Evaluation of chlorine dioxide in liquid state and in gaseous state as virucidal agent against avian influenza virus and infectious bronchitis virus

Abstract

The antiviral activity of chlorine dioxide (ClO₂) in liquid (ClO₂ gas dissolved liquid) and gaseous state against avian influenza virus (AIV) and infectious bronchitis virus (IBV) was evaluated. To evaluate the effect of ClO₂ in liquid state, suspension tests (10 ppm) and carrier tests in dropping / wiping techniques (100 ppm) were performed. In the suspension test, virus titers were reduced below the detection limit within 15 sec after treatment, in spite of the presence of an accompanying organic matter. In the carrier test by dropping technique, AIV and IBV were reduced to below the detection limit in 1 and 3 min, respectively. Following wiping technique, no virus was detected in the wiping sheets after 30 sec of reaction. Both viruses adhering to the carriers were also reduced by 3 logs, thereby indicating that they were effectively inactivated. In addition, the effect of ClO₂ gas against IBV in aerosols was evaluated. After the exposure of sprayed IBV to ClO₂ gas for a few seconds, 94.2% reduction of the virus titer was observed, as compared to the pre-treatment control. Altogether, hence, ClO₂ has an evident potential to be an effective disinfectant for the prevention and control of AIV and IBV infections on poultry farms.

Respiratory viruses of poultry, such as avian influenza virus (AIV) and avian infectious bronchitis virus (IBV), have been pathogens of highly impactful diseases in chicken farms worldwide. To reduce the occurrence of these infectious diseases, disinfection by means of appropriate disinfectants is necessary [23].

In Japan, sodium hypochlorite (NaOCl) and quaternary ammonium compounds (QACs) are commonly used in poultry farms for disinfection. NaOCl is known to have a wide spectrum [36], but it may produce trihalomethane and it exhibits weak antimicrobial activity in the presence of organic materials [4]. Furthermore, NaOCl is known to cause corrosion to metals such as stainless steel and nickel titanium [14]. QAC has less corrosion effect on metals compared to NaOCl, but previous studies have shown that the antiviral activity of QACs will be reduced not only in the presence of organic materials, but also under low temperature [6, 10].

Chlorine dioxide (ClO₂) is a strong oxidant with a broad spectrum. In addition, there is an advantage to use ClO₂ as a disinfectant in terms of its safety. From previous studies, ClO₂ is regarded to be safe for using in fresh products, since it does not leave detectable residues, due to its volatility [18]. In fact, ClO₂ is proved to be able to disinfect bacteria on vegetables [34], viruses on fruits [13], and bacteria in water [24]. ClO₂ is a genuine gas at above 11°C and is known to be effective in both the liquid state (ClO₂ gas dissolved liquid) and gaseous state. ClO₂ gas has been proven effective against various viruses in liquids [1, 39]. Furthermore, ClO₂ in the gaseous state can effectively disinfect bacteria on surfaces [22, 28]. Despite its effectiveness, a paucity of study has been done on ClO₂ applicability for poultry farms to prevent AIV and IBV infections.

The purpose of the present study is to evaluate the antiviral activity of ClO₂ in the liquid state against AIV and IBV and in the gaseous state against IBV. To check the effect against viruses in water, the suspension test using 10 parts per million (ppm) of ClO₂ in the liquid state was performed. Furthermore, carrier tests in dropping technique and wiping technique were performed to examine its effect against viruses on dry surfaces. For every experiment, 0.5% or 5% of fetal bovine serum (FBS) was added to the virus suspension to simulate the organic environment of poultry farms. In addition, IBV suspension containing 0.5% FBS was sprayed into the air and exposed to ClO₂ in the gaseous state to evaluate the latter’s effect against the virus in the air for a few seconds.

MATERIALS AND METHODS

Virus preparation

A low pathogenic AIV, A/duck/Aomori/395/04 (H7N1), isolated from a wild duck [11], was propagated in Madin-Darby Canine Kidney (MDCK) cells. Coronaviridae, Gammacoronavirus, IBV strain M41 which was kindly provided by National Institute of Animal Health (Tsukuba, Japan) [20], was propagated in primary chicken kidney cells (CK cells). These virus cultures were centrifuged at 1,750 × g for 15 min, supernatants were taken, aliquoted and then kept at −80°C until use. The titer of the stocked AIV was around 10⁷ fifty percent tissue culture infectious dose (TCID₅₀)/mL based on the Spearman-Kaerber method [35]. For IBV, titer of around 10⁷ plaque forming units (PFU/mL) was calculated by counting plaques at three days post-inoculation (dpi).

