Expression of the antimicrobial peptides in plants to control phytopathogenic bacteria and fungi.

Three antimicrobial peptides exhibiting in vitro antifungal activity were expressed in Arabidopsis to compare their in planta activity. Beta-Purothionin, cecropin B, and phor21 were expressed under an endogenous promoter with moderate-level activity and excreted extracellularly. Expression of beta-purothionin rendered the greatest antibacterial and antifungal resistance while cecropin B enhanced only antibacterial activity and phor21 did not improve antimicrobial resistance. The transgenic beta-purothionin arrested fungal growth on leaf surfaces and infection of stomata. Leaf extracts from plants producing beta-purothionin and cecropin B displayed membrane permeabilizing activity. The in planta antimicrobial activity of the tested peptides was consistent with previously reported in vitro experiments. The expression strategy allowed enhanced antifungal resistance without high-level transgene expression.

Characterization of the caprine model for ruminant brucellosis.

The relationship between man, the goat, and brucellosis is historical. Today Brucella melitensis and Brucella abortus pose a serious economic and public health threat in many countries throughout the world. Infection of pregnant goats and sheep with B. melitensis results in abortion during the third trimester of pregnancy. Although nearly eradicated in the US, bovine brucellosis is still a problem in many countries and the potential for re-infection of domestic stock from wildlife reservoirs in this country is a regulatory nightmare. Humans infected with this pathogen develop undulant fever, which is characterized by pyrexia, arthritis, osteomyelitis, and spondylitis. Although available for both organisms, currently available vaccines have problems ranging from false positive serological reactions to limited efficacy in different animal species. With the continued need for new and better vaccines, we have further developed a goat model system to test new genetically derived strains of B. melitensis and B. abortus for virulence as measured by colonization of maternal and fetal tissues, vaccine safety, and vaccine efficacy.

Pathogenicity and protective activity in pregnant goats of a Brucella melitensis Deltaomp25 deletion mutant.

The Brucella melitensis mutant BM 25, which lacks the major 25 kDa outer membrane protein Omp25, has previously been found to be attenuated in the murine brucellosis model. In the present study, the capacity of the Deltaomp25 mutant to colonise and cause abortions in the caprine host was evaluated. The vaccine potential of BM 25 was also investigated in goats. Inoculation of nine pregnant goats in late gestation with the B. melitensis mutant resulted in 0/9 abortions, while the virulent parental strain, B. melitensis 16M, induced 6/6 dams to abort (P<0.001, n=6). BM 25 also colonised fewer adults (P<0.05, n=6) and kids (P<0.01, n=6) than strain 16M. The Deltaomp25 mutant was found capable of transient in vivo colonisation of non-pregnant goats for two weeks post-infection. Owing to the ability of BM 25 to colonise both non-pregnant and pregnant adults without inducing abortions, a vaccine efficacy study was performed. Vaccination of goats prior to breeding with either BM 25 or the current caprine vaccine B. melitensis strain Rev. 1 resulted in 100 per cent protection against abortion following challenge in late gestation with virulent strain 16M (P<0.05, n=7). However, unlike strain Rev. 1, BM 25 does not appear to cause abortions in late gestation based on this study with a small number of animals. The B. melitensis Deltaomp25 mutant, BM 25, may be a safe and efficacious alternative to strain Rev. 1 when dealing with goat herds of mixed age and pregnancy status.

Oral vaccination of sexually mature pigs with Brucella abortus vaccine strain RB51.

OBJECTIVE: To develop a novel oral vaccine delivery system for swine, using the rough vaccine strain of Brucella abortus. ANIMALS: 56 crossbred pigs from a brucellosis-free facility. PROCEDURE: In 3 separate experiments, pigs were orally vaccinated with doses of 1 x 10(9) to > 1 x 10(11) CFU of strain RB51 vaccine. The vaccine was placed directly on the normal corn ration, placed inside a whole pecan, or mixed with cracked pecans and corn. RESULTS: Oral vaccination of pigs with vaccine strain RB51 resulted in a humoral immune response to strain RB51 and short-term colonization of the regional lymph nodes. CONCLUSIONS AND CLINICAL RELEVANCE: A viscous liquid such as Karo corn syrup in association with pecans that scarify the oral mucosa are necessary when placing the live vaccine directly onto corn or other food rations. Doses of > 1 x 10(11) CFU of RB51 organisms/pig in this mixture ensures 100% colonization of regional lymph nodes via the oral route. This method may allow an efficient and economical means to vaccinate feral swine for brucellosis.

