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Experimental Infection of Point-of-Lay and in-Lay Pullets with Salmonella Typhimurium
Br. vet. }. ( 1978 ), 134, 92
EXPERIMENTAL INFECTION OF POINT-OF-LAY AND IN-LAY PULLETS WITH SALMONELLA TYPHIMURIUM
By
D. D. BROWN AND T. F. BRAND
Ministry of Agriculture, Fisheries and Food, Veterinary Laboratory, Eskgrove, Lasswade, Midlothian
SUMMARY
Thirty po int-of-Iay and 27 in-lay pullets were infected orally with Salmonella typhimurium (1 x 10 10 ) and maintained individually in cages. They were slaughtered over a three-week period after inoculation. Infection produced severe clinical disease in all birds and a varied effect on egg production. Pathological changes were no ted at post-mortem examination. Bacteriological examination of tissues and all eggs laid demonstrated the presence of enteric and systemic infection including the ova ry, but produced no evidence of direct ovarian transmission or shell contaminatio n.
INTRODUCTION
Salmonella pullorum and S. gallinarum can localize in the ovary of chickens and turkeys r esulting in direct ovarian transmission (Snoeyenbos, Smyser & Roekel, 1969; Smith, 1971 ). The hatching of infected eggs and the subsequent dissemination within the incuba tor are the primary factors in the spread of pullorum disease (Wilson, 1945; Smith, 1971) and less frequently oHowl typhoid Uordan, 1956; Wilson, 1958 ). Other salmonellae, unlike S. pullorum and S. gallinarum, are motile and can penetrate the shell under favourable conditions of temperature and humidity. Shell contamination is recognized as the most frequent mode of egg transmission of these motile organisms (Mellor & Banwart, 1965; Gordon, 1971; Smith, 1971; Williams, 1972). Although S. typhimurium has been isolated from the ovaries of laying hens (Wilson, 1947; Snoeyenbos et at., 1969; Slavkov & Vaptsarova, 1970; Savov et at. , 1974) and from egg contents (Wilson, 1950; Slavkov & Vaptsarova, 1970; Savov et at., 1974), conflicting views have been expressed on the occurrence of direct ovarian transmission of this organism in the fowl. The present paper reports a study of experimental oral infection of point-of-Iay and in-lay pullets with S. typhim'lJ,rium with special reference to the incidence of direct ovarian transmission, the frequency and significance of ovarian infection and the effect on egg production.
SALMONELLA TYPHIMURIUM IN PULLETS
93
MATERIALS AND METHODS
Inoculum The strain of S. typhimurium phage type U223 used in the investigation was an isolate from a field outbreak of disease in broiler breeders. The inoculum was a saline (0 ·85%) suspension prepared from washings of moist agar slants which had been seeded with an actively motile organism and incubated for 18 h at 37°C. The suspension was standardized by Brown's opacity tube method and viable plate counts on nutrient agar indicated that the inoculum contained 1 x 1010 viable organisms per m\. The counts were carried out prior to inoculation. Birds Thirty point-of-lay and 27 in-lay specific pathogen free (SPF) Brown Leghorn birds were maintained individually in cages. Serum agglutination tests and cultural examination of cloacal swabs from all birds for the presence of salmonellae before the start of the experiment were negative. All birds were caged in the premises several weeks before the date of inoculation. The birds were inoculated orally with 1 ml of the suspension administered into the middle third of the oesophagus by means of a sterile syringe and catheter tube. Before inoculation 10 point-of-lay and 25 in-lay experimental birds and one point-of-lay and four in-lay control birds were in production. Birds were slaughtered 17 to 19 days after infection, except for the most severely affected non-laying birds which were slaughtered between the 10th and 12th day. One of the infected point-of-lay birds died on the sixth day and one of the infected in-lay group was slaughtered in a comatose condition on the seventh day. Five point-of-lay and five in-lay uninfected SPF Brown Leghorns, caged individually