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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 (1979) 35--45 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
35
INFECTION OF THE CHICKEN WITH A VIRULENT OR AVIRULENT STRAIN OF MYCOPLASMA GALLISEPTICUM ALONE AND TOGETHER W I T H N E W C A S T L E D I S E A S E V I R U S O R E. C O L I O R B O T H
M.M. AMIN* and F.T.W. J O R D A N
Sub-Department of Avian Medicine, University of Liverpool, Veterinary Field Station, 'Leahurst', Neston, Wirral L64 7TE (Great Britain) *Present address: Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh (Bangladesh) (Accepted 12 March 1978)
ABSTRACT 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. Vet. Microbiol., 4: 35--45. The influence of the virulence of M. gailisepticum (Mg) was studied in multiple infections of chickens involving Mg, Newcastle disease virus (NDV) and E. eoli. Separate groups of 3-week-old chickens were inoculated supra-conjunctivally with a virulent and an avirulent strain of Mg alone and in combination with the La Sota strain of NDV, the 01 serotype of E. coli, and both NDV and E. coli. In addition, chickens were inoculated with NDV alone, E. coli alone, and with NDV and E. coli; one group was left uninfected. Clinical signs, lesions, recovery of the pathogens and the serological response were observed for all groups for 3 weeks after infection and for those involving Mg alone and Mg together with NDV for 18 weeks. No clinical signs were seen in any of the birds; in each infected group some showed mild lesions of the trachea and air sacs without any marked difference among the groups. Although the numbers of birds examined were small, the virulent strain of Mg was more readily recovered than the avirulent, from the respiratory tract, in the first 3 weeks following multiple infections. However, the virulence of Mg had no influence on the recovery of the other pathogens. F o r the first 6 weeks after infection there was a direct relationship between the virulence of the Mg and the proportion of birds with agglutinins to the Mg rapid serum agglutination (RSA) test in single or multiple infections; multiple infections enhanced the antibody response to both virulent and avirulent mycoplasma. For NDV, multiple infections, particularly involving E. coli, enhanced the peak titre of haemagglutination-inhibition antibodies and accelerated their appearance; the virulence of the Mg had no apparent effect on this. Neither the mycoplasma nor NDV were detected in the trachea by immunofluorescence.
INTRODUCTION Concomitant infection of chickens with various combinations of two or m o r e o r g a n i s m s s u c h as N e w c a s t l e d i s e a s e v i r u s ( N D V ) , M. gallisepticum ( M g )
36
and E. coli may sometimes be synergistic in disease production (Bankowski, 1961; Fabricant, 1969; Jordan, 1972, 1975). However, Thornton (1971) failed to demonstrate synergism between a strain of M. gallisepticum and E. coli each given by a variety of routes, and Nonomura {1973) and Nonomura and Sato (1975) demonstrated interference of NDV by Mg. Of the factors influencing the outcome of multiple infections the strain of the individual organisms might be of significance. This communication compares the influence of a virulent and an avirulent strain of Mg on concomitant infections of the chicken with a vaccine strain of Newcastle disease virus and/or a strain of E. coli. The disease produced, duration of infections and pattern of serological response were observed. MATERIALS AND METHODS
Mycoplasma gallisep ticum An $6 strain of low passage (in artificial media) and high virulence, and one of high passage and low virulence were used. They were the strains described by Power and Jordan (1976) a s " M g l " and " M g 2 " and are referred to here as Mg(v) and Mg(a), respectively. Both were used for infection at a concentration of 107 CFU/ml in mycoplasma broth. For the recovery of mycoplasma from infected birds, 0.15 ml of homogenate of respiratory tissue and cloaca (see later) was plated separately onto mycoplasma agar and placed in overlay medium with sub-cultures from overlay onto plates as described by Jordan and Amin (1975). Mycoplasma isolates were identified as M. gallisepticum by immunofluorescence by the m e t h o d described by Bradbury et al. (1976).
Newcastle disease virus (ND V) This was a La Sota vaccine strain reconstituted in sterile distilled water so that one dose as indicated by the manufacturers was contained in 0.1 ml. For the recovery of NDV, respiratory tissue homogenate from each bird examined was centrifuged at 800 g for 10 min and the supernatant added to an equal volume of nutrient broth containing 100 000 IU of benzyl penicillin and 10 mg streptomycin sulphate per ml. After 30 min each individual specimen was inoculated into five 9 to 10-day-old SPF chick embryos, and further examination and identification followed the m e t h o d of Hanson (1975). A1lantoic fluid failing to agglutinate chicken erythrocytes was passaged twice through embryonated chicken eggs and tested before being discarded.
E. coli This was a culture of the 01 serotype grown in nutrient broth and used at a concentration of 104 CFU/ml.
37
The recovery of E. coli from the respiratory tissue homogenate (see later) followed the method of Soyjka (1965). Isolates were characterised biochemically as described by Sleigh (1975) and identified serologically using specific 01 K antiserum. Viable counts Viable counts for Mg and E. coli were based on the method of Miles et al. (1938). For Mg, serial lo-fold dilutions were made in mycoplasma broth, and five 0.02 ml drops from each dilution immediately transferred to mycoplasma agar medium using one plate per dilution. For E. coli, dilutions were made in MacConkey broth and drops sown on MacConkey agar. Plates for each were incubated at 37°C those for mycoplasma in a sealed container, and colony counts for Mg and E. coli were made after 10 and 2 days, respectively. Specific pathogen-free
(SPF) chickens
The chickens were hatched from eggs from the flock of the sub-department of Avian Medicine, regularly monitored and found free from a number of avian pathogens (Power and Jordan, 1976), including Newcastle disease virus, and mycoplasma. They were divided into 12 separate groups, each housed individually in isolation in rearing cages. Experimental procedure At 3 weeks of age individual groups of chickens were infected with appropriate organisms by eye drop (Table I). Each inoculum was administered in a TABLE I The experimental groups, numbers of chickens and infections Group
No. of chickens
Infections
1 2 3 4 5 6 7 8 9 10 11 12
24 24 24 24 25 23 25 24 24 23 26 23
NDV Mg(a) + NDV Mg(v) + NDV E. coli serotype 01 Mg(a) + E. coli Mg(v) + E. coli NDV + E. coli Mg(a) + NDV + E. COC Mg(v) + NDV + E. coli Uninfected
Mg(a) = avirulent strain of Mycoplasma gallisepticum; Mg(v) = virulent strain of Mycoplasma gallisepticum; NDV = La Sota vaccine strain of Newcastle disease virus; E. coli = 01 serotype of E. coli.
