Risk Assessment of Ozone Fumigation Under Vacuum to Control Potential Infestation of Coffee Berry Borer and Coffee Leaf Rust in Green Coffee Beans Imported Into Hawaii.

Studies were conducted with ozone gas fumigation under vacuum as a methyl bromide alternative against life stages of coffee berry borer (CBB) Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae: Scolytinae), and the urediniospores of coffee leaf rust (CLR), Hemileia vastatrix Berkeley & Broome (Basidiomycota: Pucciniales) in green coffee, Coffea spp. L. Fumigation with 10,000 ppm O3 gas under -25.4 mm Hg vacuum1 at 13.0 ± 3.0°C for 6.0 h killed all CBB larvae, pupae, and adults, but did not kill all CBB eggs (~15% survival). Mortality of CLR urediniospores was 100% within the first hour of the 6-h fumigation. Ozone fumigation had no adverse effects on coffee quality. Results indicated that CBB adult hitchhikers may be the only target life stage of quarantine concern, and additional studies focused on this stage. CBB adult survival and reproduction decreased significantly at moisture contents ≤20%, and F1 generation survival did not occur in green coffee at moisture contents ≤15%. As the international standard for green coffee moisture content is 9-12%, adult CBB should not survive or reproduce in exported dry green coffee. Standard industry processing of harvested coffee cherries to the green coffee stage using either mechanical- or sun-drying eliminated CBB infestations from the field. A systems approach is recommended for exporting green coffee to control CBB and CLR that includes eliminating CBB life stages with standard processing methods, reducing moisture content to 9-12% to prevent egg deposition, survival or reproduction, and O3 fumigation to ensure quarantine security against potential CBB adult hitchhikers.

Physical and antibacterial properties of edible films formulated with apple skin polyphenols.

Fruit and vegetable skins have polyphenolic compounds, terpenes, and phenols with antimicrobial and antioxidant activity. These flavoring plant essential oil components are generally regarded as safe. Edible films made from fruits or vegetables containing apple skin polyphenols have the potential to be used commercially to protect food against contamination by pathogenic bacteria. The main objective of this study was to evaluate physical properties as well as antimicrobial activities against Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella enterica of apple skin polyphenols at 0% to 10% (w/w) concentrations in apple puree film-forming solutions formulated into edible films. Commercial apple skin polyphenol powder had a water activity of 0.44 and high total soluble phenolic compounds and antioxidant capacity (995.3 mg chlorogenic acid/100 g and 14.4 mg Trolox/g, respectively). Antimicrobial activities of edible film containing apple skin polyphenols were determined by the overlay method. Apple edible film with apple skin polyphenols was highly effective against L. monocytogenes. The minimum concentration need to inactive L. monocytogenes was 1.5%. However, apple skin polyphenols did not show any antimicrobial effect against E. coli O157:H7 and S. enterica even at 10% level. The presence of apple skin polyphenols reduced water vapor permeability of films. Apple skin polyphenols increased elongation of films and darkened the color of films. The results of the present study show that apple skin polyphenols can be used to prepare apple-based antimicrobial edible films with good physical properties for food applications by direct contact.

Gelation, oxygen permeability, and mechanical properties of mammalian and fish gelatin films.

The objective of this study was to evaluate the gelation, thermal, mechanical, and oxygen permeability properties of different mammalian, warm- and cold-water fish gelatin solutions and films. Mammalian gelatin solutions had the highest gel set temperatures, followed by warm-water fish and then cold-water fish gelatin solutions. These differences were related to concentrations of imino acids present in each gelatin, with mammalian gelatin having the highest and cold-water fish gelatin having the lowest concentrations. Mammalian and warm-water fish gelatin films contained helical structures, whereas cold-water fish gelatin films were amorphous. This was due to the films being dried at room temperature (23 °C), which was below or near the gelation temperatures of mammalian and warm-water fish gelatin solutions and well above the gelation temperature of cold-water fish gelatin solutions. Tensile strength, percent elongation, and puncture deformation were highest in mammalian gelatin films, followed by warm-water fish gelatin film and then by cold-water fish gelatin films. Oxygen permeability values of cold-water fish gelatin films were significantly lower than those for mammalian gelatin films. These differences were most likely due to higher moisture sorption in mammalian gelatin films, leading to higher oxygen diffusivity.