Disinfectant preparation

One hundred ppm of ClO₂ gas dissolved in liquid−thereafter referred as “liquid ClO₂”−was prepared by adding a ClO₂ tablet, CREINS (CLOX Inc., Tokyo, Japan) to 500 mL of redistilled water (dW₂), and mixed by using magnetic stirrer until it was completely dissolved. Information from CLOX Inc. indicated that the concentration ratio of ClO₂ molecules and ClO₂⁻ ions was typically around 100:1. Ten ppm of liquid ClO₂ was used for the suspension tests, 50 ppm was used for the gaseous test and 100 ppm was used for the carrier tests. The liquid ClO₂ with each concentration were kept at 4°C and were used within 2 weeks. The concentration of liquid ClO₂ was measured each time before an experiment, using a measuring machine (CLOX Inc.). The liquid ClO₂ was diluted before measurement to adjust the concentration of 10 ppm. The diluted solution was put into the test tube, and 2 drops of glycine solution and DPD solution were added to the test tube, and mixed well. If the concentration is 10 ppm, the color of the test solution would match the color of the indicator. In the gaseous test, ClO₂ gas was generated by using a ClO₂ gas generator (CLOX Inc.). To generate around 1,500 to 2,000 parts per billion (ppb) of ClO₂ gas in air, a 50 ppm liquid ClO₂ was used in the generator. The concentration of chlorine dioxide in the text box was measured by using Interscan Portable Gas Concentration Measurer IS4000 (JMS Inc., Tokyo, Japan), and was recorded during the experiment. Chlorine dioxide concentration instruments are sent to CLOX on a regular basis for calibration and validation by the respective meter manufacturer to ensure that accuracy indicators are correct.

Suspension test

The virus suspensions were diluted 10 times by adding phosphate-buffered saline (PBS, pH 7.4), and 0.5% or 5% of FBS was added to the suspensions. To 450 µL of 10 ppm of liquid ClO₂ in a microtube, 50 µL of diluted virus was added and vortexed. After 15, 30, 60 and 90 sec, inactivating solution (0.7M N-(2-hydroxyethyl)-piperazine-N’-2-ethanesulfonic acid containing 30% FBS) [20] was added to neutralize the effect of liquid ClO₂. To test the efficacy of inactivating solution, the latter solution was added to the liquid ClO₂ before adding the virus suspension, which was indicated as 0 sec treatment.

Carrier test (dropping technique and wiping technique)

A 0.5% or 5% of FBS was added to the original virus. As non-porous carriers, 5 cm × 5 cm plastic plates were used. Carriers were microwaved for sterilization for 5 min and kept at room temperature (RT: 25 ± 2 °C) before use. One hundred microliter of virus suspension containing 0.5 or 5% of FBS was inoculated onto each carrier, and was spread out to approximately 3 cm diameter. After inoculation, the contaminated carriers were dried completely for 30–45 min in a safety cabinet. For the dropping technique, 500 µL of 100 ppm liquid ClO₂ was directly added on the carrier, and incubated for 1, 3 or 5 min in a safety cabinet. After incubation, each carrier was put into a stomacher bag containing 2 mL of stop solution and rubbed by thumb for 2 min. For the wiping technique, the inoculated area of each carrier was then wiped with a rayon sheet treated with 500 µL of 100 ppm liquid ClO₂ for 30 sec. After wiping, each rayon sheet was kept on a plastic plate for 30 sec to disinfect the viruses that transferred to the rayon sheets, and was put into a stomacher bag containing 2 mL of stop solution. The stomacher bag containing rayon sheet was treated with the Bag Mixer (MiniMix 100 W CC, Practical Japan Inc., Chiba, Japan) for 1 min. Each of the wiped carrier was put into a stomacher bag containing 2.5 mL of inactivation solution and rubbed similarly to the dropping technique.

