Treatment of fresh poultry carcases with emulsions of glycerol monocaprate (monocaprin) to reduce contamination with Campylobacter and psychrotrophic bacteria.

1. A previous study has shown that emulsions of monocaprin in citrate lactate buffer at pH 4·1-4·3 are highly active in killing Campylobacter in water, where they reduce viable bacterial counts by more than 6 log(10) colony forming units (cfu) in 1 min at a concentration of 1·25 mM (0·03%). 2. The present study was carried out to evaluate whether monocaprin emulsions could be used to kill Campylobacter on raw poultry. 3. It was shown that immersion of naturally contaminated chicken legs in 20 mM (0·5%) monocaprin emulsion at pH 4·1 for 1 min at 20°C reduced the number of Campylobacter by 2·0 to 2·7 log(10) cfu. Pre-chill dipping of whole carcases into 20 mM monocaprin emulsion in the slaughterhouse also caused a significant reduction in Campylobacter contamination. 4. Immersion in monocaprin emulsions at pH 4·1 was also assessed as a means to reduce the number of psychrotrophic spoilage bacteria. There were lower psychrotrophic bacteria counts on treated chicken parts than on untreated controls after storage at 3°C for up to 14 d. 5. Immersion in emulsions of monocaprin, which is a natural lipid classified as GRAS, may be a feasible method to reduce the number of Campylobacter and spoilage bacteria on raw poultry. This method could reduce the risk of human exposure to Campylobacter, and at the same time increase the shelf-life of poultry products.

Comparison of wild-type and subacute sclerosing panencephalitis strains of measles virus. Neurovirulence in ferrets and biological properties in cell cultures.

The neurovirulence of two wild type (wt) and seven Subacute Sclerosing Panencephalitis (SSPE) measles virus strains was tested in young adult ferrets by intracerebral (IC) inoculation of infected Vero cell suspensions. Wt strains Edmonston and Woodfolk and SSPE strains Mantooth, Halle, and LEC-S did not produce a detectable encephalitis in the ferrets, but caused a significant formation of serum antibodies against measles virus. SSPE strains LEC, IP-3, Biken, and D.R., on the other hand, were all neurovirulent in ferrets, particularly strain D.R. which caused an acute encephalitis in all inoculated animals. Strain Biken was of particular interest since it caused a subacute encephalitis in four of seven ferrets. The subacute encephalitis was characterized by a long incubation time, persistence of virus in the brain for at least 8 mo, widespread inflammatory lesions, and production of measles virus specific IgG in the brain. A study of the biological properties of the various measles virus strains showed that wt strains Edmonston and Woodfolk and SSPE strains Mantooth, Halle, and LEC-S produced free virus particles in significant titers both in Vero and ferret brain (FB) cultures. Cytopathic effect (CPE) with cell-fusion was marked in Vero cultures, whereas only minimal CPE and no cell-fusion were observed in the FB cultures. SSPE strains LEC, IP-3, Biken, and D.R., on the other hand, were mostly cell-associated in Vero and FB cultures, although atypical cell-free particles were produced by strains Biken and IP-3. All four strains showed cell-fusing activity in FB cultures, particularly strain D.R., which was the only strain that spread more actively by fusion in FB than in Vero cultures. The results are discussed in relation to the neurovirulence of the various measles virus strains in adult ferrets. Pronounced cell-fusing activity in FB cells and cell-association with minimal or no production of cell-free virus seem to be essential to establish a brain infection in the animals.

Killing of Campylobacter on contaminated plastic and wooden cutting boards by glycerol monocaprate (monocaprin).