Effects of allspice, cinnamon, and clove bud essential oils in edible apple films on physical properties and antimicrobial activities.

Essential oils (EOs) derived from plants are rich sources of volatile terpenoids and phenolic compounds. Such compounds have the potential to inactivate pathogenic bacteria on contact and in the vapor phase. Edible films made from fruits or vegetables containing EOs can be used commercially to protect food against contamination by pathogenic bacteria. EOs from cinnamon, allspice, and clove bud plants are compatible with the sensory characteristics of apple-based edible films. These films could extend product shelf life and reduce risk of pathogen growth on food surfaces. This study evaluated physical properties (water vapor permeability, color, tensile properties) and antimicrobial activities against Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes of allspice, cinnamon, and clove bud oils in apple puree film-forming solutions formulated into edible films at 0.5% to 3% (w/w) concentrations. Antimicrobial activities were determined by 2 independent methods: overlay of the film on top of the bacteria and vapor phase diffusion of the antimicrobial from the film to the bacteria. The antimicrobial activities against the 3 pathogens were in the following order: cinnamon oil > clove bud oil > allspice oil. The antimicrobial films were more effective against L. monocytogenes than against the S. enterica. The oils reduced the viscosity of the apple solutions and increased elongation and darkened the colors of the films. They did not affect water vapor permeability. The results show that apple-based films with allspice, cinnamon, or clove bud oils were active against 3 foodborne pathogens by both direct contact with the bacteria and indirectly by vapors emanating from the films.

Antibacterial effects of allspice, garlic, and oregano essential oils in tomato films determined by overlay and vapor-phase methods.

Physical properties as well as antimicrobial activities against Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes of allspice, garlic, and oregano essential oils (EOs) in tomato puree film-forming solutions (TPFFS) formulated into edible films at 0.5% to 3% (w/w) concentrations were investigated in this study. Antimicrobial activities were determined by 2 independent methods: overlay of the film on top of the bacteria and vapor-phase diffusion of the antimicrobial from the film to the bacteria. The results indicate that the antimicrobial activities against the 3 pathogens were in the following order: oregano oil > allspice oil > garlic oil. Listeria monocytogenes was less resistant to EO vapors, while E. coli O157:H7 was more resistant to EOs as determined by both overlay and vapor-phase diffusion tests. The presence of plant EO antimicrobials reduced the viscosity of TPFFS at the higher shear rates, but did not affect water vapor permeability of films. EOs increased elongation and darkened the color of films. The results of the present study show that the 3 plant-derived EOs can be used to prepare tomato-based antimicrobial edible films with good physical properties for food applications by both direct contact and indirectly by vapors emanating from the films.

Comparison of the sensitivity of the Legionella urinary antigen EIA kits from Binax and Biotest with urine from patients with infections caused by less common serogroups and subgroups of Legionella.

The detection of urinary antigen is the most widely used method to diagnose Legionnaires' disease (LD), so it is important that these assays have a high sensitivity for the disease. In this study, we compare two kits for their ability to detect urinary antigen in urine samples from patients infected with Legionella species and L. pneumophila sero- and subgroups not considered as the most common causes of LD. Urine samples (n = 33) from 30 culture-proven cases of L. pneumophila serogroup (sg) 1, subgroup non-Pontiac infection, and urine samples (n = 35) from 32 cases of non-L. pneumophila species or non-sg 1 infection were examined using the Binax EIA and Biotest EIA kits. For both groups, the overall diagnostic sensitivity of the Binax kit was significantly better than the sensitivity of the Biotest kits (P < 0.0001). For the non-Pontiac group, the sensitivity was 81.8 and 42.4%, respectively, and for the non-sg1 group, it was 51.4 and 28.6%, respectively. It was concluded that the Binax kit was more suitable for the general diagnosis of LD than the Biotest kit, but we still need urinary antigen detection methods with higher sensitivity for non-sg1 LD.