and housed in a third pen were maintained as controls, so that a comparison of egg production could be made. Daily clinical observations were made. Eggs were collected twice daily, wrapped individually in squares of aluminium foil and stored at 4°C prior to bacteriological examination within 48 h of collection. Post-mortem examination Gross pathological changes were noted post mortem and liver, spleen, caeca and ovary were examined bacteriologically. The number of ovary samples per bird varied fro m one to four depending on development, with regressed ovules a nd groups of varying sized follicles being cultured separately. Us ing a Colworth Stomacher (Seward Ltd, London), all soft tissues were pulverized in 25 ml selenite F medium in polythene bags, an approximate 10% w/v ratio being maintained. The suspensions were transferred to 7 x 1 inch sterile tubes and incubated at 37°C for 24 h prior to plating on to MacCo nkey medium and subsequent incubation at 37°C for 18 h . Bacteriological examination of eggs was carried out by thoroughly rubbing each shell with a swab moistened in selenite F medium and then placing the swab in a tube with 25 ml of this medium. The eggs were then washed in 2·5% Hyco -quat solution (Hy-co Products (Scotland ) Ltd ), thoroughly rinsed in cold water and finall y immersed in 50% ethyl alcohol for 30 s and allowed to dry in air before the yolks and whites were
~
"'" TABLE I PATT ERN OF LAY AFTER I NOCULATION WITH S. TYPHIMURIUM
In lay
Point oflay
'" iO
Controls. No. of birds
No. of birds
No. with positive ovaries
2
o
2 2
1
o
4
3
Controls. No. of birds
...... en
No. of birds
No . with positive ovaries
9 I
o o
::c
< t'1 -l
t'1 iO
Z
In-lay before inoculation Ma inta ined produ ctio n I nterru pted lay I to 9 d ays p .i. Interrupted lay 4 to 16 d ays p.i . We nt o lfl ay 1 to 6 d ays p.i.
:;
Infected
Infected
1 0 0 0
5
o o o
5
o
10
5
» ~ o
'--
c:
iO
Z
)-
Not in-tay before inoculation Fail ed to lay during experi me nt Started to lay 2 to 7 days p.i. Started to lay 16 10 17 da ys p.i . Laid o nl y dut-ing first week p .i. Total p.i . post ino(ui
0 3 I 0
14
5
30
2
o
2
1
2 13
o
r .""..
o
""
o o o
o
o
o
o
o
5
27
5
2
o
SALMONELLA TYPHIMURIUM IN
PULLETS
95
separated. The shells and yolks were placed in individual flasks contammg 100 ml selenite F medium and the yolks ruptured by means of a sterile applicator stick. The swabs, shells and yolks were cultured in a similar manner to the soft tissues . RESULTS
Clinical Depression, inappetence and diarrhoea were observed in all infected birds three to five days after infection, followed by an improvement in their clinical condition during the next few days. The majority of the point-of-Iay birds were more severely affected than the in-lay group and were a day or two slower in showing an improvement. Approximately 50% of the point-of-Iay and 25% of the in-lay birds had persistent diarrhoea during the second week ; those most severely affected and not in lay were killed 10 to 12 days after infection. A few birds in both groups continued to show evidence of diarrhoea until slaughtered at the end of the experiment. One point-of-Iay bird died and one of the in-lay group was slaughtered in a comatos.e condition six and seven days after infection respectively. The control birds showed no evidence of disease. T he patterns of lay and percentage egg production are given in Tables I and II. Egg production showed a varied response to infection which was less marked in the in-lay gro up, despite the presence of clinical disease in all birds and prolonged diarrhoea in a few. Nine of the in-lay group continued to lay well until the end of the experiment, five returned to lay after a short break in production seven to 12 days after infection and one after a break in production one to eight days after infection . Ten of this group went off lay o ne to three days after infection and did not resume production and two did not co me into lay during the experiment. A mo re severe effect on the point-of-Iay birds was noted, 14 of which did not come into lay before slaughter or death. Four birds came into production during the first week but two of these laid only one egg each while two birds started to lay near the end