Targeted destruction of androgen-sensitive and -insensitive prostate cancer cells and xenografts through luteinizing hormone receptors.

BACKGROUND: We have prepared a conjugate of a lytic peptide (hecate) and a 15-amino acid segment of the beta-chain of LH to test the concept that this conjugate will target cancer cells expressing LH receptors. METHODS: Hecate-betaLH was added in vitro to cultures of Chinese hamster ovary (CHO) cells with and without LH receptors and to prostate cancer cells in the presence or absence of steroids, follicle-stimulating hormone (FSH), epidermal growth factor (EGF), or betaLH. PC-3 xenografts were established in male athymic nude mice and treated once a week for 3 weeks with hecate-betaLH via the lateral tail vein. RESULTS: The conjugate showed concentration-dependent toxicity for the following prostate cancer cell lines: BRF 41 T>DU145>PC-3>LNCaP, according to their LH receptor capacities. Steroid removal reduced sensitivity to the drug in a reversible manner. Hecate-betaLH reduced the tumor burden in the nude mice from 60 to 12.5 mg/g body weight. CONCLUSIONS: We conclude that the hecate-betaLH conjugate selectively kills androgen-dependent and-independent prostate cancer cells both in vivo and in vitro; its toxicity depends on the number of LH receptor sites present.

Coronavirus and Pasteurella infections in bovine shipping fever pneumonia and Evans' criteria for causation.

Respiratory tract infections with viruses and Pasteurella spp. were determined sequentially among 26 cattle that died during two severe epizootics of shipping fever pneumonia. Nasal swab and serum samples were collected prior to onset of the epizootics, during disease progression, and after death, when necropsies were performed and lung samples were collected. Eighteen normal control cattle also were sampled at the beginning of the epizootics as well as at weekly intervals for 4 weeks. Respiratory bovine coronaviruses (RBCV) were isolated from nasal secretions of 21 and 25 cattle before and after transport. Two and 17 cattle nasally shed Pasteurella spp. before and after transport, respectively. RBCV were isolated at titers of 1 x 10(3) to 1.2 x 10(7) PFU per g of lung tissue from 18 cattle that died within 7 days of the epizootics, but not from the lungs of the remaining cattle that died on days 9 to 36. Twenty-five of the 26 lung samples were positive for Pasteurella spp., and their CFU ranged between 4.0 x 10(5) and 2.3 x 10(9) per g. Acute and subacute exudative, necrotizing lobar pneumonia characterized the lung lesions of these cattle with a majority of pneumonic lung lobes exhibiting fibronecrotic and exudative changes typical of pneumonic pasteurellosis, but other lung lobules had histological changes consisting of bronchiolitis and alveolitis typical of virus-induced changes. These cattle were immunologically naive to both infectious agents at the onset of the epizootics, but those that died after day 7 had rising antibody titers against RBCV and Pasteurella haemolytica. In contrast, the 18 clinically normal and RBCV isolation-negative cattle had high hemagglutinin inhibition antibody titers to RBCV from the beginning, while their antibody responses to P. haemolytica antigens were delayed. Evans' criteria for causation were applied to our findings because of the multifactorial nature of shipping fever pneumonia. This analysis identified RBCV as the primary inciting cause in these two epizootics. These viruses were previously not recognized as a causative agent in this complex respiratory tract disease of cattle.

Effect of the antimicrobial peptide, D-hecate, on trichomonads.