38 volume of 0.05 ml onto each eye and when more than one agent was involved they were given in the order named in Table I. Birds in all groups were observed daily for the first 3 weeks after infection when observations on groups 6--11 inclusive were terminated. After this time observations on groups 1--5 inclusive and group 12 continued less frequently until the termination of the experiment at 18 weeks. Immediately before infection, and at 1 and 2 weeks afterwards, 10 birds from each group were randomly selected and bled, and four birds were killed by intravenous inoculation of sodium thiopentone. At week 3 the remaining birds in groups 6--11 inclusive were bled and killed, while from groups 1--5 and 12, 10 birds were bled and four killed. Blood samples were taken from the remaining birds in groups 1--5 and 12 at weeks 6, 10 and 18 when the experiment was terminated and the birds killed. The blood samples were allowed to clot and the serum removed and stored at 0--4°C. On each occasion that birds were killed, the tracheas from two birds of each group were removed and one placed in 5% formol saline for histology and the other in an embedding c o m p o u n d 1, snap frozen in a solid carbon dioxide isopentane freezing mixture and stored at --70°C for later examination by immunofluorescence. For the recovery of infecting organisms the carcases of the remaining birds in each group were opened aseptically and a homogenate prepared from the respiratory tissue of each bird (including the nares, trachea, bronchi, lungs and air sacs) in four times its volume of nutrient broth; the homogenate was then examined for the micro-organisms with which the bird had been infected. The control groups were examined for all three organisms. In addition, for the recovery of mycoplasma, the cloaca and contents were similarly homogenized for each bird infected with this organism. For histology, 5-~ thick transverse sections of tracheas fixed in formol saline were prepared and stained by haematoxylin and eosin, while for examination by immunofluorescence, 6-~ thick, transverse, cryostat sections were cut from tracheas which had been frozen. All sections were prepared from that part of the trachea immediately distal to the larynx. Frozen sections from birds infected with Mg were stained with fluorescein-labelled antiserum to Mg prepared in rabbits and those infected with NDV were stained with labelled antiserum to NDV prepared in chickens. For chickens infected with both organisms some sections from each bird were stained with one and some with the other conjugated antiserum. Serum samples were examined for agglutinins to mycoplasma by the rapid serum agglutination test (RSA) as described by Bradbury and Jordan (1971), and for the haemagglutination inhibition titre to NDV by the/~ procedure using the micro-titre technique and 4 HA units as described by Allan and Gough (1974). i "Optimal temperature cutting compound", Lab-Tek Products Division, Miles Laborgtories inc., Napierville, Ill. 6054, U.S.A.
39 RESULTS
Clinical signs and lesions No significant clinical signs were observed. Gross lesions of excess mucus in the trachea and mild airsacculitis were seen in about half the birds in each infected group (5--12 days after infection) and a few birds showed conjunctivitis.
Histological studies These were restricted to the tracheas of birds during the first 3 weeks after infection. The cellular changes were mild in each group and included deciliation, enlargement of mucous glands, epithelial hyperplasia in a few birds, small round cell infiltration and finally the appearance of l y m p h o i d foci. Apart from these foci, which persisted, the abnormalities were not detected for longer than 16 days after infection in any bird. Multiple infection did n o t influence the severity of the reaction.
The re-isolation of organisms The recovery of Mg from the respiratory tract and cloaca is shown in Table II; mycoplasma was not recovered from any of the birds in the control group. Mg of either strain and in single or mixed infection was frequently recovered from respiratory tissue but not at all from the cloaca during the first 3 weeks after infection. The organism was recovered, however, from both tissues in most groups at 18 weeks after infection. The avirulent strain of Mg was recovered from a greater proportion of birds from the respiratory tract or cloaca when they were infected with this organism alone than in multiple infection. This did not obtain with Mg(v). Only the respiratory tract was examined for NDV and it was recovered from all groups infected with the virus up to and including 11 days after infection but n o t at 15 or 21 days or 18 weeks. Multiple infection did not influence recovery. For the 01 serotype of E. coil only respiratory tissues were examined; the organism was recovered from all birds of all groups infected with the organism up to 3 weeks after infection.
The serological response The proportion of birds with agglutinins to M. gallisepticum, in the various groups up to 6 weeks after infection, is shown in Fig. 1. Agglutinins were first observed at 3 weeks when they occurred in a higher proportion of birds infected with Mg(v) than Mg(a) either alone or in multiple infection. At 6 weeks and thereafter, all the birds infected with Mg(v) and Mg(a) + NDV showed at-
+ + + + + +
NDV NDV E. coli E. coli NDV + E. coli NDV + E. coli
2
313 212 l/2 212 212 l/2 o/2 212
2/2* 212 o/2 212 112 212 112 212
tissue
212 l/2 212 212 5/10 lO/lO l/6 7/8
3 ND ND ND ND ND ND ND ND
6
(weeks after infection)
1
Respiratory
Recovery
ND ND ND ND
10 3/3 3/4 l/2 3/5
18
(Mycoplasma were not recovered from uninfected birds). *Numerator = No. of birds from which M. gallisepticum was isolated; denominator = No. of birds examined. ND: not done, experiment terminated. Mg(a), Mg(v), NDV, and E. coli, as for Table I.
