DISINFECTION OF AIR AND DUST IN FATTENING HOUSES FOR CmCKENS BY LACTIC ACID AEROSOL

Fi§er A.: Disinfection of Air and Dust in Fattening Houses for Chickens by Lactic Acid Aero~ol. Acta vet. Brno, 47,1978: 173-183. In laboratory trials, lactic acis administered in form of aerosol in doses of 720-787 mg/m3 devitalized Pseudomonas and E. coli strains ~ithin 30 minutes, Streptococcus strains within 48 hours following the treatment. In trials carried out in housings with chickens kept on bedding, amounts Qf 1,535 mg lactic acid pel 1 mS were administered on days 15 and 25 respectively, and, of 420 mg/m3 on days 29 and 51 respectively. Decreased air contamination was registered with microbes growing on meat-peptone agar, mannitol agar with 7.5 % NaCI and Sabouraud agar till day 29 only, with microbes on Endo agar even on day 51 of a~e of the chickens. After administration of aerosol at the age of 15 to ~1 days, the number of microbes per gram dust decreased with microbes growing on all substrates used. Two different patterns of continual air disinfection by lactic acid were tested in fattening houses for chicks. Improved state of health was achieved with both of them, while the first pattern only resulted in higher glin of body mass. Pattern I showed a gain by 4 g per chicken higher and pattern II by 4 g/chicken lower than the blank turn of chickens. The differences were of no economical significance. With both programs, the mean consumption of lactic acid was 90 mg/day. With program I, there was no difference in microbial contamination of dust in experimental and control houses. Aerosol disinfection, lactic acid, fattening of chickens, bedding. Being an intermediary product of metabolism, lactic acid belongs to the preparations convenient for continual disinfection of byres with animals housed during its administration. According to Va§kov and Astafjev (1951) in Janikova (1970), vapour of lactic acid in the concentration of200 mg/m3 air showed no toxic effect on laboratory anim'tls even when systematically administered for several months. In common spaces for humans, the doses recommended for disinfection of air range around 10 mg/m3 (Karpuchin 1963). Doses of approximately 50 mg/m3 were administered in animal housings (Jarnych 1972; Vorobjev 1973) and, for food storehouses, MatyU (1958) recommended 95-150 mg/m3• The present report describes results of testing disinfection by lactic acid in laboratory trials and in fattening halls for chickens on bedding. Materials and Methods Laboratory trials were carried out in an airtight fume cupboard of 1.2 m 3• Solutions of lactic acid were sprayed by means of the apparatus Mistral forming a comparatively fast sedimehting aerosol. Carriers with microbial cultures on Petri dishes were placed on the bottom level of the fume cupboard and kept there for 30 minutes or 48 hours respectively after exposure to spraying. Then the carriers were immerged into undiluted phosphate buffer in order to interrupt the action of the

Sabouraud agar till day 29 only, with microbes on Endo agar even on day 51 of a~e of the chickens.After administration of aerosol at the age of 15 to ~1 days, the number of microbes per gram dust decreased with microbes growing on all substrates used.
Two different patterns of continual air disinfection by lactic acid were tested in fattening houses for chicks.Improved state of health was achieved with both of them, while the first pattern only resulted in higher glin of body mass.Pattern I showed a gain by 4 g per chicken higher and pattern II by 4 g/chicken lower than the blank turn of chickens.The differences were of no economical significance.With both programs, the mean consumption of lactic acid was 90 mg/day.With program I, there was no difference in microbial contamination of dust in experimental and control houses.Aerosol disinfection, lactic acid, fattening of chickens, bedding.
Being an intermediary product of metabolism, lactic acid belongs to the preparations convenient for continual disinfection of byres with animals housed during its administration.According to Va §kov and Astafjev (1951) in Janikova (1970), vapour of lactic acid in the concentration of200 mg/m 3 air showed no toxic effect on laboratory anim'tls even when systematically administered for several months.In common spaces for humans, the doses recommended for disinfection of air range around 10 mg/m 3 (Karpuchin 1963).Doses of approximately 50 mg/m 3 were administered in animal housings (Jarnych 1972;Vorobjev 1973) and, for food storehouses, MatyU (1958) recommended 95-150 mg/m 3 • The present report describes results of testing disinfection by lactic acid in laboratory trials and in fattening halls for chickens on bedding.

