El conejo como modelo para el estudio del daño pulmonar generado por el lipopolisacarido de Pasteurella haemolytica mediante la reacción de Shwartzman y el fenómeno de Arthus

Tesis (Doctorado en Medicina Veterinaria) UANL

Mannheimiose pneumônica experimentalmente induzida em bezerros pela Mannheimia (Pasteurella) haemolytica A1-cepa D153: achados do exame físico, hemograma e swabs nasal e nasofaringeano

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Isolation of an ovine pulmonary surfactant-associated anionic peptide bactericidal for Pasteurella haemolytica.

Ovine pulmonary surfactant is bactericidal for Pasteurella haemolytica when surfactant and bacteria mixtures are incubated with normal ovine serum. To isolate this component, surfactant (1 mg/ml) was centrifuged at 100,000 x gav, and the supernatant was fractionated by HPLC. Fractions were eluted with acetonitrile (10-100%)/0.1% trifluoracetic acid and tested for bactericidal activity. Amino acid and sequence analysis of three bactericidal fractions showed that fraction 2 contained H-GDDDDDD-OH, fraction 3 contained H-DDDDDDD-OH, and fraction 6 contained H-GADDDDD-OH. Peptides in 0.14 M NaCl/10 microM ZnCl2 (zinc saline solution) induced killing of P. haemolytica and other bacteria comparable to defensins and beta-defensins [minimal bactericidal concentration (MBC)50 range, 0.01-0.06 mM] but not in 0.14 M NaCl/10 mM sodium phosphate buffer, pH 7.2/0.5 mM CaCl2/0.15 mM MgCl2 (MBC50 range, 2.8-11.5 mM). Bactericidal activity resided in the core aspartate hexapeptide homopolymeric region, and MBC50 values of aspartate dipeptide-to-heptapeptide homopolymers were inversely proportional to the number of aspartate residues in the peptide. P. haemolytica incubated with H-DDDDDD-OH in zinc saline solution was killed within 30 min. Ultrastructurally, cells contained flocculated intracellular constituents. In contrast to cationic defensins and beta-defensins, surfactant-associated anionic peptides are smaller in size, opposite in charge, and are bactericidal in zinc saline solution. They are members of another class of peptide antibiotics containing aspartate, which when present in pulmonary secretions may help clear bacteria as a part of the innate pulmonary defense system.

Pasteurella Vaccination of Ewes

Pasteurella haemolytica is a major contributor to neonatal pneumonia in lambs; which continues to be a major problem. Experimentation was conducted to determine the efficacy of vaccinating pregnant ewes to reduce the incidence of pneumonia in newborn lambs. Vaccines utilized in this experimentation included three different commercial Pasteurella haemolytica vaccines intended for use in cattle and an experimental vaccine prepared in our laboratory. Only one of the commercial vaccines increased levels of anti-Pasteurella antibodies in serum of the ewes at time of lambing, but lambs from all three groups of vaccinated ewes had higher levels of antibodies than control lambs. Some lambs in all groups developed pneumonia during the neonatal period. Ewes administered the experimental vaccine had significantly higher levels of serum antibodies at lambing time. This increase was reflected in increased levels in serum of lambs from the vaccinated ewes. However, the antibodies appeared not to be protective, since as many lambs in the treatment group developed pneumonia as did in the control group.

Phase variable restriction-modification systems in Moraxella catarrhalis

A repetitive DNA motif was used as a marker to identify novel genes in the mucosal pathogen Moraxella catarrhalis. There is a high prevalence of such repetitive motifs in virulence genes that display phase variable expression. Two repeat containing loci were identified using a digoxigenin-labelled 5'-(CAAC)(6)-3' oligonucleotide probe. The repeats are located in the methylase components of two distinct type III restriction-modification (R-M) systems. We suggest that the phase variable nature of these R-M systems indicates that they have an important role in the biology of M. catarrhalis. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.

The medicine and epidemiology of bovine respiratory disease in feedlots

Bovine Respiratory Disease (BRD) results from a complex, multifactorial interaction of stressors, animal susceptibility, and respiratory pathogens. The infectious agents associated with BRD are ubiquitous among cattle populations. Typically, one or a combination of stressors are necessary to initiate BRD. Prevention of BRD should, therefore, address management procedures to minimise stressors. Administration of vaccines against BRD agents may help reduce the incidence of BRD but is unlikely to eliminate the condition. The effectiveness of antimicrobials in the treatment of BIRD depends primarily on early recognition and treatment. The use of antioxidant vitamins, minerals or other agents in the prevention and treatment of BRD warrants further research.