Tritrichomonas foetus and Trichomonas vaginalis are protozoan parasites that cause sexually transmitted diseases in cattle and humans, respectively. There is a need for new antimicrobial agents to treat or prevent trichomoniasis because there are currently no approved chemotherapeutic agents against T. foetus and resistance of T. vaginalis to metronidazole does occur. Therefore, we evaluated the effect of a novel antimicrobial peptide, D-hecate, on the viability of 6 isolates of T. foetus and T. vaginalis in vitro. Tritrichomonas foetus and T. vaginalis were grown to mid log phase (24 hr) or late log/stationary phase (48 hr). Parasites at 10(6)/ml were mixed with equal volumes of D-hecate to final concentrations of 10 microM, 20 microM. and 40 microM of D-hecate. Controls had minimal essential medium (MEM) alone. The numbers of viable parasites were determined microscopically after 10, 20, and 30 min of incubation at 37 C with D-hecate or MEM. Our results show that D-hecate killed all 6 isolates of T. foetus and T. vaginalis evaluated. The killing effect was dependent on the concentration of the peptide, incubation time, and phase of growth of the parasites. Ultrastructural studies of parasites treated with 10 microM of D-hecate revealed extensive damage to the plasma membrane of most T. foetus and T. vaginalis cells, while a few cells were distorted but remained intact. D-Hecate may be a useful chemotherapeutic agent for the treatment of trichomoniasis.

Protection against infection and abortion induced by virulent challenge exposure after oral vaccination of cattle with Brucella abortus strain RB51.

OBJECTIVES: To determine efficacy of orally administered Brucella abortus vaccine strain RB51 against virulent B abortus challenge exposure in cattle as a model for vaccination of wild ungulates. ANIMALS: 20 mixed-breed beef cattle obtained from a brucellosis-free herd. PROCEDURE: Sexually mature, Brucella-negative beef heifers were vaccinated by mixing > 10' viable RB51 organisms or diluent with their feed. Heifers were fed individually and consumed their entire ration. Each heifer received approximately 3 X 10' colony-forming units (CFU). Six weeks after oral vaccination, heifers were pasture-bred to brucellosis-free bulls. At approximately 186 days' gestation, heifers were challenge exposed conjunctively with 107 CFU of virulent B abortus strain 2308. RESULTS: Vaccination with the rough variant of B abortus RB51 did not stimulate antibodies against the O-polysaccharide (OPS) of B abortus. After challenge exposure and parturition, strain 2308 was recovered from 80% of controls and only 20% of vaccinates. Only 30% of the vaccinates delivered dead, premature, or weak calves, whereas 70% of the controls had dead or weak calves. CONCLUSIONS: Cattle vaccinated orally with the rough variant of B abortus strain RB51 develop significant (P < 0.05) protection against abortion and colonization and do not produce OPS-specific antibodies. Clinical Relevance-Results encourage further investigation into use of strain RB51 to vaccinate wild ungulates (elk and bison) orally.

Evaluation of a rough mutant of Brucella melitensis in pregnant goats.

Brucella melitensis strain VTRM1, a rough derivative of B melitensis strain 16M, is able to colonise the lymph nodes of goats, does not induce abortion in pregnant goats when used at doses leading to abortions with virulent strain 16M, and does not induce anti-O chain antibodies. However, strain VTRM1 as a single dose vaccine induces only partial protection against both infection and abortion following challenge.

Interleukin 2 promoter/enhancer controlled expression of a synthetic cecropin-class lytic peptide in transgenic mice and subsequent resistance to Brucella abortus.

The addition of an antimicrobial that can be synthesized by the mammalian immune system at the point of challenge may enhance disease resistance. A possible group of agents are cecropins, broad-spectrum antimicrobial peptides, which have been described and characterized. They are relatively non-toxic to normal cells from multicellular organisms but are toxic to a wide range of bacteria, protozoa and fungi, as well as infected and abnormal cells. Twenty-six lines of transgenic mice were produced by pronuclear injection of DNA consisting of the 5'-flanking region from -593 to +110 of the mouse interleukin 2 (IL-2) gene, Shiva 1a (a synthetic cecropinclass lytic peptide), and the SV40 polyadenylation/splice signal. A reverse-transcription PCR assay determined that two lines of transgenic mice were produced whose spleen-derived lymphocytes could be induced to transcribe and mature mRNA for Shiva 1a by exposure to 3.25 mg ml-1 of Con A. Two lines were challenged with an inoculation of 5 x 10(4) Brucella abortus strain 2308. After four weeks, there were significantly fewer B. abortus organisms in the spleens of transgenic mice than in non-transgenic control mice of the same strain (p < 0.05). Since the controlling regions of the IL-2 enhancer and the amino acid sequence of the signal peptide are highly conserved among several species, it is likely that this recombinant gene will function in other mammals.

