Laércio dos Anjos Benjamin

Characterizing the microbiota across the gastrointestinal tract of a Brazilian Nelore steer.

The gastrointestinal tracts (GIT) of herbivores harbor dense and diverse microbiota that has beneficial interactions with the host, particularly for agriculturally relevant animals like ruminants such as cattle. When assessing ruminant health, microbiological indicators are often derived from the rumen or feces. However, it is probable that ruminal and fecal microbiota do not reflect the microbial communities within the GIT of ruminants. To test this, we investigated the compartments of the GIT from a Brazilian Nelore steer and performed a 16S rRNA pyrosequencing analysis on the collected samples. Our results showed high intra-individual variation, with samples clustering according to their location in the GIT including the forestomach, small intestine, and large intestine. Although sequences related to the phyla Firmicutes and Bacteroidetes predominated all samples, there was a remarkable variation at the family level. Comparisons between the microbiota in the rumen, feces, and other GIT components showed distinct differences in microbial community. This work is the first intensive non-culture based GIT microbiota analysis for any ruminant and provides a framework for understanding how host microbiota impact the health of bovines.

Coadministration of sodium alginate pellets containing the fungi Duddingtonia flagrans and Monacrosporium thaumasium on cyathostomin infective larvae after passing through the gastrointestinal tract of horses

The predatory nematophagous fungi have been used as an alternative control of gastrointestinal nematodes of domestic animals in natural and laboratory conditions. However, it is unclear if the association of some of these species could bring some kind of advantage, from a biological standpoint. In this context, this study consisted of two tests in vitro: in assay A, the assessment of the viability of the association of pellets in sodium alginate matrix containing the fungus Duddingtonia flagrans (AC001) and Monacrosporium thaumasium (NF34) and its predatory activity on infective larvae (L3) of cyathostomin after passing through the gastrointestinal tract of horses and assay B, assessment of the cyathostomin L3 reduction percentage in coprocultures. Twelve crossbred horses, females, with a mean weight of 356 kg and previously dewormed were divided in three groups with four animals each: group 1, each animal received 50 g of pellets containing mycelial mass of the fungus D. flagrans and 50 g of pellets of the fungus M. thaumasium, associated and in a single oral dose; group 2, 100 g of pellets containing D. flagrans and 100 g of pellets containing M. thaumasium, associated and in a single oral dose; group 3, control. Faecal samples were collected from animals in the treated and control groups at time intervals of 12, 24, 36, 48, 60 and 72 h after the administration of treatments and placed in Petri dishes containing 2% water-agar (assay A) and cups for coprocultures (assay B). Subsequently, 1000 cyathostomin L3 were added to each Petri dish (assay A) and 1000 cyathostomin eggs were added to each coproculture (assay B) of fungi-treated and control groups. At the end of 15 days, there was observed that the two associations of pellets containing the fungi tested showed predatory activity after passing through the gastrointestinal tract of horses (assay A). In assay B, all the intervals studied showed reduction rate in the number of L3 recovered from coprocultures exceeding 80%. However, no difference (p>0.01) was seen in recovery of not predated L3 between the fungi-treated groups in the time intervals studied. The results obtained showed that the associations of pellets (50 or 100 g of each fungal isolate) were viable after passage through the gastrointestinal tract in horses and could be used in natural conditions.

Increased production of biofilms by Escherichia coli in the presence of enrofloxacin.

The literature has demonstrated that subinhibitory concentrations of some antimicrobials are able to induce biofilm formation by certain bacterial species. Biofilms present in the mammary glands of cattle contribute to antimicrobial resistance, resulting in the appearance of persistent mastitis and consequent great losses to the dairy sector worldwide. The present study aimed to investigate the induction of biofilm formation by enrofloxacin in Escherichia coli isolates obtained from bovine mastitis. Twenty-seven isolates were reactivated in brain heart infusion (BHI) broth supplemented with different subinhibitory concentrations of enrofloxacin. Biofilm formation in microtiter plates was measured and confirmed by scanning electron microscopy (SEM). Isolates submitted to the concentration 0.0125 mg/mL of enrofloxacin showed greater biofilm formation compared to the control (p<0.001). Biofilm formation results obtained for the other concentrations did not differ from those obtained for the control (p>0.05). Using SEM it was possible to visualize the typical architecture of biofilms. These results represent the first report of inducing the production of biofilms in the presence of enrofloxacin, a quinolone antibiotic used to treat clinical mastitis.

