceptible Holsteins. CONCLUSIONS: Levels of tick saliva-specific antibodies were not directly correlated with infestation phenotypes. However, in spite of receiving apparently lower amounts of tick saliva, tick-resistant bovines recognized more tick salivary proteins. These reactive salivary proteins are putatively involved in several functions of parasitism and blood-feeding. Our results indicate that neutralization by host antibodies of tick salivary proteins involved in parasitism is essential to control tick infestations.

Immune and biochemical responses in skin differ between bovine hosts genetically susceptible and resistant to the cattle tick Rhipicephalus microplus.

BACKGROUND: Ticks attach to and penetrate their hosts' skin and inactivate multiple components of host responses in order to acquire a blood meal. Infestation loads with the cattle tick, Rhipicephalus microplus, are heritable: some breeds carry high loads of reproductively successful ticks, whereas in others, few ticks feed and reproduce efficiently. METHODS: In order to elucidate the mechanisms that result in the different outcomes of infestations with cattle ticks, we examined global gene expression and inflammation induced by tick bites in skins from one resistant and one susceptible breed of cattle that underwent primary infestations with larvae and nymphs of R. microplus. We also examined the expression profiles of genes encoding secreted tick proteins that mediate parasitism in larvae and nymphs feeding on these breeds. RESULTS: Functional analyses of differentially expressed genes in the skin suggest that allergic contact-like dermatitis develops with ensuing production of IL-6, CXCL-8 and CCL-2 and is sustained by HMGB1, ISG15 and PKR, leading to expression of pro-inflammatory chemokines and cytokines that recruit granulocytes and T lymphocytes. Importantly, this response is delayed in susceptible hosts. Histopathological analyses of infested skins showed inflammatory reactions surrounding tick cement cones that enable attachment in both breeds, but in genetically tick-resistant bovines they destabilized the cone. The transcription data provided insights into tick-mediated activation of basophils, which have previously been shown to be a key to host resistance in model systems. Skin from tick-susceptible bovines expressed more transcripts encoding enzymes that detoxify tissues. Interestingly, these enzymes also produce volatile odoriferous compounds and, accordingly, skin rubbings from tick-susceptible bovines attracted significantly more tick larvae than rubbings from resistant hosts. Moreover, transcripts encoding secreted modulatory molecules by the tick were significantly more abundant in larval and in nymphal salivary glands from ticks feeding on susceptible bovines. CONCLUSIONS: Compared with tick-susceptible hosts, genes encoding enzymes producing volatile compounds exhibit significantly lower expression in resistant hosts, which may render them less attractive to larvae; resistant hosts expose ticks to an earlier inflammatory response, which in ticks is associated with significantly lower expression of genes encoding salivary proteins that suppress host immunity, inflammation and coagulation.

Molecular phylogeny and ultrastructure of Myxobolus cf. cuneus, a parasite of patinga hybrid and Henneguya pseudoplatystoma, a parasite of pintado hybrid.

Through morphological, histopathological and ultrastructural analysis of Myxobolus cuneus Adriano, Arana et Cordeiro, 2006 and Henneguya pseudoplatystoma Naldoni, Arana, Maia, Ceccarelli, Tavares, Borges, Pozo et Adriano, 2009 were identified infecting pacu respectively (Piaractus mesopotamicus) and hybrid pintado (Pseudoplatystoma corruscans x Pseudoplatystoma reticulatum) taken from Brazilian fish farms. The present study describes 18S rDNA sequencing of Myxobolus cf. cuneus infecting the spleen of farmed patinga, a hybrid fish resulting from the crossing of P. mesopotamicus x Piaractus brachypomus, and H. pseudoplatystoma found in farmed hybrid pintado from the state of Sao Paulo, Brazil. The study also provides new details of the host-parasite interface of M. cf. cuneus, which reveal that the plasmodial wall is composed of a single membrane connected to the plasmodium ectoplasm by numerous pinocytic canals. The plasmodia also displayed asynchronous development but had disporic pansporoblasts at different developmental stages; immature and mature spores were found at different depth levels of the plasmodium. Maximum likelihood phylogenetic analysis showed that M. cf. cuneus appeared as a sister species of Henneguya pellucida Adriano, Arana et Cordeiro, 2005 in a sub-clade composed mainly of myxosporean parasites of characiforms, and that H. pseudoplatystoma clustered in a sub-clade composed of Henneguya/Myxobolus spp. parasites of siluriform fish.

Supplementary data of Henneguya leporinicola (Myxozoa, Myxosporea) a parasite of Leporinus macrocephalus from fish farms in the state of São Paulo, Brazil.