Gas test

Aerosolized IBV containing 0.5% FBS supplemented with PBS was exposed to 1,500 to 2,000 ppb of ClO₂ gas in air for a few sec. The concentration of ClO₂ gas was measured by the machine mentioned above. The plastic test box (W 440 × D 740 × H 230 mm, around 75 L volume) used for this experiment is shown in Fig. 1. It was purchased from a local market and was placed in a safety cabinet with an upper airflow to let the generated ClO₂ gas escape. The design of this experiment is shown in Fig. 2. Three mL of IBV suspension was sprayed using a sprayer (Aerial Mist, Aelph Co., Ltd., Tokyo, Japan) from one side of the box for 3 and a half min at an even speed until the virus suspension was completely sprayed, and the sprayed viruses were collected onto rayon sheets which were attached to an aspirator placed on the other side of the box. Each of the rayon sheet was treated with 2 mL of FBS, microwaved, and then additionally treated with FBS by using a hand spray before the experiment, so as to neutralize the ClO₂ gas on rayon sheets. The aspirator was connected to a bottle filled with a sterilizing agent (mixture of QAC and food additive grade calcium hydroxide) and a suction pump (SP 40, MARKOS-MEFAR, Borezzo, Italy) equipped with high efficiently particulate air filter, similarly to the previous experiment [21]. The suction pump could collect the air sample at a speed of 35 L/min). ClO₂ gas was produced by using the machine mentioned above. Pre-treated control was performed to check whether the viruses were inactivated on the rayon sheets or not (Fig. 2). For pre-treated control, ClO₂ gas was generated before spraying viruses and the sprayed viruses were not exposed to the gas. After collecting viruses, each rayon sheet was collected to a stomaching bag containing 3 mL of maintenance medium (MM) and treated with the Bag Mixer, alike the wiping test.

Fig. 1.

Test box used for the gas test. (1) Infectious bronchitis virus (IBV) suspension containing 0.5% fetal bovine serum (FBS) was sprayed from one side of the box. (2) Chlorine dioxide (ClO₂) gas was generated and sprayed into air by ClO₂ gas generator. The concentration of the gas inside the box was observed during the experiment. (3) Viruses in the air were collected onto rayon sheets treated with FBS using an aspirator.

Fig. 2.

Design of gas test. (1) Water control. The chlorine dioxide (ClO₂) gas generator was filled with redistilled water (dW₂). (2) Pre-treated control. ClO₂ gas was generated for 9.5 min before spraying viruses, and the viruses were sprayed after switching off the generator and achieving the concentration that was low enough. (3) Virus was exposed to ClO₂ gas produced from a generator containing 50 ppm ClO₂ liquid for a few seconds.

Viral quantification

All experiments were performed at RT in triplicate. For water control, dW₂ was used instead of ClO₂, except for pre-treated control of the gaseous test. To measure the quantity of viruses exposed to the disinfectant, viral controls were prepared. Each sample was 10-fold diluted with MM, individually [7, 11, 32]. For AIV, the dilution was inoculated onto MDCK cells in MM with a final concentration of 2 μg/mL bovine pancreatic trypsin (Sigma, St. Louis, MO, USA) in a 96-well cell-culture plate. After incubating at 37°C in a 5% CO₂ incubator for three days, cytopathic effect (CPE) was observed. In addition, the supernatant was transported to v-shaped-96-well plates to evaluate the hemagglutinin activity using 0.5% chicken red blood cells. Virus titration was performed to calculate TCID₅₀. On the other hand, IBV suspension in 10-fold dilution series was inoculated to CK cells prepared in 6- well cell culture plates, in order to perform plaque assay [32]. The titer was calculated as PFU/mL, as explained above.

In addition to the titers, the mean ± standard error (SE) among the results of three data was shown. When the treated viral titer was reduced by more than 1,000 times compared to the control virus, the virucidal activity was regarded as effective in the suspension and carrier tests [15, 25]. This standard is based on the Organization for Economic Co-operation and Development standard stated on the Guidance Document on Quantitative Methods for Evaluating the Activity of Microbicides used on Hard Non-Porous Surfaces [23]. Statistical analysis was performed for the gas test using a one-way analysis of variance (ANOVA) and followed by Tukey’s post hoc test. Statistically significant differences were recognized as P<0.01.

RESULTS

Suspension test

Table 1 shows the result of suspension tests using 10 ppm liquid ClO₂ for AIV and IBV, containing 0.5% or 5% of FBS. For 0 sec treatment, the inactivation solution was added to the ClO₂ before adding virus suspension to the mixture. The titers of 0 sec treatments were similar to the controls (dW₂ controls and viral controls), which indicates the inactivation solution could neutralize the ClO₂ effect completely. Ten ppm of liquid ClO₂ could reduce both viruses to the detection limit (<1.80 log₁₀TCID₅₀/mL) within 15 sec. These results suggest that AIV and IBV in aqueous phase can be markedly affected by 10 ppm liquid ClO₂ even in the presence of organic materials.

