Avian Pathology

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Cloacal infection with Mycoplasma gallisepticum

and the effect of inoculation with H120 infectious
bronchitis vaccine virus

K.J. Macowan , C.J. Randall & T.F. Brand

To cite this article: K.J. Macowan , C.J. Randall & T.F. Brand (1983) Cloacal infection with
Mycoplasma�gallisepticum and the effect of inoculation with H120 infectious bronchitis vaccine
virus, Avian Pathology, 12:4, 497-503, DOI: 10.1080/03079458308436194

To link to this article: https://doi.org/10.1080/03079458308436194

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Avian Pathology, 12: 497-503, 1983

CLOACAL INFECTIONWITHMYCOPLASMA GALLISEPTICUM

AND THE EFFECT OF INOCULATION WITH H120 INFECTIOUS
BRONCHITIS VACCINE VIRUS
K.J. MACOWAN1, C.J. RANDALL and T.F. BRAND

Ministry of Agriculture, Fisheries and Food

Veterinary Laboratory, Eskgrove, Lasswade, Scotland

SUMMARY
Infectivity of an isolate of Mycoplasma gallisepticum of low virulence
was studied in specific pathogen free chickens from 1 day old until after
the onset of lay. Widespread cloacal infection developed and it was dis-
cussed in relation to the low serological response. The effects produced
by inoculation of H120 infectious bronchitis vaccine virus and coming
into lay were associated with changes in serological response and in-
creased cloacal cultural recovery. The possibility of ascending cloacal
infection giving rise to egg transmission was considered and compared
with other routes whereby ovules may become infected.

INTRODUCTION
Several flocks with persistent low levels of serological reactions to Mycoplasma
gallisepticum by the rapid slide agglutination test (RSAT) but weak or negative reac-
tions by the haemagglutination-inhibition test (HIT) were reported in Pennsylvania
(Mallinson and Rosenstein, 1976). Sahu and Olson (1976) isolated a mycoplasma,
strain WVU907, from such flocks and, more recently, they indicated that the agent
caused only mild clinical signs, gave rise to only low levels of haemagglutination-
inhibition (HI) antibodies and was more resistant to tylosin than typical M. galli-
septicum field strains (Sahu and Olson, 1982). Inoculation of 8-week-old chickens
by multiple routes established infection but spread to in-contact birds was not detected
culturally or serologically. Inoculation by the intra-ocular and nasal routes into 6-
week-old birds vaccinated by eye drop with combined live Newcastle disease and in-
fectious bronchitis vaccine viruses led to respiratory râles. Although no marked râles
developed in contact birds, WVU907 was recovered from two of them and 30% dev-
eloped agglutinating antibodies to M. gallisepticum.
The current investigation follows a preliminary report on the isolation of M. galli-
septicum (isolate M1869/81) from broiler breeder flocks with a low percentage (less
than 20%) of RSAT serum reactions to bothAf. synoviae andM. gallisepticum
antigens (MacOwan et al., 1982a). When sera which reacted by the RSAT toM.
gallisepticum were examined by the HIT, reactions were of low titre or negative.
Received 15 April 1983
Accepted 2 June 1983
1
Present address: Ministry of Agriculture, Fisheries and Food, Central Veterinary Laboratory,
New Haw, Weybridge, Surrey KT15 3NB, England.
498 KJ.MacOwan étal.

This communication reports the infectivity and pathogenicity of our isolate assessed
in specific pathogen free birds infected at 1 day old and kept under observation until
after the point of lay. Particular attention was directed to culture of the cloaca as
several of our field recoveries were from this site.