TABLE II EGG PRODUCTION EXPRESSED AS A PERCENTAGE FO R SIX·DAY PERIODS F ROM 12 DAYS BEFORE TO 18 DAYS AFTER INOCULATION
In -lay birds
Point- oj-lay birds
Before inoculation 12-7 days 6- 1 days After inoculation 1-6 days 7- 12 days 13- 18 days
Infected
Control
Infected
. Control
5 12
0 3
40 61
37 47
13
13 37 47
43 38 60
47 47 63
II
37
96
BRITISH VETERINARY JOURNAL, 134, 2
of the experiment. Ten birds had begun to lay before infection , one of which continued to lay well throughout the experiment and one laid rather irregularly; the other eight birds went ofT lay during the first week after infection and four of these resumed production during the second week. The five in-lay and one of the point-of-lay control birds had come into production before the start of the experiment; of the four remaining control point-of- lay birds, three started to lay within the first week and the fourth by the 17th day. Egg production of all birds from 12 days before until 18 days after inoculation is shown in Table II. A gradual rise was noted throughout the experiment in both control groups, compared to a fall in infected birds where the maximum decrease occurred seven to 12 days after infection, followed by a rise in production over the next six days.
Gross patholof:Y The most marked changes were observed, six to 10 days after infection, in the caeca. The walls were thickened, the linings congested and there was narrowing of the lumina which were either empty or contained pale or blood-stained necrotic cores. Congestion of the lining of the duodenum and upper small intestine was also noted at this time. Similar but less marked changes were observed in birds killed 12 days after infection, and in a few of those killed at 17 to 19 days there was congestion of the lining of the large intestine. Enlargement of the spleen was a feature of infection in most birds examined up to 12 days after infection and in many killed at the end of the experiment. Abnormality of the ovarian follicles was evident in six of the point-of-lay and six of the in-lay birds. The contents of some ovules were inspissated, in others the capsule was congested and one bird had elongated follicular stalks.
Bacteriolof:Y Eggs. Bacteriological examination of the shells and contents of the 25 7 eggs laid by infected birds during the experiment was negative for the presence of S. typhimurium. Tissues . The results of bacteriological examination of tissues post mortem are given in Fig. 1. S. typhimurium was recovered from the spleen, ovary, caeca and liver of 84, 32, 28 and 25% respectively of the 57 birds. There was a higher frequency of recovery early in infection with those tissues being positive in 96, 57, 65 and 52% respectively of the 27 birds examined six to 12 days after infection, compared to 76, 15, 3 and 6% respectively of the survivors 17 to 19 days after infection. All tissues of control birds were negative for the presence of salmonellae. The organism was recovered from the ovaries of 18 birds. With one exception, this involved birds with either apparently inactive ovaries or in which only regressed ovules were positive. In the one exception, a point-of-lay bird, the ovary was becoming active and the organism was recovered from one of two samples of apparently normal ovules. The majority of birds with positive regressed ovules, had gone ofT or were going ofT lay, but one point-of-lay bird was coming back into production and two were in lay; there was no recovery of the organism from other ovule samples cultured from these birds. In no case was the organism recovered from an active ovule of a bird in lay.
POINT- OF- LAY GROUP
~
t--
:s;
()
~
t--
[;: ...., ~ ::t: IN - L AY GROUP
-;2:'~----
t-I
r __ .. _
~
c:::
::t> .....
c::: :s; Z
'" c: •
o
Positive
0- died
r r
Nega tive
C - comatose when killed
Vl
~
~ No sample Fig. I. Bacteriol ogica l exam ination of tissue post mortem.
