Morphological and molecular characterization of Contracaecum australe (Nematoda: Anisakidae) parasitizing Phalacrocorax brasilianus (Aves: Phalacrocoracidae) on the north coast of Brazil.

For the first time in Brazil, Contracaecum australe is recorded parasitizing Phalacrocorax brasilianus (Aves, Suliformes, Phalacrocoracidae) from the Marine Extractive Reserve of Soure on Marajó Island, Brazilian Amazon. Its morphology revealed a body with a transversally striated cuticle, smooth or slightly cleft interlabia, lips with auricles, labial papillae, and conspicuous amphids. In males, the presence of the median papilla on the upper lip of the cloaca and spicules that reach almost half of the body of the parasite. These morphological characters, added to the number and distribution of the pre- and postcloacal papillae of the male specimens, and supported by the molecular phylogeny from the analysis of the ITS-1, 5.8S and ITS-2 genes, allowed the identification of these parasites.

Systematic and parasite-host relationship by Baruscapillaria appendiculata in Phalacrocorax brasilianus collected from Marajó Island, State of Pará, Brazil.

The genus Baruscapillaria Moravec, 1982 has six valid species recorded in birds Phalacrocoracidae, namely Baruscapillaria appendiculata Freitas, 1933, B. spiculata Freitas, 1933, B. carbonis (Dubinin & Dubinina, 1940), B. jaenschi (Johnston & Mawson, 1945), B. phalacrocoraxi (Borgarenko, 1975) and B. rudolphii Moravec, Scholz and Nasincová, 1994. Helminthological tests carried out on cormorants of the species Phalacrocorax brasilianus (Gmelin), a migratory bird that occurs in the northeast of the State of Pará, Brazil, demonstrate B. appendiculata parasitizing the cloaca of these birds, through light microscopy, scanning electron microscopy and molecular biology. These studies allowed a redescription of males and females of this nematode in these hosts and in this geographical area through integrative taxonomy. The occurrence of lesions in the cloaca caused by this nematode parasite was registered using histological analysis. This is a new geographic report for this nematode.

Performance of intron 7 of the ß-fibrinogen gene for phylogenetic analysis: An example using gladiator frogs, Boana Gray, 1825 (Anura, Hylidae, Cophomantinae).

Boana, the third largest genus of Hylinae, has cryptic morphological species. The potential applicability of b-fibrinogen intron 7 - FGBI7 is explored to propose a robust phylogeny of Boana. The phylogenetic potential of FGBI7 was evaluated using maximum parsimony, MrBayes, and maximum likelihood analysis. Comparison of polymorphic sites and topologies obtained with concatenated analysis of FGBI7 and other nuclear genes (CXCR4, CXCR4, RHO, SIAH1, TYR, and 28S) allowed evaluation of the phylogenetic signal of FGBI7. Mean evolutionary rates were calculated using the sequences of the mitochondrial genes ND1 and CYTB available for Boana in GenBank. Dating of Boana and some of its groups was performed using the RelTime method with secondary calibration. FGBI7 analysis revealed high values at informative sites for parsimony. The absolute values of the mean evolutionary rate were higher for mitochondrial genes than for FGBI7. Dating of congruent Boana groups for ND1, CYTB, and FGBI7 revealed closer values between mitochondrial genes and slightly different values from those of FGBI7. Divergence times of basal groups tended to be overestimated when mtDNA was used and were more accurate when nDNA was used. Although there is evidence of phylogenetic potential arising from concatenation of specific genes, FGBI7 provides well-resolved independent gene trees. These results lead to a paradigm for linking data in phylogenomics that focuses on the uniqueness of species histories and ignores the multiplicities of individual gene histories.

First molecular detection of Leishmania (Leishmania) infantum chagasi in a domestic cat (Felis catus) from an urban area in eastern Amazon.

