Avian Pathology

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The survival of infectious bronchitis (ib) virus in

water

F.T.W. Jordan & T.J. Nassar

To cite this article: F.T.W. Jordan & T.J. Nassar (1973) The survival of infectious bronchitis (ib)
virus in water, Avian Pathology, 2:2, 91-101, DOI: 10.1080/03079457309353787

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Avian Pathology, Vol 2, No. 2, 91—101 1973.

THE SURVIVAL OF INFECTIOUS BRONCHITIS (IB)

VIRUS IN WATER

F.T.W. JORDAN and T. J. NASSAR,*

Sub-Department of Avian Medicine,

University of Liverpool Veterinary Field Station,
»Leahurst«, Neston, Wirral, Cheshire, U. K.

SYNOPSIS

Observations on the survival of infectious bronchitis virus in 3

mains waters showed that it survived for a much shorter period
in one of them which contained a relatively high content of
copper (0.2 mg/1).
Other factors adversely affecting survival were 5 ppm of free
residual chlorine and increase in temperature over the range
of 20-35°C.
Survival of virus was prolonged in the presence of 0.1% w/v
powdered skim milk (PSM), 1,0% w/v poultry mash, 1,0% w/v
fresh poultry droppings, rust and 0,01% sodium thiosulphate. In
part this protection might have been against the high copper
content of the water used but 0.1 % PSM protected also against
the combined effect of a temperature of 35°C and 5 pmm chlorine.
Factors which seemed to exert relatively little effect on virus
survival were the nature of the container, whether of poly-
styrene, galvanized iron or glass, the pH range of 7 - 9 and
total hardness of the water.

INTRODUCTION

The administration of live virus in the drinking water of poultry is

a common method of vaccination against Virus diseases. Since the
stimulation of protection seems to be associated with adequate
quantities of live virus (Gentry and Braune, 1970) its longevity in
drinking water and factors which might influence this are important.
Cunningham and Stuart (1947) and Raggi (1958) observed the effect
Received 23 Novembar 72.
* Present Address — T. J. Nassar, P.O. Box 2395, Amman, Jordan.
92 F. T. W. Jordan and T. J. Nassar

of pH on the survival of IB virus in water, Buthola (1956) examined

its survival in distilled water and a variety of suspending media at
—35°C. Hopkins (1967) varied the nature and concentration of electro-
lytes and Gentry and Braune (1970) measured the survival of virus
in the presence of disinfectants and skim milk.
The following is a report of a more comprehensive (investigation into
the effect of some of the factors thought most likely to influence
the survival of IB Virus in the drinking water of poultry.

MATERIALS and METHODS

Virus: —Lyophilized Massachusetts strain of vaccine virus*.

Eggs:— Nine day-old embryonated chicken eggs from a specific
pathogen free flock**.

Water, additives and other factors: —

(a) Mains water from three different sources (A, B and C) : — A was
a river derived water, B was derived from an artesian well, and C
was an upland .impounded surface water. They were all chlorinated
but not fluorinated at the respective pumping stations. Water A
was sampled from a tap in the laboratory and B and C at domestic
sources, and in each case after the water had been allowed to
flow fully for at least two minutes. Quantities of no less than 10
litres were drawn in this way at each sampling.
(b) Distilled water: — Single glass distilled water, pH 5.6.
(c) WHO hard water: — Prepared by the addition of 3.04 g of calcium
chloride (CaCl22H2O) and 1.39 g of magnesium chloride
(MgCI26H2O) in 10 I of glass distilled water. The total hardness
was 342 ppm and the pH 5.6.
(d) Phosphate buffered distilled water: — Prepared by the addition
of Sorensen's phosphate buffer 0.1 M to distilled water to give
pH values of 5, 7 lind 9. The range covers the values likely to be
encountered in drînkïng water in the U. K.
(e) Physiological saline: — Prepared by the addition of 8.5 g sodium
chloride to 100 mis distilled water.
(f) Water containers: — The survival of virus i n containers of
polystyrene, galvanized iron and glass was observed using mains
water A.
(g) Chlorine content of water; < 0.02, 0.1, 1.0, 5 and 10 ppm: —
Mains water A as drawn from the tap contained < 0.02 ppm of
chlorine and the other concentrations were obtained by the
addition of the requisite amount of sodium hypo chlorite solution
to 10 I quantities of water A. The concentration of chlorine was
• Bronvirin No. 1 (H120) Glaxo Laboratories Ltd. Greenford, Middlesex.
** Free from infection with the viruses of Newcastle disease, infectious bronchities infectious
laryngotracheitis, Leukoses, Marek's disease, infectious avain encephalomyelitis, adenovirus (Phelps
strain), and also, mycoplasma and Salmonellae.
 Survival of IB virus in water 93

