Pasteurella haemolytica A1 and Bovine Respiratory Disease: Pathogenesis

The severe fibrinonecrotic pneumonia associated with pneumonic pasteurellosis usually results from colonization of the lower respiratory tract by Pasteurella haemolytica biotype A, serotype 1(A1). Despite recent research efforts, the authors lack a detailed understanding of the interactions and host response to P. haemolytica in the respiratory tract. The authors hypothesize that management and environmental stress factors or viral infection alters the upper respiratory tract (URT) epithelium allowing P. haemolytica to colonize the epithelium. Once the URT is colonized, large numbers of organisms enter the lung where they interact with alveolar macrophages. Endotoxin, released from the bacteria, crosses the alveolar wall where it activates pulmonary intravascular macrophages, endothelium, neutrophils, lymphocytes, platelets, complement, and Hageman factor leading to complex interactions of cells and mediators. It is the progression of this inflammatory response with neutrophil influx that is ultimately responsible for the pulmonary injury. Leukotoxin is a major virulence factor of P. haemolytica that allows it to survive by destroying phagocytic cells. At subcytolytic concentrations it may also enhance the inflammatory response by activating cells to produce mediators and release reactive oxygen metabolites and proteases.     

Effects of Pasteurella haemolytica A1 leukotoxin on bovine neutrophils: degranulation and generation of oxygen-derived free radicals.

To further define the role of Pasteurella haemolytica A1 leukotoxin in the pathogenesis of bovine pneumonic pasteurellosis, its in vitro effects on bovine neutrophils were investigated. Leukotoxin-containing culture supernatant, from P. haemolytica, stimulated a neutrophil respiratory burst as measured by the generation of oxygen-derived free radicals O2- and H2O2. This effect was immediate because preincubation of neutrophils with the culture supernatant for 5 min or longer substantially suppressed this respiratory burst. This suppression was due to cytolysis of the neutrophils. Prolonged incubation of neutrophils with the same culture supernatant caused further cytolysis and degranulation. Heat-inactivated P. haemolytica culture supernatant that had lost its cytotoxic properties failed to stimulate respiratory burst by neutrophils. Furthermore, the respiratory burst, cytolysis and degranulation were abrogated only by leukotoxin-neutralizing monoclonal and polyclonal antibodies, but not by antibodies against the lipopolysaccharide. These studies show that the leukotoxin component in the culture supernatant was responsible for the generation of oxygen-derived free radicals and proteolytic enzymes from neutrophils which may participate in direct lung injury.     

Variability in concentrations of zinc in serum and feed when using zinc oxide as a supplement for the prevention of facial eczema

AIMS: To determine the variability of concentrations of Zn in feed, when used as a supplement to prevent facial eczema, and to determine the variability in concentrations of Zn in serum between cows and herds that are being supplemented with ZnO in feed, using in-shed feeders or on a feed pad.

METHODS: Sixteen commercial dairy farms in the Waikato region of New Zealand were enrolled, that were supplementing cows with ZnO in the feed using either an automatic in-shed feeder (ASF) or a feed pad (FP) using a feed-out or mixer wagon. On each farm 10 cows were selected by the farmer, that were assumed to be representative of the age and liveweight of the herd. Four hours after supplement feeding, each cow was weighed and a blood sample collected for measurement of concentrations of Zn in serum. Three samples of feed were collected from each farm for Zn analysis, from the beginning, middle and end of the feed being distributed. Levene’s test for homoscedasticity was used to analyse whether there were differences in variation of individual concentrations of Zn in serum, and in the feed, between the two feeding systems. Multivariable linear regression was used to examine associations between age, feeding method or liveweight and concentrations of Zn in serum, after accounting for the variability between farms.