Structure and Mode of Action of Microplusin, a Copper II-chelating Antimicrobial Peptide from the Cattle Tick Rhipicephalus (Boophilus) microplus

Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single alpha-helical globular domain, microplusin consists of five alpha-helices: alpha 1 (residues Gly-9 to Arg-21), alpha 2 (residues Glu-27 to Asn-40), alpha 3 (residues Arg-44 to Thr-54), alpha 4 (residues Leu-57 to Tyr-64), and alpha 5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.

Serum protein concentrations in calves with experimentally induced pneumonic pasteurellosis

Foram examinados 10 bezerros da raça Holandesa com duas a quatro semanas de idade, distribuídos aleatoriamente em dois grupos, controle e infectado. Os bezerros do grupo-controle foram inoculados por via intrabronquial com 5ml de solução salina fosfato-tamponada Dulbecco (DPBSS). Os do grupo infectado receberam um inóculo contendo 5x10(9) unidades formadoras de colônia-UFC de Mannheimia (Pasteurella) haemolytica, suspensas em 5ml de DPBSS. As amostras de sangue foram colhidas 15 minutos antes e uma, duas, quatro e seis horas após a inoculação. O proteinograma sérico foi obtido por eletroforese em gel de acrilamida. As concentrações séricas das proteínas de pesos moleculares 125.000 D (ceruloplasmina), 60.000 D (a1-antitripsina), 45.000 D (haptoglobina) e 40.000 D (glicoproteína ácida) foram significativamente maiores em bezerros infectados do que nos do grupo-controle. Os resultados indicam que as concentrações das proteínas de fase aguda se elevaram mais rapidamente que o previamente imaginado. O proteinograma sérico pode ser útil no monitoramento da evolução da pneumonia de bezerros causada por M. haemolytica.

Pesquisa de vírus relacionados com doenças respiratórias em ovinos sadios e naturalmente acometidos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Apx toxins in Pasteurellaceae species from animals

Pasteurellaceae species particularly of porcine origin which are closely related to Actinobacillus pleuropneumoniae were analyzed for the presence of analogues to the major A. pleuropneumoniae RTX toxin genes, apxICABD, apxIICA and apxIIICABD and for their expression. Actinobacillus suis contains both apxICABD(var.suis) and apxIICA(var. suis) operons and was shown to produce ApxI and ApxII toxin. Actinobacillus rossii contained the operons apxIICA(var.rossii) and apxIIICABD(var.rossii). However, only the toxin ApxII and not ApxIII could be detected in cultures of A. rossii. The Apx toxins found in A. suis and A. rossi may play a role in virulence of these pathogens. Actinobacillus lignieresii, which was included since it is phylogenetically very closely related to A. pleuropneumoniae, was found to contain a full apxICABD(var.lign.) operon which however lacks the -35 and -10 boxes in the promoter sequences. As expected from these results, no expression of ApxI was detected in A. lignieresii grown under standard culture conditions. Actinobacillus seminis, Actinobacillus equuli, Pasteurella aerogenes, Pasteurella multocida, Haemophilus parasuis, and also Mannheimia (Pasteurella) haemolytica, which is known to secrete leukotoxin, were all shown to be devoid of any of the apx toxin genes and did not produce ApxI, ApxII or ApxIII toxin proteins. However, proteins of slightly lower molecular mass than ApxI, ApxII and ApxIII which showed limited cross-reactions with monospecific, polyclonal anti-ApxI, anti-ApxII and anti-ApxIII were detected on immunoblot analysis of A. equuli, A. seminis and P. aerogenes. The presence of Apx toxins and proteins that imunologically cross react with Apx toxins in porcine Actinobacillus species other than A. pleuropneumoniae can be expected to interfere with serodiagnosis of porcine pleuropneumonia.