checked in each case by the orthotolidine method.* The range

of 0.2 ppm to 1 ppm is found in drinking water in Britain, while
5 to 10 ppm was an arbitrary assessment of the chlorine which
may be present in equipment and utensils [inadequately washed
after disinfection with a hypochlorite.
(h) Temperature of water; 20, 25, 30 and 35°C: — These temperatures
covered the range to be imet in brooding stock, and were attained
by placing the container in a water bath at the required temper-
ature.
(i) Other additives to mains water A: —
(1) Powdered skim milk** (PSM) was aded to 10 litre quantities
and thoroughly mixed to give 0.1%, 0.25% and 0.5% w/v
concentrations.
(2) Poultry mash: — A commercial chicken starter mash without
antibiotic or coccidiostat was similarly added to 10 I to give
a 1% w/v concentration.
(3) Fresh poultry droppings from an SPF flock were similarly
mixed with water A, to give a 1% w/v concentration.
(4) Rust was prepared by adding 10 g of iron filings to 10 I of
water A in a glass container. After 24 hours at room tempe-
rature the resulting rusty water was decanted for use.
(5) Sodium thiosulphate, 0,01% was prepared by adding 1 g of
sodium thiosuiphate (Na2S2O45H2O) to 10 I of mains water
A. It was used because Yoshida, Shimizu, Yuasa and Koyona
(1969) reported that it prolonged the survival of Newcastle
disease vaccine virus in water.

Conditions of exposure: —
For exposure lyophilized virus was reconstituted in distilled wste r
Using a separate vial of lyophilized virus for each suspending medium
and set of conditions the reconstituted virus was diluted in 10 I of
test medium and thoroughly mixed. One litre was transferred to a
container, 5 ml was immediately sampled for virus titration, and the
remainder was exposed under the conditions required. After periods
of 1, 2, 3, 4, 5 and 6 hours, 5 ml quantities were removed and the
titre of virus determined, Exposure was lin diffused daylight in all
cases. Where the experimental design permitted, mains water A,
because it was the most convenient source of water, and a poly-
styrene container was used, and a temperature of 25°C was kept
constant. The pH, hardness,*** and chlorine content**** were me-
asured in each case at the commencement of each experiment.
* Oxoid Ltd. London S.E.1.
** This described in The examination of Waters and water supplies'. E. Winde Taylor
Churchill, London, 1958, 7th edition.
*** Hardness measured by the Schwarzenbach method.
**** Chlorine content measured by the Ortho-tolidine method.
Both these methods are described in »The examination of Waters and Water Suppliers«
E. Windle Taylor, Churchill, London, 1958, 7th edition.
94 F. T. W. Jordan and T. J. Nassar

The survival of virus was observed under the conditions indicated in

the results.
RESULTS

Survival of virus in mains water from sources A, B and C,

in polystyrene containers at 25°C.
The titres and peniods of survival are given in Table 1. The initial titre
of virus in all 3 samples was very similar but in mains water A the
titre fell rapidly and virus was found in low titre at 2 hours but not at
all at 3. In waters B and C, fall in titre was more gradual and virus was
present up to 6 hours when sampling ceased.