RESULTS: Of the 163 cows sampled, concentrations of Zn in serum were between 20–35?µmol/L in 75/163 (46 (95% CI=38–54)%) cows; were <20?µmol/L in 71/163 (44 (95% CI=36–52)%) cows, and >35?µmol/L in 17/163 (10 (95% CI=6–16)%) cows. The variation in concentrations of Zn in serum in individual cows differed between farms (p<0.001), and the variability was greater for cows fed using a FP than ASF (p<0.001). There was no difference in the variation of concentrations of Zn in feed between the two feeding methods (p=0.54), but concentrations of Zn in serum were associated with the amount of Zn offered in feed (p=0.008).

CONCLUSIONS AND CLINICIAL RELEVENCE: There was significant variability between farms in the concentrations of Zn in the serum of cows being supplemented with ZnO in feed. Only 46% of cows sampled had concentrations of Zn between 20–35?µmol/L. Effective management of facial eczema should include monitoring Zn in the feed and in serum to ensure cows are receiving the correct dose they require.     

Influence of packaging method and storage time on shear value and mechanical strength of intramuscular connective tissue of chevon

The objectives of this study were to determine the effects of storage time (ST) and packaging method (PM) on tenderness and changes in intramuscular connective tissue (IMCT) strength of chevon. Spanish does (8 mo of age, average BW 25 kg) were harvested (n = 12), chilled at 4 degrees C for 24 h, and then fabricated into 2.5-cm-thick leg, shoulder/arm, and loin/rib cuts. The cuts from six carcasses were vacuum-packed and aged at 2 degrees C for 0, 4, 8, or 12 d. To assess the influence of a packaging method that favors oxidation on postmortem tenderization, the cuts from the remaining six carcasses were placed on styrofoam trays, overwrapped with polyvinyl-chloride film, and stored at 2 degrees C for similar periods. At each ST, longissimus (LM), semimembranosus (SM), and triceps brachii (TB) muscles were assessed for Warner-Bratzler shear (WBS) values. The WBS of uncooked meat, myofibrillar fragmentation index (MFI), and collagen solubility were assessed on LM. The IMCT samples were prepared to assess changes in mechanical strengths and for scanning electron microscopy (SEM). Intact honeycomb structures of endomysium, with no muscle fiber elements, were observable under SEM. The PM or ST did not influence the mechanical strength of IMCT preparations, as measured by a texture analyzer. Collagen solubility of LM muscles also did not change during aging. For both PM, cooked meat WBS values were higher (P < 0.01) in SM and TB than in LM. In the SM samples, the average WBS values were higher (P < 0.01) at d 0 than at other ST. Although MFI of LM increased with increasing aging time (P < 0.05), changes in WBS over ST were minimal in TB and LM samples. The WBS of uncooked LM decreased sharply up to 8 d postmortem in both PM (P < 0.05). However, there was no PM x ST interaction to indicate any adverse influence of packaging on tenderization of chevon. The results suggest that aging chevon cuts for more than 4 d may not result in significant additional improvement in tenderness.     

Microsatellite Marker Based Assessment of Genetic Diversity among Cultivars, Landraces and Wild Relatives in Rice

India being the primary center of origin for rice had a very large treasure of local land races, most of which are out of cultivation today. The exact genetic potential and their differences from commercial varieties and the magnitude of heterogeneity still present in them are not well catalogued. Hence the need to characterize the available land races has become imminent in the modem day concept of crop improvement （Rezai and Frey, 1990）. The present study addresses the utility of SSR markers in divulging the genetic relationships at molecular level among the major component factions office gennplasm viz., local cultivars, land races and wild species collected from a wide range of agro-geographical regions of Indian subcontinent.     

Fat extraction from acid- and base-hydrolyzed food samples using accelerated solvent extraction

This paper describes a new in-cell method for pursuing accelerated solvent extraction (ASE) prior to lipid analysis from food samples. It is difficult to pursue direct ASE with acid- or base-hydrolyzed samples due to the corrosive nature of the reagents and material limitations. In this study ion exchange based materials were used to remove acid or base reagents in-cell without compromising the recovery of lipids. The performance data are presented here for the new methods for lipid extraction for a variety of food samples and compared to the Mojonnier method. NIST Standard Reference Materials (SRM-1546 and SRM-1849) were used to validate the ASE methods. Excellent fat recoveries were obtained for the ASE methods. The new methods presented here enhance the utility of ASE and eliminate labor intensive protocols.     

