Pathogenesis of Mycoplasma meleagridis in diethylstilboestrol treated Turkey embryos and poults

J. COMP. PATH.

1971.

235

VOL. 81.

PATHOGENESIS OF MYCOPLASMA DIETHYLSTILBOESTROL TREATED AND POULTS

MELEAGRIDIS IN TURKEY EMBRYOS

BY

R. REIS*, N. ofEpiahniolo~

Department

and R.

MATZER*,

YAMAMOTO

and Prcvmtive Medicinc, University of California, Davis, California 95616

INTRODUCTION

It has been established that congenital transmission of Mycoplasma meleagridis in turkeys is directly related to genital infection of the female (Yamamoto, Bigland and Peterson, 1966; Mohamed, Chema and Bohl, 1966; Kumar and Pomeroy, 1969). While semen from infected males constitutes an important source of such infections (Yamamoto, 1967), the female may harbor the organism in its reproductive tract even prior to mating (Yamamoto et al., 1966). Cultures taken from the vagina of virgin hens at sexual maturity in naturally infected flocks have shown infection rates ranging from 15 to 67 percent (Yamamoto et al., 1966; Mohamed and Bohl, 1967). A recent study has demonstrated that one source of oviduct infection of the virgin female originates from the bursa of Fabricius. Since the organism invades the bursa of Fabricius during embyronic development (Reis and Yamamoto, 1970), it was postulated and proven that such a localized infection could result in an ascending infection of the oviduct at puberty upon perforation of the cloacal-vaginal occluding membrane (Matzer and Yamamoto, 1970). However, it is also possible that infection of the oviduct may occur during its formation and development by the blood stream or by extension of infection from the diseased air sac in contact with the fimbria. While Reis and Yamamoto (1970) showed that the organism is disseminated widely in various tissues of the turkey during embyronic development, the oviduct was not examined as a possible site of infection since it was felt that cultures could not be taken from the immature oviduct due to its minute size without the possibiliq of cross-contamination from adjacent tissues. The present study was designed to explore the possibility of an endogenous mode of infection of the oviduct at an early age. In order to obviate the technical difficulty of sampling from the immature oviduct, the infected embryos and poults were inoculated with diethylstilboestrol to develop this organ prematurely.

MATERIALS

AND

METHODS

The oviduct isolate (Yamamoto and Ortmayer, 1967) of recently passaged through turkey embryos, was used. The inocula for embryo or poult infections were prepared from cultures grown on agar plates by procedures described previously (Yamamoto and Bigland, 1966).

Mycophma M. meleagridis,

culture.

* Fellows of the Rockefeller Foundation. Permanent address: Dr. Reis - Escola De Veterinaria, Universidade Federal De Minas Gerais, Caixa Postal 576, Belo Horizonte, Brazil. Dr. MatzerFacultad De Medicina Veterinaria Y Zootecnia, Universidad De San Carlos De Guatemala, Guatemala, Centro America. B

236

Mycoplasma

meleagridis

IN TURKEY

EMBRYOS

AND

POULTS

Hormone. Diethylstilboestrol in vegetable oil* (25 mg. per ml.) or diethylstilboestrol powder? were used. The powder was dissolved in 95 percent ethyl alcohol (4 mg. in 0.2 ml.) and the latter diluted in 1.8 ml. of 0.85 per cent. saline (Kar, 1947). The final solution contained 2 mg. of hormone per ml. Fertile eggs and pouEts. These were obtained from a mycoplasma-free flock of turkeys developed at this station (Yamamoto, 1967). Isolation and serologic procedures. The media and method for the isolation of M. meleagridis from infected tissues have been described previously (Bigland and Yamamoto, 1964). However, when cultures were taken from heavily contaminated sources such as the cloaca, polymyxin B sulphatet was added to the enrichment broth (100 units per ml.). Other studies have shown this antibiotic to be helpful when used together with penicillin (1000 units per ml.) and thallium acetate (1 :4000) in the isolation of M. meleagridis under such circumstances (Ortmayer and Yamamoto, 1969 unpublished). The agglutination test procedure was similar to that described by Mohamed and Bohl (1968). RESULTS