Table 1. The survival of virus in mains water from sources A, B and C in

polystyrene containers at 25°C.

Total Chlorine
Source of Survival and titre: of virus PH hardness content
water (EIDM log,0/0.1m1) ppm ppm

0 1 2 3 4 5 6 (h)
A 4.2 1.8 1.4 0 0 0 0 8.0 135 <0.02
B 3.8 3.5 3.0 2.8 2.6 2.2 2.0 7.6 248 0.05
C 4.0 3.7 3.6 3.2 3.3 3.3 3.2 7.6 30 <0.02

This was a consistent finding in repeated experiments.

Differences in ipH, total hardness and chlorine content are shown in
Table 1.

Survival of virus in mains water A in containers of polystyrene,

zinc-galvanized iron and pyrex* glass at 25"C.
There was little difference but virus survived in higher titre and for
the longest period in glass and for the shortest period in the galva-
nized container (Table 2).

Table 2. Ihe survival of virus in mains water A, in containers of polystyrene, glass

and zinc- galvanized iron at 25°C.

Total Chlorine
Survival and titre of virus pH hardness content
Container (EIDso logl0/0.1rn1) ppm ppm

0 1 2 3 4 5 6 (h)

Poly-
styrene 4.2 1.8 1.4 0 0 0 0 7.6 112 <0.02
Glass 4.2 2.4 1.5 •< 1.0 0 0 0 7.7 108 <0.02
Zince-
galvanized
iron 3.8 2.0 0 0 0 0 0 7.6 113 <0.02

* Pyrex glass — A. Jobling & Co. Ltd., Sunderland, England.

 Survival of IB virus in water 95

The pH, total hardness and chlorine content of the suspension are
shown in Table 2.

Survival of virus in phosphate buffered distilled water at pH,

5, 7 and 9, in polystyrene containers at 25°C.
The pH remained constant throughout for each of the 3 values and
virus survived in each for the period of examination but at pH 7 and 9
there was a less rapid fall in titre (Table 3).

Table 3. The survival of virus in phosphate buffered distilled water at pH values

of 5, 7 and 9 in polystyrene containers at 25°C.

Survival and titre of virus

pH (EUDso log,0/0.1ml)

0 1 2 3 4 5 6 (h)

5 3.5 3.3 2.5 2.3 2.2 1.8 1.8

7 3.5 3.3 3.2 3.0 3.0 3.2 2.8
9 3.3 3.2 3.2 3.2 3.2 2.8 3.0

Survival of virus in distilled water, W.H.O. hard water and

0.85% saline in polystyrene containers at 25°C.
In all 3 diluents (Table 4) the virus survived for at least 6 hours, but
the titre of virus in 0.85% saline fell less rapidly than in either
distilled water or WHO hard water. The pH varied from 5.5 to 6.

Table 4. The survival of virus in distilled water, WHO hard water and 0.85% saline
in polystyrene containers at 25°C.

Total
Diluent Survival and titre of virus pH hardness
(E log,0/0.1m1) ppm

0 1 2 3 4 5 6 (h)

Distilled
water 3.5 3.3 3.0 2.4 1.6 1.5 1.2 5.5 0
WHO hard
water 3.5 3.5 2.5 1.6 1.5 1.5 <1.0 5.6 342
Normal
saline 3.5 3.2 3.2 3.2 3.2 3.2 3.2 6.0 —
96 F. T. W. Jordan and T. J. Nassar

Survival of virus in mains water A containing < 0.02, 0.1, 1.5

and 10 ppm of free residual chlorine in polystyrene containers at 25°C.
At concentrations of chlorine of 1 ppm and below, virus was recovered
at 2 but not at 3 hours (Table 5). At a concentration of 5 ppm it was

Table 5. The survival of virus in mains water A. containing 0.02, 0.1, 1, 5 and 10
ppm chlorine, in polystyrene containers at 25°C.