Mg(v) Mg(a) Mg(v) Mg(a) Mg(v) Mg(a) Mg(v)
M&4
Infection
o/2 o/2 o/2 o/2 o/2 o/2 012
o/2
1
3 o/2 o/2 o/2 o/2 O/10 O/10 O/S O/8
o/3 o/2 o/2 012 012 o/2 012 012
616 o/2 o/2 o/2 ND ND ND ND
6
016 O/Z o/2 o/2
10
313 314 o/2 l/5
18
at 3 weeks of age with an avirulent
2
infected
Cloaca
Recovery of M. gallisepticum from the respiratory tract and cloaca of chickens strain of M. gallisepticum in single, dual and multiple infection
TABLE II or virulent
41 Group
Proportion 1
(%)of
2
chlckens
3
4
wth 5
agglutinlns
(weeks after infection)
6
Mg(o)
Mg(v)
Mg (al+ NDV
-
-
Mg (v)+ NDV
-
-
,RR% ElBR%
Mg (a)+ Q&i
-
-
0
-
El
!?cti
-
- -
Mg (a)+ NDVt E.coli Mg(vH NDV+ E.coli
agglutinins
%
El
>.76-loo
B
51-75
q
26-50
0
l-25
-
0
El R
/
I2
-(No
\
in birds
not
infected
with
mycoplasma)
Fig. 1. Agglutinins to M. gallisepticum by the rapid serum agglutination test following infection with Mg(a) or Mg(v) in single, dual or multiple infections. Mg(a), avirulent M. gdisepticum; Mg(v), virulent M. gallisepticum; NDV, La Sota vaccine strain of Newcastle disease virus; E. coli, 01 serotype of E. coli. No. of birds examined: - during the first 3 weeks after infection the serum from 10 to 14 birds/group was examined: thereafter that from five to eight birds.
glutinins but all birds infected with Mg(a) alone did not show agglutinins until the 18th week. HI titres to Newcastle disease virus are shown in Table III. E. coli accelerated the appearance and enhanced the peak HI titres. The dual infection of NDV and Mg(a) or Mg(v) had a significant but less marked influence in this respect and there appeared to be no difference in the effect of the two strains of mycoplasma. Immunofluorescence
studies
Examination of tracheal sections by immunofluorescence for Mg and NDV failed to detect the presence of either organism in any of the material examined. DISCUSSION
It is not surprising that in the single infections clinical signs and gross lesions were absent or only mild and transitory. For M. gallisepticum this is supported by the observations of Adler et al. (1962) and Corstvet and Sadler (1966a) who
42 TABLE III HI titres of NDV-infected c h i c k e n s Infection
Weeks after infection
1 NDV NDV Mg(v) + NDV NDV + E. coli Mg(a) + NDV + E. coil Mg(v) + NDV + E. coil Mg(a) +
4.2* 4.6 4.3 9.4 8.9 9.5
2
3
6
6.5
6.4
7.1
9.1
6.3 9.6 10 9.6
9.2 9.7 9.4 8.7
6.3 7.2 7.0 ND ND ND
There were no positive NDV HI titres in groups u n i n f e c t e d w i t h NDV or in infected groups before infection. ND: n o t d o n e , e x p e r i m e n t terminated.
Mg(a), Mg(v), NDV and E. coli, as for Table I. * G e o m e t r i c m e a n titre expressed as log 2. infected chicken several weeks old by the infra-orbital sinus and tracheal routes, respectively. F.T.W. Jordan (unpublished observations 1969) has also f o u n d th at when infection is by the upper respiratory tract, clinical signs are more likely to occur in very y o u n g chicks. For NDV the explanation may be that the strain used was mild, that the i nf l am mat ory response was receding by the first examination at 7 days and t hat perhaps relatively little virus entered the trachea, since Burnstein and Bang (1958) have shown that when the B1 strain o f NDV was given intranasally in small volumes little appeared in the trachea. F o r E . c o l i the failure of organisms to enter the trachea in large numbers may also be the reason for absence o f gross lesions, since Fabricant and Levine {1962) and T h o r n t o n (1971) have shown t hat this organism (serotype 02) given alone, intratracheally, may cause air sac lesions. Th at dual and triple infection can be synergistic in disease product i on has been shown for Mg and NDV (Corstvet and Sadler, 1966b; Sato et al., 1970), Mg a n d E . c o l i (Gross, 1958; Fabricant and Levine 1962) and Mg, NDV and E . c o l i (Gross, 1961). However, in all these cases none of the organisms was given by the intraocular r out e but by aerosol, intratracheally or into the air sac so that larger numbers of organisms gained access to the lower respiratory tissues. In this respect it is of interest that Suzuki et al. (1971), giving Mg and the B1 strain o f NDV intranasally to day-old chicks, found no clinical signs or gross lesions and our results are in accord with this. The histological appearance of tracheal sections supports the mild nature of the reaction. It is of interest t hat a similar histopathological response was obtained with all three organisms either alone or in multiple infection. Suzuki et al. (1971) obtained similar results with Mg and NDV. Recovery o f Mg(v) and Mg(a) was obtained from respiratory tissues from almost all the birds examined during the first 3 weeks after single infection.