98
BRITISH VETERINARY JO U RNAL, 134,2
DISC USSION
Experimental oral infection with 1 x 10 10 viable S. typhimurium org\lnisms resulted in severe clinical disease in all birds three to five days after infection, with one dea th a nd one killed in a comatose condition during the first week. The point-of-lay birds were more severely affected than the in-lay group and were one to two days longer in showing recovery. The clinical disease was more severe than previously observed with experimental infection of six-month-old Brown Leghorn cockerels after administration of a similar inoculum prepared from the same strain of S. typhimurium (Brown, Ross & Smith, 1975), but was comparable to that observed in hens naturally infected with S. typhimurium in which depression, inappe tence, diarrhoea, but no mortality was reported (Kashiwazaki et al., 1966). Severe clinical disease was also reported by Mundt & Tugwell (1958 ) up to four days after the intravenous inoculation of adult layers with other motile salmonellae. In co ntrast with the controls which laid well during the investigation, infection had a varied effect on egg production in both groups (Table I) . Apart from two of the inlay birds which did not come into production, the remainder of this group showed three main types of response; those which continued to lay well despite the presence of clinical disease, others which went off lay completely and those which had a break in production during the second week . A more severe effect was observed in the pointof- lay birds, half of which did not come into lay; those which were in lay before inoculation showed a varied response to infection (Table I). Because of the small number of controls and the removal of some non-laying infected birds during the second week for bacteriological examination, only a tentative comparison is made of egg production figures. Both control groups showed a gradual rise in production throughout the experiment, while a reduction was noted in the infected groups, this being most marked during the seven to 12-day period after infection, when a 23% fall was recorded from the in-lay birds (Table 11). Surviving infected birds showed a subseq uent rise in production during the 13th to 18th days . A gradual drop in production was reported by Kashiwazaki et al. (1966) in a flock showing evidence of clinical disease associated with natural S. typhimurium infection . Mundt & Tugwell ( 1958 ), following intravenous inoculation of layers with other motile salmonellae, reported variation in egg production ranging from normal lay to a maximum fall of 2 1% depending on the seroLype. The most marked gross pathological changes were seen in birds which were killed or had died six to 12 days after infection and involved the caecum, duodenum and spleen, although splenic enlargement was still a feature of infection in birds killed 17 to 19 days after inoculation. These findings are consistent with those previously observed in adult cockerels slaughtered three to nine days after oral administration of a similar inoculum (Brown et al., 1975). Distorted ovules were noted in birds in varying stages of lay and similar observations on fowls naturally infected with S. typhimurium are reported by Wilson ( 1947 ) and Kashiwazaki et al. ( 1966). Systemic infection was confirmed post mortem in 49 of the 57 birds (84%). The spleen was the most frequent site of recovery, being positive in 84% of cases, in contrast with previous experimental infection of cockerels with S. typhimurium (Brown et al. , 1975 ) when caecal recovery was predominant and more frequent recovery from the liver
SALMONELLA TYPHIMURIUM IN P ULLETS
99