Influence of the preservation period in silica-gel on the predatory activity of the isolates of Duddingtonia flagrans on infective larvae of cyathostomins (Nematoda: Cyathostominae)

The continued maintenance of nematophagous fungi predatory activity under laboratory conditions is one of the basic requirements for a successful biological control. The purpose of this study was to evaluate the influence of time on the preservation of the fungus Duddingtonia flagrans (AC001 and CG722) stored in silica-gel for 7 years and their subsequent predatory activity on cyathostomin L(3) larvae in 2% water-agar medium (2% WA). Samples of the isolates AC001 and CG722, originating from vials containing grains of silica-gel sterilized and stored for 7 years, were used. After obtaining fungal conidia, the predation test was conducted over 7 days on the surface of 9.0 cm Petri dishes filled with 2% WA. In the treated groups each Petri dish contained 500 cyathostomin L(3) and conidia of fungal isolates in 2% WA. In the control group (without fungi) the plates contained 500 L(3) in 2% WA. The experimental results showed that isolated AC001 and CG722 were efficient in preying on cyathostomin L(3) (p<0.01) compared to control (without fungus). However, no difference was observed (p>0.01) in the predatory activity of the fungal isolates tested. Comparing the groups, there was a significant reductions of cyathostomin L(3) (p<0.01) of 88.6% and 78.4% on average recovered from the groups treated with the isolates AC001 and CG722, respectively, after 7 days. The results of this test showed that the fungus D. flagrans (AC001 and CG722) stored in silica-gel for at least 7 years maintained its predatory activity on cyathostomin L(3).

Scanning electron microscopy of Ancylostoma spp. dog infective larvae captured and destroyed by the nematophagous fungus Duddingtonia flagrans

The interaction between the nematode-trapping fungus Duddingtonia flagrans (isolate CG768) against Ancylostoma spp. dog infective larvae (L(3)) was evaluated by means of scanning electron microscopy. Adhesive network trap formation was observed 6h after the beginning of the interaction, and the capture of Ancylostoma spp. L(3) was observed 8h after the inoculation these larvae on the cellulose membranes colonized by the fungus. Scanning electron micrographs were taken at 0, 12, 24, 36 and 48 h, where 0 is the time when Ancylostoma spp. L(3) was first captured by the fungus. Details of the capture structure formed by the fungus were described. Nematophagous Fungus Helper Bacteria (NHB) were found at interactions points between the D. flagrans and Ancylostoma spp. L(3). The cuticle penetration by the differentiated fungal hyphae with the exit of nematode internal contents was observed 36 h after the capture. Ancylostoma spp. L(3) were completely destroyed after 48 h of interaction with the fungus. The scanning electron microscopy technique was efficient on the study of this interaction, showing that the nematode-trapping fungus D. flagrans (isolate CG768) is a potential exterminator of Ancylostoma spp. L(3).

Platelet activation: ultrastructure and morphometry in platelet-rich plasma of horses

O objetivo desse estudo foi investigar a capacidade de ativacao do plasma rico em plaquetas (PRP) por substâncias farmacologicas, assim como verificar a necessidade ou nao dessa ativacao para uso terapeutico. O PRP foi obtido de quatro equinos mesticos higidos, machos castrados, com 13 a 16 anos (15±1anos) de idade, e processado para observacao e quantificacao da morfologia plaquetaria mediante a utilizacao da microscopia eletronica de transmissao. Todas as amostras de PRP foram ativadas com cloreto de calcio (CaCl2) a 10%, trombina bovina pura ou associada a CaCl2. O controle (PRP puro) nao foi ativado farmacologicamente. Nas amostras de PRP puro, 49% das plaquetas foram classificadas como ativacao incerta, 41% em repouso, 9% totalmente ativada e 1% com dano irreversivel. O tratamento com CaCl2 a 10% proporcionou uma distribuicao de 54% de plaquetas com ativacao incerta, 24% totalmente ativada, 20% em repouso, e 2% como com dano irreversivel. Amostras tratadas com trombina bovina apresentaram morfologia plaquetaria que nao se enquadraram na classificacao adotada, apresentando forma irregular com emissao de grandes pseudopodes filamentosos, aspecto de rompimento e grânulos inteiros no citoplasma remanescente e meio extracelular. Houve efeito do tratamento sobre a morfologia plaquetaria (P=0,03). O CaCl2 a 10% e um adequado agente ativador de plaquetas. Entretanto, nos casos onde se faz necessario o uso de PRP na forma mais liquida, recomenda-se o uso do PRP puro, que alem de apresentar uma adequada porcentagem de plaquetas totalmente ativadas, tambem possui importante quantidade do tipo em repouso, que pode ser ativado por substâncias presentes no tecido lesionado.