AIMS: Contamination in the kitchen with foodborne bacteria is a risk factor in human exposure to these pathogens, an important route being transfer of bacteria from contaminated cutting boards and other surfaces to humans. The aim of this study was to test microbicidal emulsions of glycerol monocaprate (monocaprin) against Campylobacter on contaminated cutting boards. METHODS AND RESULTS: Plastic and wooden cutting boards, soiled with meat juice heavily contaminated with Campylobacter, were treated for 2 min with emulsions of monocaprin (MC) made in water or in buffer at low pH. Viable Campylobacter counts were reduced below the detectable level on plastic board surfaces after treatment with MC emulsions with or without 1.25% washing-up liquids (WUL). The counts were also greatly reduced on wooden boards (P < 0.05). CONCLUSIONS: Monocaprin emulsions and mixtures of MC emulsions and WUL may be useful as sanitizers/disinfectants in kitchens and in other food preparing and processing facilities. SIGNIFICANCE AND IMPACT OF THE STUDY: Cleaning with MC emulsions with or without WUL may reduce the risk of human exposure to Campylobacter.

Effect of glycerol monocaprate (monocaprin) on broiler chickens: an attempt at reducing intestinal Campylobacter infection.

Attempts have been made by several workers to prevent or to reduce colonization of Campylobacter in the intestines of broiler chickens by adding antibacterial agents to their food, but the results have varied. Monocaprin, the 1-monoglyceride of capric acid, has been found to be very active in killing Campylobacter in vitro. It was therefore studied whether Campylobacter infection in chickens would be affected by adding emulsions of monocaprin to their drinking water and feed. It was found that treatment with monocaprin in water and feed did not prevent spread of Campylobacter from artificially infected to noninfected 24-d-old chickens, but Campylobacter counts in cloacal swabs were significantly reduced, particularly during the first 2 d of treatment. There was also a significant reduction in the Campylobacter counts in cloacal swabs of naturally infected 36-d-old broilers that were treated for 3 d prior to slaughter. Addition of monocaprin to drinking water and feed 2 to 3 d before slaughter might therefore be considered as a means of reducing Campylobacter infection in broilers, possibly in conjunction with other antibacterial agents such as short-chain organic acids. Further studies are needed to determine whether this would reduce carcass contamination.

Development of a virucidal cream containing the monoglyceride monocaprin.

The lipid monocaprin (1-monoglyceride of capric acid) has been shown to be effective against enveloped viruses such as herpes simplex virus HSV in vitro. As it is known that HSV can develop resistance to acyclovir which is the most common treatment used, it was considered to be of interest to formulate a cream containing the lipid monocaprin as the active substance against HSV. The aim of this study was to develop an o/w-emulsion (cream) containing monocaprin and to evaluate the effects of formulation variables on the virucidal activity of monocaprin as well as the in vitro release rate of the monoglyceride from the formulations. The results show that release rate and extent of monocaprin release as well as the microbicidal properties of the the o/w-emulsion formulations are affected by the proportion of the oil phase and the amount of carbomer in the aqueous phase. Reducing the oil volume fraction increased antiviral effect and release of monocaprin from the formulation.

Ultrastructural and histological studies of brains of ferrets inoculated with subacute sclerosing panencephalitis: similarities to human disease.

Similarities to human subacute sclerosing panencephalitis (SSPE) were revealed in a study of the brains of ferrets inoculated with a cell associated measles virus originally isolated from an SSPE patient. The similarities were greatest in animals that showed neurological signs 3-4 months after inoculation and had high titers of neutralizing antibodies against measles virus. These included dense core particles, nuclear bodies, alterations of basement membranes of small blood vessels, plasma cells, distorted myelin, and rod-like structures in some nuclei. Other abnormalities seen were Hirano bodies, tubular aggregates in cisternae of endoplasmic reticulum and clusters of cytoplasmic tubules. No cells containing viral inclusion bodies were observed by electron microscopy but cell cultures of the brains of these animals always yielded abundant measles virus nucleocapsids in typical SSPE multinucleated syncytia. These findings suggest that the ferret is a suitable animal model for the study of the pathogenesis of SSPE.

Quantitation of measles-specific IgG. Its presence in CSF and brain extracts of patients with multiple sclerosis.