reported by Kas hiwazaki et al. (1966). After oral inoculation of layers with other motile salmonellae, Turnbull & Snoeyenbos (1974) reported recovery from the spleen at nine, but not at 18 days, although frequent recoveries were obtained from various levels of the intesti nal tract. In the present experiment the organism was recovered from the ovary of 18 birds. This involved birds either with apparently inactive ovaries or in wh ich only regressed ovules were positive; most of those showing regression of ovules ha d go ne off lay, but two were in lay and one was coming in to lay. On one occasion on ly, the organism was recovered from one of two samples of apparently normal ovules of a bird which was coming in to lay. Isolation of S. typhimurium from regressed ovules of fowls has also been reported by Wilson (1947 ) and other reports illustrate the occurrence of ovarian infection in the fowl with this serotype and other motile salmonellae (Clarenburg & Romijn, 1954; Mundt & Tugwell, 1958; Kashiwazaki et al., 1966; Snoeyenbos et ai., 1969 ; Slavkov & Vaptsarova, 1970 ; Savov et at., 1974), but none o f these workers relate ovule infection with direct ovarian transmission. Some reports do not state if infection involved quiescent, active or regressed o'vules, factorsof potential significance in considering this mode of transmission. Desp ite severe clinical disease, systemic infection and the recovery of the organism post mortem from the ovaries of birds which had laid, on no occasion was the organism recovered from an active ovule of a bird which was in lay, nor from any of the 257 wh ole eggs examined . Following inoculation of caged layers with other motile salmonellae, Mellor & Banwart (1965 ) and Brown, Ross & Smith (1976 ) also failed to recover the organisms from the shell or contents of eggs laid during the experiment. The presence of S. typhimurium and other motile salmonellae in egg contents has been reported (Wilson, 1945, 1950; Gordon & Tucker, 1965 ; Smyser et ai., 1966; Forsythe, Ross & Ayres, 1967; Olesiuk et ai., 1969; Slavkov & Vaptsarova, 1970; Cox et ai. , 1973 ; Savoy et at., 1974). As some of these workers have also recorded isolation of the organisms from the shell, the possibility of shell penetration must be considered as a potential r oute of infection of egg contents in such cases. Wilson (1950) reported yo lk infection in three eggs examined within minutes of lay from a fowl naturally infected with S. typhimurium and concluded that direct ovarian transmission had occurred . The literature, however, suggests that this is a rare occurrence in the fowl with motile salmonellae (Gordon, 1971; Smith, 1971; Williams, 1972). Cox et ai. (1973) found no evidence to support this mode of transmission with S. typhimurium and similar findings with other motile salmonellae are reported (Gibbons & Moore, 1946 ; Buxton & Gordon, 1947; Clarenburg & Romijn, 1954; Mundt & Tugwell, 1958; Mellor & Banwart, 1965). Whereas it is accepted that the challenge of experimental and natural field infection cannot be equated, the findings of the present in~estigation, obtained under conditions designed to preclude the possibility of shell contamination and subsequent shell penetration, produced no evidence of direct ovarian transmission of S. typhimurium and suggest the alternative of early regression of infected ovules. ACKNOWLEDGEMENTS
We wish to thank Dr J. G. Ross and Mr R. D. Lapraik for their helpful criticism during the preparation of the paper and the staff of the Bacteriology Section for their technical assistance.
100
BRITISH VETERINARY JOURNAL, 134,2
REFERENC ES