Materials and Methods
Laboratory trials were carried out in an airtight fume cupboard of 1.2 m 3 • Solutions of lactic acid were sprayed by means of the apparatus Mistral forming a comparatively fast sedimehting aerosol.Carriers with microbial cultures on Petri dishes were placed on the bottom level of the fume cupboard and kept there for 30 minutes or 48 hours respectively after exposure to spraying.Then the carriers were immerged into undiluted phosphate buffer in order to interrupt the action of the lactic acid, and, after 10 minutes transferred to meat-peptone broth or, in the case of E. coli strains, to Chejfec substrate.During exposure, the carriers were protected by a layer of 2% agar.From half of them the protective layer has been shaken off either in the liquid substrate or in the phosphate buffer.Results were read after 48 hours of incubation at 37°C.
The microbial strains used were fowl strains of E. coli, Staphylococcus, Pseudomonas and Bacillus from the collection of the Institut de pathologie aviaire I.N.R.A. at Nouzilly, C.RZ.V. Tours, France.After 24 hours of growth on BHI gelatine broth, the E. coli strain had a mean of 9.4 x 108, the Staphylococcus strain 9.1 x lOs, the Pseudomonas strain 1.2 x 10 8 and the Bacillus strain 3.2 x 10' microbes/ml substrate.Resistance against phenol after 5 and 10 minutes (+, -) corresponded to dilutions 1 : 109 with E. coli and Staphylococcus, 1 : 154 with Pseudotnonas and 3 : 4 with Bacillus.The strains E. coli, Staphylococcus and Bacillus tested by the method of Valikov (1952) showed to be thermoresistant to temperature of 60 DC for 35 minutes and to survive the following 6 days.Thermoresistance of Pseudotnonas has nct been examined.
For each microbial strain, five sterile filter paper discs of different shape were placed on a Petri dish.Volumes of 0.05 ml of 24 hrs broth cultures diluted up to 10 -, were administered on each paper carrier.After drying in the incubator, one half of the carriers with the microbes tested were covered by a layer of 2 % meat-peptone agar.As soon as the agar cover congelated, all paper carriers on Petri dishes to be examined were placed in the fume cupboard, while control paper carriers were left in the laboratory.
The field trial with aerosol of lactic acid was carried out in one division of a fattening hall for 3,760 chickens, having a space of 755 m 3 , and belonging to the farm Raspach near Tfebon.The aerosol of particles around 60 I' was produced by two fogging devices Klimatex.The experiment was performed in the summer period.In the course of 6 -7 hours of discontinual aerosol administration the behaviour of chickens.was under permanent observation, and, the ventilation was kept permanently operating to maintain temperature and humidity within tolerable limits.Consequently, the total amount of lactic acid rose to 6.2 g/m3 with chickens aged 15 and 25 day<1, and to 3.6 g/m3 with chickens aged 29 and 51 days.In proportion to intensity of ventilation, the concentrations achieved with every administration were 307 mg/m 3 lactic acid with chickens of 15 and 25 days, and, 84 mg/m 3 with chickens of 29 and 51 days.With five discontinual administrations in the course of disinfection, there was a total of 1,535 mg/m 3 and 420 mg/m3 respectively of lactic acid exerting influence on air and dust in the division with chickens.
Air samples were collected on 5 posts at the.level of bedding prior to disinfection and one hour after the last administration of aerosol.The substrates used were meat-peptone agar, Endo agar, mannitol agar with 7.5 % NaCl and Sabouraud agar.The mean number of microbes per 1 m 3 air was calculated by the formula of Spurny et at.(1961).
Simultaneously, 2-4 samples of dust were collected from the surface of electric hens, ve.ntilators and heaters.They were wiped with sterile paper pads and inserted into sterile test tubes.In the laboratory, the dust samples were processed by the method of successive diluting with saline of pH 7.0, and, 0.1 ml of each dilution were inoculated indifferent substrates.Cultures on meat--peptone agar and Endo agar were kept for 24 hours, cultures on.mannitol agar with 7.5 % Nael for 72 hours in the incubator at 37°C, cultures on Sabouraud agar for 5 days at room ~mperature.Then colonies were counted and numbers of microbes/I g dust calculated.