Antibacterial activity against E. coli O157:H7, physical properties, and storage stability of novel carvacrol-containing edible tomato films.

Edible films containing plant antimicrobials are gaining importance as potential treatment to extend product shelf life and reduce risk of pathogen growth on contaminated food surfaces. The main objective of the present study was to evaluate the antimicrobial activities, storage stabilities, and physical-chemical-mechanica1 properties of novel edible films made from tomatoes containing carvacrol, the main constituent of oregano oil. The antimicrobial activities against E. coli O157:H7 and the stability of carvacrol were evaluated during the preparation and storage of tomato-based films made by 2 different casting methods, continuous casting and batch casting. Antimicrobial assays of tomato films indicated that optimum antimicrobial effects occurred with carvacrol levels of approximately 0.75% added to tomato purees before film preparation. HPLC analysis of the films indicated that the carvacrol concentrations and bactericidal effect of the films remained unchanged over the storage period of up to 98 d at 5 and 25 degrees C. Carvacrol addition to the tomato puree used to prepare the films increased water vapor permeability of tomato films. The continuous method for casting of the films appears more suitable for large-scale use than the batch method. This 1st report on tomato-based edible antimicrobial tomato films suggests that these films have the potential to prevent adverse effects of contaminated food and promote human health associated with the consumption of tomatoes.

Immunization with recombinant modified vaccinia Ankara (rMVA) constructs encoding the HA or NP gene protects ponies from equine influenza virus challenge.

Two novel recombinant strains of modified vaccinia Ankara (rMVA) for the vaccination of horses against equine influenza virus were developed, and preliminary evidence of their immunogenicity in ponies was demonstrated [Breathnach CC, Rudersdorf R, Lunn DP. Use of recombinant modified vaccinia Ankara vectors for equine influenza vaccination. Vet Immunol Immunopathol 2004:98;127-36]. The present study assessed the protective efficacy of these rMVA strains in ponies, examined the advantage of combining rMVA vaccination with a DNA priming dose, and investigated the protection resulting from equine influenza nucleoprotein (NP) versus haemagglutinin (HA) vaccination. Twenty yearling ponies, seronegative for equine influenza virus, were divided into four groups of five. Group 1 and Group 2 ponies were vaccinated using a DNA prime-rMVA boost vaccination regimen, with HA- or NP-expressing vectors, respectively. Group 3 ponies were vaccinated with rMVA-HA only. Group 4 ponies served as unvaccinated controls. Vaccines were administered on days 0, 42 and 70, and all ponies were challenge infected with influenza virus on day 100. Antigen-specific antibody and cellular immune responses to each vaccination regimen were monitored throughout the experiment. Both groups of HA-vaccinated ponies were significantly protected from clinical disease following challenge infection, demonstrating the efficacy of rMVA vaccination with or without a DNA prime. NP-vaccination provided more limited protection from clinical disease. The protective post-vaccinal immune responses were characterized by antigen-specific IgGa, IgGb and IgA antibodies which were induced both in serum and in nasal secretions. Virus-specific lymphoproliferative and IFN-gamma mRNA responses were also elicited by each vaccination regimen. These data demonstrate that vaccination of horses with rMVA alone, or as part of a prime-boost regimen, is an effective means of inducing protective immunity to influenza virus infection, and also indicate that NP-specific immune responses can contribute to protection of horses.

Regional antibody and cellular immune responses to equine influenza virus infection, and particle mediated DNA vaccination.