Background: Domestic cats have been implicated as accidental hosts of Leishmania sp. However, in recent years, the recurrent description of new cases in endemic and nonendemic areas draw attention to the potential epidemiological role of cats as reservoir hosts. Although dogs are considered urban reservoirs, cats could act as a secondary natural reservoirs in these areas. Thus, feline leishmaniasis has become an emerging disease in several countries worldwide. Case presentation: This study aimed to describe the first case of feline leishmaniasis in a stray animal that presented lesions compatible with the disease in Belém, Pará, Brazil, an important urban area in eastern Amazon. Serological tests for Leishmania infantum (ELISA and IFA) were nonreactive, whereas histopathological examination indicated infectious dermatitis caused by Leishmania spp. or Toxoplasma gondii. Cytopathological study of lesion aspirate confirmed the presence of Leishmania sp. amastigotes within macrophages. Finally, molecular analyses revealed that the feline infection was caused by Leishmania (Leishmania) infantum chagasi. Conclusion: To the best of the authors' knowledge, this study reports the first case of natural infection by Leishmania (Leishmania) infantum chagasi in a feline from eastern Amazon. These findings suggest domestic cats as potential secondary reservoir hosts of Leishmania spp. in Belém, which reinforces the importance of further epidemiological investigation of feline leishmaniasis, especially in urban areas with human cases.

Virtual screening and repurposing of approved drugs targeting homoserine dehydrogenase from Paracoccidioides brasiliensis.

Paracoccidioidomycosis is a systemic mycosis endemic in Latin America, and one of the etiological agents of the disease is Paracoccidioides brasiliensis. Currently, available treatments present adversities, such as duration, side effects, and drug interactions. In search of new therapy possibilities, this study evaluates drugs approved for use against the homoserine dehydrogenase enzyme, by an in silico approach, which performs an important biosynthesis phase for the fungus and is not present in the human body. The three-dimensional structure of the homoserine dehydrogenase enzyme from Paracoccidioides brasiliensis was obtained by homology modeling. The model was validated, and simulations were performed for virtual screening of molecules of drugs approved from the Drugs-libs database by the MTiOpenScreen web server. Molecular dynamics in three replicas were used for four drugs with better results, and in two more molecules as a control, the HS9 with inhibition against enzyme and HON which shows inhibition against mold structure. Based on the results of molecular dynamics and the comparison of binding free energy, the drug that obtained the best result was Bemcentinib. In comparison with the controls, it presented a highly relevant affinity with - 44.63 kcal/mol, in addition to good structural stability and occupation of the active site. Therefore, Bemcentinib is a promising molecule for the inhibition of PbHSD protein (homoserine dehydrogenase of Paracoccidioides brasiliensis) and a therapeutic option to be investigated.

Kudoa rousseauxii n. sp. (Cnidaria: Multivalvulida) Infects the Skeletal Muscles of the Freshwater Fish Brachyplatystoma rousseauxii in the Amazon River.

PURPOSE: Members of the genus Kudoa Meglitsch, 1947 are known to infect the muscles of commercially important fishes worldwide, including those in the order Siluriformes. This paper describes the occurrence of a new species of Kudoa in the catfish Brachyplatystoma rousseauxii based on morphological study and molecular analysis of the ribosomal RNA gene (SSU rDNA). METHODS: Fifteen specimens of Brachyplatystoma rousseauxii were purchased from fishing zones near Mosqueiro Island, Belém, Pará, Brazil. After necropsy, tissue samples and cysts were analyzed using a stereomicroscope, and fresh slides were viewed under a light microscope to confirm parasitic infection. The tissue fragments were removed and processed for molecular and histological analyses. RESULTS: Microscopic pseudocysts were found in the epaxial region of skeletal muscle fibers in 80% of the analyzed specimens. The myxospores were quadrangular with four shell valves (SV), pyriform polar capsules (PC), and internal symmetry. Phylogenetic analyses indicated that the new species formed a cluster with the species previously described in the Amazon, being close to two freshwater species. CONCLUSIONS: Morphological differences and molecular data of SSU rDNA support that Kudoa rousseauxii n. sp. is a new species that infects B. rousseauxii, a freshwater fish with intense migratory cycles that is widely captured and consumed in the Amazon.