MATERIALS AND METHODS

Cultural examination
The procedures for isolation of mycoplasma from experimental birds were similar to
those described previously (MacOwan et al., 1982b). To reduce contamination in pri-
mary cultures the medium was modified by the replacement of penicillin with ampi-
cillin at a final concentration of 1 mg/ml.
Inoculum
The broth culture of M1869/81 M. gallisepticum used as inoculum had been derived
by sub-inoculation of a single colony and millipore membrane filtration. This proce-
dure was repeated four times and the culture was also passaged on media free of
bacterial inhibitors (Anon, 1979). The inoculum contained more than 1 x 10 s colony
forming units/ml.
Serological procedures
The methods used for the preparation of antigen, hyperimmune rabbit antiserum, and
for growth-inhibition and immunofluorescence tests have been described previously
(MacOwan et al., 1982b).
For the RSAT commercially produced M. gallisepticum and M. synoviae antigens
(Burroughs Wellcome) were used according to the manufacturers' instructions and,
where stated, similar antigens from Salsbury Laboratories and Intervet were also em-
ployed. The HIT for antibodies to M. gallisepticum was carried out at the Poultry
Department, Central Veterinary Laboratory, Weybridge, Surrey, using the procedures
described by Timms (1967).
Experimental design
Birds were obtained from the laboratory specific pathogen free (SPF) flock of Brown
Leghorns for which repeated serological testing over a period of years has failed to
detect antibody to M. gallisepticum, M. synoviae, reovirus and the viruses of infectious
bronchitis (IB), infectious bursal disease, infectious laryngotracheitis and Newcastle
disease (ND). All infected experimental birds were kept in accommodation with ven-
tilation by filtered air, while the controls were housed separately.
Of 26 chicks selected from the same hatch at 1 day old, six were kept as unexposed
controls and 20 were reared separately as the infected group (Text-fig.1). Within this
group eight were inoculated intranasally with two drops of culture delivered by a 19
gauge needle, five were infected by rubbing a swab soaked in culture on the cloacal
orifice and the remaining seven were kept as in-contacts. At 108 days post inoculation
(p.i.) the 20 birds were divided into two groups of 10 and housed in separate rooms
so that they contained as nearly as possible representative numbers of both sexes ino-
culated by each method (Text-fig.l). One group which contained four cockerels was
inoculated intranasally with 1 x 106 infectiousunitsoftheH120IB vaccine virus
while the other, which included three cockerels, was given no further treatment.
All the birds were bled at 3 weeks of age and weekly thereafter for serological exam-
ination by the RSAT. Serum samples collected on days 70,136,155,186 and 213
were also examined by the HIT.
 Cloacal Mycoplasma gallisepticum and vaccine virus 499

26 chicks
at I day old

controls infected with M1869/81

6 chicks M. gallisepticum
20 chicks

day 108

infected with infectious not treated

bronchitis vaccine further
10 chickens 10 chickens
Tex t:flg. 1, Experim en tal design..

For cultural examination cloacal swabs were taken from all birds on days 35, 57,
140 and 155, when they came into lay. At 177 days of age both the trachea and the
cloaca were swabbed and tissues from these sites were sampled again for culture
when all the birds were killed at 225 days of age. Other tissues cultured at post-
mortem examination included turbinate and testes.
Fertile eggs from all the experimental birds were incubated for 10-14 days before
harvesting for cultural examination. Yolk sac and allantoic fluid from each embry-
onated egg were separately cultured for mycoplasma.

RESULTS
Infection with M. gallisepticum alone
At 35 days of age the organism was recovered from the cloacas of 16 of the 20 birds:
all of those cloacally inoculated, five of the eight intranasally inoculated and six of
the seven in-contacts. However, by day 57, cultural examination was positive for only
three cloacally inoculated birds, one of which was cockerel number 254. By the RSAT
all sera were negative to the Burroughs Wellcome antigens and this result was confirm-
ed when the samples on day 63 and day 70 were also tested with antigens supplied by
Salsbury Laboratories and Intervet. In addition the sera from all birds collected on
day 70 were examined by the HIT and were free of antibodies toAf. gallisepticum.
On day 79 in-contact hen number 248 had a weak RSAT reaction to M. gallisepticum
which persisted throughout the experiment. On day 108, when the birds were divided
into two groups, this bird was amongst those inoculated with vaccine virus. After day
108, in the group given no further treatment, one in-contact hen became a consistent
reactor toM. gallisepticum in the RSAT while the samples from a second cloacally
inoculated bird alternated between weakly positive and negative (Table 1). No clinical
signs were observed during the experiment and no lesions were found at postmortem
examination, nor were cultural recoveries achieved from swabs taken during the ex-
periment or from tissues sampled at postmortem examination.
Infection with M. gallisepticum followed by IB vaccine virus
Following inoculation with IB vaccine virus a further four birds became consistent
500 KJ.MacOwanefaZ.
Table 1. Cultural and serological examination of 10 birds infected at
1 day old with M1869/81 M. gallisepticum and given no
further treatment.

