Susceptibility of Vaccinia Virus to Chemical Disinfectants

Abstract

Vaccinia virus (VACV) is the cause of bovine vaccinia (BV), an emerging zoonotic disease that affects dairy cows and milkers. Some chemical disinfectants have been used on farms affected by BV to disinfect cow teats and milkers' hands. To date, there is no information about the efficacy of disinfectants against VACV. Therefore, this study aimed to assess the virucidal activity of some active disinfectants commonly used in the field. Sodium hypochlorite, quaternary ammonium combined with chlorhexidine, and quaternary ammonium combined with glutaraldehyde were effective in inactivating the virus at all concentrations tested. Iodine and quaternary ammonium as the only active component were partially effective. The presence of bovine feces as organic matter and light decreased the effectiveness of sodium hypochlorite. These results show that an appropriated disinfection and asepsis of teats and hands may be helpful in the control and prevention of BV and other infections with VACV.

Introduction

Bovine vaccinia (BV) is an emerging zoonosis caused by Vaccinia virus (VACV), an orthopoxvirus. The disease is characterized by the appearance of lesions on the teats of cows and on the hands of milkers. This disease has serious veterinary, public health, and economic effects. In Brazil, VACV has been consistently identified as the causative agent of several outbreaks of BV in many states since the late 1990s.^1–7 In addition to cattle and humans, in Brazil, VACV has also been isolated and/or identified in other species such as rodents and monkeys.^8,9

One of the measures used to control and prevent this disease within the herd is the use of disinfectants and antiseptics on the teats and udders of cows. Both pre-dipping and post-dipping procedures are used, in addition to treating the hands of the milkers. The use of disinfectants reduces the viral load, which decreases the risk of disease transmission to humans and animals, thus decreasing the number of cases.⁶ Because it is easy to buy and handle, inexpensive, and has low toxicity to living tissue, bleach, whose active ingredient is sodium hypochlorite, is the most widely used disinfectant in the field. It is common to use a solution prepared from one part household bleach to three parts water to disinfect human hands and the teats and udders of dairy cows.

In addition to bleach, other disinfectants such as iodine, quaternary ammonium, and chlorhexidine have also been used on farms affected by the disease. Despite being a widespread practice, there are no data about the sensitivity of VACV to different disinfectants. The objective of this study was to evaluate the sensitivity of VACV to different disinfectants commonly used in the field. The titer of active chlorine in a 1:4 dilution of bleach subjected to conditions commonly encountered in the field, such as light and the presence of organic matter (bovine feces), was also examined.

Materials and methods

Cells.

Vero cells were obtained from the American Type Culture Collection (Manassas, VA) (catalog no. CCL-81). Cells were grown as described by Abrahão and others.¹⁰

Virus.

Vaccinia virus Guarani (VACV-GP2), which was isolated from lesions on the teats of a cow from an outbreak of BV in Guarani, Minas Gerais, Brazil in 2001, was used. It was previously characterized by using serologic and molecular procedures as VACV.³

Titration.

Assays were conducted to determine the titers of viral stocks and virus controls during the tests with disinfectants. They were also used to calculate reduction in the viral titer (original titer – final titer) in tests using disinfectants. All titration procedures were performed as described by Abrahão and others¹⁰ and Campos and Kroon.¹¹

Calculation of reduction in viral titer.

Reduction in viral titers in response to disinfectants was represented as a percentage and a log₁₀ value. The percentage of reduction in viral titer was calculated by using the formula¹² % Reduction in viral titer = T before – T after × 100, where T before is the viral titer (plaque-forming units [PFU]/mL) in the no disinfectant virus control and T after is the average viral titer (PFU/mL) of eight samples after treatment with disinfectant. The SD was also calculated. The logarithmic reduction was calculated by using the formula Log reduction = T before (log₁₀) – T after (log₁₀).

Disinfectants.