Adoptive transfer of granulomatous inflammation to Brugia antigens in jirds.

Brugia pahangi infections of jirds (Meriones unguiculatus) produce a granulomatous inflammatory response within the lymphatic vessels. Granulomas that form around beads coated with soluble adult antigens embolized in the lungs have been used to measure this response. Similar lesions were observed in naive jirds receiving lymph node cells and splenocytes from animals with acute infections. This was not the case with cells from chronically infected jirds that were hyporesponsive to implanted antigen-coated beads. Passively transferred immune sera collected during the acute and chronic periods of infection did not transfer this response. Lymph node cells but not splenocytes obtained from chronically infected jirds induced a down regulation of this response in animals during the acute period. These results indicate that this inflammatory response in the jird is cell mediated and that adoptive transfer in jirds is feasible. The induction of the down-regulated state may also be mediated by cells, but not serum factors.

Light-microscopic studies of 3T3 cell plasma membrane alterations mediated by melittin.

Various light microscopic techniques were used to study the effect of melittin, a major toxic constituent of honey bee venom, on plasma membranes of 3T3 mouse fibroblasts. Bright-field light microscopy and Trypan Blue dye exclusion were used to demonstrate changes in membrane permeability after exposure to melittin. Differential interference contrast (DIC) microscopy showed that membrane vesiculation induced by melittin was dose dependent. Using both fluorescent lipid and glycoprotein markers, we found that membrane vesicles were primarily composed of lipids. A sequence of events associated with vesicle formation was depicted by DIC and fluorescence microscopy. Confocal laser scanning fluorescence microscopy demonstrated a translocation of membrane glycoproteins from the plasma membrane to the cytosol following melittin treatment. The significance of membrane vesiculation and translocation of membrane glycoproteins in damaged cells is discussed.

A Brucella melitensis high-temperature-requirement A (htrA) deletion mutant is attenuated in goats and protects against abortion.

It has been previously demonstrated that a Brucella melitensis high-temperature-requirement A (htrA) deletion mutant is more susceptible to oxidative killing in vitro than the parental strain and is attenuated in mice. To evaluate the contribution of the B melitensis HtrA protease to virulence in ruminants, the capacity of the B melitensis htrA mutant RWP5 to produce abortion in goats was compared to that of the virulent parental strain 16M. Experimental infection with strain 16M caused abortion in eight of 12 pregnant nannies, while none of the 12 nannies inoculated with RWP5 aborted. Furthermore, intramuscular injection of fetuses in utero with RWP5 led to colonisation of the fetus with subsequent colonisation of the nanny, but no abortion was observed. Nannies vaccinated with RWP5 showed complete protection against abortion when challenged with 16M during the third trimester of pregnancy. However, these animals were not protected from colonisation by 16M. The results presented here clearly indicate that the B melitensis htrA gene product contributes to pathogenesis in goats, but the utility of B melitensis htrA mutants as vaccines in this host appears to be limited.

Protection of BALB/c mice against homologous and heterologous species of Brucella by rough strain vaccines derived from Brucella melitensis and Brucella suis biovar 4.