Table 1. Virucidal activity of 10 ppm of chlorine dioxide (ClO₂) toward avian influenza virus (AIV) and infectious bronchitis virus (IBV) in aqueous phase containing 0.5 and 5% fetal bovine serum (FBS).

Virus		AIV		IBV
FBS (%)		0.5	5.0	0.5	5.0
Control		6.42 ± 0.18	5.92 ± 0.14	5.71 ± 0.01	5.80 ± 0.01
CLOX-10a	0 sec	6.38 ± 0.07	6.38 ± 0.07	5.93 ± 0.02	6.02 ± 0.02
	15 sec	<1.80 ± 0.00	<1.80 ± 0.00	<2.00 ± 0.00	<2.00 ± 0.00
	30 sec	<1.80 ± 0.00	<1.80 ± 0.00	<2.00 ± 0.00	<2.00 ± 0.00
	1 min	<1.80 ± 0.00	<1.80 ± 0.00	<2.00 ± 0.00	<2.00 ± 0.00
dW2	1 min	6.97 ± 0.07	6.30 ± 0.00	5.89 ± 0.01	5.89 ± 0.02

^a Chlorine dioxide liquid at 10 ppm of total chlorine. Figures are shown as mean ± SE from replicated 3 times. Viral titer was measured for AIV as log₁₀ 50% tissue culture infective dose (TCID₅₀)/mL, and for IBV as log₁₀ plaque forming unit (PFU)/mL. Viral titer <1.80 log₁₀TCID₅₀/mL and <2.00 log₁₀PFU/mL indicates the virus was inactivated to undetectable level.

Carrier test using dropping technique

Table 2 shows the results of carrier tests. Each of the plastic carrier was inoculated with 100 µL of AIV or IBV suspension, containing 0.5% or 5% of FBS. The concentration of liquid ClO₂ was 100 ppm. With dW₂ treatment for 5 min, lower than 2 log reduction of virus titers from the original virus titers were observed. These results indicate that 5 min of incubation at RT did not bring about an effect on the virus titer. By contrast, ClO₂ could inactivate AIV within 1 min and IBV within 3 min on dry plastic carriers to the detection limit (<1.90 log₁₀TCID₅₀/mL and 2.10 log₁₀PFU/mL, respectively), which means ClO₂ was the key factor for the reduction of virus titer.

Table 2. Virucidal activity of 100 ppm of chlorine dioxide (ClO₂) toward avian influenza virus (AIV) and infectious bronchitis virus (IBV) containing 0.5 and 5% fetal bovine serum (FBS) on carriers with the wiping and dropping techniques.

Virus			AIV		IBV
FBS (%)			0.5	5.0	0.5	5.0
Control			7.08 ± 0.18	7.58 ± 0.07	6.86 ± 0.03	6.63 ± 0.01
CLOX-100a	Dropping-carrier	1 min	<1.90 ± 0.00	<1.90 ± 0.00	2.62 ± 0.43*	2.90 ± 0.47*
		3 min	<1.90 ± 0.00	<1.90 ± 0.00	<2.10 ± 0.00	<2.10 ± 0.00
		5 min	<1.90 ± 0.00	<1.90 ± 0.00	<2.10 ± 0.00	<2.10 ± 0.00
	Wiped-carrier	30 secb	1.98 ± 0.07*	2.40 ± 0.12*	<2.10 ± 0.00	<2.10 ± 0.00
	Wiping-sheet	1 minc	<1.90 ± 0.00	<1.90 ± 0.00	<2.10 ± 0.00	<2.10 ± 0.00
dW2	Dropping-carrier	5 min	5.53 ± 0.09	6.03 ± 0.09	5.90 ± 0.19	5.65 ± 0.17
	Wiped-carrier	30 sec	2.40 ± 0.20*	4.53 ± 0.18*	2.67 ± 0.35*	2.32 ± 0.26*
	Wiping-sheet	1 min	6.15 ± 0.18	6.57 ± 0.07	6.06 ± 0.39	5.93 ± 0.07

