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.

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.

Virucidal activities of medium- and long-chain fatty alcohols and lipids against respiratory syncytial virus and parainfluenza virus type 2: comparison at different pH levels.

Recent studies have shown that some lipids and fatty alcohols have microbicidal activities against a broad variety of pathogens. In this study, virucidal activities of fatty acids, monoglycerides and fatty alcohols were tested against respiratory syncytial virus (RSV) and human parainfluenza virus type 2 (HPIV2) at different concentrations, times and pH levels. The most active compounds were mixed with milk products and fruit juices and the mixtures tested for virucidal effects. The aim was to determine which compounds are the most active against these respiratory viruses and could possibly be used in pharmaceutical formulations or as additives to milk products or juice. Several compounds caused a significant inactivation of virus, and there was generally a good agreement between the activities against RSV and parainfluenza virus. By changing the pH from 7 to 4.2, the virucidal activities of some of the compounds were greatly increased, i.e., they inactivated virus in a shorter time and at lower concentrations. The most active compound tested was 1-monoglyceride of capric acid, monocaprin, which also showed activity against influenza A virus and significant virucidal activities after addition to milk products and fruit juices, even at a concentration as low as 0.06-0.12%. The significant virucidal activities of fatty alcohols and lipids on RSV and parainfluenza virus demonstrated in this in vitro study raise the question of the feasibility of using such compounds as ingredients in pharmaceutical dosage forms against respiratory infections caused by these viruses, and possibly other paramyxo- and myxoviruses.

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.

Virucidal effect of lipids on visna virus, a lentivirus related to HIV.

Natural lipids and fatty alcohols show virucidal activities against enveloped viruses. A virucidal profile of these compounds against visna virus (VV), a lentivirus related to HIV, or against other viruses of the genus Lentivirus has not been established before and could help elucidate how lipids inactivate enveloped viruses and assist in the development of virucidal drugs. The activity profile for VV may not exactly reflect the profile for HIV or for the lentivirus subgroup in general, but the results for VV are in agreement with earlier studies, which have shown that lipids become generally more virucidal at low pH.

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.

Effect of buffers on the properties of microbicidal hydrogels containing monoglyceride as the active ingredient.

Hydrogel formulations containing the monoglyceride monocaprin have shown potent microbicidal activity against several sexually transmitted viruses and bacteria. It is recommended that formulations for preventing infection in the vagina have a low pH as the HIV virus is inactivated at low pH. The object of the work was to investigate how incorporation of buffers into the hydrogel formulations affects physicochemical properties and microbicidal activity of the active substance. Two series of gels were formulated using carbomer (Carbopol 934) and sodium carboxymethylcellulose (NaCMC) as gel-forming agents. The presence of buffers in the gels caused a lowering in gel viscosity, with carbomer gels being more sensitive to buffer presence than NaCMC gels. To obtain viscosity similar to that of a gel without buffer, the amount of polymer needs to be increased. An increase in the amount of NaCMC by 60-70% is needed to obtain the same viscosity as in gel without buffers; but for carbomer, the amount of polymer needs to be doubled. It appears that the effect of maleate buffer on NaCMC gel formation is greater than that of the citrate/lactate buffer; but for carbopol gels, the effects of the buffer systems tested on gel viscosity were equal. The virucidal activity of NaCMC gel buffered with citrate/lactate buffer against herpes simplex virus type 1 and HIV was not reduced by the presence of buffer. The results show that the presence of buffers in the hydrogel formulations affects gel viscosity, but the virucidal effect of the active compound, monocaprin, is not diminished.

In vitro killing of Candida albicans by fatty acids and monoglycerides.

The susceptibility of Candida albicans to several fatty acids and their 1-monoglycerides was tested with a short inactivation time, and ultrathin sections were studied by transmission electron microscopy (TEM) after treatment with capric acid. The results show that capric acid, a 10-carbon saturated fatty acid, causes the fastest and most effective killing of all three strains of C. albicans tested, leaving the cytoplasm disorganized and shrunken because of a disrupted or disintegrated plasma membrane. Lauric acid, a 12-carbon saturated fatty acid, was the most active at lower concentrations and after a longer incubation time.

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.

Hydrogels containing monocaprin prevent intravaginal and intracutaneous infections with HSV-2 in mice: impact on the search for vaginal microbicides.