Efect

of Hormone

on Oviduct

Development

The primary aim of this experiment was to asses the effect of various doses of diethylstilboestrol on oviduct development, and secondarily to obtain preliminary data on the possibility of recovering mycoplasma from the oviduct of infected poults following hormone treatment. A group of 66 embryos of 14 days incubation were injected by the yolk sac or allantoic cavity with 2.5 or 5 mg. of diethylstilboestrol in oil. At the time of hormone injection approximately half of each treatment group was inoculated by the yolk sac with 1.6 x lo* organisms (subgroups of 6 to 9 embryos each). Four uninoculated embryos were included to compare the size of the oviduct of treated and untreated poults (Table 1). Approximately half of the poults which hatched from each treatment group were necropsied at 1 day of age. The remaining poults were necropsied at 19 days of age following two intramuscular injections of hormone at doses similar to the initial doses (i.e., 2.5 or 5.0 mg.) at 1 and 8 days of age for cumulative doses of 7.5 or 15 mg., respectively. As shown in Table 1 the poult yield for all groups was low. However, since the 23 embryos which hatched from all treatment groups consisted of 12 females and 11 males, it seems that the hormone treatment did not contribute to excessive mortality of one sex over the other. Examination of poults at 1 day of age. Five females and 6 males were examined (Table 1). Of the 3 females injected with 2.5 mg. of hormone by the yolk sac or allantoic cavity routes, both the right and left oviducts were enlarged (1 to 3 cm. in length and 0.3 cm. in diameter). The oviduct was slighty larger (4 to 5 cm. in length and 0.5 cm. in diameter) in the poult injected by the yolk sac with 5 mg. of hormone (Fig. 1). In the case of the untreated poult the immature oviduct was of such minute size that it required careful manipulation and dissection of various organs and tissues before it could be identified (Fig. 2). The cloacalvaginal occluding membrane wzs intact in all of these poults. Pathological changes *H.C.BurnsPharmaccuticals. t Mann

Research

2 Pfizer

Lab.

Laboratories,

Inc

R. REIS et al. TABLE EXPRRIMRNTAL EMBRYONIC

1

DRSION To STUDY THE EFPRCT OF DIETHYLSTILBOESTROL WHEN INJECTRD AND EARLY POSTRMBRYONIC LIFE ON OVIDUCT DEVRLOPMENT OF TURKEY

Hormone' Groups

237

Dose in mg.

.Numbcr of

Route of injection

Embryos injected

DURING POULTS

Number of poulti necropsiedate

Poults hatched

I day F

19 day M

F

M

0 1

t

0 1

A

0 1

0

0

HZroups 5 through 8 were injected at 14 days embryonic development with 1.6 x 10’ organisms per embryo by the yolk sac to obtain preliminary data on oviduct localization. bathe hormone was injected at 14 days embryonic cavity (AC).

development

cThe poults necropsied at 19 days of age were given two additional of age (see text) ; F = female, M = male.

by the yolk sac (YS) or allantoic doses of hormone at 1 and 8 days

Not injected.

l

TABLE ISOLATION

Inoculated

2

OF Mycopkxsma mcleagridis

PROM

DAY-OLD

POULTS

at 14 days embryonic development with 2.1 x 10s organisms and 5 mg. of diethylstilboestrol in oil by the yolk sac route Myoph.ma

isolationb

Oviduct sizea Pol&no.

L&$2

1

large

: 4

kg large large large large large

z zi

0 The ocduding

Right small large small large small small large large

Oviduct Abdominal cavity

+++ +++ +++ +++ +++ +++ +++ +++

IleUm

Cloaca

Jw

+ +3 +-

+++ -I-++ +T+ +++ ++i+++ +++

+ t+ + ++ -

Right

ALz ’

n ++

-I

n

t-

z +

:

T++

: +

membrane was intact in all poults.

* Mycoplasma growth on artificial medium was graded as - , f , + + , and + + + for no growth, slight, moderate or heavy growth, respectively; n = not done and c = fungal contamination.