Free
residual Total
chlorine Survival and titre of virus pH hardness
ppm (EID» Iogio/0.1m1) value ppm

0 1 2 3 4 5 6 (h)

<0.02 4.2 1.8 1.4 0 0 0 0 7.6 108

0.1 3.8 2.0 <1.0 0 0 0 0 7.7 90
1.0 3.7 1.8 <1.0 0 0 0 0 7.7 100
5.0 2.5 1.6 0 0 0 0 0 7.8 96
10.0 0 0 0 0 0 0 0 8.0 96

detected at 1 but not at 2 hours and from water containing 10 ppm

of chlorine it was inot possible to recover virus at all.
The duration of survival and titres of virus in water A containing less
than 0.02 ppm chlorine was the same in other experiments where
virus was exposed under the same conditions (Tables 1, 2, 5, 6, 7, 8.).
In this experiment the pH rose from 7.6 to 8.0 for the mains water
containing < 0.02 ppm chlorine to that containing 10 ppm. The total
hardness was similar throughout.

Survival of virus in mains water A at 20, 25, 30 and 35°C in

polystyrene containers.
At 20°C virus could be recovered after 5 but not after 6 hours. (Table
6). Recovery at 25°C confirmed the earlier observations on survival

Table 6. The survival of virus in mains water A, in polystyrene containers, at

temperatures of 20, 25, 30 and 35°C.

Total Chlorine
Temperature Survival and titre of virus pH hardness content
°C (EID5„ Iog,o/1m1) value ppm ppm

0 1 2 3 4 5 6 (h)
20 3.7 2.7 2.4 1.5 1.0 < 1.0 0 7.6 113 <0.02
25 4.2 1.8 1.4 0 0 0 0 7.6 112 <0.02
30 3.5 1.7 0 0 0 0 0 7.6 104 <0.02
35 3.5 0 0 0 0 0 0 7.6 104 <0.02
 Survival of IB virus in water 97

in this water under these conditions, while at 301C it could be recov-

ered after 1 but not after 2 hours and at 35°C it was recovered imme-
diately after exposure but not after 1 hour.

Survival of virus in mains water A and mains water A together with

various additives in polystyrene containers at 25°C.
The presence of powdered skim milk (PSM) at all concentrations
prolonged the survival of! virus to at least 6 hours (Table 7). With

Table 7. The survival of virus in mains water A and mains water A together with
various additives in polystyrene containers at 25°C.

Total Chlorine
Suspending Survival and titre of virus pH hardness content
medium (EIC>*, Iog,o/0.1m1) value ppm ppm
0 1 2 3 4 5 6 (h)

Mains Water A 4.2 1.8 1.4 0 0 0 0 7.6 112 <0.02

0.1% powdered
skim milk (PSM) 3.7 3.5 3.5 3.6 3.3 2.5 2.8 7.6 84 <0.02
0.25% PSM 3.5 3.2 3.4 3.0 2.2 2.2 2.8 7.6 84 <0.02
0.5% PSM 3.6 3.2 3.0 2.5 2.5 2.3 2.3 7.6 84 <0.02
1.0% poultry mash 3.6 3.5 3.0 3.0 2.5 2.3 2.0 7.3 84 <0.02
1.0% poultry
droppings 3.6 3.5 3.0 3.0 2.5 2.3 2.0 7.3 84 <0.02
Rust 4.0 3.7 3.6 3.5 3.5 3.2 3.0 7.6 81 <0.02
0.01% sodium
thiosulphate 3.5 3.2 1.6 1.5 1.0 < 1.0 0 7.7 86 <0.02

the addition of 1 % poultry starter mash, fresh droppings or rust, the

viability of virus was similarly prolonged. With sodium thiosulphate
(Na2S2O45H2O) at a concentration of 0.01%, virus survived for 5 but
not 6 hours.