First molecular detection of canine herpesvirus 1 (CaHV-1) in the Eastern Brazilian Amazon.

BACKGROUND: Canine herpesvirus type 1 (CaHV-1) infects dogs and is associated with neonatal deaths and reproductive, ocular, neurological, and respiratory problems. In Brazil, reports of CaHV-1 have been restricted to the southeast and south regions, particularly in municipalities in the state of Rio Grande do Sul. OBJECTIVES: To assess the presence and variability of CaHV-1 in canine populations in the state of Pará, North Brazil. METHODS: Biological samples from 159 dogs from 4 municipalities in the State of Pará were evaluated using polymerase chain reaction and phylogenetic analyses, with the target being the viral enzyme, thymidine kinase. RESULTS: CaHV-1 was detected in 13 dogs (8.2%), with 2 animals being from the municipality of Santa Bárbara do Pará, 8 from Algodoal Island, 2 from Salinópolis, and one from Capanema. The study sequences revealed 100% identity among themselves and 64% to 100% identity with the other nucleotide sequences from Australia, Brazil, United Kingdom, and United States, including 100% identity with the 2002 isolate from Australia. The 1996 isolate from France was grouped in a branch that was different from the sequence of this study. CONCLUSIONS: This study presents the first molecular detection of CaHV-1 in dogs from the Amazon region in northern Brazil. The nucleotide identity between the strains and cytosine insertion in the sequences isolated in this study suggests at least 2 strains of CaHV-1 circulating in Brazil (Pará and BTU-1).

In silico improvement of the cyanobacterial lectin microvirin and mannose interaction.

Lectins that bind to HIV envelope glycoprotein can inhibit virus-cell fusion and be used for rational drug design. This paper presents the results of an in silico approach to improve affinity interaction between the cyanobacterial lectin microvirin and its ligand Manα(1-2)Man. Comparative modeling and molecular dynamics tools were used. Additionally, the alanine scanning webserver was used to study the importance of protein residues in the binding site and to guide mutant production. The model obtained presented two homologous domains designated as domains A and B, each consisting of a single strand with triple and antiparallel ß-sheets of (ß1-ß3 and ß6-ß8). Disulfide bonds between the cysteines (Cys60-Cys80, Cys63-Cys78 and Cys8-Cys24) were also found. The highly conserved binding site, including residues Asn44, Ile45, Asp46, Gln54, Asn55, Glu58, Thr59, Gln81, Thr82 and Met83. The RMSD values of the di-mannose and the interaction site were very stable during the molecular dynamics. Calculations of the occupation time of the hydrogen bonds were made for the residues that showed interaction in the complex lectin and ligand. The residue that contributed most to the interaction with Manα(1-2)Man was Asn55. After validation, the model generated remained stable during the entire simulation. Despite its structural similarity with the template we used, our mutant (Thr82Arg) showed a higher affinity interaction with Manα(1-2)Man. Communicated by Ramaswamy H. Sarma.

Morphological diagnosis of Setaria labiatopapillosa in domestic bovids from Marajó Island, Brazil.

The genus Setaria is known worldwide for parasiting wild and domestic mammals, with 43 species registered, of which five occur in the American continent. The objective of this study was to characterize the occurrence, morphology and morphometry of S. labiatopapillosa in B. taurus and B. bubalis in Marajó Island, Brazil. Carcass inspections of 420 cattle were carried out in two slaughterhouses in Pará. The nematodes were found on the white and serous viscera of the small intestine, being cleaned and discussed for analysis by light microscopy and scanning electron microscopy. The morphological and morphometric characteristics are compatible with S. labiatopapillosa parasitizing both species, with a prevalence of 25% (B. bubalis) and 24% (B. taurus). Information obtained by scanning electron microscopy was added to the description of the species, in addition to expanding the distribution of this parasite in the national territory.

Myxobolus freitasi n. sp. (Myxozoa: Bivalvulida), a parasite of the brain of the electric knifefish in the Brazilian Amazon region.