^a Chlorine dioxide liquid at 100 ppm of total chlorine. ^b Carriers were wiped for 30 sec, and immediately transferred into a stomacher bag containing inactivation solution. ^c After 30 sec of wiping, the sheets were kept for 30 sec. Figures are shown as mean ± SE from replicated 3 times. Viral titer was measured for AIV as log₁₀ TCID₅₀/mL, and for IBV as log₁₀PFU/mL. Single asterisk indicates effective viral reduction (≥3 log₁₀TCID₅₀/mL or log₁₀PFU/mL). Viral titer <1.90 log₁₀TCID₅₀/mL and <2.10 log₁₀PFU/mL indicates the virus was inactivated to undetectable level.

Carrier test using wiping technique

Table 2 shows the result of carrier test using the wiping technique. For both AIV and IBV, no virus was detected on wiping sheets after 1 min (30 sec of wiping and 30 sec of reaction). For AIV, viruses on the carriers were effectively inactivated, showing a higher than 3 log reduction of virus titer, even in the 5% FBS. Moreover, the IBV titer after 30 sec of wiping with ClO₂−absorbed rayon sheet was lower than the detection limit. These results indicate that liquid ClO₂ is effective for wiping disinfection, affecting the viruses not only on surfaces but also on the wiping sheets.

Gas test

Table 3 shows the result of the gas test. The concentration of ClO₂ gas inside the box was kept around 1,500 to 2,000 ppb by the generator containing 50 ppm liquid ClO₂. Figure 3 shows the concentration of ClO₂ gas inside the test box during one of the experiments. IBV suspension containing 0.5% of FBS was sprayed into the air using a sprayer (Aerial Mist, Aelph Co., Ltd., Tokyo, Japan). After 3 and a half min of an exposure of sprayed IBV to chlorine dioxide gas for a few sec, the virus titer was reduced by 94.2% compared with the pre-control titer. Comparing the virus titers of gas-treated and pre-treated by Tukey test, it was shown that the reduction was statistically significant (P<0.01). Comparing the virus titers of dW₂ control and pre-treated control, there was no statistical significance observed. Therefore, it is certain that the viruses were inactivated in air by chlorine dioxide gas within a few sec, rather than on the rayon sheets.

Table 3. Virucidal activity of chlorine dioxide (ClO₂) gas toward aerosolized infectious bronchitis virus (IBV) containing 0.5% fetal bovine serum (FBS) within a few seconds.

	Concentration (ppb)a	Post	Preb	RF (%)c
ClO2 gas	1,500–2,000	3.57 ± 0.19	4.81 ± 0.14	94.25
dW2		4.83 ± 0.11	NTd	NCe

Figures are shown as mean ± SE from replicated 3 times. Viral titer was measured for IBV as log₁₀ PFU/mL. ClO₂ gas generator was contained with dW₂ instead of ClO₂ liquid. ^a Concentration of ClO₂ gas inside the box during the experiment was recorded and shown in Fig. 3. ^b ClO₂ gas was generated before spraying IBV (6.22 ± 0.07 PFU/mL). ^c Reduction factor (RF) was calculated by comparing the titer of pre-control and that of post treatment virus. ^d Not tested. ^e Not calculated.

Fig. 3.

Chlorine dioxide gas concentration inside the test box during the gas test. ^a Since chlorine dioxide gas concentration could be only measure up to 1,999 ppb, the concentration over 1,999 ppb was recorded as 1,999 ppb. ^b Pre-treated control. Chlorine dioxide gas was generated before spraying infectious bronchitis virus (IBV).

DISCUSSION

Both IBV and AIV cause large economical losses to poultry industry. The key concerning IBV is its exceptional infectivity, and its spread by aerogenic transmission via infected chickens, or contaminated equipment and materials [9]. Although IBV infection does not cause a fatal disease, it can be considerably harmful for chicken farmers, since the infection is known to cause decreased productivity [4, 29]. Currently, live attenuated vaccine is widely used to prevent IBV infection [33]. However, the extreme variability and rapid evolution of IBV have been obstacles to controlling the infection by vaccines only [5]. On the other hand, the spread of infection of highly pathogenic AIV (HPAIV) is not prevented by vaccination but by culling all of the chickens in HPAIV positive farms in Japan. In 2022 to 2023 season, the number of chickens culled due to outbreaks of highly pathogenic avian influenza (HPAI) has already reached over 17 million. The important measure to prevent and control HPAI outbreaks is to improve sanitation, since one of the main transmission risks of AIV is the poultry supply chain [37]. Furthermore, surface disinfection is crucial since previous study has revealed that viruses can survive on surfaces for days, or even months [16, 19]. In fact, a previous study has shown that AIV was detected on various surfaces in AIV positive farms [17]. In addition, one study has shown that AIV was detected inside, outside of, and downwind from infected duck and chicken facilities in France [26]. To prevent the infections of IBV and AIV in poultry farms, it is important to treat the environment, such as surfaces and air, using proper disinfectants with adequate concentration and in an appropriate way to effectively reduce major or residual viral contamination [2, 8].