Henneguya leporinicola is a parasite of the gill filament of Leporinus macrocephalus, a characiform fish belonging to the Anostomidae family, which is of major economic importance. Despite the damage it causes in fish, little is known about this parasite. Therefore, a study was undertaken with fourteen specimens of L. macrocephalus taken from fish farms in the state of Sao Paulo. The fish were collected and examined searching for lesions and/or myxosporean plasmodia. One of the specimens (7.14%) contained white elongated plasmodia in the gill filament. The mature spores had elongated bodies with polar capsules of equal size and a caudal length greater than body length. Morphological characteristics identified the parasite as H. leporinicola. Molecular analysis of the 18S rDNA sequence resulted in a 1954 bp, demonstrating significant genetic differences with previously described species of Henneguya/Myxobolus. Phylogenetic analysis comparing the 18S rDNA sequence of H. leporinicola with other species, previously described in South America, and the 20 closest species as indicated by BLASTn Max Score showed H. leporinicola as a basal branch of a subclade composed by Henneguya spp. parasite of characiform hosts.

Haplotypes of the bovine IgG2 heavy gamma chain in tick-resistant and tick-susceptible breeds of cattle.

Bovines present contrasting, heritable phenotypes of infestations with the cattle tick, Rhipicephalus (Boophilus) microplus. Tick salivary glands produce IgG-binding proteins (IGBPs) as a mechanism for escaping from host antibodies that these ectoparasites ingest during blood meals. Allotypes that occur in the constant region of IgG may differ in their capacity to bind with tick IGBPs; this may be reflected by the distribution of distinct allotypes according to phenotypes of tick infestations. In order to test this hypothesis, we investigated the frequency of haplotypes of bovine IgG2 among tick-resistant and tick-susceptible breeds of bovines. Sequencing of the gene coding for the heavy chain of IgG2 from 114 tick-resistant (Bos taurus indicus, Nelore breed) and tick-susceptible (B. t. taurus, Holstein breed) bovines revealed SNPs that generated 13 different haplotypes, of which 11 were novel and 5 were exclusive of Holstein and 3 of Nelore breeds. Alignment and modeling of coded haplotypes for hinge regions of the bovine IgG2 showed that they differ in the distribution of polar and hydrophobic amino acids and in shape according to the distribution of these amino acids. We also found that there was an association between genotypes of the constant region of the IgG2 heavy chain with phenotypes of tick infestations. These findings open the possibility of investigating if certain IgG allotypes hinder the function of tick IGBPs. If so, they may be markers for breeding for resistance against tick infestations.

Ensaios de padronização do teste ELISA para diagnósticoda cisticercose bovina utilizando antígenos de larvade Taenia crassiceps / ELISA test standardization for the diagnosis of bovine cysticercosis employing Taenia crassiceps metacestode antigens

Os antígenos total e vesicular de Taenia crassiceps e soros-controle de bovinos positivos e negativos para a cisticercose foramcomparados por meio de alguns parâmetros de padronização do teste ELISA, visando avaliar o seu desempenho no diagnósticoda referida doença. Os parâmetros avaliados foram: diferentes concentrações dos antígenos, diluições de soros e conjugados,marcas de placa e substâncias bloqueadoras das placas. As diluições 1:25 de soro e 1:2.500 de conjugado para o antígenode líquido vesicular e 1:5.000 para o antígeno total, o leite desnatado e as maiores concentrações dos antígenos testadas (1,2 e 4 µg/orifício) foram os critérios de padronização que proporcionaram maior diferenciação entre soros positivos e negativos,conseqüentemente, melhor desempenho do teste ELISA.

Using the cyst fluid and the total antigens of Taenia crassiceps and positive and negative bovine control sera, some standardizationparameters of one ELISA were compared, in order to evaluate its performance in the diagnosis of the bovine cysticercosis. Thereferred parameters were different antigen concentrations, dilutions of sera and conjugated, plate marks and blocking substances.The 1:25 dilution sera and 1:2.500 dilution conjugated for the cyst fluid antigen and 1:5.000 for the total antigen, the skimmedmilk and the largest antigens concentrations (1, 2 and 4 µg/well) improved the differentiation among positive and negative sera,consequently, they were the best criteria for the bovine cysticercosis determination employing the ELISA.

Comparative evaluation of different immunoassays for the detection of Taenia solium cysticercosis in swine with low parasite burden.

