Effect of carbohydrates on the adhesion of Bordetella bronchiseptica to the respiratory epithelium in rabbits.

This study proposes an ecological approach for preventing respiratory tract infections caused by Bordetella bronchiseptica in mammals using a mixture of carbohydrates. In an in vivo study, 51-day-old New Zealand rabbits were treated with a solution containing 1 × 107 CFUs of B. bronchiseptica and 250 µg of one of the following carbohydrates: N acetylglucosamine (GlcNAc), N acetylgalactosamine (GalNAc), alpha methyl mannose (AmeMan), alpha methyl glucose (AmeGlc) and sialic acid (Neu5AC). Positive (B. bronchiseptica) and negative (Physiological Saline Solution (PSS)) controls were included. Animals treated with GlcNAc or AmeGlc showed no clinical signs of infection and exhibited a significant reduction (p < 0.05) in the severity of microscopic lesions evaluated in the nasal cavity and lung compared with the positive controls. Additionally, the presence of bacteria was not detected through microbiological isolation or PCR in the lungs of animals treated with these sugars. Use of a mixture of GlcNAc and AmeGlc resulted in greater inhibition of microscopic lesions, with a significant reduction (p < 0.05) in the severity of these lesions compared to the results obtained using individual sugars. Furthermore, the bacterium was not detected through microbiological isolation, Polymerase Chain Reaction (PCR) or indirect immunoperoxidase (IIP) in this group.

Intranasal instillation of Pasteurella multocida lipopolysaccharide in rabbits causes interstitial lung damage.

In order to characterize the in vivo lesions in the nasal cavities and lungs, twenty-eight rabbits were intranasally instilled with lipopolysaccharide (LPS) from P. multocida and then divided into seven groups according to euthanasia time. The nasal cavities and the lungs were processed for light microscopy, lectin histochemistry and transmission electron microscopy. Increased goblet cell activation and neutrophil infiltration were relevant changes in the nasal cavity. A predominantly interstitial pattern of diffuse alveolar damage and bronchopneumonic foci were the main lesions found in the lungs. LPS was found in the cytoplasm of ciliated cells, goblet cells, glandular cells, venular endothelial cells and neutrophils in the nasal cavity and in club cells, capillary endothelial cells and neutrophil in the lung. This study demonstrates that the LPS is able to cause lesions in the upper and lower respiratory tract, it binds to and is internalized by respiratory epithelial cells. Furthermore, it also traverses the intercellular spaces to reach the blood vessels, where it binds to and is internalized by neutrophil and red blood cells. These cells may then travel to the lungs where the LPS induces typical diffuse alveolar damage. This route of lung interstitial damage, to our knowledge, has not been described for this molecule or any known pathogen.

The effect of carbohydrates on the adherence of Pasteurella multocida to the nasal respiratory epithelium.

Pasteurella multocida subsp. multocida is responsible for different diseases that generate great economic losses in farm animal. The effectiveness of immunization against those bacteria are variable and the use of antibiotics is questioned; for that reason, we investigated the potential inhibitory effect of different carbohydrates on the adherence in vivo of P. multocida to the rabbit respiratory epithelium as an alternative for the prevention of respiratory infections. Rabbits were intranasally and intratracheally inoculated with a solution containing 200 µl of 1x107 CFU of P. multocida that was previously mixed with 250 µg /200 µl of N-acetylglucosamine, alphamethylglucoside, alphamethylmannoside, N-acetylgalactosamine or sialic acid. The animals that received N-acetylglucosamine, alphamethylglucoside or alphamethylmannoside individually or a mixture of these three carbohydrates plus the bacterium, showed a significant decrease (P <0.05) of the clinical symptoms, microscopic and macroscopic lesions in the nasal septa and in the lungs; also, the number of adhered bacteria to the nasal epithelium were also significantly reduced. This research demonstrates for the first time that such an approach could convert into a method for prevention of P. multocida infection in rabbits that is ecologically and economically safe and effective.

Content of the Saponin Protodioscin in Brachiaria spp. from the Eastern Plains of Colombia.

Protodioscin is used as a marker of saponin content that could cause hepatotoxicity in ruminants. In Brachiaria spp. from two regions of the Colombian Eastern Plains (east mountain range of the Andean-"piedemonte" and Ariari River Valley) were determined this metabolite at 14 and 28 days post-cutting under different climatic conditions. No protodioscin was detected in B. dictyoneura or B. humidicola. In B. brizantha, B. decumbens and B. ruziziensis x B. decumbens x B. brizantha (hybrid), protodioscin content corresponded to an interaction between species, post-cutting time and season. Concentrations ≥1% (minimum toxic level) were recorded in B. decumbens and the hybrid, and to a lesser extent in B. brizantha. The concentration of protodioscin was higher at 28 days, when the pastures are suitable for consumption. B. brizantha accumulated the lowest saponin concentration, whereas the hybrid had the highest levels, particularly in the "piedemonte" and during drought (3.37%). Dry season favored the protodioscin concentration in B. decumbens (in river valley) and in the hybrid (in "piedemonte"). In the latter, there was a positive correlation with temperature and a negative with humidity, which are typical characteristics of dry periods. This is the first report of protodioscin content in the hybrid.