We have previously demonstrated that hemagglutinin (HA) gene vaccination and influenza virus infection generate protective antibody responses in equids. However, these antibody responses differ substantially in that particle mediated DNA vaccination does not induce an immunoglobulin A (IgA) response. A study was performed to investigate the regional immunoregulatory mechanisms associated with these different immune responses. Ponies were either vaccinated with equine HA DNA vaccines at skin and mucosal sites, infected with influenza virus or left untreated and influenza-specific antibody responses and protection from challenge infection was studied. In a subset of ponies, lymphocytes from peripheral blood (PBLs), nasopharyngeal mucosal tissue, or lymph nodes (LNLs) were collected for measurement of influenza virus-specific lymphoproliferative responses, local antibody production and IL-2, IL-4 and IFN-gamma mRNA production by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). DNA vaccination and influenza virus infection induced humoral immunoglobulin Ga (IgGa) and immunoglobulin Gb (IgGb) production and lymphoproliferative responses that were positively correlated with IFN-gamma mRNA production. However, there were marked differences in immune response in that only influenza infection induced an IgA response, and the regional distribution of lymphoproliferation, IFN-gamma and antibody responses. Responses to DNA vaccination occurred in PBLs and in lymph nodes draining DNA vaccination sites, while influenza virus infection induced responses in PBLs and hilar LNLs. In summary, common features of immune responses to either influenza virus infection or DNA vaccination were virus-specific IgGa, IgGb and IFN-gamma responses, which are associated with protection from infection, even when the regional distribution of these immune responses varied depending on the site of immune encounter.

Mucosal co-administration of cholera toxin and influenza virus hemagglutinin-DNA in ponies generates a local IgA response.

We have previously demonstrated that equine influenza virus hemagglutinin (HA) DNA vaccination protects ponies from challenge infection, and induces protective IgGa and IgGb responses. However, this approach does not induce a nasal IgA response. The objective of this study was to examine the value of cholera toxin (CT) administration as an adjuvant for intranasal HA DNA vaccination, and to measure protection 3 months after DNA vaccination. After an immunogenic dose of CT was determined, ponies were immunized on two occasions by intranasal administration of HA DNA and cholera toxin, or HA DNA alone. Ponies in both groups received two additional HA DNA particle mediated vaccinations at skin and mucosal sites. Antibody responses, and protection from challenge infection 3 months after the last vaccination were studied and compared to an influenza virus naive control group. Ponies in both vaccination groups produced virus-specific IgG antibodies in serum following vaccination and showed clinical protection from challenge infection 3 months after the last vaccination. Co-administration of CT plus HA DNA vaccination induced a nasal IgA response. In addition, analysis of antibody titers in nasal secretions indicated local production of nasal IgGb, which was amplified by CT administration.

Immunization of pigs against influenza virus infection by DNA vaccine priming followed by killed-virus vaccine boosting.

In a previous study of particle-mediated DNA vaccination of pigs, it was found that administration of an influenza virus hemagglutinin (HA) gene elicited low levels of virus-specific antibody, but did not provide significant protection from challenge infection (as evidenced by virus shedding in nasal secretions). However, the vaccinated pigs developed high antibody titers after exposure to the challenge virus, suggesting strong priming of humoral immune responses by DNA vaccination. In the present study, pigs given a conventional, inactivated influenza virus vaccine 4 weeks after a priming dose of HA DNA developed higher levels of virus-specific serum antibodies and were protected from challenge virus infection to a significantly greater degree than pigs that received two doses of DNA vaccine.

Isolation and characterization of H4N6 avian influenza viruses from pigs with pneumonia in Canada.

In October 1999, H4N6 influenza A viruses were isolated from pigs with pneumonia on a commercial swine farm in Canada. Phylogenetic analyses of the sequences of all eight viral RNA segments demonstrated that these are wholly avian influenza viruses of the North American lineage. To our knowledge, this is the first report of interspecies transmission of an avian H4 influenza virus to domestic pigs under natural conditions.