Commercial disinfectants with the following active ingredients were tested: chlorine, polyvinylpyrrolidone, iodine, quaternary ammonium (benzalkonium chloride), glutaraldehyde, and chlorhexidine gluconate. The tested concentrations were recommended by the manufacturers on product labels according to its use as a disinfectant or an antiseptic (Table 1). All disinfectants were tested for 1, 5, and 30 minutes. Disinfectants and theirs recommended dilutions were selected if they contained active ingredients commonly used for disinfection of the teats during pre-dipping and post-dipping or for disinfection of surfaces in the dairy stables. The selected disinfectants, with the exception of the glutaraldehyde-based disinfectant, are also antiseptics if used at the dilutions recommended by the manufacturer.

Table 1.

Disinfectants used, active ingredients, and directions for use according to the manufacturer for inactivation of vaccinia virus^*

Product	Active ingredient	% Active ingredient in product	Recommended dilution	Indications	% Concentration of active ingredient in test
1	Active chlorine	2 to 2.5 w/v	1:4	Dilution recommended for antisepsis of teats and milker's hands	0.5 to 0.625 v/v active chlorine
2	Iodophor	11.25 w/v	1:250	Disinfection and washing of udders of cows and milkers hands	0.045 v/v iodophor
			1:1,200	Washing and sterilization of equipment, tanks, cans, milk tanks, slaughterhouses and meatpackers	0.009 v/v iodophor
			1:2,000	Prevention of disease through drinking water of animals	0.005 v/v iodophor
3	Quaternary ammonium (benzalkonium chloride)	12.5 w/v	1:500	Disinfection of poultry environments such as warehouses, processing rooms of chickens and eggs, stables, milking parlors, hutches, pens, kennels and other animal housing	0.025 v/v quaternary ammonium
			1:1,000	External use in animals and disinfection of utensils, hands and equipment used for milking, artificial insemination and other uses at poultry industry, livestock, fish farms, and kennels	0.0125 v/v quaternary ammonium
4	Quaternary ammonium	30 w/v	1:2000	Spraying and disinfecting poultry litter, poultry, incubator chambers, trays, equipment in general, maternity, nursery, milking machines, udders, milker's hands, footbath	0.015 v/v quaternary ammonium
			1:3000	Disinfection in general (uniform, walls and floors), washing and asepsis of wounds and interdigital dermatitis	0.01 v/v quaternary ammonium
5	Chlorhexidine gluconate	0.2 w/v	1:20	Disinfection of instruments, machinery and agricultural and veterinary facilities	0.01 v/v chlorhexidine gluconate
	Quaternary ammonium	0.5 p/v			0.025 v/v quaternary ammonium
6	Glutaraldehyde	10.625 v/v	1:1,500	Disinfection of equipment, tools, environment of dairy industry, fish and meat	0.007 v/v
	Quaternary ammonium (benzalkonium chloride)	15 v/v			0.01 v/v quaternary ammonium

^*w/v = weight/volume; v/v = volume/volume; p/v = parts/volume.

Testing the effectiveness of disinfectants.

The protocol of the European Committee for Standardization (no. EN 14675, 2006) was used to determine the virucidal activity of disinfectants and antiseptics used in veterinary medicine.¹³ This test includes variables that simulate the conditions of product application in the field, such as the use of hard water to perform the dilutions of the disinfectants.

Briefly, eight parts of each disinfectant to be tested at the dilutions described in Table 1 and one part of hard water were added to viral suspensions containing 10⁶ PFU/mL. At this stage, the concentration of disinfectant in the test was 1.25 times higher than recommended by the manufacturer to compensate for the dilution of the disinfectant during the test (80%). The mixture was kept at room temperature for 1, 5, and 30 minutes. After each period, each virus and disinfectant reaction was diluted 100-fold (10⁻² dilution) in minimal essential medium and kept on ice until completion of the remaining dilutions up to 10⁻⁴. An aliquot of 300 μL of each dilution was inoculated into VACV-infected Vero cell monolayers in six-well plates. For each test, a negative control was performed by replacing the disinfectant with phosphate-buffered saline (PBS). The plates were incubated at 37°C and after 60 hours, the cells were fixed and stained with crystal violet solution. Evaluation of the effectiveness of the disinfectant was performed by comparing the viral titer in the control well with those in the test wells after the incubation period. Each concentration of disinfectant was tested in eight replicates. The disinfectant was considered effective if the viral titer was reduced by at least 4 log₁₀ or 99.99% on viral titer reduction.¹³