A total of 30 specimens of the Amazonian electric knifefish, Brachyhypopomus beebei Schultz, 1944 (Gymnotiformes: Hypopomidae), were collected from the Peixe-Boi River in the state of Pará, Brazil (1°06'59" S; 47°18'26" W). Fragments of the brain tissue were extracted for analysis via optical microscopy, and 18 specimens (60%) presented microparasites of the genus Myxobolus, with unequal capsules. The spores were 18.6 µm (17.7-19.8 µm) long and 8.6 µm (8.4-9.0 µm) wide; the largest polar capsule was 13.0 µm (12.4-13.4 µm) long and 5.6 µm (5.3-6.0 µm) wide, and the smallest capsule was 5.0 µm (4.5-5.3 µm) long and 2.5 µm (2.3-2.6 µm) wide. Infected brain fragments were extracted for histological processing and staining with hematoxylin-eosin and Ziehl-Neelsen. Some fragments were conserved in ethanol for molecular genetics analysis. A partial sequence of the 18S DNA gene was obtained from the spores, which did not correspond to any other sequences deposited in GenBank, although it did form a clade with other Myxobolus parasites of the nervous system. The morphological data, together with molecular phylogeny, supported the designation of a new species Myxobolus freitasi n. sp.

Current Knowledge on Microviridin from Cyanobacteria.

Cyanobacteria are a rich source of secondary metabolites with a vast biotechnological potential. These compounds have intrigued the scientific community due their uniqueness and diversity, which is guaranteed by a rich enzymatic apparatus. The ribosomally synthesized and post-translationally modified peptides (RiPPs) are among the most promising metabolite groups derived from cyanobacteria. They are interested in numerous biological and ecological processes, many of which are entirely unknown. Microviridins are among the most recognized class of ribosomal peptides formed by cyanobacteria. These oligopeptides are potent inhibitors of protease; thus, they can be used for drug development and the control of mosquitoes. They also play a key ecological role in the defense of cyanobacteria against microcrustaceans. The purpose of this review is to systematically identify the key characteristics of microviridins, including its chemical structure and biosynthesis, as well as its biotechnological and ecological significance.

Polymorphism of the leptin gene in buffalo breed groups from eastern Amazon.

The objective of this work was to characterize genetically some buffalo herds raised in Varzea (VA) and Terra-Firme (upland) (TF) ecosystems through polymorphism of the intron 2 of the leptin gene ( LEP-1620). Two hundred seventy-nine animals from four distinct populations were evaluated using the PCR-RFLP method for LEP-1620 polymorphism (SNP) of the leptin gene with restriction enzyme BsaAI. The animal samples were sorted into 4 groups, according to their breed and environmental origin: Mediterranean TF, Murrah TF, Mediterranean VA and Crossbreed VA. Two alleles (A and G) were detected and their frequencies were analyzed. Allele A frequency ranged from 0.395 (Mediterranean TF) to 0.850 (Murrah TF), with AA genotype ranged from 0.114 (Murrah TF) to 0.700 (Mediterranean TF). The observed and expected heterozygosities ranged from 0.268 (Mediterranean TF) to 0.562 (Murrah TF), and 0.255 (Mediterranean TF) to 0.478 (Murrah TF), respectively. The Hardy-Weinberg probabilities were greater than 0.05. The Crossbred herd in Varzea was the only population with significant inbreeding and the Shannon index ranged between 0.423 (Mediterranean TF) and 0.671 (Murrah TF).

Genomic screening and molecular dynamics simulations of cyanovirin-N homologs from cyanobacteria phylum.