Genetic characterization of H3N2 influenza viruses isolated from pigs in North America, 1977-1999: evidence for wholly human and reassortant virus genotypes.

Since 1998, H3N2 viruses have caused epizootics of respiratory disease in pigs throughout the major swine production regions of the U.S. These outbreaks are remarkable because swine influenza in North America had previously been caused almost exclusively by H1N1 viruses. We sequenced the full-length protein coding regions of all eight RNA segments from four H3N2 viruses that we isolated from pigs in the Midwestern U.S. between March 1998 and March 1999, as well as from H3N2 viruses recovered from a piglet in Canada in January 1997 and from a pig in Colorado in 1977. Phylogenetic analyses demonstrated that the 1977 Colorado and 1997 Ontario isolates are wholly human influenza viruses. However, the viruses isolated since 1998 from pigs in the Midwestern U.S. are reassortant viruses containing hemagglutinin, neuraminidase and PB1 polymerase genes from human influenza viruses, matrix, non-structural and nucleoprotein genes from classical swine viruses, and PA and PB2 polymerase genes from avian viruses. The HA proteins of the Midwestern reassortant swine viruses can be differentiated from those of the 1995 lineage of human H3 viruses by 12 amino acid mutations in HA1. In contrast, the Sw/ONT/97 virus, which did not spread from pig-to-pig, lacks 11 of these changes.

Virologic and serologic surveillance for human, swine and avian influenza virus infections among pigs in the north-central United States.

Influenza virus infection in pigs is both an animal health problem and a public health concern. As such, surveillance and characterization of influenza viruses in swine is important to the veterinary community and should be a part of human pandemic preparedness planning. Studies in 1976/1977 and 1988/1989 demonstrated that pigs in the U.S. were commonly infected with classical swine H1N1 viruses, whereas human H3 and avian influenza virus infections were very rare. In contrast, human H3 and avian H1 viruses have been isolated frequently from pigs in Europe and Asia over the last two decades. From September 1997 through August 1998, we isolated 26 influenza viruses from pigs in the north central United States at the point of slaughter. All 26 isolates were H1N1 viruses, and phylogenetic analyses of the hemagglutinin and nucleoprotein genes from 11 representative viruses demonstrated that these were classical swine H1 viruses. However, monoclonal antibody analyses revealed antigenic heterogeneity among the HA proteins of the 26 viruses. Serologically, 27.7% of 2,375 pigs tested had hemagglutination-inhibiting antibodies against classical swine H1 influenza virus. Of particular significance, however, the rates of seropositivity to avian H1 (7.6%) and human H3 (8.0%) viruses were substantially higher than in previous studies.

Genetic characterization of an H1N2 influenza virus isolated from a pig in Indiana.

An H1N2 influenza virus was isolated from a pig during an outbreak of respiratory disease and abortion on an Indiana farm in November 1999. Results of phylogenetic analyses indicate that this virus is a reassortant between a recent classical H1 swine virus and the reassortant H3N2 viruses that have emerged among American pigs since 1998.

Systemic and mucosal immune responses to H1N1 influenza virus infection in pigs.