The control of viral titers was also examined in the presence of PBS at the previously determined disinfectant concentrations. After 30 minutes, serial dilutions of PBS were made in minimal essential medium from 10⁻¹ to 10⁻⁴. An aliquot (300 μL) of each dilution was inoculated into each well of the cell culture plate, and viral titers were determined as described.

Preparation of hard water.

Hard water, a solution of MgCl₂, CaCl₂, and NaHCO₃, was prepared according to protocol EN14675: 2006.¹³ Water hardness was measured by the amount of CaCO₃ in grams/liter or parts per million. In this test, the water contained CaCO₃ at a concentration of 300 mg/L.

Control of cytotoxicity.

This test was designed to evaluate the possible cytotoxic effect of the disinfectants at the recommended dilutions. It was conducted as described,¹³ except for the replacement of the virus by PBS.

Determination of active chlorine in a 1:4 bleach dilution.

The amount of chlorine present in the 1:4 solution of bleach used in the tests was determined by using the iodometric method.¹⁴ Legislation defines the percentage of active chlorine in commercial bleach to range between 2.0% and 2.5%, with an acceptable range between 1.75% and 2.75% weight/weight.¹⁵ Although bleach labels indicate 2.0–2.5% active chlorine, this amount can be altered by the storage time. However, for the solution used in this test, it was expected that the concentrations would range from 0.5% to 0.625% active chlorine in the dilute solution, corresponding to 0.25% of the total concentration specified on the label.

Titration of active chlorine subjected to light and presence of organic matter.

The titrations were determined according to a published protocol.¹⁵ The test was repeated three times, and the mean and SD of concentrations of chlorine was determined in each titration. The titrations were performed at 48 hours (T1), 96 hours (T2), and 144 hours (T3) at the same time of the day.

To assess the influence of light and evaporation on the concentration of active chlorine, different storage containers were used. The solution was stored in an open 10-liter bucket or three properly closed two-liter bottles made of ethyl polyethylene (PET bottles). One bottle was green, one was transparent, and one was covered with aluminum foil before exposure to the environment.

Aliquots of the contaminated bleach stock solution were inoculated with 1%, 5%, 10%, and 20% solutions of bovine feces to evaluate the influence of organic matter in the concentration of active chlorine. These samples were stored at 4°C, after which titration of active chlorine was performed as described above.

Statistical design.

Each disinfectant was tested for two factors (concentration and time of action). Eight samples were taken for every possible combination further analyzed for each concentration of disinfectant. Thus, for all experiments, in addition to testing the main effect of the factors (concentration and time of action) and the interaction between them, the consistency of the replicates was also evaluated.

Analysis of the significance of the experiment was performed by using a generalized linear model composed of factors of interest (concentration and time of action). The main goal was to test the significance of these factors in reducing viral load and the possible effect of the interaction between them. The significance cutoff value was P < 0.05. The effect of the eight samples of each possible combination was tested. The alpha level of significance assumed for this analysis was 0.05 (5%).

Results

Sensitivity of VACV-GP2 to iodophor-based disinfectants.

Disinfectant 2, an iodophor-based compound with iodine as the active ingredient, did not reduce viral titers by at least 4 log₁₀ (or 99.99%) at concentrations of 0.005% and 0.009%. However, at a concentration of 0.045% iodine, it reduced the viral titer by 100% after 1, 5, or 30 minutes (Figure 1). The initial viral titer was 2.9 × 10⁵ PFU/mL.