238

Mycofilasma meleagridis IN TURKEY

EMBRYOS

AND

POULTS

of a protruding cloaca and increased elimination of urates were observed in all of the hormone treated poults including the 6 males. Samples for culture were not taken from these poults necropsied at 1 day of age. Examination of poults at 19 days of age. Of the 13 poults examined 5 were recognized as males at necropsy and consequently eliminated from the study. The distribution of the 8 females among the various treatment groups is shown in Table 1. There was no difference grossly in the development of the oviduct of poults injected with a cumulative dose of 7.5 or 15 mg. The oviducts were welldeveloped so that various anatomical segments could be easily differentiated. The vaginal-occluding membrane of all poults did not appear to be altered grossly. MycopIasma was recovered from the uterus, distal to the occluding membrane, of the two poults (groups 6 and 7, Table 1) which had been inoculated with M. meleagridis during embryonic life. Isolations were made also from the sinus, trachea, lungs, air sacs, ileum, cloaca and bursa of Fabricius, and airsacculitis was observed in both poults. Distribution

of the Organism

in Yolk Sac Infected-Hormone

Treated Embryos

In order to simulate the natural infection (Reis and Yamamoto, 1970) fifty turkey embryos of 14 days incubation were inoculated simultaneously by the yolk sac with 2.1 x 10’ organisms and 5 mg. of diethylstilboestrol in oil. The poults were killed immediately after hatching and the females, which consisted of 8 poults, were examined. Three male poults and 39 dead embryos were discarded from the experiment. As shown in Table 2 the oviducts of all poults were enlarged, but the occluding membranes were intact. Similar to the preliminary findings, severe airsacculitis was observed in all poults and the organism was recovered from the abdominal cavity, ileum, cloaca and oviduct of most of the poults. Distribution of the Organism in Air Sac Infected-Hormone Treated Poults The aim of this experiment was to study more accurately the possibility of a descending type of infection of the oviduct by injecting the organism into the air sac adjacent to the oviduct. Nine female poults (4 or 11 days old) were inoculated by the left posterior thoracic sac with 5 x 10’ organisms. Starting on the third day following injection, 6 of the poults were inoculated intramuscularly at daily intervals with a 2 mg. dose of diethylstilboestrol in alcohol-saline solution for cumulative doses ranging from 20 to 28 mg. among individual poults. All poults were bled and necropsied between 13 and 24 days following infection. Table 3 shows that cumulative hormone doses of 20 to 28 mg. per bird resulted in marked development of the oviduct. The occluding membrane was ruptured in only 1 poult which had been injected with a cumulative dose of 28 mg. over a 14 day period. Figs. 3 and 4 show the oviducts of 3 of these poults. The antibody response in both the infected-hormone treated and untreated poults were similar with respect to the time of appearance and to the level of titres (Table 3). As in the previous trials gross changes in the hormone treated poults included cloaca1 blockage and excess mates in the ureter and vent. Five of the 6 infected-hormone treated poults yielded the organism from their

A@

afh

infection

Total hormone dose (mg.)*

OF

ooiduct si2.F Tr

TABLB

3

Lu

LAS

LEFT

Cl

BF

Va

Myco@mu~ isolatiotr’

IN TURKEY POIJLTS INFECTED BY THE DAILY DlEl’HYUTILBOESTROL INJECTIONS

Mycopkasma mcleagridis

Ut

AIR

Oviduct

THORACIC

Ma

Fi

SAC FOLLOWED

Tube Air agg utination &It& titer

BY

a Age of Pot&s at the time mycoplasma injection. b See tent for hormone injection schedule. cE = enlarged; R = rudimentary; the occluding membrane was ruptured only in poult no. 5. fl Tr = trachea; Lu = lung; LAS = left abdominal air sac; Cl = cloaca; BF = bursa Fabricius; Va = vagina; Ut = uterus; Ma = magnum; and Fi = fimbria. Cultures also were taken from the sinus, ileum, and joints; only one poult (no. 9) yielded the organism from sinus and ileum. + = positive isolation; - = negative isolation; n = not done. 8 Air sac lesions were scored as -, 1 +, 2+, or 3+ for no lesions, mild, moderate, and severe, respectively.

Bird M.

Necro&y dnvs

PATHOGENESIS

240

Mycoplasma meleagridis IN TURKEY

EMBRYOS

AND

POULTS

air sacs adjacent to the oviduct as well as from other sites; the 6th poult in this group responded with agglutinins, but failed to yield the organism from any tissues sampled (Table 3). In spite of the rather consistent air sac infection only 2 of the 6 poults harboured the organism in their oviducts. In one of them the organism was localized only in the distal end (fimbria) of the oviduct; the cloaca and bursa of Fabricius failed to yield the organism and the occluding membrane was intact (No. 4). In the other poult the organism was isolated from both the proximal (vagina and uterus) and distal (fimbria), but not from the midportion (magnum) of the oviduct; the cloaca and bursa of Fabric& were infected and the occluding membrane was ruptured (No. 5). The 3 infected poults not treated with hormone (7, 8, and 9) yielded the organism from various respiratory and intestinal sites. The oviducts were not cultured due to their rudimentary size (Table 3). DISCUSSION