Influence of Insulin-like Growth Factor I (IGF-I) on the survival and the in vitro development of caprine preantral follicles

The aim of this study was to investigate the effects of the insulin-like growth factor -I (IGF-I) on survival, activation (transition from primordial to primary follicles) and growth of caprine preantral follicles cultured in vitro. Fragments of ovarian cortex were cultured for one and seven days in the absence or presence of IGF-I (0, 50 and 100ng/ml). The non-cultured and cultured tissues were processed and analyzed by histology and transmission electron microscopy. The culture for one day in a medium with 100ng/ml of IGF-I showed 86.7% of morphologically normal follicles. These results were similar (P>0.05) to the percentage of normal follicles found in the control (96.7%). It was also found that this medium increased the percentage of follicular activation (developing follicles) with one day of culture. The oocyte and follicular diameters remained similar to the control by culturing for one day in a medium containing 100ng/ml of IGF-I. The ultrastructural analysis did not confirm the integrity of the follicular fragments in a medium containing IGF-I (100ng/ml) after one and seven days of culture. In conclusion, this study demonstrated that the addition of 100 ng/ml of IGF-I in the culture medium enables the development of preantral follicles of goats with one day of culture. However, it is not sufficient to maintain the follicular integrity and the follicular survival rate after seven days of culture.

Survival of Pochonia chlamydosporia in the gastrointestinal tract of experimentally treated dogs.

The predatory capacity of the nematophagous fungus Pochonia chlamydosporia (isolate VC4) after passage through the gastrointestinal tract of dogs was assessed in vivo against Toxocara canis eggs. Twelve dogs previously wormed were divided into two groups of six animals and caged. The treatments consisted of a fungus-treated group (VC4) and a control group without fungus. Each dog of the fungus-treated group received a single 4 g dose of mycelial mass of P. chlamydosporia (VC4). Fecal samples from animals of both groups (treated and control) were collected at five different times (6, 12, 24, 36, and 48 h) after fungal administration, and placed in Petri dishes. Each Petri dish of both groups for each studied time interval received approximately 1000 T. canis eggs. Thirty days after the fecal samples were collected, approximately one hundred eggs were removed from each Petri dish of each studied time interval and evaluated by light microscopy (LM) and scanning electron microscopy (SEM). Microscopy examination of plates inoculated with the fungus showed that the isolate VC4 was able to destroy the T. canis eggs with destruction percentages of 28.6% (6 h), 29.1% (12 h), 32.0% (24 h), 31.7% (36 h), and 37.2% (48 h). These results suggest that P. chlamydosporia can be used as a tool for the biological control of T. canis eggs in feces of contaminated dogs.

Pathological and histometric analysis of the gills of female Hyphessobrycon eques (Teleostei:Characidae) exposed to different concentrations of the insecticide Dimilin®

Female individuals of Hyphessobrycon eques were exposed to Diflubenzuron (Dimilin(®)) in order to determine whether exposure to sublethal levels of this insecticide causes changes in gill morphology. Fish were exposed to 0.01, 0.1 and 1.0mgL(-1) for 96h and 17 days and then submitted to pathological and histometric evaluation. Pathological lesions, such as hyperplasia, lamellar fusion, vascular congestion, secondary lamellar disarray, vasodilatation, hemorrhage and increased lamellar epithelium, were significantly more common in the gills of fish exposed to Dimilin(®) than the control. Histometric analysis documented significant changes in blood vessel diameter, primary lamellae width and secondary lamellae length, and the appearance of hemorrhage foci in all concentrations tested. Even at low Dimilin(®) concentrations, the histopathological alteration index was mild to moderate, thereby indicating that the function of this tissue was compromised. These findings indicate that indiscriminate use of Dimilin(®) can adversely affect the structural integrity of the gills of H. eques, which can cause numerous problems for fish farming systems.

Histological and Histometric Evaluation of the Liver in Astyanax Bimaculatus (Teleostei: Characidae), Exposed to Different Concentrations of an Organochlorine Insecticide

To investigate possible morphological changes to the liver tissue of lambaris, Astyanax bimaculatus (Linnaeus, 1758), females were exposed to treatments of sublethal concentrations of the insecticide Thiodan® for 96 hr. Treatments included three sublethal concentrations of 1.15, 2.3, and 5.6 μg L−1 of Thiodan® and a control group without insecticide. The action of Thiodan® at sublethal concentrations did not affect the morphological structure of the liver as a whole, but changes in isolated locations of the hepatic parenchyma were observed. Glycogen depletion, nuclear and cytoplasmic deformation, nuclear and cytoplasmic hypertrophy, hyperemia, and cellular degeneration in liver cells at the different concentrations studied were recorded. These observed changes in the livers were greater in groups exposed to Thiodan® in comparison to the control group. Furthermore, there was a change in the diameter of the nuclei and cytoplasm of hepatocytes in the different treatments. The groups exposed to Thiodan® also exhibited a larger number of hepatocyte nuclei and a reduction in the amount of cytoplasm. We conclude that for the exposure period and concentrations of Thiodan® analyzed, the morphology of hepatic tissue had a cellular adaptive response. Anat Rec, 298:1754–1764, 2015.