Modulation of Immune Response in Lambs

Experiments using different types of antigen-adjuvant preparations were conducted in outbred sheep to compare effects of adjuvants on immune responses. Trinitrophenyl-ovalbumin (TNP-ovalbumin) incorporated in a preparation with nonionic block copolymers elicited high antibody titers to both ovalbumin and TNP. Different humoral immune responses were observed when Pasteurella haemolytica lipopolysaccharide (LPS) was added to the preparations. Responses to ovalbumin and TNP were reduced when Pasteurella haemolytica LPS was added to copolymer L121. The antibody titers to ovalbumin or TNP were not affected by the addition of LPS to the preparation containing copolymer L180.5. Lymphocyte proliferation assays demonstrated high stimulation indices at day 17 to ovalbumin by lymphocytes from lambs receiving preparations containing copolymers without Pasteurella haemolytica LPS.

Detection of RTX toxin genes in gram-negative bacteria with a set of specific probes

The family of RTX (RTX representing repeats in the structural toxin) toxins is composed of several protein toxins with a characteristic nonapeptide glycine-rich repeat motif. Most of its members were shown to have cytolytic activity. By comparing the genetic relationships of the RTX toxin genes we established a set of 10 gene probes to be used for screening as-yet-unknown RTX toxin genes in bacterial species. The probes include parts of apxIA, apxIIA, and apxIIIA from Actinobacillus pleuropneumoniae, cyaA from Bordetella pertusis, frpA from Neisseria meningitidis, prtC from Erwinia chrysanthemi, hlyA and elyA from Escherichia coli, aaltA from Actinobacillus actinomycetemcomitans and lktA from Pasteurella haemolytica. A panel of pathogenic and nonpathogenic gram-negative bacteria were investigated for the presence of RTX toxin genes. The probes detected all known genes for RTX toxins. Moreover, we found potential RTX toxin genes in several pathogenic bacterial species for which no such toxins are known yet. This indicates that RTX or RTX-like toxins are widely distributed among pathogenic gram-negative bacteria. The probes generated by PCR and the hybridization method were optimized to allow broad-range screening for RTX toxin genes in one step. This included the binding of unlabelled probes to a nylon filter and subsequent hybridization of the filter with labelled genomic DNA of the strain to be tested. The method constitutes a powerful tool for the assessment of the potential pathogenicity of poorly characterized strains intended to be used in biotechnological applications. Moreover, it is useful for the detection of already-known or new RTX toxin genes in bacteria of medical importance.

Epithelial antibiotic induced in states of disease

Epithelial defensins provide an active defense against the external microbial environment. We investigated the distribution and expression of this class of antimicrobial peptides in normal cattle and in animals in varying states of disease. β-defensin mRNA was found to be widely expressed in numerous exposed epithelia but was found at higher levels in tissues that are constantly exposed to and colonized by microorganisms. We observed induction in ileal mucosa during chronic infection with Mycobacterium paratuberculosis and in bronchial epithelium after acute infection with Pasteurella haemolytica. It has been proposed that expression of antimicrobial peptides is an integral component of the inflammatory response. The results reported here support this hypothesis and suggest that epithelial defensins provide a rapidly mobilized local defense against infectious organisms.

Ocorrência de Mannheimia haemolytica e de Pasteurella multocida em ovinos sadios e com enfermidade respiratória

Frequency of Mannheimia haemolytica and Pasteurella multocida in the respiratory tract of lambs in the region of Botucatu, SP, Brazil, was studied. Nasopharingeal and oropharingeal swabs were obtained from 262 animals: 180 from healthy and 82 from animals with respiratory diseases. M. haemolytica was the most prevalent (47%), followed by the association of M. haemolytica and P. multocida (27%), and P. multocida (11%). Animals with respiratory disease presented higher occurrence of P. multocida in the nasopharynx as compared to healthy animals (P<0.05). No significant difference in isolation rate of M. haemolytica, P. multocida, and association of these microorganisms in the oropharynx of healthy and affected animals was observed.

Susceptibilidade in vitro a antimicrobianos da Mannheimia haemolytica e da Pasteurella multocida isoladas de ovinos sadios e com doenças respiratórias

Pasteurella multocida and Mannheimia haemolytica (P. haemolytica) are associated with ovine respiratory diseases. With the purpose of observe the susceptibility in vitro of these organisms against antimicrobials, were collected samples of nasopharingeal (n=180) and oropharingeal (n=82) from ovines healthy and with respiratory disease. Among the antimicrobials tested, the sensibility was greater for enrofloxacin (100%) and florfenicol (100%), for both bacteria. The greater resistance indices for M. haemolytica and P. multocida were observed with tetracyclin (15.64% and 17.65% respectively) and penicillin (1.82% and 4.2%).