Figure 1.

Sensitivity of Vaccinia virus VACV-GP2 to disinfectant 2. Shown are reduction of % mean and SD VACV-GP2 titer after treatment with disinfectant 2 at concentrations of 0.045%, 0.009%, and 0.005% of active agent. From a reduction of 99.99%, effectiveness of concentration of active ingredients was evaluated. Values above the bars represent average reduction of eight samples in log₁₀/SD.

Sensitivity of VACV-GP2 to quaternary ammonium compound-based disinfectants.

Disinfectant 3, a 0.0125% quaternary ammonium solution, reduced the virus titer by 4 log₁₀ (99.99%) only after 30 minutes of incubation. However, at a concentration of 0.025%, it reduced the viral titer by at least 4 log₁₀ (99.99%) after 5 minutes (Figure 2). The initial virus titer was 2.1 × 10⁶ PFU/mL.

Figure 2.

Sensitivity of Vaccinia virus VACV-GP2 to disinfectant 3. Shown are reduction of % mean and SD VACV-GP2 titer after treatment with disinfectant 3 at concentrations of 0.0125% and 0.025% of the active ingredient. From the reduction of 99.99%, effectiveness of concentration of active ingredients was evaluated. Values above the bars represent average reduction of eight samples in log₁₀/SD.

Disinfectant 4 was a 0.015% quaternary ammonium solution that was effective against the virus at all times tested. At a concentration of 0.01%, this disinfectant reduced the viral titer by 100% after the maximum time tested (30 minutes). At a concentration of 0.01%, length of time of the treatment influenced the disinfectant effectiveness, and there was a progressively increased reduction in viral titer as the time of incubation increased. However, the maximal decrease was only achieved after 30 minutes (Figure 3).

Figure 3.

Sensitivity of Vaccinia virus VACV-GP2 to disinfectant 4. Shown are reduction of % mean and SD VACV-GP2 titer after treatment with disinfectant 4 at concentrations of 0.01% and 0.015% of the active ingredient. From the reduction of 99.99%, effectiveness of concentration of active ingredients was evaluated. Values above the bars represent average reduction of eight samples in log₁₀/SD.

Sensitivity of VACV-GP2 to a chlorhexidine gluconate and quaternary ammonium–based disinfectant.

Disinfectant 5, which contained 0.01% chlorhexidine and 0.025% quaternary ammonium, was completely effective against the virus at 1, 5 and 30 minutes.

Sensitivity of VACV to a glutaraldehyde and quaternary ammonium–based disinfectant.

Disinfectant 6, which contained 0.007% glutaraldehyde and 0.01% quaternary ammonium, was effective against VACV at 1, 5, and 30 minutes. The initial titer of the virus was 5.61 × 10⁵ PFU/mL.

Sensitivity of VACV-GP2 to bleach at a 1:4 dilution.

Bleach at 1:4 dilution (0.636% active chlorine) was able to completely reduce the viral titer after 1, 5, and 30 minutes of treatment. No cytotoxic effect was observed on the cell monolayer in control wells. The initial titer of the virus was 5.61 × 10⁵ PFU/mL.

Concentration of active chlorine in a bleach solution in the presence of light.

The average value of three chlorine measurements in a 1:4 solution of bleach through the iodometric method was 0.636% volume/volume (SD = 0.075). If one considers the dilution factor of the bleach, this concentration should be between 0.5% and 0.625%. However, the amount of chlorine measured was higher than the minimum recommended.¹⁵

The iodometric titration showed that the concentration of active chlorine in the bleach solution in the bottles and bucket decreased after 48 hours of exposure (T1) even without the presence of organic matter, as shown in Figure 4. However, the bleach solution kept in the transparent PET bottles showed a greater decrease compared with the other containers. After 96 hours (T2 and T3), the comparative decrease in the concentration of active chlorine in the solution kept at the green and transparent PET bottles became more evident than at T1. The concentration of active chlorine in the bucket had a slight decrease.

