Phagocytotic activity and gene expression of leukocytes isolated from Astyanax lacustris.

The constant intensification of aquaculture has considerable increased the stress levels of farmed fish and, consequently, the number and intensity of diseases outbreaks. Thus, studies on fish immune response, especially regarding the interaction of fish leukocytes with potential pathogens and xenobiotics are of great importance in order to develop new prophylactic and curative strategies. We isolated leukocytes from the head kidney of Astyanax lacustris-an important Neotropical fish species for aquaculture and a potential model for Neotropical aquaculture research-using a Percoll centrifugation protocol. The isolated leukocytes were incubated with lipopolysaccharide (LPS), and the expression of genes IL-1ß, IL-8, LysC, and LysG were measured. We assessed the phagocytotic activity of leukocytes using Congo red-dyed yeast, a novel and cost-effective protocol that has been developed in this study. The isolated leukocytes responded to LPS induction, exhibiting strong IL-1ß and IL-8 upregulation, two of the most important pro-inflammatory interleukins for vertebrates immune reponse. The optimal concentration of yeast for the phagocytic assay was 106 cells mL-1, resulting in acceptable phagocytic capacity (PC) but without excess of yeasts during the counting process, ensuring a high precision and accuracy of the method. To the best of our knowledge, the present study is the first to investigate the in vitro gene expression and phagocytic activity of leukocytes isolated from A. lacustris. Our findings will serve as a reference for future studies on the immunology and toxicology of Neotropical fish.

Phylogenetic and host-parasite relationship analysis of Henneguya multiplasmodialis n. sp. infecting Pseudoplatystoma spp. in Brazilian Pantanal wetland.

A new species of the genus Henneguya (Henneguya multiplasmodialis n. sp.) was found infecting the gills of three of 89 specimens (3.3%) of Pseudoplatystoma corruscans and two of 79 specimens (2.6%) of Pseudoplatystoma reticulatum from rivers in the Pantanal wetland, Brazil. Partial sequencing of the 18S rDNA gene of the spores obtained from one plasmodium from the gills of P. corruscans and other one from the gills of P. reticulatum, respectively, resulted in a total of 1560 and 1147 base pairs. As the spores of H. multiplasmodialis n. sp. resemble those of Henneguya corruscans, which is also a parasite of P. corruscans, sequencing of the 18S rDNA gene of the spores of H. corruscans found on P. corruscans caught in the Brazilian Pantanal wetland was also provided to avoid any taxonomic pendency between these two species, resulting in 1913 base pairs. The sequences of H. multiplasmodialis n. sp. parasite of P. corruscans and P. reticulatum and H. corruscans did not match any of the Myxozoa available in the GenBank. The similarity of H. multiplasmodialis n. sp. obtained from P. corruscans to that from P. reticulatum was of 99.7%. Phylogeny revealed a strong tendency among Henneguya species to form clades based on the order and/or family of the host fish. H. multiplasmodialis n. sp. clustered in a clade with Henneguya eirasi and H. corruscans, which are also parasites of siluriforms of the family Pimelodidae and, together with the clade composed of Henneguya spp. parasites of siluriforms of the family Ictaluridae, formed a monophyletic clade of parasites of siluriform hosts. The histological study revealed that the wall of the plasmodia of H. multiplasmodialis n. sp. were covered with a stratified epithelium rich in club cells and supported by a layer of connective tissue. The interior of the plasmodia had a network of septa that divided the plasmodia into numerous compartments. The septa were composed of connective tissue also covered on both sides with a stratified epithelium rich in club cells. Inflammatory infiltrate was found in the tissue surrounding the plasmodia as well as in the septa.

Host-parasite-environment relationship, morphology and molecular analyses of Henneguya eirasi n. sp. parasite of two wild Pseudoplatystoma spp. in Pantanal Wetland, Brazil.

A new myxosporean species, Henneguya eirasi n. sp., is described parasitizing the gill filaments of Pseudoplatystoma corruscans and Pseudoplatystoma fasciatum (Siluriformes: Pimelodidae) caught in the Patanal Wetland of the state of Mato Grosso, Brazil. The parasite formed white, elongated plasmodia measuring up to 3mm. Mature spores were ellipsoidal in the frontal view, measuring 37.1 ± 1.8 µm in total length, 12.9 ± 0.8 µm in body length, 3.4 ± 0.3 µm in width, 3.1 ± 0.1 µm in thickness and 24.6 ± 2.2 µm in the caudal process. Polar capsules were elongated and equal in size, measuring 5.4 ± 0.5 µm in length and 0.7 ± 0.1 µm in width. Polar filaments had 12-13 coils. Histopathological analysis revealed that the parasite developed in the sub-epithelial connective tissue of the gill filaments and the plasmodia were surrounded by a capsule of host connective tissue. The plasmodia caused slight compression of the adjacent tissues, but no inflammatory response was observed in the infection site. Ultrastructure analysis revealed a single plasmodial wall connected to the ectoplasmic zone through numerous pinocytotic canals. The plasmodial wall exhibited numerous projections and slightly electron-dense material was found in the ectoplasm next to the plasmodial wall, forming a line just below the wall. Partial sequencing of the 18S rDNA gene of H. eirasi n. sp. obtained from P. fasciatum resulted in a total of 1066 bp and this sequence did not match any of the Myxozoa available in the GenBank. Phylogenetic analysis revealed the Henneguya species clustering into clades following the order and family of the host fishes. H. eirasi n. sp. clustered alone in one clade, which was the basal unit for the clade composed of Henneguya species parasites of siluriform ictalurids. The prevalence of the parasite was 17.1% in both fish species examined. Parasite prevalence was not influenced by season, host sex or host size.