Day of experiment 35 57 70 79-225

Cloacal recovery 8 1 NDa 0
RSAT reactions 0 0 0 2b
HIT reactions ND ND 0 0

a Not done.
b One alternated between weakly positive and negative.

Table 2. Cultural and serological examination of 10 birds infected at

1 day old with strain M1869/81 M. gallisepticum and at 108 days
inoculated with H120 infectious bronchitis vaccine.
Day of experiment 35 57 70 79 136 140 155a 177 186 213 225
Cloacal cultural 8 2 NDb ND ND 1 4 6(0c) ND ND 7(0d)
recoveries
Positive RSAT 0 0 0 1 5 ND 6 8 8 8 8
reactions
Positive HIT ND ND 0 ND 0 ND I e
ND l+5 e
3 ND
reactions

a Point of lay.
b Not done.
c Results of trachéal swab culture.
d Results of turbinate, upper trachea and testes cultures.
e Inhibition resulting in an atypical reaction.

reactors by day 136 (Table 2). At the point of lay (day 155) one other bird had
developed a serum agglutination reaction and two of the remaining four birds be-
came consistent reactors by day 177. Occasional weak RSAT reactions were detec-
ted to M. synoviae antigen at irregular intervals but only in the sera which reacted
more strongly withM. gallisepticum.
At the point of lay serum samples were examined by the HIT also and an atypical
inhibitory effect or a 'ring' of cell deposit was observed in one sample at a titre of
1/10. By day 186 the sera of five birds reacted in the same atypical manner at titres
of 1/5 to 1/10 while a typical positive reaction was observed in the serum of a sixth
bird at a titre of 1/5 to 1/10. A further test on day 213 showed that the typical re-
action had persisted and two further birds, which had 'ring' reactions in the previous
test, had become typical reactors at a titre of 1/5.
When all the birds were swabbed, on day 140, cloacal infection was detected in
cockerel 254 only. On day 155 cloacal recoveries were achieved from this bird and
three hens (number 248, an intranasally and a cloacally inoculated bird), while by
day 177 isolates were obtained from the same site in two further birds. Cockerel
number 254 was the only bird to be culturally positive throughout the experiment
and one of the only two birds in the group inoculated with vaccine virus which failed
to develop a consistent RSAT response to M. gallisepticum.
 Cloaca] Mycoplasma gàllisepticum and vaccine virus 501

No clinical signs developed and no lesions were observed at postmortem examination

although mycoplasma were recovered from cloacal specimens of seven of the 10 birds.
All the mycoplasma recovered reduced the pH of broth cultures and were sensitive
to 1.5% digitonin. Serologically the hyperimmune serum to M1869/81 prepared in
rabbits combined strongly with colonies in the indirect epi-immunofluorescence test
and specifically inhibited colony growth.
The allantoic fluid and yolk from 31 embryonated eggs laid by the group inoculated
with vaccine virus and from 34 eggs laid by the group given no further treatment were
individually examined culturally but no mycoplasma were recovered.
Control group
The six control birds remained clinically, serologically and culturally negative through-
out the experiment and no mycoplasma were recovered from any tissue following
postmortem examination.