Influenza is a common respiratory disease in pigs, and since swine influenza viruses are zoonotic pathogens, they also pose human health risks. Pigs infected with influenza virus mount an effective immune response and are protected from subsequent challenge, whereas the currently available, inactivated-virus vaccine does not consistently confer complete protection to challenge. To develop and evaluate new vaccination strategies, it is imperative to fully understand the immune responses that are associated with protection following natural infection. Therefore, we have evaluated the phenotype and kinetics of immune responses to primary and re-challenge infection with H1N1 swine influenza virus in the pig. Through the use of isotype-specific antibody secreting cell ELISPOT assays, interferon-gamma ELISPOT assays and isotype-specific ELISAs on serum, nasal wash and bronchoalveolar lavage samples, we defined the humoral and cellular immune responses, both locally in the respiratory tract and systemically, to this viral infection. Virus-specific serum IgG, IgA, and HI titers all peaked 2-3 weeks after primary infection and did not substantially increase after re-challenge. The predominant virus-specific isotype in serum was IgG. Pigs responded with virus-specific IgG and IgA in both the upper (nasal washes) and lower (bronchoalveolar lavages) airways; IgA was the predominant isotype in both sites. Despite the fact that the pigs were completely protected from re-challenge, the antibody titers in the nasal washes increased. Results of the antibody-secreting cell ELISPOT assays demonstrated that the numbers of both IgG and IgA secreting cells in the nasal mucosa were dramatically higher than in any other tissue examined. In contrast, IFN-gamma secreting cells were predominantly localized to the spleen and tracheobronchial lymph nodes. These data will be helpful in the future development and evaluation of novel vaccines.

DNA vaccination against influenza viruses: a review with emphasis on equine and swine influenza.

The influenza virus vaccines that are commercially-available for humans, horses and pigs in the United States are inactivated, whole-virus or subunit vaccines. While these vaccines may decrease the incidence and severity of clinical disease, they do not consistently provide complete protection from virus infection. DNA vaccines are a novel alternative to conventional vaccination strategies, and offer many of the potential benefits of live virus vaccines without their risks. In particular, because immunogens are synthesized de novo within DNA transfected cells, antigen can be presented by MHC class I and II molecules, resulting in stimulation of both humoral and cellular immune responses. Influenza virus has been used extensively as a model pathogen in DNA vaccine studies in mice, chickens, ferrets, pigs, horses and non-human primates, and clinical trials of DNA-based influenza virus vaccines are underway in humans. Our studies have focused on gene gun delivery of DNA vaccines against equine and swine influenza viruses in mice, ponies and pigs, including studies employing co-administration of interleukin-6 DNA as an approach for modulating and adjuvanting influenza virus hemagglutinin-specific immune responses. The results indicate that gene gun administration of plasmids encoding hemagglutinin genes from influenza viruses is an effective method for priming and/or inducing virus-specific immune responses, and for providing partial to complete protection from challenge infection in mice, horses and pigs. In addition, studies of interleukin-6 DNA co-administration in mice clearly demonstrate the potential for this approach to enhance vaccine efficacy and protection.

Antibody responses to DNA vaccination of horses using the influenza virus hemagglutinin gene.

Equine influenza virus infection remains one of the most important infectious diseases of the horse, yet current vaccines offer only limited protection. The equine immune response to natural influenza virus infection results in long-term protective immunity, and is characterized by mucosal IgA and serum IgGa and IgGb antibody responses. DNA vaccination offers a radical alternative to conventional vaccines, with the potential to generate the same protective immune responses seen following viral infection. Antigen-specific antibody isotype responses in serum and mucosal secretions were studied in ponies following particle-mediated delivery of hemagglutinin (HA)-DNA vaccination on three occasions at approximately 63-day intervals. One group of four ponies were vaccinated at skin and mucosal sites and the another group were vaccinated at skin sites only. All ponies were subjected to a challenge infection 30 days after the third vaccination. Skin and mucosal vaccination provided complete protection from clinical signs of infection, while skin vaccination provided partial protection; DNA vaccination provided partial protection from viral shedding. DNA vaccination generated only IgGa and IgGb antibody responses, which occurred with a higher frequency in the skin and mucosa vaccinated ponies. No mucosal IgA response was generated prior to challenge infection and IgA responses were only detected in those ponies which shed virus postchallenge. These results demonstrate that HA-DNA vaccination induces IgG(a) and IgG(b) antibody responses which are associated with protection in the absence of mucosal IgA responses. In addition, additional DNA vaccinations of mucosal sites increased protection and the frequency of seroconversion in ponies.