OBJECTIVE: To evaluate stable rough mutants derived from Brucella melitensis 16M and B suis 2579 (biovar 4) as vaccines against homologous and heterologous Brucella spp in the BALB/c mouse model. DESIGN, ANIMALS, AND PROCEDURE: Rough mutants VTRM1 and VTRS1 were obtained from B melitensis 16M and B suis 2579, respectively, by allelic exchange of rfbU gene encoding mannosyltransferase with a Tn5-disrupted rfbU gene. Mice were vaccinated with VTRM1 or VTRS1 and challenge exposed 8 weeks later. RESULTS: VTRM1 and VTRS1 replicated extensively in the spleen during the first 3 weeks of infection, then decreased rapidly. Antibodies specific for the O polysaccharide were not detected in sera of mice inoculated with either rough strain. Vaccination with VTRM1 or VTRS1 induced protection against virulent strains of B abortus (2308), B melitensis (16M), B suis biovar 1 (750), and B suis biovar 4 (2579). VTRM1 also protected against B ovis (PA) and against 4 field isolates of B abortus from bison or elk. VTRS1 conferred protection against 4 field isolates of B suis biovar 4 from reindeer. Vaccines prepared from live VTRM1 or VTRS1 provided significantly greater protection than that afforded by vaccines of killed cells in QS-21 adjuvant. Vaccination with VTRM1 containing VTRS1 gave minimal protection against the antigenically unrelated Listeria monocytogenes, thus demonstrating the immunologic specificity of protection against Brucella spp. CONCLUSIONS AND CLINICAL RELEVANCE: Results encourage evaluation, in primary host species, of VTRM1 and VTRS1, along with RB51, as alternative vaccines to strain 19, Rev 1, or other smooth phase vaccines.

Characterization of bovine pulmonary and serum antibody responses after parenteral or intrapulmonary vaccination with live Pasteurella haemolytica.

Pulmonary and serum antibody responses were evaluated in eight calves vaccinated [four intrapulmonary-right diaphragmatic lobe (IP) and four subcutaneous (SC)] with Pasteurella haemolytica A1 (Ph-1) impregnated agar beads and eight respective sham-vaccinated calves. Experimental and sham groups were challenged in both diaphragmatic lobes with Ph-1 34-37 d after vaccination (DAV) and necropsied 6 d after challenge (DAC; 40-43 DAV). IgG antibodies contained in fluids from the diaphragmatic lobes of vaccinated calves had different patterns of antigen specificity compared with IgG antibodies in analogous sera. Using ELISA, anti-Ph-1 IgA and IgG antibody concentrations were significantly higher (P < 0.05) in lung lavage fluids from the IP group before and after challenge compared to the SC and sham groups. The IP and SC groups developed IgA, IgG and IgM antibody titers in nonvaccinated lung lobes after vaccination and challenge. The IP and SC groups exhibited significantly (P < 0.05) smaller pulmonary lesions than the sham groups and pulmonary IgG and IgA antibodies were associated with increased protection.

Brucella abortus differs in the multiplication within bovine chorioallantoic membrane explants from early and late gestation.

The ability of Brucella to infect and grow within extraplacentomal chorioallantoic explants (CAMs) derived from early and late gestational cattle was compared. Following inoculation of CAMs with equal numbers of strain 2308 B. abortus, the infectivity was approximately the same in CAMs from both ages, however, bacterial replication was significantly greater in late gestational CAMs than in early gestational CAMs. Co-culture of both early and late gestation CAMs or culture of both types of CAMs in the presence of tissue culture media collected from either early or late B. abortus inoculated CAMs failed to alter B. abortus growth rates and/or cytopathic effects.

Behaviour of a high-temperature-requirement A (HtrA) deletion mutant of Brucella abortus in goats.

Previous studies have shown that high-temperature-requirement A (HtrA) mutants of Brucella abortus are more sensitive to oxidative killing in vitro, are less able to survive in cultured murine macrophages and are attenuated in BALB/c mice. To measure the effect of an HtrA mutation on the virulence of B abortus in ruminants, pregnant goats in late gestation were exposed to infection by the conjunctival route with B abortus 2308 or an isogenic htrA mutant, PHE1. Infection with either 2308 or PHE1 resulted in abortion, but the serological responses to infection were consistent with 2308 but variable with PHE1. Strain 2308 was recovered post mortem both from aborted fetuses and infected dams, whereas PHE1 was recovered from neither. Nevertheless, short term studies revealed that PHE1 could be recovered from infected goats for up to two weeks after infection, suggesting that although the HtrA mutation may change the colonising ability of B abortus, the virulence of the mutant in pregnant goats is not reduced.