43 However, when given with NDV, or E. coli or both there was a reduction in the proportion of birds from which Mg(a) could be recovered. This did n o t occur with Mg(v). The number of birds examined was too small for the results to be significant but it would seem that the avirulent strain of Mg was less able to establish itself in the respiratory tract early in infection, in the face of competition from either NDV of E. coli or both. In contrast with this, Corstvet and Sadler (1966b) and Sato et al. (1970) found that dual infection of Mg with NDV enhanced the recovery of Mg but the route of infection was different and the virulence of the strain of Mg was n o t indicated. The failure to recover either strain of Mg from the cloaca during the first 3 weeks after infection emphasises the slow invasion of the tissues by this organism. Its recovery at 18 weeks after infection suggests that it can establish itself there and that this may be a possible source of spread. The recovery of NDV and E. coli was not influenced by concomitant infection with either or both of the other organisms. The apparent lack of influence of Mg on the recovery of NDV is similar to the observations of Corstvet and Sadler (1966b) and the failure to recover NDV from the trachea after 11 days following infection is in keeping with general findings when utilizing the method of recovery already described (Hanson, 1975). The recovery of E. coli for at least up to 3 weeks is in keeping with the findings of Harry and Hemsley (1965) who found that the organism would survive for at least 12 weeks in the trachea. Agglutinins against mycoplasma were found in a higher proportion of birds infected alone with Mg(v) than Mg(a) at 3, 6 and 10 weeks after infection. This was not seen by Varley and Jordan (1978) in chicks up to 28 days of age but this might be explained by the relative immunological immaturity at this age. Multiple infections involving Mg stimulated the antibody response to this organism as shown by a higher proportion of birds with agglutinins after infection compared with single infection. This was noted at 3 weeks for Mg(v) and at 4 weeks for Mg(a) and NDV. This is also probably associated with greater tissue invasion in multiple infection. For the avirulent strain of Mg which seemed to be initially inhibited in the trachea in multiple infection, the enhanced tissue invasion might have occurred in the ocular region. The earlier and enhanced antibody response to NDV in multiple infections was most marked when E. coli was involved. It is probable that the adjuvent effect of this organism is due to its endotoxin as indicated by Johnson and Hoekstra (1967). Concomitant infection with Mg also had an adjuvent effect in this respect although it was not described by Corstvet and Sadler (1966b) and Suzuki et al. (1971) when the mycoplasma was given intratracheally and intranasally, respectively. The inability to detect either Mg or NDV by immunofluorescence may be due to a variety of factors, such as the relatively small area of the trachea examined, the small number of organisms present and the infrequency of examination.
44 ACKNOWLEDGEMENTS
This work was undertaken when one of us (M.M.A.) was in receipt of a grant from the British Egg Marketing Board. We wish to thank Mrs L y n d a Moore for technical assistance.
REFERENCES
Adler, H.E., McMartin, D.A. and Ortmayer, H., 1962. The effect of infectious bronchitis virus on chickens infected with Mycoplasma gallisepticum. Avian Dis., 6: 267--274. Allan, W.H. and Gough, R.E., 1974. A standard haemagglutination inhibition test for Newcastle disease. 1. A comparison of macro and micro methods. Vet. Rec., 95: 120--123. Bankowski, R.A., 1961. Respiratory disease complex of chickens in the United States. Br. Vet. J., 117: 306--315. Bradbury, J.M. and Jordan, F.T.W., 1971. The influence of pH of the culture medium on the sensitivity of Mycoplasma gallisepticum antigens for use in certain serological tests. J. Hyg., 69: 593--606. Bradbury, J.M., Oriel, C.A. and Jordan, F.T.W., 1976. Simple method for immunofluorescent identification of mycoplasma colonies. J. Clin. Microbiol., 3: 449--452. Burnstein, T. and Bang, F.B., 1958. Infection of the upper respiratory tract of the chick with a mild (vaccine strain) of Newcastle disease virus. 1. Initiation and spread of the infection. Bull. Johns Hopkins Hosp., 102: 127--134. Corstvet, R.E. and Sadler, W.W., 1966a. A comparative study of single and multiple respiratory infections in the chicken: single infection (with Mycoplasma gallisepticum and Newcastle disease virus). Am. J. Vet. Res., 27: 1721--1733. Corstvet, R.E. and Sadler, W.W., 1966b. A comparative study of single and multiple respiratory infections in the chicken: Multiple infections (with Mycoplasma gallisepticum, Newcastle disease virus, and infectious bronchitis virus). Am. J. Vet. Res., 27: 1703-1720. Fabricant, J., 1969. Avian mycoplasma. In: L. Hayfliek (Editor), The Mycoplasmatales and the L Phase of Bacteria. North Holland Publishing Co., Amsterdam, pp. 621--641. Fabricant, J. and Levine, P.P., 1962. Experimental production of complicated chronic respiratory disease infection ("Air sac" disease). Avian Dis., 6: 13--23. Gross, W.B., 1958. Symposium on chronic respiratory disease of poultry. II. The role of E. coil in the cause of chronic respiratory disease and certain other respiratory diseases. Am. J. Vet. Res., 19: 448--463. Gross, W.B., 1961. The development of "air sac disease". Avian Dis., 5: 431--439. Hanson, R.P., 1975. Newcastle disease. In: S.B. Hitchner, C.H. Domermuth, H.G. Purchase and J.E. Williams (Editors), Isolation and Identification of Avian Pathogens. Arnold Printing Corporation, Ithaca, New York, U.S.A., pp. 160--173. Harry, E.G. and Hemsley, L.A., 1965. The relationship between environmental contamination with septicaemia strains of Escherichia coil and their incidence in chickens. Vet. Rec., 77: 241--245. Johnson, A.G. and Hoekstra, G., 1967. Acceleration of the primary antibody response. In: R.T. Smith, R.A. Good and P.A. Miescher (Editors), Ontogeny of Immunity. University of Florida Press, Gainesville, Florida, U.S.A., pp. 187--190. Jordan, F.T.W., 1972. The epidemiology of disease of multiple aetiology: The avian respiratory disease complex. Vet. Rec., 90: 556---562. Jordan, F.T.W., 1975. I m m u n i t y to mycoplasma infections of the respiratoru system in domestic fowls and turkeys (protection by vaccination and natural infection). Proc. 47th Syrup. Int. Assoc. Biol. Standard. Develop. Biol. Standard., 28: 590--596.