Another testing of lactic acid aerosol was carried out in the course of two fattening turns in double-halls for chickens kept on bedding at Zidlochovice.The capacity of the halls was 10,000 chickens each.The halls were equipped with the device Funki and environmental conditions were controled automatically.Fodder for both feeding lines was supplied from a common storage tank.One hall was selected for the experiment, the second served for control.Chickens were delivered to both halls from one hatchery at the same time.
During the first tum, chickens of 14 to 50 days of age vere housed in the halls.Three fogging devices Klimatex were installed in the experimental hall.With chickens aged 14 and 15 days:, 1,350 mg/m 3 lactic acid were sprayed each day.During the following period of 34 days, 90 mg!m 3 were administered daily.The Klimatexes were operated 6 times for 30 minutes daily while the ventilators were swit.::hedoft'.During the second tum, lactic acid was sprayed as soon as the first day of life ofthe chickens.From day 1 to 7, the amount sprayed was 45 mg/m 3 , from day 8 to 49, it was 90 mg/m 3 • The state of health of chickens was under control by post-mortem examinations of dead birds, by inspection in the slaughterhouse at Modfice, by examination of a part of t-lood sera from slaugh tered chickens for precipitating antibodies against adenoviruses by means of the FA V -f (CELO) antigen, and, by evaluation of mortality and growth rates in the course of fattening.
With the first turn, samples of dust were collected from equipments in both halls, and, examined by the method of successive diluting for quantity of microbes growing on MPA, Endo agar, mannitol agar with 7.5 % NaCl, an4 on Czapek-Dox agar.' """' " ..n
From Tab. 1 it is evident that all strains protected by agar layer survived 30 minutes and 48 hours following disinfection in spite of the high doses of 720 to 787 mg/m3 of lactic acid sprayed.Without protection, Pseudomonas strains only were devitalized, E. coli partly, and, after 40 hours Staphylococcus also.The Bacillus strain remained resistant to the aerosol of lactic acid.From Fig. 1 it can be seen that disinfection of air in the hall by lactic acid aerosol administered in the large amounts of 1.535 mg/m 3 and 420 mg/m3 was efficient against microbes growing on MPA, mannitol agar with salt and Sabouraud agar till 4 weeks only (chickens aged 29 days).With microbes growing on Endo agar, their quantity decreased successively (1.2 X 10 3 , 7.7 X 10 3 , 7.8 X 10 3 , 2.4 x 10 3 ) from day 15 to day 51.Compared with the numbers prior to disinfection and at 7-8 weeks (chickens 51 days of age), more than 60 % of microbes were devitalized by lactic acid aerosol.Fig. 2 illustrates that at day 51, microbial contamination of air was substantially higher than at days 29, 25, and 15, but, disinfection by aerosol of lactic acid resulted in spite of that in devitalization of the microbes examined.Tab. 2 demonstrates that 7.3 % of blood sera showed antibodies against adenoviruses in the control hall while no positive reactions were registered in the experimental hall (III).Neither were observed in the slaughterhouse pathological changes due to leucosis, Marek's disease, mycoplasmosis or other diseases (II).
Working indicators were less convincing, though mortality was lower and body mass gain a little higher than in the control hall (IV).
From Tab. 3 it can be seen that the lactic acid administered ha:l no effect on the number of microbes/g dust.Obviously the administered dose of 15 mg/m3 (90 mg/6 m 3 ) which sedimented on the surface of equipments, displayed no dis-  As it results from Tab. 4, most indicative was the effect of lactic acid aerosol when the presence of precipitating antibodies against adenoviruses was tested in blood serum samples.Here again, there were fewer positive reactions in the experimental hall than in the control hall (III: 17.5 %: 32.1 %).Inspection of slaughtered chickens also showed a better situation in the experimental hall (II) than in the control hall (1.79 %: 2.08 %).Nevertheless, body mass gain was higher with controls (1.42 kg) than with chickens from the experimental hall (IV: 1.38 kg).