Survival of virus in mains water A, in polystyrene containers,

at temperatures of 25 and 35°C, with free residual chlorine at
<0.02 ppm and 5.0 ppm, and with and without PSM.
Table 8 shows the protective effect on the virus of 0.1 % PSM since
even when exposure occurred at 35°C in the presence of 5 ppm of
chlorine the virus survived for at least 6 hours. In contrast, in the
absence of PSM, the virus survived at 35°C fin the presence of as
little as <0.02 ppm of chlorine for less than 1 hour.and at a tempera-
ture of 25 C and <0.02 ppm chlorine for 2 but not 3 hours.
98 F. T. W. Jordan and T. J. Nassar

Table 8. The survival of virus in mains water A in polystyrene containers, at

temperatures of 25°C and 35°C with free residual chlorine at < 0.02 ppm
and 5 ppm and with and without powdered skim milk

Tempe- Total
rature Chlorine PSM Survival and titre of virus pH hardness
°C ppm % 0 1 2 3 4 5 6 (h) pmm

•15 <0.02 — 4.2 1.8 1.4 0 0 0 0 8.0 135

25 <0.02 0.1 3.5 3.2 3.4 3.0 2.2 2.2 2.8 7.6 84
25 5.0 — 2.5 1.6 0 0 0 0 0 7.8 96
25 5.0 0.1 3.2 3.0 2.8 2.8 2.8 2.5 2.5 7.8 100
35 <0.02 — 3.5 0 0 0 0 0 0 7.6 104
S5 <0.02 0.1 3.5 3.0 2.8 2.6 2.5 2.5 2.4 7.7 105
35 5.0 0.1 3.3 3.0 2.8 2.8 2.5 2.3 2.0 7.8 100

DISCUSSION

The choice of the 3 mnains waters in this study wi<s arbitrary but a
surprising feature was the relatively short survival or virus in water
A. At first it was considered that this might have been associated
with hardness or pH but was eventually found to be associated with
its relatively high content of copper (0.2 mg/l.) (Jordan and Nassar,
1971). Thus in assessing the observations on virus survival in water
A throughout these experiments the adverse effect of the high copper
content must be borne in mind.
The prevalence of water with a copper, Content as high as (0.2 mg/l.)
is not known but it is recognised as a possible occurrence parti-
cularly where the water flows through much new copper piping.
The slightly shorter period of survival of virus in the metal container
and the more rapid fall <in virus titre in the metal and polystyrene
container compared with glass might possibly be associated with the
release from these containers of small quantities of substances which
are toxic to the virus or, alternatively the release of protective ions
from the glass.
The progressively shorter period of virus survival with increasing
temperature confirms the observations of Hofstad (1956), Quiroz and
Hanson (1958) and Estola (1966). Limited virus survival with rapid
fall in titre at the higher temperatures have practical implications for
the administration of live vaccine virus in drinking water to young
chicks. Nevertheless in the absence of high quantities of copper the
virus might have survived longer at these temperatures.
Values of pH within the range, 5—9, likely to be encountered in
mains water (in the U. K., do not have a marked influence on virus
survival. Least fall in titre, however, occured at pH 9. This is in accord
with the observations of Raggi (1958) who found little appreciable
difference in titre of Massachusetts strain of IB virus exposed under
 Survival of IB virus in water gg

similar conditions at pH 6.3 to 7.85, and Cunningham and Stuart (1947)