45 Jordan, F.T.W. and Amin, M.M., 1975. The isolation of mycoplasma from avian species. In: K. Jurmanova and Z. Dreslerova (Editors), Proceedings of the 3rd Conference on Taxonomy and Physiology of Animal Mycoplasma, Brno, Czechoslovakia, pp. 85--95. Miles, A.A., Misra, S.S. and Irwin, J.O., 1938. The estimation of the bactericidal power of the blood. J. Hyg., 38: 732--749. Nonomura, I., 1973. Interference of M. gallisepticum with the multiplication of Newcastle disease virus with chicken tracheal organ cultures. Natl. Inst. Anim. Health Q., 13: 105--111. Nonomura, I. and Sato, S , 1975. Interference of M. gallisepticurn with the multiplication of Newcastle disease virus in chickens. Avian Dis., 19: 603--607. Power, J. and Jordan, F.T.W., 1976. A comparison of the virulence of three strains of Mycoplasma galli~epticum and one strain of Mycoplasma gallinarum in chicks, turkey poults, tracheal organ cultures and embryonated eggs. Res. Vet. Sci., 21: 41--46. Sato, S., Nonomura, I., Shimizu, F., Shoya, S. and Horiuchi, T., 1970. Mixed infection with M. gaUisepticum and the BI strain of Newcastle disease virus in chickens. Natl. Inst. Anim. Health Q., 10: 58---65. Sleigh, J.D., 1975. Escherichia, Klebsiella, Proteus and other enterobacteria. In: R. Cruickshank, J.P. Duguid, B.P. Marmion and R.H.A. Swain (Editors), Medical Microbiology, 12th edn. Churchill, Livingstone, Edinburgh, London and New York, N.Y., pp. 428--439. Soyjka, W.T., 1965. Escherichia coli in Domestic Animals and Poultry. Commonwealth Agricultural Bureaux, Farnham Royal, Slough, 231 pp. Suzuki, K., Omuro, M., Sato, S., Kuniyasu, C. and Goroya, K., 1971. Influence of Newcastle disease and infectious bronchitis live vaccines on chickens infected with Mycoplasma gallisepticum. Natl. Inst. Anita. Health Q., 11: 94--99. Thornton, G.A., 1971. Failure of M. gallisepticum infection to predispose chickens to coliform pericarditis. Br. Vet. J., 127: 163--172. Varley, J. and Jordan, F.T.W., 1978. The response of chickens to experimental infection with strains of M. gaUisepticurn of different virulence and M. gallinarum. Avian Pathol., I: 157--170.
35
INFECTION OF THE CHICKEN WITH A VIRULENT OR AVIRULENT STRAIN OF MYCOPLASMA GALLISEPTICUM ALONE AND TOGETHER W I T H N E W C A S T L E D I S E A S E V I R U S O R E. C O L I O R B O T H
M.M. AMIN* and F.T.W. J O R D A N
Sub-Department of Avian Medicine, University of Liverpool, Veterinary Field Station, 'Leahurst', Neston, Wirral L64 7TE (Great Britain) *Present address: Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh (Bangladesh) (Accepted 12 March 1978)
ABSTRACT 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. Vet. Microbiol., 4: 35--45. The influence of the virulence of M. gailisepticum (Mg) was studied in multiple infections of chickens involving Mg, Newcastle disease virus (NDV) and E. eoli. Separate groups of 3-week-old chickens were inoculated supra-conjunctivally with a virulent and an avirulent strain of Mg alone and in combination with the La Sota strain of NDV, the 01 serotype of E. coli, and both NDV and E. coli. In addition, chickens were inoculated with NDV alone, E. coli alone, and with NDV and E. coli; one group was left uninfected. Clinical signs, lesions, recovery of the pathogens and the serological response were observed for all groups for 3 weeks after infection and for those involving Mg alone and Mg together with NDV for 18 weeks. No clinical signs were seen in any of the birds; in each infected group some showed mild lesions of the trachea and air sacs without any marked difference among the groups. Although the numbers of birds examined were small, the virulent strain of Mg was more readily recovered than the avirulent, from the respiratory tract, in the first 3 weeks following multiple infections. However, the virulence of Mg had no influence on the recovery of the other pathogens. F o r the first 6 weeks after infection there was a direct relationship between the virulence of the Mg and the proportion of birds with agglutinins to the Mg rapid serum agglutination (RSA) test in single or multiple infections; multiple infections enhanced the antibody response to both virulent and avirulent mycoplasma. For NDV, multiple infections, particularly involving E. coli, enhanced the peak titre of haemagglutination-inhibition antibodies and accelerated their appearance; the virulence of the Mg had no apparent effect on this. Neither the mycoplasma nor NDV were detected in the trachea by immunofluorescence.