BROWN , D. D ., ROSS,]. G. & SMITH, A. F. G. (1975 ). Res. vet. Sci. 18, 165. BROWN, D. D., ROSS,]. G. &SMITH, A. F. G. (1976). Res. vet. Sci . 20, 237. BUXTON, A. & GORDON, R. F. ( 1947).]. Hyg. , Camb. 45, 265 . CLARENB URG, A. & ROMIJN, C . (1954 ). Proc. 10th Wld Poultry Congr. p. 214 . Cox, N. A., DAVIS, B. H., WATTS, A. B. & COLMER, A. R . (1973 ). Poultry Sci. 52, 661. FORSYTHE, R . H., Ross, W.]. & AYRES,]. c. (1967 ). Poultry Sci. 46, 849. GIBBONS, N . E. & MOORE, R . L. (1946 ). Poultry Sci. 2,115. GORDON, R . F. (1971). Vet. A. p. 95 . Bristol:]. Wright & Sons Ltd. GORDON, R . F. & TUCKER,]. F. (1965 ). Br. Poultry Sci. 6, 251. JORDAN, F. T. W. (1956 ). Poultry Sci. 35,1019. KASHIWAZAKI, M., AOKI, S., HORI UCH I, T., SHOYA, S. & NAM IOKA, S. (1966 ). Natn 1mt. Anim. Hlth Q" Tokyo 6, 144. MELLOR, D . B. & BANWART, G. j. ( 1965). ]. Food Sci. 30, 333. M UNDT,]. 0. & T UGWELL, R. L. (1958 ). Poultry Sci. 37, 415. OLESI UK, 0. M., CARLSON, V. L. , SNOEYENBOS, G. H. &SMYSER, c. F. ( 1969 ). Avian Dis. 13,500. SAVOY, D. , VRIGAZOV, A., DIMITROV, N. & GROMKOV, N. (1974 ). In Salmonelite i salmoneloz.ite v Bulgariya p. 265. Sofia, Bulgaria: Academy of Sciences. SLAVKOV, I. & VAPTSAROVA , M. (1970 ). Vet . Med. Nauki, Sofia 7,103 . SMI TH, H. WILLIAMS ( 1971 ). Poultry Disease and World Economy, ed. R. F. Gordon and B. M. Freemen, p. 37. Edinburgh: Longman for British Poultry Science . SMYSER, C. F., ADINARAYANAN, N., RO EKEL, H . VAN & SNOEYENBOS, G. H. (1960). Avian Dis. 10, 314 . SNOEYENBOS, G. H., SMYSER, C. F. & RO EKEL, H. VAN ( 1969 ). Avian Dis. 13,668. TURNBULL, P. C. B. & SNOEYENBOS, G. H. ( 1974). Avian Dis. 18, 153. WILLIAMS,]. E. (1972). Diseases of Poultry, 6th edn, p. 135, ed. M . S . Hofstad and others. Ames , Iowa: State University Press. WILSON,]. E . (1945 ). Vet . Rec. 57,411. WILSO N, ]. E. ( 1947). Br. vet.}. 103, 101. WILSON,]. E. (1950). Vet. Rec. 62,449. WILSON, j. E. ( 1958). Agric. Rev., Lond. 3, 23.
(AcceptedfOT publication 22 March 1977)
EXPERIMENTAL INFECTION OF POINT-OF-LAY AND IN-LAY PULLETS WITH SALMONELLA TYPHIMURIUM
By
D. D. BROWN AND T. F. BRAND
Ministry of Agriculture, Fisheries and Food, Veterinary Laboratory, Eskgrove, Lasswade, Midlothian
SUMMARY
Thirty po int-of-Iay and 27 in-lay pullets were infected orally with Salmonella typhimurium (1 x 10 10 ) and maintained individually in cages. They were slaughtered over a three-week period after inoculation. Infection produced severe clinical disease in all birds and a varied effect on egg production. Pathological changes were no ted at post-mortem examination. Bacteriological examination of tissues and all eggs laid demonstrated the presence of enteric and systemic infection including the ova ry, but produced no evidence of direct ovarian transmission or shell contaminatio n.
INTRODUCTION
Salmonella pullorum and S. gallinarum can localize in the ovary of chickens and turkeys r esulting in direct ovarian transmission (Snoeyenbos, Smyser & Roekel, 1969; Smith, 1971 ). The hatching of infected eggs and the subsequent dissemination within the incuba tor are the primary factors in the spread of pullorum disease (Wilson, 1945; Smith, 1971) and less frequently oHowl typhoid Uordan, 1956; Wilson, 1958 ). Other salmonellae, unlike S. pullorum and S. gallinarum, are motile and can penetrate the shell under favourable conditions of temperature and humidity. Shell contamination is recognized as the most frequent mode of egg transmission of these motile organisms (Mellor & Banwart, 1965; Gordon, 1971; Smith, 1971; Williams, 1972). Although S. typhimurium has been isolated from the ovaries of laying hens (Wilson, 1947; Snoeyenbos et at., 1969; Slavkov & Vaptsarova, 1970; Savov et at. , 1974) and from egg contents (Wilson, 1950; Slavkov & Vaptsarova, 1970; Savov et at., 1974), conflicting views have been expressed on the occurrence of direct ovarian transmission of this organism in the fowl. The present paper reports a study of experimental oral infection of point-of-Iay and in-lay pullets with S. typhim'lJ,rium with special reference to the incidence of direct ovarian transmission, the frequency and significance of ovarian infection and the effect on egg production.