Figure 4.

Concentration of active chlorine in a solution of sodium hypochlorite (bleach) in the presence of light.

Concentration of active chlorine in a bleach solution in the presence of organic matter (bovine feces).

Iodometric titration of the bleach solution in the presence of 1%, 5%, 10%, and 20% organic matter (bovine feces) demonstrated a decrease in the levels of active chlorine at the moment of the dilution (T0). However, at feces concentrations of 5%, 10%, and 20%, the reduction of active chlorine was more intense (Figure 5). Analysis over time (T1, T2, and T3) showed that the decrease in active chlorine was progressive and was time and concentration related.

Figure 5.

Concentration of active chlorine in a solution of sodium hypochlorite (bleach) in the presence of organic matter (bovine feces). OM = organic matter.

As shown in Figures 4 and 5, the reduction in the amount of active chlorine in the prepared solutions was significantly higher in the presence of organic matter compared with solutions exposed to light or the environment during the same period. Moreover, the higher the concentration of feces and the longer the exposure time, the greater the decrease in chlorine (Figure 5). All solutions shown in Figure 4 maintained levels of active chlorine up to 5 g/L during the seven-day test period. As shown in Figure 5, the solution that contained 1% feces maintained a sufficient amount of active chlorine for only 24 hours. The other concentrations of feces reduced the levels of active chlorine to less than 5 g/L at the time of the dilution (T0).

Discussion

In this study, disinfectant 1, whose active ingredient was sodium hypochlorite at the 1:4 dilution commonly used in the field, was able to prevent viral multiplication after 1-, 5-, and 30-minute exposures. This bleach dilution, containing approximately 5,000 parts per million or 0.5% active chlorine, has been used to control BV in the field. This concentration is sufficient to inactivate most enveloped viruses, including VACV.¹⁶ Although the concentration of chlorine in the bleach solution used in this experiment was somewhat higher than that prescribed by law, this average reflects only one batch from one particular brand of bleach. However, the 1:4 dilution can still be safely used as a hand or teat antiseptic to control the spread of BV.

Tanabe and Hotta evaluated the effectiveness of disinfectants against smallpox virus (orthopoxvirus).¹⁷ Among the tested disinfectants, sodium hypochlorite was the most effective, reducing 100% of the initial viral titer at the concentration of 0.1%. The hypochlorite acts against viruses by disrupting viral capsid and nucleic acid.¹⁸

Disinfectant 2, which contained 0.045% iodine as the active ingredient, was effective against VACV-GP2, and this concentration is recommended by the manufacturer for use as a hand or cow udder antiseptic (Table 1). However, use of Disinfectant 2, which at a concentration of 0.009% is recommended for use in disinfection of tanks and other dairy milking equipment, was not effective in inactivating the virus and therefore should not be used for this purpose.

The Centers for Disease Control and Prevention (Atlanta, GA) recommends using 0.0075% iodine for 10 minutes to disinfect surfaces that contain smallpox virus. However, this concentration should be increased when the surfaces vary or if higher disinfection is desired, such as in hospital environments.¹⁹ In this study, 0.009% iodine, which is higher than the concentration recommended by the Centers for Disease Control and Prevention, was not effective in the inactivation of VACV-GP2.

Disinfectant 3, which contained 0.025% quaternary ammonium, was not effective against VACV after a one-minute incubation. However, the same concentration of quaternary ammonium was able to reduce the viral titer by at least 4 log₁₀ of the initial titer after 5 minutes and 30 minutes. Tanabe and Hotta evaluated the effectiveness of disinfectants against the smallpox virus in cell cultures.¹⁷ They found that 0.001% and 0.025% quaternary ammonium were not effective disinfectants at 1, 3, 5, and 10 minutes. In the present study, 0.025% quaternary ammonium was not effective against VACV after a one-minute incubation.