Hydrogel formulations containing the 1-monoglyceride of capric acid (monocaprin) possess potent in vitro microbicidal activity against HIV and HSV, Chlamydia trachomatis and Neisseria gonorrhoeae. These formulations were studied to determine whether they prevent intracutaneous and intravaginal infections of mice with HSV-2, a virus that is in vitro as sensitive to the virucidal action of the compound as is HIV. In mice intravaginal infection with HSV-2 and the associated mortality was prevented completely when the infection was carried out in the presence of a 20 mM monocaprin containing gel formulation. Similarly, virtually complete protection of lesion development and associated mortality was observed when mice were infected intracutaneously with HSV-2 in the presence of gels containing 10 or 20 mM monocaprin. No irritation or toxicity was observed following application of the gel to the skin or the vaginal mucosa. Hydrogel formulations of monocaprin could thus be pursued as vaginal microbicides for the prevention of sexual transmission of HSV, HIV and other infectious pathogens.

Development and evaluation of microbicidal hydrogels containing monoglyceride as the active ingredient.

A number of medium-chain saturated and long-chain unsaturated fatty acids and their monoglycerides were tested against herpes simplex virus (HSV-1) to determine which lipids were most active during a short incubation time. The aim was to find which lipid would be preferable as the active ingredient in a virucidal hydrogel formulation for the purpose of preventing transmission of pathogens to mucosal membranes, particularly sexually transmitted viruses, such as herpes simplex virus and human immunodeficiency virus (HIV), and bacteria, such as Chlamydia trachomatis and Neisseria gonorrheae. The main strategy was that the formulations would be fast-acting, killing large numbers of virus or bacteria on contact in a short time, preferably causing at least a 10000-fold reduction in virus/bacteria titer in 1-5 min. Monocaprin, the 1-monoglyceride of capric acid, and lauric acid were found to be most active of all the lipids tested, causing a greater than 100000-fold reduction in virus titer in 1 min at a concentration of 20 mM. When tested at a concentration of 10 mM for 1 min, monocaprin was still fully active whereas lauric acid had no or negligible activity. It was concluded that monocaprin was most suitable as the active ingredient in a fast-acting virucidal gel formulation, and several hydrogel formulations containing monocaprin were tested. Formulations where the monoglyceride was dissolved in glycofurol were found to be active against HSV-1. The hydrogel formulations containing 20 mM monocaprin were highly virucidal in vitro and caused a greater than 100000-fold (HSV-1) inactivation of virus in human semen in 1 min. Formulations in dilution 1:10 were cytotoxic in monolayers of CV-1 cells, but they were 10-100 fold less cytotoxic than a commercial product which contains 2% nonoxynol-9.

In vitro susceptibilities of Neisseria gonorrhoeae to fatty acids and monoglycerides.

The susceptibility of Neisseria gonorrhoeae to several medium-chain fatty acids and their 1-monoglycerides was tested at a short inactivation time of 1 min. The results indicate that monocaprin, a monoglyceride of capric acid (10 carbon atoms, no double bonds), causes the fastest and most effective killing of all strains of N. gonorrhoeae tested.

Naturally occurring mutations within 39 amino acids in the envelope glycoprotein of maedi-visna virus alter the neutralization phenotype.

Infectious molecular clones have been isolated from two maedi-visna virus (MVV) strains, one of which (KV1772kv72/67) is an antigenic escape mutant of the other (LV1-1KS1). To map the type-specific neutralization epitope, we constructed viruses containing chimeric envelope genes by using KV1772kv72/67 as a backbone and replacing various parts of the envelope gene with equivalent sequences from LV1-1KS1. The neutralization phenotype was found to map to a region in the envelope gene containing two deletions and four amino acid changes within 39 amino acids (positions 559 to 597 of Env). Serum obtained from a lamb infected with a chimeric virus, VR1, containing only the 39 amino acids from LV1-1KS1 in the KV1772kv72/67 backbone neutralized LV1-1KS1 but not KV1772kv72/67. The region in the envelope gene that we had thus shown to be involved in escape from neutralization was cloned into pGEX-3X expression vectors, and the resulting fusion peptides from both molecular clones were tested in immunoblots for reactivity with the KV1772kv72/67 and VR1 type-specific antisera. The type-specific KV1772kv72/67 antiserum reacted only with the fusion peptide from KV1772kv72/67 and not with that from LV1-1KS1, and the type-specific VR1 antiserum reacted only with the fusion peptide from LV1-1KS1 and not with that from KV1772kv72/67. Pepscan analysis showed that the region contained two linear epitopes, one of which was specific to each of the molecularly cloned viruses. This linear epitope was not bound by all type-specific neutralizing antisera, however, which indicates that it is not by itself the neutralization epitope but may be a part of it. These findings show that mutations within amino acids 559 to 597 in the envelope gene of MVV virus result in escape from neutralization. Furthermore, the region contains one or more parts of a discontinuous neutralization epitope.

Hydrogels containing monocaprin have potent microbicidal activities against sexually transmitted viruses and bacteria in vitro.