Henneguya pseudoplatystoma n. sp. causing reduction in epithelial area of gills in the farmed pintado, a South American catfish: histopathology and ultrastructure.

The present study is part of an ongoing investigation into the characteristics of Myxozoan parasites of Brazilian freshwater fish and was carried out using morphology, histopathology and electron microscopy analysis. A new Myxosporea species (Henneguya pseudoplatystoma) is described causing an important reduction in gill function in the farmed pintado (a hybrid fish from a cross between Pseudoplatystoma corruscans and Pseudoplatystoma fasciatum), which is a commercially important South American catfish. From a total of 98 pintado juveniles from fish farms in the states of São Paulo and Mato Grosso do Sul (Brazil), 36 samples (36.7%) exhibited infection of the gill filaments. Infection was intense, with several plasmodia occurring on a same gill filament. The plasmodia were white and measured up to 0.5mm in length; mature spores were ellipsoidal in the frontal view, measuring 33.2+/-1.9 microm in total length, 10.4+/-0.6 microm in body length, 3.4+/-0.4 microm in width and 22.7+/-1.7 microm in the caudal process. The polar capsules were elongated, measuring 3.3+/-0.4 microm in length and 1.0+/-0.1 microm in width and the polar filaments had six to seven turns. Histopathological analysis revealed the parasite in the connective tissue of the gill filaments and lamella. No inflammatory process was observed, but the development of the plasmodia reduced the area of functional epithelium. Ultrastructural analyses revealed a single plasmodial wall, which was in direct contact with the host cells and had numerous projections in direction of the host cells as well as extensive pinocytotic canals. A thick layer (2-6 microm) of fibrous material and numerous mitochondria were found in the ectoplasm. Generative cells and the earliest stage of sporogenesis were seen more internally. Advanced spore developmental stages and mature spores were found in the central portion of the plasmodia.

Light, electron microscopy and histopathology of Myxobolus salminus n. sp., a parasite of Salminus brasiliensis from the Brazilian Pantanal.

In this report, we describe the morphology and histopathology of Myxobolus salminus n. sp., a parasite of the gill filaments of wild Salminus brasiliensis (dourado) from the Brazilian Pantanal. The small polysporic plasmodia were approximately 100 microm in diameter and the development was asynchronous. The mature spores were oval to pear shaped and had a smooth wall. The spore measurements were (mean+/-S.D., with range in parentheses): length 10.1+/-0.4 microm (9.6-10.5), width 6.1+/-0.4 microm (5.8-6.6) and thickness 5.0+/-0.6 microm (4.7-5.3). The polar capsules were elongated and of equal size: length 4.6+/-0.2 microm (4.3-4.8) and width 1.7+/-0.1 microm (1.5-1.9). The histological analysis revealed numerous plasmodia in the blood vessels of the gill filaments. The site of parasite development was the wall of the large-caliber blood vessel of the gill filament, with progressive growth towards the lumen, resulting in the obstruction of blood flow, congestion and perivascular edema. The ultrastructural study revealed that the plasmodial wall was composed of two membranes, had numerous pinocytic canals and was in direct contact with the basement membrane of the vessel. The development of the parasite was asynchronous, with mature spores, immature spores and young developmental stages randomly distributed throughout the plasmodium. The prevalence of the parasite was 4.4%, with male and female fish being infected.

Myxobolus cordeiroi n. sp., a parasite of Zungaro jahu (Siluriformes: Pimelodiade) from Brazilian Pantanal: morphology, phylogeny and histopathology.