who found that the Beaudette strain of IB Virus at 4°C, survived longest
at pH 8.5 in phosphate buffer but for shorter periods at pH above and
below this.
Hopkins (1967) showed that the inactivation rate of IB virus in water
at constant temperature is related to the type and concentration of
an ions and demonstrated that IB virus was more stable in solutions
of electrolytes than in distilled water. This would explain our ob-
servations of enhanced virus survival in normal saline (0.15M sodium
chloride) over distilled and WHO hard water (0.0027M'Calcium chlo-
ride and 0.0013M magnesium chloride). It seems probable therefore
that other causes of hardness if in adequate amount would similarly
support virus survival.
Chlorine inactivates micro organisms by oxidation and 10 ppm has been
shown to be effective (In the inactivation of a wide range of viruses
such as adeno virus, in less than 1 minute, Coxsackie in 1—3 minutes,
infectious hepatitis in 30 minutes, and poliomyelitis in 10 minutes,
(Trueman, 1971). Our results confirm this and indicate that concen-
trations of 1 ppm and below have little adverse effect on IB virus
survival. In contrast Gentry and Braune (1970) found that as little
as 1 ppm of chlorine reduced the survival of IB virus in distilled water
after 30 minutes at room temperature. However in their experiments,
distilled water alone appeared to have a markedly adverse effect
on virus survival.
Powdered skim milk, poultry mash, fresh poultry droppings, rust and
to a lesser extent 0.01% sodium thiosulphate added to ware A ,at
25°C, all enhanced virus survival. The protection provided may have
been exaggerated because the relatively high copper content in this
water limited survival in the control system to 2 hours.
PSM was protective to virus in mains water A not only at 25°C, but
also at 35°C and even in the presence off5 ppm of chlorine. Gentry
and Braune also (1970) observed the protective effect of PSM for IB
virus in the presence of chlorine or a quaternary ammonium disinfect-
ant. Furthermore Winterfield and Seadale (1956) and Beard (1971)
working with Newcastle disease, and Ellery and Howes (1971) with
infectious Jaryngotracheitis virus, found PSM to be protective to virus
exposed in aqueous suspension. There is much published evidence
for the toxic effect of copper on bacteria, moulds and fungi (Kushner
1971) but not on viruses. However, in mains water A the high copper
content seemed to be a potent virucidal factor but PSM afforded
considerable protection to IB virus under these conditions. There are
several possible explanations for this. Perhaps the most likely one
is the direct combination of copper with the sulphydryl groups of the
protein constituents of the milk. Other explanations include the chel-
ating action of milk on cooper, the coating effect of millk partiels,
displacement of copper by minerals in the milk and perhaps reaction
of copper with phosphates in the milk resulting in the removal of
copper by precipitation. The protective effect of PSM on virus against
high temperature is not understood, but is probably in part associated
with its effect on copper. The organic content of PSM probably rapidly
 F
100 - T. W. Jordan and T. J. Nassar

reduces chlorine and protects the virus from this substance in this
way. It would seem probable that poultry mash and droppings may
enhance virus survival by the same means as PSM. However, Winter-
field and Seadale (1956) record that food and faeces invariably
depressed the survival of Newcastle disease virus in water but in
their observations the environmental temperature was 37°C and
bacterial growth at this temperature might well have been respons-
ible for virus depletion.
The protective effect of rust 6p virus in the presence of copper is
probably associated with the displacement of copper by iron, while
sodium thiosulphate perhaps prolongs survival by simply increasing
molarity although it was recommended by Yoshida, Shimizu, Yuasa
and Koyona (1969) to neutralise residual chlorine.

Acknowledgements
We wish to thank Mr. M. S. Savage for technical assistance and the A. R. C. for
financial help.

REFERENCES

Beard, C. W., (1971). Newcastle Disease Virus: Evaluation of an avirulent enteric