Twenty of 41 patients with multiple sclerosis (MS) and five of 30 patients with other neurological diseases had measles antibody titers in their CSF when examined in hemagglutination inhibition and neutralization tests. Quantitation of measles-specific IgG in CSF of patients with MS with significant measles antibody titers showed that less than 5% of the total IgG was measles virus specific. In contrast, using the identical quantitation procedures, 30% to 60% of the total IgG in CSF of patients with subacute sclerosing panencephalitis was found to be measles specific. Thus, only a very minor portion of the total IgG in CSF of patients with MS is synthesized against measles virus. When measles hemagglutination inhibition, complement fixation, and neutralizing antibody titers were measured in individual oligoclonal IgG bands isolated from neutral pH brain extracts of two patients with MS, low titers were present in all the oligoclonal bands rather than being restricted to any single band.

Serological identification of viral antigens after electrophoretic transfer.

The method described permits detection of specific antibodies to antigenic material, the protein components of which can first be separated by electrophoresis and then transferred to a supporting medium where their reactivity with antibody may be demonstrated using peroxidase-labelled anti-Ig. Results are described for measles virus and measles-like virus isolated from subacute sclerosing panencephalitis (SSPE) brain. Dilutions, incubation time and conditions, detection with peroxidase-labelled immunoglobulins and selection of substrate are described and discussed.

Measles virus-specific immunoglobulin D antibody in cerebrospinal fluid and serum from patients with subacute sclerosing panencephalitis and multiple sclerosis.

Quantitation of measles-specific immunoglobulin D (IgD) antibody was carried out in cerebrospinal fluid (CSF) and serum samples from 18 patients with subacute sclerosing panencephalitis (SSPE), 12 patients with multiple sclerosis (MS) and seven normal controls with high measles antibody titers in serum, using polyclonal and monoclonal antibodies specific for human IgD and enzyme-linked immunosorbent assay. Measles-specific IgD activity was significantly higher in CSF and serum from SSPE patients compared to that found in patients with MS or normal controls. The IgD antibody to measles virus was not due to high levels of measles-specific IgG since significant measles IgD activity was found after eluting IgG from SSPE serum. The increased level of measles-specific IgD found in SSPE sera is consistent with the levels observed in patients with acute and chronic viral infections.

Light-chain profiles in sera from patients with subacute sclerosing panencephalitis.

Sera from 2 subacute sclerosing panencephalitis (SSPE) patients were absorbed with a concentrated preparation of measles virus. Measles-specific IgG was eluted from the precipitates containing measles antigen-antibody complex. These IgGs, when subjected to immunofixation after isoelectric focusing showed a number of oligoclonal bands with one type of light-(L) chain. In urea polyacrylamide gel electrophoresis, the reduced and alkylated measles-specific IgG showed 1-3 homogeneous L-chain bands, whereas IgG isolated from unabsorbed sera and IgG isolated from supernatants of SSPE sera after absorption with measles virus showed a diffuse L-chain band. It can be concluded that in SSPE, measles virus is responsible for the synthesis of L-chain with restricted heterogeneity.

Killing of Gram-positive cocci by fatty acids and monoglycerides.

The susceptibilities of three Gram-positive cocci to medium-chain saturated and long-chain unsaturated fatty acids and their one-monoglycerides were studied. The bacteria were incubated with equal volumes of lipid solutions for 10 min. Lauric acid, palmitoleic acid and monocaprin reduced the number of CFU by 6.0 log10 or greater at 5 mM concentration for streptococci of group A (GAS) and group B (GBS). When further compared at lower concentrations and after longer incubation time monocaprin proved to be the most active. Capric acid showed the highest activity against Staphylococcus aureus at 10 mM. However, at lower concentrations monocaprin was the only lipid that showed significant activity against S. aureus. The mode of action of monocaprin against GBS was studied by a novel two-color fluorescent assay of bacterial viability and by electron microscopy. The results indicate that the bacteria are killed by disintegration of the cell membrane by the lipid, leaving the bacterial cell wall intact. The highly lethal effect of monocaprin indicates that this lipid might be useful as a microbicidal agent for prevention and treatment of infections caused by these bacteria.