Seven swine were experimentally infected with Taenia solium eggs and blood samples from each animal were periodically collected. At the end of the experiment (t140) the animals did not show clinical aspects of cysticercosis or parasites in tongue inspection. All animals were slaughtered and cut into thin slices in searching for cysts. The number of cysts found in each animal varied from 1 to 85. Enzyme-linked immunosorbent assay (ELISA) tests for antibody (Ab) detection and for antigen (Ag) detection were performed, which presented respectively 71 and 57% of positivity. By immunoblot (IB), using 18/14(T. crassiceps Ag) or lentil-lectin-purified glycoproteins from T. solium Ag (LLGP) as Ag, five (71%) and six (86%) animals were positive, respectively. The association between Ag-ELISA with any IB (18/14 or LLGP) allowed the detection of all animals at 140 days post-experimental infection (days p.e.i.). The use of IB 18/14 combined to the Ag-ELISA allowed the detection of all animals since 70 days p.e.i., and the association between IB LLGP and Ag-ELISA allowed the detection of all animals since 112 days p.e.i. While all animals could be considered healthy by conventional screening tests, the use of immunoassays for detecting Ab and Ag showed better accuracy; therefore it would be more useful than usual clinical examination for screening cysticercosis in slightly infected pigs.

Comparative evaluation of different immunoassays for the detection of Taenia solium cysticercosis in swine with low parasite burden

Seven swine were experimentally infected with Taenia solium eggs and blood samples from each animal were periodically collected. At the end of the experiment (t140) the animals did not show clinical aspects of cysticercosis or parasites in tongue inspection. All animals were slaughtered and cut into thin slices in searching for cysts. The number of cysts found in each animal varied from 1 to 85. Enzyme-linked immunosorbent assay (ELISA) tests for antibody (Ab) detection and for antigen (Ag) detection were performed, which presented respectively 71 and 57 percent of positivity. By immunoblot (IB), using 18/14(T. crassiceps Ag) or lentil-lectin-purified glycoproteins from T. solium Ag (LLGP) as Ag, five (71 percent) and six (86 percent) animals were positive, respectively. The association between Ag-ELISA with any IB (18/14 or LLGP) allowed the detection of all animals at 140 days post-experimental infection (days p.e.i.). The use of IB 18/14 combined to the Ag-ELISA allowed the detection of all animals since 70 days p.e.i., and the association between IB LLGP and Ag-ELISA allowed the detection of all animals since 112 days p.e.i. While all animals could be considered healthy by conventional screening tests, the use of immunoassays for detecting Ab and Ag showed better accuracy; therefore it would be more useful than usual clinical examination for screening cysticercosis in slightly infected pigs.

Metacercárias de Ascocotyle (Phagicola) longa Ransom, 1920 (Digenea: Heterophyidae), em Mugil platanus, no estuário de Cananéia, SP, Brasil / Ascocotyle (Phagicola) longa Ransom, 1920 (Digenea: Heterophylidae) metacercariae, in Mugil platanus in estuarin of Cananéia, SP, Brazil

Estudou-se no estuário de Cananéia, litoral do estado de São Paulo, Brasil, a presença de Ascocotyle (Phagicola) longa Ransom, 1920 (Digenea: Heterophylidae), trematódeo, em tainha (Mugil platanus, Günther, 1880). Foram realizadas amostragens a fresco e cortes histológicos em tecidos provenientes de 61 exemplares. A presença de metacercárias foi comprovada em 100 por cento dos peixes examinados, com a seguinte distribuição: coração (21,6 por cento), fígado (19,7 por cento) e rins (58,6 por cento). As alterações histológicas no tecido cardíaco foram caracterizadas pela presença de um granuloma parasitário formado por uma cápsula de tecido conjuntivo ao redor do parasita, levando a um edema generalizado. Para determinar a infecção em alevinos de tainha da espécie M. platanus, foram capturados 100 exemplares nas entradas de córregos da região estuarino-lagunar. Esses peixes foram eviscerados para a pesquisa de metacercárias pelo exame a fresco, verificando-se que 100 por cento dos alevinos não estavam infectados.

The present research was carried out the coast of the state of São Paulo, Brazil, when it was studied the presence of Ascocotyle (Phagicola) longa Ransom, 1920 (Digenea: Heterophylidae), trematode, in mullet (Mugil platanus, Günther, 1880). Fresh samples and histological tissues sections from 61 animals were obtained. The results showed presence of metacercariae of A. (P.) longa in 100 percent of the fish examined, with the following distribution: heart (21.6 percent), liver (19.7 percent) and kidneys (58.6 percent). Histological alterations in cardiac tissues were characterized by the presence of a parasitic granuloma formed by a connective tissue capsule around the parasite, leading to generalized edema. To determine the infection in juvenile grey mullets of the M. platanus species, 100 of them were captured in the stream entrances of the estuary region, each fish was individually sacrificed and fresh mounts were prepared to determine the presence of metacercariae, demonstrating that 100 percent of the juveniles were not infected.

Recombinant expression of Taenia solium TS14 antigen and its utilization for immunodiagnosis of neurocysticercosis.