Disinfectant 4, which contained 0.015% quaternary ammonium, reduced the viral titer by at least 4 log₁₀ at all times tested. However, the 0.025% concentration of the same active component in disinfectant 3, which is higher than that in disinfectant 4, was not effective. The fact that better results were obtained with disinfectant 4 at lower concentrations of active ingredients may be attributed to the addition of a surfactant in its formulation (polyethylene oxide). Although this agent has no virucidal action, it is a nonionic surfactant with detergent properties.

Disinfectant 5, which contained 0.01% chlorhexidine and 0.025% quaternary ammonium, was completely effective against the virus at 1, 5, and 30 minutes. McDonnell and Russell reported that chlorhexidine is more efficient against enveloped viruses than against non-enveloped viruses, and it works by disrupting the lipid chains of the viral envelope.¹⁶ Although the 0.05% concentration tested in this experiment is recommended by the manufacturer for cleaning agricultural machinery and facilities, it can also be used for disinfection of teats because this concentration is not toxic to tissues. Jones highlights the ability of 0.55% chlorhexidine to form a protective barrier on the teats, which protects them more effectively than treatment with 1% iodophor.²⁰

Disinfectant 6, which contained 0.007% glutaraldehyde and 0.01% quaternary ammonium, was effective against VACV at all tested times. Testing the efficiency of 2% glutaraldehyde against VACV in dermal wounds in rabbits, Schümann and Grossgebauer found that it was effective only when the scabs were treated for 90 minutes with the disinfectant at this high concentration.²¹ Although its use is restricted to disinfecting surfaces and footbaths, and it cannot be directly used on living tissues, glutaraldehyde is recommended because of its broad spectrum of action, which includes enveloped and non-enveloped viruses.¹⁶

In the present study, there was a higher reduction in the concentration of active chlorine in sodium hypochlorite solutions in PET bottles (transparent and green), which enables greater light penetration (Figure 4). The mechanism of reduction of active chlorine in bleach solutions by ultraviolet light has not yet been elucidated.¹⁸

The presence of organic matter, especially in higher concentrations and for longer exposure times, tended to reduce the concentration of potentially active chlorine (Figure 5). Organic matter may decrease the effectiveness of the hypochlorite solution because of the reaction of chlorine with nitrogen compounds to form chloramines.¹⁸

Bleach is a cheap and easy to acquire disinfectant. Moreover, it can be used for either the disinfection of facilities and equipment, or asepsis of cows' teats and milkers' hands. Furthermore, when used at the proper dilution, it does not leave toxic products in the milk and is also known to be efficient in controlling other diseases such as mastitis. For these reasons, it is a popular disinfectant used by farmers. However, on farms, is it is common to see inadequate storage of bleach, which could cause a decrease in its germicidal effectiveness. Other commercial products in addition to the bleach can also be used as chlorine sources. However, the different initial concentrations of commercial chlorine on the market make it difficult to recommend a standard dilution for use as an antiseptic or disinfectant for surfaces and milking utensils. The use of a higher concentration of chlorine can lead to lesions on the teats of the cows and on the hands of the milkers, making them ultimately more susceptible to VACV infection.

The results of the present study show that hypochlorite and quaternary ammonium combined with chlorhexidine or glutaraldehyde could be considered for use as recommended products for the control of BV because of the susceptibility of VACV to these disinfectants. Among the disinfectants tested, sodium hypochlorite is the cheapest, but to be effective, it is recommended to be stored in opaque containers, protected from sunlight and discarded whenever contamination with organic matter occurs.

The use of effective disinfectants against VACV, as shown in this study, represents an important step in the control and prevention of BV and other infections with VACV. These disinfectants may also be used by health professionals for disinfection when dealing with VACV-infected patients. They may also be used to avoid laboratory-acquired infections because VACV is a common virus in laboratory-based research.