OBJECTIVE: To investigate the in vitro microbicidal and cytocidal potency of monocaprin dissolved in pharmaceutical hydrogel formulations and to evaluate their potential use as vaginal microbicides against sexually transmitted pathogens such as herpes simplex virus type 2 (HSV-2), human immunodeficiency virus type 1 (HIV-1), Chlamydia trachomatis, and Neisseria gonorrhoeae. METHODS: Gel formulations were mixed with equal volumes of virus/bacteria suspensions in culture medium and incubated for 1 and 5 minutes. The reduction in virus/bacteria titre was used as a measure of microbicidal activity. Similarly, gels were mixed with human semen to study their effect on leucocytes. The toxicity of the gels was tested in rabbits by the standard vaginal irritation test. RESULTS: Gels containing 20 mM of monocaprin caused a greater than 100,000-fold inactivation of HSV-2 and Neisseria in 1 minute and of Chlamydia in 5 minutes. Similarly, the gels caused a greater than 10,000-fold inactivation of HIV-1 in semen in 1 minute. They caused more than a 10,000-fold reduction in the number of viable leucocytes in semen in 1 minute. No toxic effect on the vaginal mucosa of rabbits was observed after daily exposure for 10 days. CONCLUSIONS: Hydrogels containing monocaprin are potent inactivators of sexually transmitted viruses and bacteria in vitro. This simple lipid seems to be a feasible choice as a mucosal microbicide for prevention of sexually transmitted infections. It is a natural compound found in certain foodstuffs such as milk and is therefore unlikely to cause harmful side effects in the concentrations used.

In vitro inactivation of Chlamydia trachomatis by fatty acids and monoglycerides.

The antichlamydial effects of several fatty acids and monoglycerides were studied by incubating Chlamydia trachomatis bacteria with equal volumes of lipid solutions for 10 min and measuring the reduction in infectivity titer compared with that in a control solution without lipid. Caprylic acid (8:0), monocaprylin (8:0), monolaurin (12:0), myristic acid (14:0), palmitoleic acid (16:1), monopalmitolein (16:1), oleic acid (18:1), and monoolein (18:1) at concentrations of 20 mM (final concentration, 10 mM) had negligible effects on C. trachomatis. In contrast, lauric acid (12:0), capric acid (10:0), and monocaprin (10:0) caused a greater than 10,000-fold (>4-log10) reduction in the infectivity titer. When the fatty acids and monoglycerides were further compared at lower concentrations and with shorter exposure times, lauric acid was more active than capric acid and monocaprin was the most active, causing a greater than 100, 000-fold (>5-log10) inactivation of C. trachomatis at a concentration of 5 mM for 5 min. The high levels of activity of capric and lauric acids and particularly that of monocaprin are notable and suggest that these lipids have specific antichlamydial effects. The mode of action of monocaprin was further studied by removal of the lipid by centrifugation before inoculation of Chlamydia onto host cells and by electron microscopy. The results indicate that the bacteria are killed by the lipid, possibly by disrupting the membrane(s) of the elementary bodies. A 50% effective concentration of 30 microgram/ml was found by incubation of Chlamydia with monocaprin for 2 h. The rapid inactivation of large numbers of C. trachomatis organisms by monocaprin suggests that it may be useful as a microbicidal agent for the prevention of the sexual transmission of C. trachomatis.

Marked inhibitory activity of masked aryloxy aminoacyl phosphoramidate derivatives of dideoxynucleoside analogues against visna virus infection.

Lipophilic masked aryloxyaminoacylphosphoramidate derivatives of 2',3'-dideoxynucleoside (ddN) analogues with potent anti-HIV activity (i.e., stavudine [d4T], azidothymidine [AZT], dideoxycytidine [ddC], 3'thio-2',3'-dideoxy cytidine [3TC], dideoxyadenosine [ddA], and 2',3'-didehydro-2',3'-dideoxyadenosine [d4A]) activity were evaluated for their activity against visna virus (VV) in sheep choroid plexus (SCP) cells. The activity of several prodrug derivatives against VV proved markedly superior to that of the corresponding free ddN analogues. In particular, the d4A and ddA prodrug derivatives were exquisitely inhibitory in this model system (50% effective concentration [EC50], < or = 0.003 microM), and their anti-VV potency exceeded by at least 200-fold the antiviral potency of the corresponding free nucleosides. Marked differences were noted in the anti-VV potencies of several of the test compounds depending on the nature of the amino acid linked to the 5'-phosphate moiety, the nature of the nucleoside, or both. In view of the stability of the prodrugs in lamb serum, the VV infection model in lambs may be considered highly useful for investigating the in vivo antiretroviral efficacy of these type of drugs, particularly the d4T, ddA, and d4A prodrug derivatives.

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.