Assessment of Pasteurella multocida A Lipopolysaccharide, as an Adhesin in an In Vitro Model of Rabbit Respiratory Epithelium.

The role of the P. multocida lipopolysaccharide (LPS) as a putative adhesin during the early stages of infection with this bacterium in the respiratory epithelium of rabbits was investigated. By light microscopy and double enzyme labeling of nasal septa tissues, the amount of bacteria attached to the respiratory epithelium and the amount of LPS present in goblet cells at different experimental times were estimated. Transmission electron microscopy (TEM) and LPS labeling with colloidal gold particles were also used to determine the exact location of LPS in the cells. Septa that were challenged with LPS of P. multocida and 30 minutes later with P. multocida showed more adherent bacteria and more severe lesions than the other treatments. Free LPS was observed in the lumen of the nasal septum, forming bilamellar structures and adhering to the cilia, microvilli, cytoplasmic membrane, and cytoplasm of epithelial ciliated and goblet cells. The above findings suggest that P. multocida LPS plays an important role in the process of bacterial adhesion and that it has the ability of being internalized into host cells.

Inhibition of Pasteurella multocida Adhesion to Rabbit Respiratory Epithelium Using Lectins.

This study aimed to evaluate the ability of a panel of lectins to inhibit the ability of Pasteurella multocida to adhere to and affect the rabbit respiratory epithelium. Nasal septa from rabbit fetuses were cultured with various lectins before the addition of P. multocida. The percentage of bacteria adhering to the epithelium was evaluated semiquantitatively by indirect immunoperoxidase (IIP) staining. The goblet cells (GCs) were counted in semithin sections stained with toluidine blue and served as the main morphological criterion to evaluate the inhibitory effect of the lectins. The lectins PNA, WGA, RCA120, and DBA significantly inhibited the adhesion of P. multocida to the ciliated epithelium (P < 0.05) and prevented the pathogen-induced increase in the number of GCs (P < 0.05) compared with those of positive control tissues. In addition, VVA, SJA, UEA I, DSL, SBA, and ECL significantly inhibited the increase in GCs compared with that of the control tissues. The results suggest that less aggressive therapeutic strategies, such as treatment with lectins, may represent alternative approaches to control bacterial respiratory infections.

Interaction of Bordetella bronchiseptica and Its Lipopolysaccharide with In Vitro Culture of Respiratory Nasal Epithelium.

The nasal septa of fetal rabbits at 26 days of gestation were harvested by cesarean section of the does while under anesthesia and then exposed to Bordetella bronchiseptica or its lipopolysaccharide (LPS) for periods of 2 and 4 hours. A total of 240 explants were used. The tissues were examined using the Hematoxylin & Eosin technique. Then, semithin sections (0.5 µm) were stained with toluidine blue and examined with indirect immunoperoxidase (IPI) and lectin histochemistry. The most frequent and statistically significant findings were as follows: (1) cell death and increased goblet cell activity when exposed to bacteria and (2) cell death, cytoplasmic vacuolation and infiltration of polymorphonuclear leukocytes when exposed to LPS. The lesions induced by the bacterium were more severe than with LPS alone, except for the cytoplasmic vacuolation in epithelial cells. IPI stained the ciliated border of the epithelium with the bacterium more intensely, while LPS lectin histochemistry preferentially labeled the cytoplasm of goblet cell. These data indicate that B. bronchiseptica and its LPS may have an affinity for specific glycoproteins that would act as adhesion receptors in both locations.

Use of sodium chloride and zeolite during shipment of Ancistrus triradiatus under high temperature

The use of sodium chloride (0.5 g/L and 1 g/L) and zeolite (22.7 g/L) during shipment (48 h) of Ancistrus triradiatus at high temperatures (between 24.5 and 34ºC) were evaluated. Several water quality parameters (dissolved oxygen, pH, conductivity, and total ammonia) were measured before and after shipment. Glycemia was measured before shipment and at 24 and 48 h after shipment. After shipment, a resistance test was carried out in a high concentration of sodium chloride, and mortality was recorded after shipment, and 7 days post-shipment. While the two evaluated substances increased survival of A. triradiatus challenged by high temperatures during shipment, the best result was obtained with 1 g/L of sodium chloride.

O uso de cloreto de sódio (0,5 g/L e 1 g/L) e zeolita (22,7 g/L) foram avaliados durante o transporte (48 h) de Ancistrus triradiatus em altas temperaturas (entre 24,5 e 34ºC). Os seguintes parâmetros foram monitorados: pH, oxigênio dissolvido, condutividade e amônia antes e depois do transporte. Também foi mensurada a concentração de glicose no sangue antes do transporte e 0, 24 e 48 h após o transporte. Foi realizado um teste de resistência a altas concentrações de cloreto de sódio após o transporte, sendo registrada a mortalidade no final do transporte e após 7 dias. As duas substâncias testadas aumentam a sobrevivência de A. triradiatus a altas temperaturas durante o transporte, porém o melhor resultado foi obtido com o uso de 1 g/L cloreto de sódio.