The aim of this study was to determine whether M. meleagridis infection of the oviduct of the virgin turkey could occur by an endogenous route early in the life of the bird. In approaching this study it was necessary to overcome a major technical problem of accurately sampling from the immature oviduct of the bird. The idea that diethylstilboestrol might be used advantageously for this purpose was based on the work of Kar (1947) who showed that administration of this hormone in doses of 4 to 15 mg. in young chickens significantly developed the size of the immature oviduct. Kar further showed that the perforation of the occluding membrane was governed by the dose and period of hormone injection. The present study indicates that the oviduct of the immature turkey responds similarly to that of the chicken under the influence of diethylstilboestrol. When the hormone was injected in doses of 2.5 or 5 mg. during embryonic development and the poults were examined at the time of the hatch, their oviducts were significantly enlarged and their occluding membranes were intact. Additional injections after hatching for cumulative doses of 7.5 or 15 mg. further enlarged the oviduct without grossly affecting the occluding membrane. The intramuscular administration in young poults of diethylstilboestrol in 2 mg. doses at daily intervals for cumulative doses ranging from 20 to 24 mg. also resulted in enlarged oviducts without rupture of the occluding membranes. However, under this injection regime a cumulative dose of 28 mg. appeared to be the critical level which could cause dissolution of the occluding membrane. While the study was not designed specifically to determine the possible deleterious side effects of the hormone, two obvious changes included increased urate elimination and a protuding cloaca in all hormone treated embryos and poults. While the former observation suggests kidney malfunction, the latter change was believed to be due to mechanical displacement of the cloaca by the enlarged oviduct. It is possible, however, that the hormone may have altered the invasiveness of the organism or the integrity of the oviduct tissue. With respect to the first possibility, in vitro studies indicated that this hormone at levels of 5 or 10 micrograms per ml. of medium did not enhance or depress the growth of M. meleagridis (Matzer and Yamamoto, 1970 unpublished). Further-

R. -1s

et al.

241

more, in the present study the hormone apparently did not alter the immune response to infection. Some evidence which suggests that the treatment did not alter the oviduct tissue is reported by Kar (1947) who did not observe gross or microscopic changes in the oviduct of young chickens inoculated with 15 mg. of diethylstilboestrol. Another study has shown that oestrogens protected and stimulated the Miillerian duct of the chicken embyro while androgens induced necrosis of this organ (Scheib, 1963). Nevertheless, since conclusive evidence is not available which shows that the hormone in itself did not influence the pathogenesis of the organism, the following interpretation of the results of the study must be tempered accordingly. The oviducts of poults injected with diethylstilboestrol during embryonic or early postembryonic life were easily sampled due to their large size. While the air sacs and abdominal cavity were heavily infected, problems of cross-contamination from such sources were unlikely during sampling from the oviduct lumen. Thus, the high incidence of infection of the oviduct of poults, infected by the yolk sac during embryonic development, in which the occluding membranes were intact, suggests that infection of thii organ could occur during embryonic life of the poult by a haematogenous route or by extension of infection by direct contact with infected air sac tissue at the distal end of the oviduct. While the cloaca of these poults was infected it would appear unlikely that an ascending infection of the oviduct occurred from such a site due to the natural barrier presented by the occluding membrane. The importance of the occluding membrane in preventing the organism present in the cloaca from gaining access to the oviduct at its proximal end was recently demonstrated (Matzer and Yamamoto, 1970). The isolation of the organism from the oviduct of only 2 of 6 poults following infection by the air sac and intramuscular hormone injections suggests that the organism apparently does not invade the oviduct as consistently from such an infection site as that resulting from yolk sac infection during embryonic development. These results are in agreement with earlier studies which indicate that the organism does not localize readily in the oviduct of semimature or mature hens injected by the air sac (Yamamoto, 1967 ; Kumar and Pomeroy, 1969). In regard to the mode of oviduct infection following air sac inoculation of the organism, the distribution of the organism in one of the oviduct infected poults suggests a direct extension of infection from the infected air sac to the fimbria. The mode of oviduct infection in the other poult also could be explained as a descending type of infection from the infected air sac. However, since the occluding membrane was ruptured in this poult and the organism was present in most areas of the oviduct, the cloaca and bursa of Fabricius it is possible to visualize an ascending type infection as described by Matzer and Yamarnoto (1970). Since other studies have shown that the organism can invade the bursa of Fabricius by a haematogenous route (Reis and Yamamoto, 1970; Reis, DaSilva and Yamamoto, 19,70), it is possible that the organism infected the bursa by the blood stream following air sac inoculation, with subsequent extension of infection into the cloaca. It should be emphasized again that the conclusions reached in this study may be equivocal since they are based on the assumption that the hormone treatment did not influence the pathogenesis of the organism. However, it is hoped that the