INTRODUCTION Concomitant infection of chickens with various combinations of two or m o r e o r g a n i s m s s u c h as N e w c a s t l e d i s e a s e v i r u s ( N D V ) , M. gallisepticum ( M g )
36
and E. coli may sometimes be synergistic in disease production (Bankowski, 1961; Fabricant, 1969; Jordan, 1972, 1975). However, Thornton (1971) failed to demonstrate synergism between a strain of M. gallisepticum and E. coli each given by a variety of routes, and Nonomura {1973) and Nonomura and Sato (1975) demonstrated interference of NDV by Mg. Of the factors influencing the outcome of multiple infections the strain of the individual organisms might be of significance. This communication compares the influence of a virulent and an avirulent strain of Mg on concomitant infections of the chicken with a vaccine strain of Newcastle disease virus and/or a strain of E. coli. The disease produced, duration of infections and pattern of serological response were observed. MATERIALS AND METHODS
Mycoplasma gallisep ticum An $6 strain of low passage (in artificial media) and high virulence, and one of high passage and low virulence were used. They were the strains described by Power and Jordan (1976) a s " M g l " and " M g 2 " and are referred to here as Mg(v) and Mg(a), respectively. Both were used for infection at a concentration of 107 CFU/ml in mycoplasma broth. For the recovery of mycoplasma from infected birds, 0.15 ml of homogenate of respiratory tissue and cloaca (see later) was plated separately onto mycoplasma agar and placed in overlay medium with sub-cultures from overlay onto plates as described by Jordan and Amin (1975). Mycoplasma isolates were identified as M. gallisepticum by immunofluorescence by the m e t h o d described by Bradbury et al. (1976).
Newcastle disease virus (ND V) This was a La Sota vaccine strain reconstituted in sterile distilled water so that one dose as indicated by the manufacturers was contained in 0.1 ml. For the recovery of NDV, respiratory tissue homogenate from each bird examined was centrifuged at 800 g for 10 min and the supernatant added to an equal volume of nutrient broth containing 100 000 IU of benzyl penicillin and 10 mg streptomycin sulphate per ml. After 30 min each individual specimen was inoculated into five 9 to 10-day-old SPF chick embryos, and further examination and identification followed the m e t h o d of Hanson (1975). A1lantoic fluid failing to agglutinate chicken erythrocytes was passaged twice through embryonated chicken eggs and tested before being discarded.
E. coli This was a culture of the 01 serotype grown in nutrient broth and used at a concentration of 104 CFU/ml.
37
The recovery of E. coli from the respiratory tissue homogenate (see later) followed the method of Soyjka (1965). Isolates were characterised biochemically as described by Sleigh (1975) and identified serologically using specific 01 K antiserum. Viable counts Viable counts for Mg and E. coli were based on the method of Miles et al. (1938). For Mg, serial lo-fold dilutions were made in mycoplasma broth, and five 0.02 ml drops from each dilution immediately transferred to mycoplasma agar medium using one plate per dilution. For E. coli, dilutions were made in MacConkey broth and drops sown on MacConkey agar. Plates for each were incubated at 37°C those for mycoplasma in a sealed container, and colony counts for Mg and E. coli were made after 10 and 2 days, respectively. Specific pathogen-free
(SPF) chickens
The chickens were hatched from eggs from the flock of the sub-department of Avian Medicine, regularly monitored and found free from a number of avian pathogens (Power and Jordan, 1976), including Newcastle disease virus, and mycoplasma. They were divided into 12 separate groups, each housed individually in isolation in rearing cages. Experimental procedure At 3 weeks of age individual groups of chickens were infected with appropriate organisms by eye drop (Table I). Each inoculum was administered in a TABLE I The experimental groups, numbers of chickens and infections Group
No. of chickens
Infections
1 2 3 4 5 6 7 8 9 10 11 12
24 24 24 24 25 23 25 24 24 23 26 23
NDV Mg(a) + NDV Mg(v) + NDV E. coli serotype 01 Mg(a) + E. coli Mg(v) + E. coli NDV + E. coli Mg(a) + NDV + E. COC Mg(v) + NDV + E. coli Uninfected
Mg(a) = avirulent strain of Mycoplasma gallisepticum; Mg(v) = virulent strain of Mycoplasma gallisepticum; NDV = La Sota vaccine strain of Newcastle disease virus; E. coli = 01 serotype of E. coli.
38 volume of 0.05 ml onto each eye and when more than one agent was involved they were given in the order named in Table I. Birds in all groups were observed daily for the first 3 weeks after infection when observations on groups 6--11 inclusive were terminated. After this time observations on groups 1--5 inclusive and group 12 continued less frequently until the termination of the experiment at 18 weeks. Immediately before infection, and at 1 and 2 weeks afterwards, 10 birds from each group were randomly selected and bled, and four birds were killed by intravenous inoculation of sodium thiopentone. At week 3 the remaining birds in groups 6--11 inclusive were bled and killed, while from groups 1--5 and 12, 10 birds were bled and four killed. Blood samples were taken from the remaining birds in groups 1--5 and 12 at weeks 6, 10 and 18 when the experiment was terminated and the birds killed. The blood samples were allowed to clot and the serum removed and stored at 0--4°C. On each occasion that birds were killed, the tracheas from two birds of each group were removed and one placed in 5% formol saline for histology and the other in an embedding c o m p o u n d 1, snap frozen in a solid carbon dioxide isopentane freezing mixture and stored at --70°C for later examination by immunofluorescence. For the recovery of infecting organisms the carcases of the remaining birds in each group were opened aseptically and a homogenate prepared from the respiratory tissue of each bird (including the nares, trachea, bronchi, lungs and air sacs) in four times its volume of nutrient broth; the homogenate was then examined for the micro-organisms with which the bird had been infected. The control groups were examined for all three organisms. In addition, for the recovery of mycoplasma, the cloaca and contents were similarly homogenized for each bird infected with this organism. For histology, 5-~ thick transverse sections of tracheas fixed in formol saline were prepared and stained by haematoxylin and eosin, while for examination by immunofluorescence, 6-~ thick, transverse, cryostat sections were cut from tracheas which had been frozen. All sections were prepared from that part of the trachea immediately distal to the larynx. Frozen sections from birds infected with Mg were stained with fluorescein-labelled antiserum to Mg prepared in rabbits and those infected with NDV were stained with labelled antiserum to NDV prepared in chickens. For chickens infected with both organisms some sections from each bird were stained with one and some with the other conjugated antiserum. Serum samples were examined for agglutinins to mycoplasma by the rapid serum agglutination test (RSA) as described by Bradbury and Jordan (1971), and for the haemagglutination inhibition titre to NDV by the/~ procedure using the micro-titre technique and 4 HA units as described by Allan and Gough (1974). i "Optimal temperature cutting compound", Lab-Tek Products Division, Miles Laborgtories inc., Napierville, Ill. 6054, U.S.A.