isolate as a viable vaccine. Avian Diseases, 15: 334-342.
+Buthala, D. A., (1956). Some properties of the avian bronchitis virus. Ph. D. Thesis,
Iowa State College.
+Cunningham, C. H. and Stuart, H. O., (1947). Cultivation of the virus of infectious
bronchitis of chiokens in embryonated chicken eggs. American Journal of
Veterinary Research, 8: 209-212.
Ellery, B. W., and Howes, D. W., (1971). lnactivation of infectious laryngotracheitis
virus by disinfectants. Australian Veterinary Journal, 47: 330-333.
+Estola,T., (1966). Studies on the infectious brofliehitis virus of chiokens isolated
in Finland. Acta Veterinaria Scandinavia. Supplementum 18: 1-111.
+Gentry, R. F., and Braune, M. O., (1970). Prevention of virus inactivation during
drinking water vaccination of poultry. Proceedings of XIV World Poultry
Congress, Madrid, 1970, pp 137-183.
+Hofstad, M. S., (1956). Stability of avian infectious bronchitis virus at 56°C. Cornell
Veterinarian, 46: 122-128.
+Hopkins, S. R., (1967). Thermal stability of infectious bronchitis virus in the
presence of salt solutions. Avian Diseases, 11: 261-267.
+Jordan, F. T. W., and Nassar, T. J., (1971). The influence of copper on the survival
of infectious bronchitis vaccine virus in water. Veterinary Record, 89. 609.
+Kushner, D. J., (1971). Influence of solutes and ions on micro organisms. In:
Inhibition and destruction of the microbial cell, pp. 259-283. Edited by
Hugo, W. B., Academic Press, London and New York.
+Quiroz, C. A., and Hanson, R. P., (1958). Physical — Chemical treatment of inocula
as a means of separating and lidentifying avian viruses. Avian Diseases,
2: 94-98.
+Raggi, L G., (1958). A note on survival time of an 'infectious bronchitis virus at
various pH in distilled water. Poultry Science, 37: 1464-1465.
+Trueman, R. J., (1971). The Halogens. In: Inhibition and destruction of the microbial
cell, pp. 137-183. Edited by Hugo, W. B., Academic Press, London and
New York.
+Winterfield, R. W., and Seadale, E. H., (1956). Newcastle disease immunization
studies. I Viability of Newcastle disease virus administered as a vaccine
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Yoshida, K., Shimizu, F., Yuasa, N., and Koyano, H., (1969). Inactivating effect of
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Résumé

Des observations sur la survie du virus de la bronchite infectieuse

dans l'eau de trois conduites principales ont montré qu'elle est bien
plus courte dans celle dont la teneur en cuivre est relativement haute
(0,2mg/1).
D'autres facteurs exerçant une influence défavorable sur la survie
sont: 5 ppm de chlore libre résiduel, et une élévation de la tempéra-
ture de 20 à 35°C.
La survie du virus est plus longue en présence de 0,1 % de poudre de
lait écrémé, de 1 % d'aliment complet pour volaille, de 1 % d'excré-
ments de volaille, de rouille, et de 0,01% d'hyposulfite de soude. On
pourrait penser que cette protection pouvait, en partie, être due à
une action contre le cuivre, mais 0,1% de poudre de lait écrémé
protège aussi contre les effets réunis d'une température de 35°C et
de 5 ppm du chlore.
Semblent avoir peu d'influence sur la survie du virus: la nature du
récipient, qu'il soit en polystyrène, en tôle galvanisée, ou en verre;
une variation du pH entre 7 et 9, et la crudité de l'eau.

Zusammenfassung

Die Bestimmung der Überlebensrate eines Virus der infektiösen

Bronchitis in Leitungswasser von dreierlei Zusammensetzungen
zeigten, daß die Lebensdauer des Virus in Wasser mit einem relativ
hohen Kupfergehalt (0,2 mg/1) viel kürzer war.
Andere Faktoren, die das Überleben ungünstig beeinflußten, waren
5 ppm freies Restchlor und eine Temperaturzunahme in dem Bereich
20 bis 35°C.
Das Vorhandensein von 0,1% Gew./Vol. Trookenmagermilch (TMM),
1,0% Gew/Vol. Hühnerfutter, 1,0% Gew./Vol. frischem Hühnerkot,
Rost und 0,1% Natriumthiosulfat verlängerte die Lebensdauer des
Virus. Diese Substanzen dürften zum Teil als Schutz gegen den hohen
Kupfergehalt gewirkt haben. 0,1% TMM hat jedoch auch gegen die
kombinierte Wirkung einer Temperatur von 35°C und 5 ppm Chlor aus-
reichend geschützt.
Von relativ geringem Einfluß auf das Überleben des Virus waren die
Beschafferheit des Behälters (ob aus Polystyrol, verzinktem Eisen
oder Glas), der Bereich des pH-Wertes von 7 — 9 und die Gesamthöhe
des Wassers.