Discussion
Laboratory trials concerning the effect of lactic acid on 24 hrs microbial cultures already showed that even the comparatively high dosis of 720-787 mg/m3 devitalized only microbes with the level of resistance of Pseudomonas and E. coli strains.By longer exposure in unprotected environment, microbial species on the level of resistance of Staphylococcus were devitalized as well.From Tab. 1 it is also evident that dilutions of 4 % were less effective than dilutions of 0.4 %.With 0.4 % dilutions administered, the amount of lactic acid was by 50-60 mg higher, the volume of dilution was 10 times larger and so was the amount of aerosol sedimented on the bottom of the exposed Petri dish containing the microbes tested.This appears to be the decisive factor when Staphylococcus strains without protective agar layer were devitalized after 48 hrs exposure following administration of aerosol.The conclusion is supported by results with 5 % and 7.5 % dilutions of lactic acid, not recorded in the Table .The amounts of 833.3 mg and 2.108 mg/m3 respectively resulted after 30 minutes of exposure in the first case only in devitalization of the Pseudomonas strain and that with the highest dilution of 10-4 , and in the second case, in devitalization of Pseudomonas and E. coli strains with dilutions of 10-4 to 10-2 • Microbial species protected by an agar layer, could not be devitalized after 30 minutes of• exposure, regardless to the degree of dilution, as had been demonstrated in supplementary trials by FiSer (1977).
Results of the field trial are illustrated on Fig. 1 and 2. A partial decrease of microbial contamination of air and dust could be registered not earlier than after 5 separated administrations of aerosol sprayed in comparatively large amounts of 1.535 mg and 420 mg/m 3 respectively.The term "partial decrease" is to be underlined since effectivity was registered in per cent, and consequently, the decrease in number of microbes ranged within one mathematical order.Thus, even after disinfection by aerosol, the microbial contamination of air and dust in the hall with fattened chickens remained on a high level when compared with values noted prior to disinfection and at different days of examination (days 15, 25.,29.and 51. of age of chickens).In addition, dust samples wiped from the surface of electrical hens and heaters after disinfection were wet and pasty so that a prolonged effect of sedimented aerosol on the microflora of the secondarily fallen dust could be assumed.The best cumulative effect was observed after the third disinfection (at the age of 29 days of the chickens housed).Already prior to this disinfection, sporulates only were grown on MP A from dust, and, the number of microbes on mannitol agar with salt and Sabouraud agar was by two mathematical orders lower than with chickens aged 15 days.Neither before nor after this disinfection, microbes from dust were grown on Endo agar (Fig. 2).Microbial contamination of the bedding which is the primary source of dustiness, was not examined presently.However, experience with dynamics of microbes in bedding in the course of fattening chickens (FiSer 1977), and, with difficulty in disinfecting bedding by gaseous formaldehyde (FiSer 1978), permits to assume that aerosol of lactic acid has no effect on the contamination of bedding.From results reported by Cole et al. ( 1968) the conclusion can be made that sedimented lactic acid exerts a favourable effect on the organism of chickens.In agreement with the results in Fig. 1 are also the results described by Devos (1971) who achieved decrease in airborne microflora not longer than to the fifth week of life of chickens, when different preparations were sprayed in halls for fattening chickens.Unlike ours, his chickens had a higher final body mass while their feed consumption index was lower.
From our two field trials, only chickens in the first one were heavier than controls to the end of the fattening turn.In both trials, nevertheless, continual disinfection by aerosol oflactic acid resulted in an improved state of health of chickens.Their mortality was lower, few blood serum samples showed the presence of precipitation antibodies against adenoviruses, less pathological changes were detected by inspection in the slaughterhouse.The importance of lactic acid aerosol for controlling infectious laryngotracheitis and for improving generally the state of health in poultry has been emphasized by J arnych (1972).Thus, disinfection by lactic acid in the form of aerosol is a recommendable procedure but, according to our present results, a higher dose than 90 mgjm 3 and interrupted administration have to be considered.In order to improve the effect on the microflora of air and dust, amounts of 300-400 mgjm 3 appear to be necessary.

Fig. 1 .
Fig. 1.Microbial contamination of 1 m 3 air in the fattening hall for chickens on bedding prior (100 %) and following (X %) disinfection of air by aerosol of lactic acid.x-axis:age of chickens in days y-axis: numbers of microbes (in %).100 % corresponds to values prior to disinfection which are noted above the columns in the figure++ disinfection carried out without collecting samples of dust + Fig. 2.

Table 1
EfI'ect of disinfection by lactic acid aerosol in

Table 2
State of health aDd working Indicators.TurD 1 -fatteD1q of chic:k_

Table 3
Microbial contamination of 1 II dust in the experimental and control haD.

Table 4
State of health and workiDtr lnclicators.Turn 2 -fattening of chickens I. Post-mortem: In consequence of experience from turn 1 no post -monem examination carried out II. Inspection in slaughterhouse:.