From the results of the suspension tests, we can expect that low concentration of liquid ClO₂ is capable of sufficiently inactivating both AIV and IBV in water. For suspension test and carrier test, virus suspension which contains 0.5% or 5% FBS was used to simulate the organic situation of poultry farms. From previous studies, it is known that the presence of organic matters does reduce the virucidal effects of chlorine−based disinfectants [12, 14]. Unlike these studies, liquid ClO₂ in 10 ppm showed an adequate virucidal activity even in the presence of 5% FBS. This indicates, notably, that chlorine dioxide is less effected by organic materials, as compared to other chlorine-based disinfectants.

In the carrier test, the dropping and wiping techniques were performed to evaluate the virucidal activity of liquid ClO₂ against viruses on dry surfaces when applied in different forms. By the dropping technique, viruses on carriers were inactivated to the detection limit within 1 min and 3 min for AIV and IBV, respectively, in the presence of 5% FBS using 100 ppm liquid ClO₂. Furthermore, after performing the wiping method, an adequate reduction of viruses in both carriers and rayon sheets was observed. These data show that liquid ClO₂ has a remarkable potential to be used as a disinfectant towards contaminated surfaces.

In the gas test, ClO₂ gas exhibited strong virucidal activity against IBV in air within a few seconds. Sprayed IBV was collected onto the rayon sheets placed in a distance of 53 cm from the sprayer. Comparing the virus titer of pre-treated control and gas treatment, the reduction rate was 94.2%. By performing a Tukey test, statistical significance was observed between the titers of pre-treated and gas-treated virus. This indicates that ClO₂ gas could effectively inactivate IBV in air within a few seconds.

Through our experiments, we have provided new insights into the prevention and control concept of AIV and IBV infections in poultry farms. Currently, NaOCl is mainly used as a chlorine-based disinfectant in poultry farms. Due to its poor persistence as a consequence of its high volatility, NaOCl is used to disinfect drinking water and slaughtered chickens [27]. While it is regarded to have a strong virucidal activity, there are some disadvantages which are noted above in the introduction. On the other hand, though, ClO₂ produces less trihalomethane compared to other chlorine-based disinfectants, due to its incapability of developing halogenation reaction [31]. In a previous study, ClO₂ has been shown to be effective against non-enveloped viruses on surfaces, as well [30]. In addition, the present study shows that ClO₂ is not only effective against viruses in water and on surfaces, but also in the air, even with the presence of organic matter. Therefore, ClO₂ has good potential to become a new chlorine–based disinfectant for poultry farms.

From the results of the present study, the introduction of ClO₂ is apparently beneficial for poultry farmers. Yet, further studies are required to put it into practice. In the previous study, the virus used for the gas test was IBV since it is famous for its high infectivity through air. However, the mode of AIV air transmission −particularly, yet not merely, in reference to HPAIVs−is fairly evident, in that there are various studies demonstrating such feasibility by air samplings [3, 26, 38]. Therefore, a similar experiment using AIV should be performed to test the effect of ClO₂ gas against AIV in air, as well.

This research aimed to evaluate the efficacy of ClO₂ in liquid state and in gaseous state for preventing and controlling outbreaks of avian influenza and avian infectious bronchitis. From the results of suspension tests and carrier tests, it stems that ClO₂ in liquid state is effective against both viruses in water or dry surfaces, even with the presence of organic materials (FBS). From the result of the gas test, it stems that ClO₂ in gas state can effectively inactivate IBV in air within a second in the presence of FBS. On the whole, these findings indicate that ClO₂ can be used resultfully as a new virucidal agent against those two destructive diseases in poultry farms.

Potential of Conflicts of Interest

The authors have nothing to disclose.