DISCUSSION
Cloacal infection with M1869/81 was established in 16 of the 20 experimental chicks
including those challenged cloacally, intranasally and by contact. For the first 10
weeks no serological response was detected by the RSAT using commercialM. galli-
septicum antigens or by the HIT. Later in the experiment few serological reactions
were detected in birds which were not inoculated with vaccine virus. Only one became
a consistent serum agglutination reactor by day 136 and another gave irregular results
alternating between positive and negative.
This poor serological response was not unexpected in view of the earlier studies in the
U.S.A. with strain WVU907 (Mallinson and Rosenstein, 1976; Sahu and Olson, 1976).
They reported an improvement in sensitivity of the RSAT with antigen prepared to
the infecting organism but because of the low levels of HI antibodies detectable use
of the agar gel precipitin test was recommended (Sahu and Olson, 1982). Variation
in the serological response following infection of chickens with different M. gallisep-
ticum strains is well recognised and particularly evident using the HIT (Lin and Kleven,
1982). In part this may have accounted for the poor performance of the HIT, particu-
larly later in the experiment when eight of the 10 birds inoculated with vaccine virus
had developed RSAT reactions.
Recovery of M. gàllisepticum from the cloaca has been reported following inoculation
of 18-day-old embryonated eggs and after infection of 3-week-old chickens (Amin and
Jordan, 1979; Varley and Jordan, 1978). These authors considered infection at this
site could be a possible source of spread of the agent. In our study there was a high
level of cloacal infection early in the experiment and also latterly in the group inocu-
lated with vaccine virus. These findings suggest that cloacal culture would be a useful
diagnostic procedure in flocks with low levels of RSAT and HIT reactors toM. gàlli-
septicum antigen. Even in infections with more virulent strains, cloacal culture could
prove to be a valuable method of isolation, in addition to culture of the respiratory
tract.
Sahu and Olson (1982) observed serological and cultural evidence of in-contact spread
only when birds were concurrently infected with ND and IB virus vaccines. In con-
trast, prior to inoculation with vaccine virus, the mycoplasma was isolated from the cloa-
ca of six of the seven contact birds in our experiment on day 35. Indeed the organism
persisted in the cloaca without giving rise to detectable agglutinating or HI antibodies
which suggested that at this site infection may have stimulated only low levels of
502 K.J. MacOwan et al.
humoral antibodies.
After intranasal inoculation with the H120 IB vaccine virus there was, within 28
days, a rise in the number of serum agglutination reactors from one to five birds,
although at this time cloacal infection was detected in only one bird. It seemed that
inoculation with vaccine virus did not encourage further transmission of M1869/81
but stimulated the existing infection to produce a serological response. From the
group infected with M1869/81 alone there were no cultural recoveries after day 57,
but as this group differed only in freedom from vaccine virus, it is probable a sero-
logical response to M1869/81 would have developed following IB virus infection.
After onset of lay the increased multiplication of M1869/81 in the vaccine virus
infected birds was reflected by an increase in cloacal recoveries and a rise in the num-
bers of RSAT reactors from six to eight. The sexual activity of the cockerel which
had remained positive by cloacal culture throughout the experiment also may have
contributed to this rise in cloacal recoveries and RSAT reactors.
When the birds were killed, eight of the 10 in the group which had received vaccine
virus were consistent serum agglutination reactors toM. gallisepticum antigen and
from seven of these the organism was recovered from the cloaca. No recoveries were
achieved from the respiratory tract. Indeed attempts to demonstrate trachéal infec-
tion after the point of lay (on day 177) were unsuccessful although at this time
eight were RSAT reactors, and cloacal cultures from six birds yielded the organism.
The cloaca proved to be the principal site of infection although the respiratory tract
may have been infected, albeit with so few organisms that they were not recoverable
by the cultural methods employed.
During egg transmission it has been generally considered that the ovule becomes in-
fected in the oviduct from contiguity with affected abdominal air sacs, although bac-
teraemia has been considered as an alternative source of infection (Roberts and
McDaniel, 1967). These authors also suggested that the oviduct might become infec-
ted at mating since M. gallisepticum had been isolated from semen (Yoder and
Hofstad, 1964). In our work the absence of detectable egg transmission associated
with the widespread cloacal infection would seem to favour the first two hypotheses.
However, the long interval between infection and onset of lay may have permitted
development of local immune defences which prevented ascending infection. Where-
as in breeding birds it remains probable that a recently introduced infection with
active proliferation of cloacal organisms could give rise to an ascending infection and
subsequent egg transmission.
Acknowledgements
We are grateful to Mr. J.W. Macdonald and staff of the Avian Microbiology section
for providing the H120 IB vaccine virus, and we thank Mrs. M. Atkinson and Mrs. M.
Bell for technical assistance with mycoplasma culture and serology. We are indebted
to Dr. Janet Thain of the Central Veterinary Laboratory, Weybridge, Surrey, for
the examination of sera by the haemagglutination-inhibition test. Mr. A.R. Hunter is
thanked for constructive criticism and helpful comment in the preparation of this
paper.