The morphological effects of two antimicrobial peptides, hecate-1 and melittin, on Escherichia coli.

The effects of the 26 amino acid, cationic, amphipathic, antibacterial peptide melittin and hecate-1, a 23 amino acid analog of it, on the gram negative bacterium Escherichia coli were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and freeze-fracture. Both peptides killed virtually all bacteria at the peptide concentration and cell density used. TEM and SEM revealed aggregates of bacteria entangled with material extruded from the bacterial surfaces. SEM revealed irregular bacterial surfaces with bleb-like projections. TEM and freeze-fracture indicate that the bacterial inner and outer membranes, as well as the peptidoglycan layer between, were extensively damaged. The cytoplasmic contents of the cells, however, did not appear radically disturbed, providing little evidence for osmotically induced cytolysis.

Brugia pahangi: differential granulomatous reactivity of infected jirds (Meriones unguiculatus) to fractions of adult worm extract.

Soluble extracts of adult Brugia pahangi (SSE) were fractionated by lectin affinity chromatography, followed by reversed phase HPLC. The immunologic and in vivo inflammatory reactivity of the resulting fractions were compared in jirds with acute and chronic infections of B. pahangi. When separated by SDS-PAGE, all fractions possessed bands which were recognized in Western blots by antibodies from jirds with both acute and chronic infections. Fractions were coupled to sized Sepharose beads that were subsequently embolized into the lungs of infected and uninfected control jirds. Granulomas were induced by SSE, the lectin column eluate, and HPLC fractions E, F, and G in acutely infected jirds. These reactions were significantly reduced in chronically infected jirds. HPLC fractions B, C, and D did not elicit an in vivo inflammatory response. A perivascular infiltrate of eosinophils and mononuclear cells was also observed in lungs of acutely infected jirds which received granuloma-inducing coated beads but not in lungs of similar jirds which received beads that did not induce this inflammatory response. Proliferative responses of splenocytes stimulated with SSE or the lectin eluate and lymph node cells and splenocytes stimulated with HPLC fractions B, C, or D corresponded to the in vivo granulomatous response to homologously coated beads. Correlations between in vivo inflammatory responses and in vitro proliferative responses were not seen using other fractions in these assays. These data indicate that varying degrees of granulomatous inflammation are induced by different filarial proteins mixtures and that the in vivo granuloma induction by antigen-coated beads will be useful in the identification of specific proteins involved in the induction, maintenance, and regulation of filariae-elicited inflammatory reactions. Although the size of these granulomas corresponds to severity of granulomatous inflammatory responses visualized within the jird lymphatics during the course of infection, the reaction does not correlate in all instances to lymphoproliferative responses of cells from peripheral lymph nodes or the spleen. Distinct differences between antibody and granulomatous reactivity to some fractions were noted.

Vaccination with Brucella abortus rough mutant RB51 protects BALB/c mice against virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis.

Vaccination of BALB/c mice with live Brucella abortus RB51, a stable rough mutant, produced protection against challenge with virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis. Passive-transfer experiments indicated that vaccinated mice were protected against B. abortus 2308 through cell-mediated immunity, against B. ovis PA through humoral immunity, and against B. melitensis 16M through both forms of immunity. Live bacteria were required for the induction of protective cell-mediated immunity; vaccination with whole killed cells of strain RB51 failed to protect mice against B. abortus 2308 despite development of good delayed-type hypersensitivity reactions. Protective antibodies against the heterologous species were generated in vaccinated mice primarily through anamnestic responses following challenge infections. Growth of the antigenically unrelated bacterium Listeria monocytogenes in the spleens of vaccinated mice indicated that nonspecific killing by residual activated macrophages contributed minimally to protection. These results encourage the continued investigation of strain RB51 as an alternative vaccine against heterologous Brucella species. However, its usefulness against B. ovis would be limited if, as suggested here, epitopes critical for protective cell-mediated immunity are not shared between B. abortus and B. ovis.