The phylum cyanobacteria are one of the most ancient groups of organisms on the planet and are well recognized due to its wide distribution, ecological role, and biotechnological potential. Cyanobacterial lectins are being extensively explored due to their antiviral activity, mainly because of their capacity of inhibiting HIV strains from infecting human cells by gp120 and gp41 binding. Cianovirin-N from Nostoc ellipsosporum was the first lectin isolated with this property. Since then, various homologs have been discovered and characterized. In this article, we present results of a genomic screening to find cyanovirin-N homologs (CVNH) in all cyanobacteria genomes available in the GenBank, resulting in 155 CVNH proteins with 63 presenting significant identity differences of cyanovirin-N. Homology modeling and molecular dynamics were employed to characterize 18 unexplored models and their functional capacity of binding to Manα(1-2)Man. Results presented here support the hypothesis of multiple ligand recognition for the CVNH family and may help to understand the function of these lectins for the producer cyanobacteria. Additionally, the theoretical results observed here justify carrying out experimental investigations that can expand the therapeutic potential of cyanobacterial lectins.

Incidence of viral hepatitis in Brazil from 2009 to 2018: an epidemiological study of confirmed cases of viral hepatitis

Abstract INTRODUCTION: Viral hepatitis is a major public health problem. It is necessary to understand the epidemic, verifying the combination of biological and demographic characteristics. METHODS: This is an analytical ecological and epidemiological study. Confirmed case data from the Notification Disease Information System (SINAN) were used. RESULTS: From 2009-2018, SINAN confirmed 404,003 viral hepatitis cases in Brazil, with 12.49%, 37.06%, and 48.28% cases of hepatitis A, B, and C, respectively. CONCLUSIONS: In Brazil, 4,296 deaths were associated with viral hepatitis, of which 36.66% were associated with acute hepatitis B. The proportional distribution of cases varied among the five Brazilian regions.

High-quality draft genome sequence of pantanalinema sp. GBBB05, a cyanobacterium from cerrado biome

Cyanobacteria comprise one of the oldest and most diverse phyla in the Bacteria domain and are recognized for their importance in the biosphere evolution. Members of this phylum can be found in a wide variety of environments reflecting their photosynthetic ability, adaptability to various environmental conditions, and diversified metabolism. Such characteristics make cyanobacteria one of the preferred targets for research on bioactive compounds and new enzymes (Schirrmeister et al., 2011; Dittmann et al., 2015). Pantanalinema was described as a new genus of the Leptolyngbyaceae cyanobacterial family by a polyphasic approach, which included morphological characteristics, 16S rRNA gene phylogeny, 16S-23S ITS rRNA secondary structures, and physiological characteristics such as adaptability to pH variations (Vaz et al., 2015). This genus has been described only in Brazilian biomes such as the Pantanal and the Amazon, the first isolates being found in a lake. These Pantanalinema isolates were characterized by their ability to grow over a wide pH range (pH 4 to 11) as well as to modify the culture medium pH around neutrality (pH 6 to 7.4). Due to these characteristics, it is thought that this genus can occupy a variety of ecological niches, such as alkaline or slightly acidic water bodies (Vaz et al., 2015; Genuário et al., 2017). Taxonomic classification of Pantanalinema isolates requires the use of molecular markers as this genus is morphologically very similar to the recently described genus Amazoninema, which, in turn, has comparable morphology to other genera of the Leptolyngbyaceae family (Genuário et al., 2018). In this work, we report the genome sequence of a new Pantanalinema strain, named GBBB05, which was isolated from the Brazilian Cerrado biome. This is the first genome assembly for the Pantanalinema genus, which, along with the analyses provided here, is expected to enhance our understanding of this genus’s metabolic potential

Myxobolus freitasi n. sp. (Myxozoa: Bivalvulida), a parasite of the brain of the electric knifefish in the Brazilian Amazon region / Myxobolus freitasi n. sp. (Myxozoa: Bivalvulida), parasita do cérebro do peixe-faca elétrico na Amazônia brasileira