Transmission of measles virus encephalitis to ferrets by intracardiac inoculation of a cell-associated SSPE virus strain.

Ferret fibroblasts infected with a cell-associated strain of subacute sclerosing panencephalitis virus were inoculated into the hearts of ferrets in order to study whether the virus can spread from the blood to the brain in this animal model. Five of 21 inoculated ferrets developed encephalitis 5-7 days later and were sacrificed. Sick animals showed inflammatory lesions in the brain, both perivascular cuffings and infiltration of inflammatory cells in the choroid plexus and meninges. Virus was isolated in cell cultures from various parts of the brain and virus antigen was found by immunostaining, particularly in the cortex. Virus was not detected in inflammatory cells by immunostaining but in situ hybridization with a cDNA probe demonstrated measles virus RNA in neurons and glia cells surrounding perivascular inflammatory cuffings and in a lymph node of one ferret. Ferrets inoculated into the heart with cell-associated SSPE virus seem to be a suitable animal model to study how the virus spreads from the blood to the brain.

Inhibition of visna virus replication and cytopathic effect in sheep choroid plexus cell cultures by selected anti-HIV agents.

Several anti-HIV agents were tested against visna virus replication and cytopathic effect (CPE) in sheep choroid plexus cell cultures. Sulphated polysaccharides (i.e., dextran sulphate, pentosan polysulphate and heparin) and plant lectins, which inhibit viral adsorption and fusion, were found to be 10- to 40-fold less active against visna virus than against HIV. Bicyclam derivatives were at least 250-fold less active against visna virus and the highly HIV-1 specific TIBO derivatives were without a significant inhibitory effect on visna virus at subtoxic concentrations. In contrast, several 2',3'-dideoxynucleosides and acyclic nucleoside phosphonate analogues, which inhibit reverse transcription, were found to be very effective inhibitors of visna virus replication and viral CPE in cell culture.

Inactivation of enveloped viruses in human bodily fluids by purified lipids.

Antimicrobial lipids are found in mucosal secretions and are one of a number of nonimmunologic and nonspecific protective factors found at mucosal surfaces. Lipids can inactivate enveloped viruses, bacteria, fungi, and protozoa. Lipid-dependent antimicrobial activity at mucosal surfaces is due to certain monoglycerides and fatty acids that are released from triglycerides by lipolytic activity. Medium chain length antiviral lipids can be added to human blood products that contain HIV-1 and HIV-2 and reduce the cell-free virus concentration by as much as 11 log10 TCID50/ml. The presence of lipids does not interfere with most clinical assays performed on human blood samples. Antimicrobial lipids can disrupt cell membranes and therefore lyse leukocytes which potentially carry virus. Genital mucosal epithelial cells should be protected from damage by the mucous layer. Preliminary studies indicate that lipids decrease sperm motility and viability suggesting that lipids may potentially be used as combination spermicidal and virucidal agents.

Inactivation of visna virus and other enveloped viruses by free fatty acids and monoglycerides.

Human milk was found to become antiviral during storage at 4 degrees C because of the release of fatty acids by lipolysis. The stored milk caused more than a 10,000-fold inactivation of visna virus during incubation at 37 degrees C for 30 minutes. Medium-chain saturated and long-chain unsaturated fatty acids inactivated visna virus and other enveloped viruses causing more than a 3000-fold to 10,000-fold reduction in virus titer. 1-Monoglycerides and ethers of medium-chain fatty acids were more antiviral than the corresponding free fatty acids. Antiviral fatty acids were found to affect the viral envelope, causing leakage and, at higher concentrations, a complete disintegration of the envelope and the viral particles. Lipids commonly found in natural products could possibly be used as antiviral agents against enveloped viruses.

Subacute sclerosing panencephalitis antibodies against measles virus polypeptides.