Protein digestibility-corrected amino acid scores for bean and bean-rice infant weaning food products

Vegetable proteins are an integral part of infant weaning diets in Latin America. Protein quality in plant-based products, however, is constrained by amino acid composition and intrinsically present antinutritional factors. The goal of this study was to improve bean protein quality by utilizing fermentation and germination processing. The objectives were to determine if protein quality, as measured by Food and Agricultural Organization (FAO) approved True Protein Digestibility (TPD) and Protein Digestibility-Corrected Amino Acid Scores (PDCAAS), of formulated bean-based weaning products could be improved upon fermentation and germination and if protein quality could be further improved when processed beans were combined with cooked rice. Results showed that the highest TPD and PDCAAS values were obtained for cooked germinated beans combined with rice. The TPD values for products ranged from 80 to 91%, and the PDCAAS values were 0.38-0.51. There was no significant increase (P < 0.05) of either TPD or PDCAAS values upon fermentation. Germination increased TPD of cooked bean products; this increase was not, however, accompanied by an increase in PDCAAS. When combined with rice, the PDCAAS values for all bean products improved significantly, thus supporting the concept of cereal-legume complementation. In conclusion, this study showed the range of PDCAAS in processed black bean and bean-rice infant weaning food products. The potential for incorporation of these products into the diets of weaning age Latin American children would, however, be confirmed only after validation with growth or metabolic balance studies in human infants.     

Foliar absorption — penetration of the cuticular membrane and nutrient uptake by isolated leaf cells

Summary Foliar absorption consists of penetration of a cuticular membrane and uptake by living cells within the leaf. A detailed analysis of nutrient absorption by these two systems has revealed that ionic penetration of the cuticular membrane is by diffusion, and that the coupling between active transport and metabolism is at the cellular level. Urea penetrates the cuticular membrane and is absorbed by leaf cells much more rapidly than are nutrient ions. Furthermore, urea facilitates penetration and absorption of other materials simultaneously applied. Penetration of nutrient ions through cuticular membranes has been localized by microautoradiography as occurring around stomatal pores and along periclinal cell walls. Chelation of metals such as iron, manganese and zinc reduces the rate of foliar absorption, but increases the translocation of the absorbed nutrient. At the cellular level the nutrient ion is absorbed and the ligand is excluded.

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Zusammenfassung Blattabsorption erfolgt durch Eindringen in Kutikularschichten der Membran und durch Aufnahme durch die lebenden Zellen innerhalb des Blattes. Eine detaillierte Analyse der N?hrstoffabsorption nach diesen beiden Systemen zeigte, da? die Ionen-Aufnahme über die Kutikularmembran durch Diffusion und da? die Koppelung des aktiven Transports mit dem Stoffwechsel im Zellbereich erfolgt. Harnstoff dringt durch die Kutikularmembran ein und wird viel schneller als N?hrstoff-Ionen durch die Blattzellen absorbiert. Weiterhin erleichtert Harnstoff das Eindringen und die Absorption anderer, gleichzeitig applizierter Substanzen. Die Fixierung der Eindringungsstelle von N?hrstoff-Ionen in Kutikularmembrane wurde durch Mikroautoradiographie ermittelt. Das Eindringen erfolgt um die Stomata-?ffnungen und entlang der periklinalen Zellw?nde. Metallgelate von Eisen, Mangan und Zink mindern die Zellabsorption, vermehren aber die Translokation der absorbierten N?hrstoffe. Im Bereich der Zellen werden N?hrstoff-Ionen absorbiert, aber Verbindungen ausgeschlossen.