This work is part of an ongoing investigation into the characteristics of Myxozoan parasites of freshwater fish in Brazil and was carried out using morphology, histopathology and molecular analysis. A new Myxosporea species (Myxobolus cordeiroi) is described infecting the jaú catfish (Zungaro jahu). Fifty jaú specimens were examined and 78% exhibited plasmodia of the parasite. The plasmodia were white and round, measuring 0.3-2.0mm in diameter and the development occurred in the gill arch, skin, serosa of the body cavity, urinary bladder and eye. The spores had an oval body and the spore wall was smooth. Partial sequencing of the 18S rDNA gene resulted in a total of 505bp and the alignment of the sequences obtained from samples in different organs revealed 100% identity. In the phylogenetic analysis, the Myxobolus species clustered into two clades-one primarily parasites of freshwater fish and the other primarily parasites of marine fish. M. cordeiroi n. sp. was clustered in a basal position in the freshwater fish species clade. The histological analysis revealed the parasite in the connective tissue of the different infected sites, thereby exhibiting affinity to this tissue. The plasmodium was surrounded by an outer collagen capsule of fibers with distinct orientation from the adjacent connective tissue and an inner layer composed of delicate collagen fibrils-more precisely reticular fibers. The development of the parasite in the cornea and urinary bladder caused considerable stretching of the epithelium.

Imunodiagnóstico da cisticercose em suíno experimentalmente infectado com ovos de Taenia solium, utilizando antígeno de escólex de Cysticercus cellulosae / Immunodiagnosis of cysticercosis in swine experimentally infected with Taenia solium eggs, using antigen of Cysticercus cellulosae scolex

Colheu-se sangue de sete suínos infectados com ovos de Taenia solium, semanalmente, durante 140 dias, para realizar ELISA no soro, utilizando antígeno de escólex (Es-Tso) de C. cellulosae. Em todos os animais, após o 21º dia pós-infecção, houve incremento significativo de anticorpos IgG, que assim se mantiveram até o final do experimento. A sensibilidade do ELISA variou entre 87,5 e 100 por cento. A necropsia, foram identificados 238 cisticercos. Seis suínos apresentaram pelo menos um cisto no coração, língua ou masseter. Não se observou correlação entre concentração de anticorpos e número de cisticercos identificados.

Blood samples from seven swines infected with eggs of Taenia solium, were collected weekly during a period of 140 days. The ELISA was carried out in serum, using antigen from Cysticercus cellulosae scolex (Es-Tso). The antibody levels for all animals significantly increased and maintained constant from the 21th day post-infection to the end of the experiment. The sensitivity of the ELISA test averaged between 87.5 percent and 100 percent. At the necropsy, 238 cysticerci were identified. Six swines presented at least one cysticercus in one of the organs: heart, tongue or masseter. No correlation between concentration of antibodies and number of identified cysticerci at necropsy, was observed.

Effect of Cysticercus cellulosae on neutrophil function and death.

Neutrophils, eosinophils and macrophages interact with invading parasites and naive hosts. The initial reaction of leukocytes is the generation of reactive oxygen species (ROS). The cytotoxic effects of extracts derived from intact Cysticercus cellulosae and from the scolex or membrane fractions on neutrophils were examined. DNA fragmentation of neutrophils was observed when cells were incubated with an extract from the intact metacestode; however, the addition of antioxidant enzymes to the incubation medium had a protective effect. The scolex and membrane extracts did not affect DNA fragmentation of neutrophils. Hydrogen peroxide production of neutrophils incubated with metacestode fractions from C. cellulosae increased by 190% (total extract), 120% (scolex) or 44% (membrane). An increase in antioxidant catalase activity (28%) concomitant with the increased production of ROS was observed in neutrophils incubated with metacestode fractions, which could be an attempt at self-protection. ROS production by neutrophils in the presence of the intact cysticerci extract did not alter phagocytosis. In contrast, the scolex and membrane fractions increased the phagocytic capacity of neutrophils by 44 and 28%, respectively. The results showed that the extract from intact C. cellulosae was toxic for neutrophils via ROS production, leading to DNA fragmentation and inhibition of phagocytic capacity, but neutrophils are able to protect themselves against oxidative stress by via catalase activity.

Oocistos de Cryptosporidium spp nas fezes de bovinos em Montes Claros, Minas Gerais / Oocysts of Cryptosporidium spp in the feces of bovines, in Montes Claros, MG, Brazil

In January 1990, fecal samples from two dairy and five beef cattle farms located in Montes Claros, MG, Brazil were examined by using the centrifugation-flotation method in saturated sugar solution. Thirteen (27,1 per cent), of 48 two month-old calves were positive for oocysts of Cryptosporidium muris, found in one beef cattle and two dairy farms. Six (6,6 per cent) out of 91 cows were positive for C. parvum oocysts. This species of coccidium was observed in one dairy and two beef cattle farms. All cows were negative for C. muris

Localizaçäo do Dipetalonema reconditum (Grassi, 1890) (Nematoda filariidae) de Canis familiaris / Localization of Dipetalonema reconditum (Grassi, 1890) (Nematoda filariidae) de Canis familiaris

Foram necropsiados 24 cäes com exame de sangue positivo para microfilarias de Dipetalonema sp. Verificou-se que 93,9 por cento dos parasitos foram encontrados no tecido subcutâneo e 6,1 por cento nas vísceras. A localizaçäo dos helmintos variou com os regimes anatômicos, sendo o tórax o local de maior frequência