Antibodies to measles virus proteins in SSPE, MS and control human sera were compared using measles virus strain Edmonston, productive SSPE strain Mantooth and non-productive SSPE strain DR. The viral antigens were subjected to transfer electrophoresis, incubated with sera and localized after a second incubation with peroxidase labeled immunoglobulins. High levels of antibodies to all measles viral proteins are present in SSPE sera and CSF and to a lesser degree in MS and control sera although the non-productive SSPE strains lack one viral protein (M protein) present in wild type measles virus strains and the productive SSPE strains.

Treatment of visna virus infection in lambs with the acyclic nucleoside phosphonate analogue 9-(2-phosphonylmethoxyethyl)adenine (PMEA).

Nucleoside and nucleotide analogues, which are inhibitors of human immunodeficiency virus reverse transcriptase, are highly active inhibitors of visna virus replication in cell cultures. One such analogue, the acyclic nucleoside phosphonate PMEA, has also been found to have a prophylactic effect on visna virus infection in lambs. In the present study, lambs were injected subcutaneously with 10 mg/kg PMEA three times a week starting 4 weeks after inoculation with visna virus, when brain infection had been established. After 3 weeks of treatment there was a reduction in the amount of virus isolated from blood cells of PMEA-treated lambs compared to controls and during the remaining 7 months of drug treatment there was significantly less virus isolated from the blood cells of treated lambs than of controls. Antibody response against visna virus was also slower in the treated than in the untreated control group. On the other hand, there was no difference in the amount of virus isolated from various organs of the two groups and the severity of CNS lesions in sheep treated with PMEA for 8 months was comparable to that found in untreated controls, even though PMEA reached concentrations in the cerebrospinal fluid which were well in excess of the EC50 value of the drug for visna virus.

Measles antibodies and immunoglobulins in sera from patients with subacute sclerosing panencephalitis (SSPE) and from an infected ferret.

Cellulose acetate electrophoresis of sera from 2 patients with SSPE revealed a slow-moving and a fast-moving gamma-globulin band. The IgG fractions from sera were isolated by the combination of starch block electrophoresis, Sephadex gel filtration and isoelectric focusing techniques. The purity of each protein was tested by immunoelectrophoresis using a potent goat anti-whole human serum. The isolated IgG preparations were tested in HI, CF and N tests against measles virus envelope, measles virus nucleocapsid and whole measles virus, respectively. The results showed that isolated IgG fractions, having different electrophoretic mobilities, contained high titers of HI, CF and N antibodies, and these titers were proportional to the protein concentration of IgG. Although both IgG fractions appeared to show an increase of K-type chain, neither fraction was devoid of L-type chain. These results indicate that the homogeneous IgG bands observed in the SSPE serum were not monoclonal as they failed to show selective increase of one type of light chain and uneven distribution of measles antibody activity. These findings were compared with the results from a ferret, which developed encephalitis 2 months after intracerebral inoculation with cell-associated measles virus derived from the brain of an SSPE patient. The electrophoretic pattern of the ferret serum was similar to that of the human. High titers of measles HI and N antibodies were seen in the isolated slow- and fast-moving IgG's. The similarities observed in the human and ferret IgG's, with respect to homogeneous bands in electrophoresis and measles HI and H antibody activities, suggest that the ferret may be a useful animal model for studying the immune mechanism of SSPE.

Bound antibody in multiple sclerosis brains.

Autopsy tissues of white and grey matter from brains of MS patients and controls were homogenized in PBS and centrifuged at 100,000 x g for 1 h. The residues were thoroughly washed with PBS and further extracted at acidic and alkaline pH. The eluates contained a low but significant amount of IgG, indicating that bound IgG was present in MS brains. A greater yield of IgG was obtained with alkaline pH buffer than with acidic pH buffer. In contrast, sections from 2 of 3 neurologic controls and 3 of 4 non-neurologic control failed to show detectable level of bound IgG extractable at acidic pH. These results might suggest the presence of antigen-antibody complexes in MS brains. Isoelectric focusing analysis demonstrated oligoclonal IgG bands in low pH eluates from MS brains and these bands were partly similar to those seen in neutral (unbound) pH brain extracts.