REFERENCES
Amin, M.M. and Jordan, F.T.W. (1979). Infection of the chicken with a virulent or avirulent
strain of Mycoplasma gallisepticum alone and together with Newcastle disease virus or
E. coli or both. Veterinary Microbiology, 4: 35-45.
 Cloacal Mycoplasma gallisepticum and vaccine virus 503

Anon. (1979). International Committee on Systematic Bacteriology. Sub-Committee on the

taxonomy of Mollicutes. Proposal of minimal standards for descriptions of new species
of the class Mollicutes. International Journal of Systematic Bacteriology, 29: 172-180.
Lin, M. Y. and Kleven, S.H. (1982). Cross-immunity and antigenic relationships among five strains
of Mycoplasma gallisepticum on young Leghorn chickens. Avian Diseases, 26: 496-507.
MacOwan, K.J., Randall, C.J. and Brand, T.F. (1982a). Low levels of Mycoplasma gallisepticum
or M. synoviae agglutination reactors. Veterinary Record, 110: 366.
MacOwan, K.J., Randall, C.J., Jones, Helen G.R. and Brand, T.F. (1982b). Association of Myco-
plasma synoviae with respiratory disease of broilers. Avian Pathology, 11: 235-244.
Mallinson, E.T. and Rosenstein, M. (1976). Clinical, cultural and serologic observations on avian
mycoplasmoses in two breeder flocks. Avian Diseases, 20: 211-215.
Roberts, D.H. and McDaniel, J.W. (1967). Mechanism of egg transmission of Mycoplasma galli-
septicum. Journal of Comparative Pathology, 77: 439-442.
Sahu, S.P. and Olson, N.O. (1976). Use of the agar gel precipitin test to evaluate broiler breeder
and commercial layer flocks for Mycoplasma gallisepticum infection. Avian Diseases, 20:
563-573.
Sahu, S.P. and Olson, N.O. (1982). Characterisation of an isolate of Mycoplasma WVU907 which
possesses common antigens to Mycoplasma gallisepticum. Avian Diseases, 25: 943-953.
Timms, L. (1967). Isolation and identification of avian mycoplasma. Journal of Medical Labora-
tory Technology, 24: 79-89.
Varley, J. and Jordan, F.T.W. (1978). The response of chickens to experimental infection with
strains of M. gallisepticum of different virulence and M. gallinarum. Avian Pathology, 7:
157-170.
Yoder, H.W. and Hofstad, M.S. (1964). Characterisation of avian mycoplasma. Avian Diseases,
8: 481-512.

RESUME
Infection cloacale par Mycoplasma gallisepticum et
effet de l'inoculation par le virus vaccinal H120 de la
Bronchite infectieuse chez la poule
Le pouvoir infectieux d'une souche de M. gallisepticum possédant une faible viru-
lence a été étudié chez des poulets exempts d'organismes pathogènes spécifiés depuis
l'âge d'un jour jusqu'à l'entrée en ponte. Le développement de l'infection cloacale est
discuté dans ses relations avec la faible réponse sérologique. Les effets produits par
l'inoculation du virus vaccinal H120 de la bronchite infectieuse au moment de l'en-
trée en ponte ont été associés avec des modifications des réponses serologiques et une
augmentation du réisolement de,M. gallisepticum au niveau cloacal. La possibilité
d'une infection cloacale ascendante conduisant à une transmission par l'oeuf est en-
visagée et comparée avec d'autres voies d'inoculation par lequel les ovules deviennent
infectés.

ZUSAMMENFASSUNG
Eine kloakale Infektion mit Mycoplasma gallisepticum und die Wirkung
der Inokulation des Impfstammes H120 des Infektiösen Bronchitis Virus
Die Infektiosität eines M. gallisepticum-stammes von niederer Virulenz für SPF-
Hühner im Alter von 1 Tag bis zum Legebeginn wurde untersucht. Es entwickelte
sich vielfach eine kloakale Infektion. Sie wird im Zusammenhang mit der geringen
serologischen Reaktion diskutiert. Die durch die Inokulation des Impfstammes H120
des Infektiösen Bronchitisvirus und durch Beginn der Legetätigkeit ausgelösten Ef-
fekte führen zu Veränderungen der serologischen Reaktionen und zu einer Erhöhung
der kloakalen Isolationsrate. Es wird die Möglichkeit einer aufsteigenden Kloakenin-
fektion als Ursache einer Eiübertragung in Betracht gezogen und mit anderen Infek-
tionswegen verglichen, die zur Infektion der Ovula führen können.