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Résumé L'absorption foliaire consiste en une pénétration à travers la membrane cuticulaire, et l'assimilation par les cellules vivantes à l'intérieur de la feuille. Une analyse détaillée de l'absorption des éléments nutritifs par ces deux systèmes a révélé que la pénétration des ions à travers la membrane cuticulaire se fait par diffusion, et que le couplage entre le transport actif et le métabolisme se fait au niveau cellulaire. L'urée pénètre à travers la membrane cuticulaire et est absorbée par les cellules de la feuille beaucoup plus rapidement que ne le sont les ions nutritifs. D'autre part l'urée facilite la pénétration et l'absorption d'autres substances appliquées simultanément. La pénétration des ions nutritifs à travers la membrane cuticulaire a été localisée par microautoradiographie comme se faisant autour des pores stomatiques, et le long des parois des cellules périclinales. La chélation de métaux tels que fer, manganèse, zinc, réduit la vitesse d'absorption foliaire mais augmente la vitesse de transport de l'ion nutritif absorbé. Au niveau de la cellule, l'ion nutritif est absorbé, et le complexant est exclu.

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Report No. COO-888-51 in cooperation with the Division of Biology and Medicine of the United States Atomic Energy Commission, Contract AT (11-1)-888. Journal Article No. 3692 of the Michigan Agricultural Experiment Station.     

Characterization of Mannheimia (Pasteurella) haemolytica leukotoxin interaction with bovine alveolar macrophage beta2 integrins

Mannheimia (Pasteurella) haemolytica, the etiologic agent of bovine pneumonic mannheimiosis, produces an exotoxic leukotoxin. The leukotoxin (LktA) is a member of the RTX (repeats in toxin) family of bacterial cytolysins and is distinguished from other toxins by its unique target cell specificity to ruminant leukocytes occurring through binding to a specific receptor. We have demonstrated previously that the beta2 integrin LFA-1 is a receptor for LktA in bovine leukocytes and is involved in leukotoxin-induced biological effects. However the subunits within LFA-1 involved in binding to LktA, and post-binding signaling leading to cellular activation have not been well characterized. The purpose of our study was to pinpoint these precise subunits on bovine alveolar macrophages and to characterize their interaction with LktA. The results in this study indicate that although LktA can efficiently bind to the CD18 subunit of both LFA-1 and Mac-1, post-binding signaling events including elevation of intracellular calcium and CD18 tail phosphorylation are only observed through LFA-1. Furthermore, LktA also binds to the CD11a subunit of LFA-1. LktA binding to CD11a could be inhibited by a small molecule inhibitor of the I(inserted)-domain, the major ligand binding interface on CD11a. I-domain inhibition significantly blunts LktA-induced intracellular calcium elevation and tyrosine phosphorylation of the CD18 tail. Based on our results we suggest that LFA-1 serves as the functional leukotoxin receptor on bovine alveolar macrophages.     

An Efficient Micropropagation Protocol for High-Frequency Plantlet Regeneration from Liquid Culture of Nodal Tissues in a Medicinal Plant,Turnera ulmifolia L.

A highly efficient, stable, and cost-effective micropropagation protocol for the conservation of a medicinal plant Turnera ulmifolia L. was established from nodal tissues via multiple axillary shoot proliferation on using Murashige and Skoog’s (MS) liquid nutrient medium. To begin with, nodal explants were placed on agar gelled medium amended with 2.0 mg L^?1 6-benzylaminopurine (BAP) and 0.1 mg L^?1 indole-3 acetic acid (IAA) for shoot induction. Subsequently, elongation of regenerated shoots could be possible on liquid MS medium supplemented with 0.5 mg L^?1 BAP and Kin (kinetin) each along with 0.1 mg L^?1 IAA where high frequency of regeneration in terms of number of shoots (47.2 shoots/explant) was achieved. Furthermore, long and healthy shoots (4?5 cm in length) were rooted on agar gelled half-strength of MS medium supplemented with 2.0 mg L^?1 indole-3 butyric acid (IBA). Finally, in vitro regenerated plantlets were gradually acclimatized in the greenhouse and transferred to the field successfully.