242

Mycophma

meleagridis IN TURKEY

EMBRYOS

AND

POULTS

findings and interpretations will stimulate other approaches which might be more definitive in the study of the pathogenesis of M. meleagridis in genital infections. A study of juvenile and semimature turkeys under more natural conditions of infection could be undertaken after more reliable dissection and cultural procedures are developed. If the present findings prove valid, one should also be able to detect infection of the oviduct of virgin females prior to the perforation of the occluding membrane. SUMMARY

The pathogenesis of Mycoplasma meleagridis in the oviduct of young turkeys was studied. Diethylstilboestrol was administered to infected embryos and poults to develop the oviduct prematurely to facilitate sampling at an early age. The organism was isolated at a high frequency from the oviducts of one-day old poults, infected during embryonic development, in which the cloacal-vaginal occluding membranes were intact. The results thus suggested that infection of the oviduct could occur by an endogenous route during embryonic life. The organism was also recovered from the oviducts of young turkeys infected by the air sac, suggesting that infection of the oviduct could occur at its distal end by contact with the infected air sac. However, the incidence of oviduct localization in such an infection system was minimal when compared to that of embryonic yolk sac infection. ACKNOWLEDGMENT

This

investigation

was supported

by Public

Health

Service

Grant

AI-07805-03.

REFERENCES

Bigland, C. H., and Yamamoto, R. (1964). AZ,& Dis., 8, 53. Kar, A. B. (1947). Poultry Sci., 26, 352. Kumar, M. C., and Pomeroy, B. S. (1969). Am??. J. vef. Res., 30, 1423. Matzer, N., and Yamamoto, R. (1970). A&n Dis., 14, 321. Mohamed, Y. S., Chema, S., and Bohl, E. H. (1966). Ibid., 10, 347. Mohamed, Y. S., and Bohl, E. H. (1967). Ibid., 11, 634; (1968). Ibid., 12, 554. Reis, R., DaSilva, J. M. L., and Yamamoto, R. (1970). Ibid., 14, 117. Reis, R., and Yamamoto, R. (1970). Amer. J. vet. Res., Scheib, D. (1963). Ciba Foundation Symposium. Edited by deReuck, A. V. S. and Cameron, M. P., Little Brown and Co.; Boston, Mass. Yamamoto, R., and Bigland, C. H. (1966). Amer. J. vet. Res., 27, 326. Yamamoto, R., Bigland, C. H., and Peterson, I. L. (1966). Poultry Sk., 45, 1245. Yamamoto, R. (1967). N.Y. Acad. Sci., 143, 225. Yamamoto, R., and Ortmayer, Herrad B. (1967). Auian Dis., 11, 288. [Received

for publication,

October

13th, 19691

R.

REIS

et al.

Fig. 1. One day old poult inoculated with 5.0 mg. of diethylstilboestrol by the yolk sac at 14 days R -= right oviduct; B = bursa of Fabric&; embryonic development. 1, --. left oviduct; Re _ rectum. Fig. 2. One day old nontreated poult. Note the small size of the left oviduct as compared to that of the hormone treated poult shown in Fig. 1. Fig. 3. Poult, 30 days old, necropsied 19 days after infection by the left posterior thoracic air sac and injected intramuscularly with 28 mg. of diethylstilboestrol. Note the enlarged oviduct (0~) and the severe thoracic air sac lesion (As). See Table 3: poult no. 4 for necropsy results. Fig. 4. Left: Oviduct of a 28 day old poult injected intramuscularly with 24 mg. of hormone (Poult no. 6 in Table 3). Note the intact occluding mcmbranr. Right: Oviduct of a 28 day old poult injected with 28 mg. of hormone (Poult no. 5 in Table 3). Note the perforated occluding memhrane. The hursa of Fabricius is shown adjarcnt to each oviduct. l-0 face*age241