Abstract A total of 30 specimens of the Amazonian electric knifefish, Brachyhypopomus beebei Schultz, 1944 (Gymnotiformes: Hypopomidae), were collected from the Peixe-Boi River in the state of Pará, Brazil (1°06'59" S; 47°18'26" W). Fragments of the brain tissue were extracted for analysis via optical microscopy, and 18 specimens (60%) presented microparasites of the genus Myxobolus, with unequal capsules. The spores were 18.6 µm (17.7-19.8 µm) long and 8.6 µm (8.4-9.0 µm) wide; the largest polar capsule was 13.0 µm (12.4-13.4 µm) long and 5.6 µm (5.3-6.0 µm) wide, and the smallest capsule was 5.0 µm (4.5-5.3 µm) long and 2.5 µm (2.3-2.6 µm) wide. Infected brain fragments were extracted for histological processing and staining with hematoxylin-eosin and Ziehl-Neelsen. Some fragments were conserved in ethanol for molecular genetics analysis. A partial sequence of the 18S DNA gene was obtained from the spores, which did not correspond to any other sequences deposited in GenBank, although it did form a clade with other Myxobolus parasites of the nervous system. The morphological data, together with molecular phylogeny, supported the designation of a new species Myxobolus freitasi n. sp.

Resumo Um total de 30 espécimes do peixe-faca elétrico da Amazônia, Brachyhypopomus beebei Schultz, 1944 (Gymnotiformes: Hypopomidae), foram coletados no rio Peixe-Mani, no estado do Pará, Brasil (1 ° 06'59 "S; 47 ° 18 ' 26 "W). Fragmentos de tecido cerebral foram extraídos para análise em microscopia óptica, sendo que 18 espécimes (60%) apresentavam microparasitos do gênero Myxobolus, com cápsulas desiguais. Os esporos apresentavam 18,6 µm (17,7-19,8 µm) de comprimento e 8,6 µm (8,4-9,0 µm) de largura; a maior cápsula polar tinha 13,0 µm (12,4-13,4 µm) de comprimento e 5,6 µm (5,3-6,0 µm) de largura, e a menor cápsula tinha 5,0 µm (4,5-5,3 µm) de comprimento e 2,5 µm (2,3-2,6 µm) de largura. Fragmentos cerebrais infectados foram extraídos para processamento histológico e coloração com hematoxilina-eosina e Ziehl-Neelsen. Alguns fragmentos foram conservados em etanol para análise genética molecular. Dos esporos, foi obtida uma sequência parcial do gene 18S do DNA, que não correspondeu a nenhuma outra sequência depositada no GenBank, embora tenha formado um clado com outros parasitas do gênero Myxobolus do sistema nervoso. Os dados morfológicos, juntamente com a filogenia molecular, apoiaram a designação de uma nova espécie Myxobolus freitasi n. sp.

Incidence of viral hepatitis in Brazil from 2009 to 2018: an epidemiological study of confirmed cases of viral hepatitis.

INTRODUCTION: Viral hepatitis is a major public health problem. It is necessary to understand the epidemic, verifying the combination of biological and demographic characteristics. METHODS: This is an analytical ecological and epidemiological study. Confirmed case data from the Notification Disease Information System (SINAN) were used. RESULTS: From 2009-2018, SINAN confirmed 404,003 viral hepatitis cases in Brazil, with 12.49%, 37.06%, and 48.28% cases of hepatitis A, B, and C, respectively. CONCLUSIONS: In Brazil, 4,296 deaths were associated with viral hepatitis, of which 36.66% were associated with acute hepatitis B. The proportional distribution of cases varied among the five Brazilian regions.

Cotylophoron marajoensis n. sp. (Digenea: Paramphistomidae) a parasite of Bubalus bubalis on Marajó Island, Pará, Brazilian Amazon.

The genus Cotylophoron belongs to the Paramphistomidae family and its definitive hosts are ruminants in general. This work describes the presence of a new species of the gender, a parasite in the rumen and reticulum of Bubalus bubalis, on Marajó Island in the Eastern Brazilian Amazon, using of light microscopy, scanning electronic microscopy and molecular biology techniques. One hundred and ten animals were analyzed, of which 4.54% were parasitized by flukes in their adult forms. The helminths were found fixed to the ruminal mucosa and present Liorchis-type pharynx, Cotylophoron-type genital sucker, oblique testicles larger than the ovary, uterus in rings full of eggs and Cotylophoron-type acetabulum. These morphologic characters do not fit into any previously described species. Thus, it is proposed that this is a new species in the genus Cotylophoron. The present work expands the record of parasitism by helminths in Bubalus bubalis, this being the first record of trematoda from the genus Cotylophoron for this host in the Brazilian Amazon.