39 RESULTS
Clinical signs and lesions No significant clinical signs were observed. Gross lesions of excess mucus in the trachea and mild airsacculitis were seen in about half the birds in each infected group (5--12 days after infection) and a few birds showed conjunctivitis.
Histological studies These were restricted to the tracheas of birds during the first 3 weeks after infection. The cellular changes were mild in each group and included deciliation, enlargement of mucous glands, epithelial hyperplasia in a few birds, small round cell infiltration and finally the appearance of l y m p h o i d foci. Apart from these foci, which persisted, the abnormalities were not detected for longer than 16 days after infection in any bird. Multiple infection did n o t influence the severity of the reaction.
The re-isolation of organisms The recovery of Mg from the respiratory tract and cloaca is shown in Table II; mycoplasma was not recovered from any of the birds in the control group. Mg of either strain and in single or mixed infection was frequently recovered from respiratory tissue but not at all from the cloaca during the first 3 weeks after infection. The organism was recovered, however, from both tissues in most groups at 18 weeks after infection. The avirulent strain of Mg was recovered from a greater proportion of birds from the respiratory tract or cloaca when they were infected with this organism alone than in multiple infection. This did not obtain with Mg(v). Only the respiratory tract was examined for NDV and it was recovered from all groups infected with the virus up to and including 11 days after infection but n o t at 15 or 21 days or 18 weeks. Multiple infection did not influence recovery. For the 01 serotype of E. coil only respiratory tissues were examined; the organism was recovered from all birds of all groups infected with the organism up to 3 weeks after infection.
The serological response The proportion of birds with agglutinins to M. gallisepticum, in the various groups up to 6 weeks after infection, is shown in Fig. 1. Agglutinins were first observed at 3 weeks when they occurred in a higher proportion of birds infected with Mg(v) than Mg(a) either alone or in multiple infection. At 6 weeks and thereafter, all the birds infected with Mg(v) and Mg(a) + NDV showed at-
+ + + + + +
NDV NDV E. coli E. coli NDV + E. coli NDV + E. coli
2
313 212 l/2 212 212 l/2 o/2 212
2/2* 212 o/2 212 112 212 112 212
tissue
212 l/2 212 212 5/10 lO/lO l/6 7/8
3 ND ND ND ND ND ND ND ND
6
(weeks after infection)
1
Respiratory
Recovery
ND ND ND ND
10 3/3 3/4 l/2 3/5
18
(Mycoplasma were not recovered from uninfected birds). *Numerator = No. of birds from which M. gallisepticum was isolated; denominator = No. of birds examined. ND: not done, experiment terminated. Mg(a), Mg(v), NDV, and E. coli, as for Table I.
Mg(v) Mg(a) Mg(v) Mg(a) Mg(v) Mg(a) Mg(v)
M&4
Infection
o/2 o/2 o/2 o/2 o/2 o/2 012
o/2
1
3 o/2 o/2 o/2 o/2 O/10 O/10 O/S O/8
o/3 o/2 o/2 012 012 o/2 012 012
616 o/2 o/2 o/2 ND ND ND ND
6
016 O/Z o/2 o/2
10
313 314 o/2 l/5
18
at 3 weeks of age with an avirulent
2
infected
Cloaca
Recovery of M. gallisepticum from the respiratory tract and cloaca of chickens strain of M. gallisepticum in single, dual and multiple infection
TABLE II or virulent
41 Group
Proportion 1
(%)of
2
chlckens
3
4
wth 5
agglutinlns
(weeks after infection)
6
Mg(o)
Mg(v)
Mg (al+ NDV
-
-
Mg (v)+ NDV
-
-
,RR% ElBR%
Mg (a)+ Q&i
-
-
0
-
El
!?cti
-
- -
Mg (a)+ NDVt E.coli Mg(vH NDV+ E.coli
agglutinins
%
El
>.76-loo
B
51-75
q
26-50
0
l-25
-
0
El R
/
I2
-(No
\
in birds
not
infected
with
mycoplasma)
Fig. 1. Agglutinins to M. gallisepticum by the rapid serum agglutination test following infection with Mg(a) or Mg(v) in single, dual or multiple infections. Mg(a), avirulent M. gdisepticum; Mg(v), virulent M. gallisepticum; NDV, La Sota vaccine strain of Newcastle disease virus; E. coli, 01 serotype of E. coli. No. of birds examined: - during the first 3 weeks after infection the serum from 10 to 14 birds/group was examined: thereafter that from five to eight birds.