In order to evaluate the potential use of TS14 antigen in an enzyme-linked immunosorbent assay (ELISA) for immunodiagnosis of neurocysticercosis (NC), its open reading frame (ORF) was amplified by RT-PCR from mRNA isolated from Taenia solium cysticerci. The ORF was subcloned into the expression vector pET-28a, and was used to transform Escherichia coli BL21 (DE3) cells to produce TS14 antigen. The His-tagged expressed protein was purified on a nickel affinity column. Using the HISTS14 as antigen, ELISA was positive for 100% of cerebrospinal fluid (CSF) and 97% of serum samples from NC patients. No positive results were observed with sera and CSF samples from control groups. Cross-reactivity with sera from patients with schistosomiasis and Chagas' disease was not observed. Serum samples from patients with taeniasis were evaluated and 2 of 13 cases showed reactivity in this assay. Our data indicate the usefulness of HISTS14 in ELISA for an accurate and rapid assay for diagnosis of NC and seroepidemiological studies.

Effect of Cysticercus cellulosae fractions on the respiratory burst of pig neutrophils.

Neutrophils, eosinophils and macrophages are cells that interact with invading parasites and naive hosts have been shown to have anti-parasitic activity. The initial reaction of these leukocytes is the generation of reactive oxygen species (ROS) to play in parasite expulsion. The present work was carried out to study the effect of total extract, scolex and membrane fractions from Cysticercus cellulosae on respiratory burst by pig neutrophils. Hydrogen peroxide (H2O2) production by neutrophils incubated with metacestode fractions from C. cellulosae showed an increase of: 190% (total extract), 120% (scolex) and 44% (membrane). High antioxidant catalatic activity (33%, 28%, 28% by total extract, scolex and membrane, respectively) was observed in neutrophils incubated with metacestode fractions, which could be an attempt at self-protection. Scolex and membrane fractions increased the phagocytic capacity of neutrophils (44% and 28%, respectively). On the other hand, total cysticerci did not alter the phagocytosis, possibly due to modifications in membrane function, caused by high ROS production from neutrophils in the presence of total cysticerci. Total fraction from C. cellulosae is toxic for neutrophils as shown by the decrease in phagocytic capacity, probably caused by high levels of ROS formation. The difference in toxicity of total extract, scolex and membrane fractions on neutrophils can be explained by the presence of an antigenic effect of the vesicular fluid in the total extract of C. cellulosae.

Effect of Cysticercus cellulosae fractions on the respiratory burst of pig neutrophils

Neutrófilos, eosinófilos e macrófagos são células que interagem com os parasitas no corpo do hospedeiro desenvolvendo atividade antiparasitária. A reação inicial destes leucócitos é a geração de espécies reativas de oxigênio (ERO) a fim de expulsar os parasitas. No presente trabalho estudou-se o efeito da fração total, de escolex e de membrana de Cysticercus cellulosae sobre a explosão respiratória de neutrófilos de suínos. A produção de peróxido de hidrogênio (H2O2) pelos neutrófilos incubados com as frações de C. cellulosae apresentou acréscimo de 190% (extrato total), 120% (escolex) e 44% (membrana). Alta atividade de catalase (33%, 28% e 28% para extrato total, escolex e membrana respectivamente) foi observada nos neutrófilos incubados com as frações de metacestodeo, podendo representar a própria proteção celular do neutrófilo. Frações de escolex e de membrana aumentaram a capacidade fagocitária dos neutrófilos (44% e 28%, respectivamente). Por outro lado, a fração total do cisticerco não alterou a capacidade fagocitária dos neutrófilos, o que pode estar relacionada com modificações na função da membrana celular causadas pela alta produção de ERO na presença da fração total. O extrato total de C. cellulosae é tóxico para os neutrófilos, indicada pela diminuição da capacidade fagocitária, provavelmente pela indução de alto nível de ERO. A diferença de toxicidade do extrato total, de escolex e de membrana para os neutrófilos pode ocorrer pelo efeito antigênico presente no fluido vesicular no extrato total de C. cellulosae.

Sensibilizaçäo alérgica à Blatella germanica (Insecta: Dyctyoptera) em pacientes com asma e rinite na cidade de Säo Paulo, Brasil / Allergic sensitization to Blatella germanica (Insecta: Dyctyoptera) in patients with asthma and rhinitis in Säo Paulo city, Brazil

Em 203 pacientes com história de asma e rinite e com crises respiratórias ao contato com pó domiciliar, foram realizados testes cutâneos com B. germanica, pesquisa de IgE específica pelo método ELISA ("Enzyme-Linked Immunosorbent Assay") e prova de Immunoblotting. Em 37, as provas cutâneas foram positivas, e em 14 foram demonstrados anticorpos IgE específicos em concentraçöes baixas ou moderadas e apenas dois com nível elevado. Pela prova de "Immunoblotting", em oito soros, os anticorpos reconheceram fraçöes alergênicas presentes no extrato total. Sete pacientes apresentaram também anticorpos para a P. americana, em concentraçöes semelhantes àquelas determinadas para a B. germanica.