A new Piper nigrum cysteine proteinase inhibitor, PnCPI, with antifungal activity: molecular cloning, recombinant expression, functional analyses and molecular modeling.

MAIN CONCLUSION: A new Piper nigrum cysteine proteinase inhibitor, PnCPI, belonging to group I of phytocystatins, with inhibitory activity against papain and growth of Fusarium solani f. sp. piperis, was isolated and characterized. Previous studies (de Souza et al. 2011) have identified a partial cDNA sequence of putative cysteine proteinase inhibitor differentially expressed in roots of black pepper (P. nigrum L.) infected by F. solani f. sp. piperis. Here, we aimed to isolate the full-length cDNA and genomic sequences of the P. nigrum cysteine proteinase inhibitor gene, named PnCPI. Sequence analyses showed that the PnCPI gene encodes a deduced protein of 108 amino acid residues with a predicted molecular mass of 12.3 kDa and isoelectric point of 6.51. Besides the LARFAV-like sequence, common to all phytocystatins, PnCPI contains three conserved motifs of the superfamily cystatin: a glycine residue at the N-terminal region, the QxVxG reactive site more centrally positioned, and one tryptophan in the C-terminal region. PnCPI, belonging to group I of phytocystatins, showed high identity with cystatins isolated from several plant species. Sequence analyses also revealed no putative signal peptide at the N-terminal of PnCPI, as well as no introns within the genomic sequence corresponding to the PnCPI coding region. Molecular modeling showed the ability of PnCPI to interact with papain, while its inhibitory activity against this protease was confirmed after heterologous expression in Escherichia coli. The effects of heat treatments on the inhibitory activity of recombinant PnCPI, rPnCPI, were evaluated. In addition, rPnCPI exhibited in vitro activity against F. solani f. sp. piperis, revealing a new cystatin with the potential antifungal application. The identification of PnCPI as a functional cystatin able to inhibit the in vitro growth of F. solani f. sp. piperis indicates other factors contributing to in vivo susceptibility of black pepper to root rot disease.

Additional characterization of the adult worm Mammomonogamus laryngeus (Railliet, 1899) and the tissue lesions caused by the infection in buffaloes.

Mammomonogamus spp. are parasites with curious characteristics, such as the "Y" shape that results from male and female maintaining the permanent copulation position and the controversial presence or absence of spicules. These nematodes are hematophagous and cause damage to the upper respiratory tracts of cattle, sheep, goats, deer, wild yaks, and orangutans. Human infection is rare and most cases until now have been in the Caribbean Islands or in Brazil, and mainly in farmworkers but recently there have been reports affecting tourists. In the present work, the parasites were recovered from the laryngopharynx and larynx region of Bubalus bubalis on the island of Marajó, Pará, Brazil. Different microscopy methodologies were applied (bright field, fluorescence, and scanning electron microscopy) to explore the ultrastructural details of the anterior end, genital structures and the host tissue damage caused by the nematodes. The well-developed mouth is an important structure in the identification of these nematodes and used by the parasite to adhere to the host's tissue. Different methodologies in microscopy and molecular biology contributed to a detailed morphological description and showed the phenotypic position of Mammomonogamus laryngeus. Light and scanning electron microscopy (SEM) showed details of the papillae, amphids, festoons, ribs, and teeth. Fluorescence microscopy enabled a detailed characterization of different structures, such as the bursal rays and SEM enabled the visualization of the specialized features of the cuticle surface in the male and female. Histopathological analyses, cryofracture and environmental SEM experiments of the infected tissues were carried out in order to investigate the lesions resultant from the parasitism. In addition, the parasite couples were submitted to cryofracture and these results revealed details of the reproductive structures of both sexes, including the male spicule.