glutinins but all birds infected with Mg(a) alone did not show agglutinins until the 18th week. HI titres to Newcastle disease virus are shown in Table III. E. coli accelerated the appearance and enhanced the peak HI titres. The dual infection of NDV and Mg(a) or Mg(v) had a significant but less marked influence in this respect and there appeared to be no difference in the effect of the two strains of mycoplasma. Immunofluorescence
studies
Examination of tracheal sections by immunofluorescence for Mg and NDV failed to detect the presence of either organism in any of the material examined. DISCUSSION
It is not surprising that in the single infections clinical signs and gross lesions were absent or only mild and transitory. For M. gallisepticum this is supported by the observations of Adler et al. (1962) and Corstvet and Sadler (1966a) who
42 TABLE III HI titres of NDV-infected c h i c k e n s Infection
Weeks after infection
1 NDV NDV Mg(v) + NDV NDV + E. coli Mg(a) + NDV + E. coil Mg(v) + NDV + E. coil Mg(a) +
4.2* 4.6 4.3 9.4 8.9 9.5
2
3
6
6.5
6.4
7.1
9.1
6.3 9.6 10 9.6
9.2 9.7 9.4 8.7
6.3 7.2 7.0 ND ND ND
There were no positive NDV HI titres in groups u n i n f e c t e d w i t h NDV or in infected groups before infection. ND: n o t d o n e , e x p e r i m e n t terminated.
Mg(a), Mg(v), NDV and E. coli, as for Table I. * G e o m e t r i c m e a n titre expressed as log 2. infected chicken several weeks old by the infra-orbital sinus and tracheal routes, respectively. F.T.W. Jordan (unpublished observations 1969) has also f o u n d th at when infection is by the upper respiratory tract, clinical signs are more likely to occur in very y o u n g chicks. For NDV the explanation may be that the strain used was mild, that the i nf l am mat ory response was receding by the first examination at 7 days and t hat perhaps relatively little virus entered the trachea, since Burnstein and Bang (1958) have shown that when the B1 strain o f NDV was given intranasally in small volumes little appeared in the trachea. F o r E . c o l i the failure of organisms to enter the trachea in large numbers may also be the reason for absence o f gross lesions, since Fabricant and Levine {1962) and T h o r n t o n (1971) have shown t hat this organism (serotype 02) given alone, intratracheally, may cause air sac lesions. Th at dual and triple infection can be synergistic in disease product i on has been shown for Mg and NDV (Corstvet and Sadler, 1966b; Sato et al., 1970), Mg a n d E . c o l i (Gross, 1958; Fabricant and Levine 1962) and Mg, NDV and E . c o l i (Gross, 1961). However, in all these cases none of the organisms was given by the intraocular r out e but by aerosol, intratracheally or into the air sac so that larger numbers of organisms gained access to the lower respiratory tissues. In this respect it is of interest that Suzuki et al. (1971), giving Mg and the B1 strain o f NDV intranasally to day-old chicks, found no clinical signs or gross lesions and our results are in accord with this. The histological appearance of tracheal sections supports the mild nature of the reaction. It is of interest t hat a similar histopathological response was obtained with all three organisms either alone or in multiple infection. Suzuki et al. (1971) obtained similar results with Mg and NDV. Recovery o f Mg(v) and Mg(a) was obtained from respiratory tissues from almost all the birds examined during the first 3 weeks after single infection.
43 However, when given with NDV, or E. coli or both there was a reduction in the proportion of birds from which Mg(a) could be recovered. This did n o t occur with Mg(v). The number of birds examined was too small for the results to be significant but it would seem that the avirulent strain of Mg was less able to establish itself in the respiratory tract early in infection, in the face of competition from either NDV of E. coli or both. In contrast with this, Corstvet and Sadler (1966b) and Sato et al. (1970) found that dual infection of Mg with NDV enhanced the recovery of Mg but the route of infection was different and the virulence of the strain of Mg was n o t indicated. The failure to recover either strain of Mg from the cloaca during the first 3 weeks after infection emphasises the slow invasion of the tissues by this organism. Its recovery at 18 weeks after infection suggests that it can establish itself there and that this may be a possible source of spread. The recovery of NDV and E. coli was not influenced by concomitant infection with either or both of the other organisms. The apparent lack of influence of Mg on the recovery of NDV is similar to the observations of Corstvet and Sadler (1966b) and the failure to recover NDV from the trachea after 11 days following infection is in keeping with general findings when utilizing the method of recovery already described (Hanson, 1975). The recovery of E. coli for at least up to 3 weeks is in keeping with the findings of Harry and Hemsley (1965) who found that the organism would survive for at least 12 weeks in the trachea. Agglutinins against mycoplasma were found in a higher proportion of birds infected alone with Mg(v) than Mg(a) at 3, 6 and 10 weeks after infection. This was not seen by Varley and Jordan (1978) in chicks up to 28 days of age but this might be explained by the relative immunological immaturity at this age. Multiple infections involving Mg stimulated the antibody response to this organism as shown by a higher proportion of birds with agglutinins after infection compared with single infection. This was noted at 3 weeks for Mg(v) and at 4 weeks for Mg(a) and NDV. This is also probably associated with greater tissue invasion in multiple infection. For the avirulent strain of Mg which seemed to be initially inhibited in the trachea in multiple infection, the enhanced tissue invasion might have occurred in the ocular region. The earlier and enhanced antibody response to NDV in multiple infections was most marked when E. coli was involved. It is probable that the adjuvent effect of this organism is due to its endotoxin as indicated by Johnson and Hoekstra (1967). Concomitant infection with Mg also had an adjuvent effect in this respect although it was not described by Corstvet and Sadler (1966b) and Suzuki et al. (1971) when the mycoplasma was given intratracheally and intranasally, respectively. The inability to detect either Mg or NDV by immunofluorescence may be due to a variety of factors, such as the relatively small area of the trachea examined, the small number of organisms present and the infrequency of examination.
44 ACKNOWLEDGEMENTS
This work was undertaken when one of us (M.M.A.) was in receipt of a grant from the British Egg Marketing Board. We wish to thank Mrs L y n d a Moore for technical assistance.
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