SALMONELLA TYPHIMURIUM IN PULLETS
93
MATERIALS AND METHODS
Inoculum The strain of S. typhimurium phage type U223 used in the investigation was an isolate from a field outbreak of disease in broiler breeders. The inoculum was a saline (0 ·85%) suspension prepared from washings of moist agar slants which had been seeded with an actively motile organism and incubated for 18 h at 37°C. The suspension was standardized by Brown's opacity tube method and viable plate counts on nutrient agar indicated that the inoculum contained 1 x 1010 viable organisms per m\. The counts were carried out prior to inoculation. Birds Thirty point-of-lay and 27 in-lay specific pathogen free (SPF) Brown Leghorn birds were maintained individually in cages. Serum agglutination tests and cultural examination of cloacal swabs from all birds for the presence of salmonellae before the start of the experiment were negative. All birds were caged in the premises several weeks before the date of inoculation. The birds were inoculated orally with 1 ml of the suspension administered into the middle third of the oesophagus by means of a sterile syringe and catheter tube. Before inoculation 10 point-of-lay and 25 in-lay experimental birds and one point-of-lay and four in-lay control birds were in production. Birds were slaughtered 17 to 19 days after infection, except for the most severely affected non-laying birds which were slaughtered between the 10th and 12th day. One of the infected point-of-lay birds died on the sixth day and one of the infected in-lay group was slaughtered in a comatose condition on the seventh day. Five point-of-lay and five in-lay uninfected SPF Brown Leghorns, caged individually and housed in a third pen were maintained as controls, so that a comparison of egg production could be made. Daily clinical observations were made. Eggs were collected twice daily, wrapped individually in squares of aluminium foil and stored at 4°C prior to bacteriological examination within 48 h of collection. Post-mortem examination Gross pathological changes were noted post mortem and liver, spleen, caeca and ovary were examined bacteriologically. The number of ovary samples per bird varied fro m one to four depending on development, with regressed ovules a nd groups of varying sized follicles being cultured separately. Us ing a Colworth Stomacher (Seward Ltd, London), all soft tissues were pulverized in 25 ml selenite F medium in polythene bags, an approximate 10% w/v ratio being maintained. The suspensions were transferred to 7 x 1 inch sterile tubes and incubated at 37°C for 24 h prior to plating on to MacCo nkey medium and subsequent incubation at 37°C for 18 h . Bacteriological examination of eggs was carried out by thoroughly rubbing each shell with a swab moistened in selenite F medium and then placing the swab in a tube with 25 ml of this medium. The eggs were then washed in 2·5% Hyco -quat solution (Hy-co Products (Scotland ) Ltd ), thoroughly rinsed in cold water and finall y immersed in 50% ethyl alcohol for 30 s and allowed to dry in air before the yolks and whites were
~
"'" TABLE I PATT ERN OF LAY AFTER I NOCULATION WITH S. TYPHIMURIUM
In lay
Point oflay
'" iO
Controls. No. of birds
No. of birds
No. with positive ovaries
2
o
2 2
1
o
4
3
Controls. No. of birds
...... en
No. of birds
No . with positive ovaries
9 I
o o
::c
< t'1 -l
t'1 iO
Z
In-lay before inoculation Ma inta ined produ ctio n I nterru pted lay I to 9 d ays p .i. Interrupted lay 4 to 16 d ays p.i . We nt o lfl ay 1 to 6 d ays p.i.
:;
Infected
Infected
1 0 0 0
5
o o o
5
o
10
5
» ~ o
'--
c:
iO
Z
)-
Not in-tay before inoculation Fail ed to lay during experi me nt Started to lay 2 to 7 days p.i. Started to lay 16 10 17 da ys p.i . Laid o nl y dut-ing first week p .i. Total p.i . post ino(ui
0 3 I 0
14
5
30
2
o
2
1
2 13
o
r .""..
o
""
o o o
o
o
o
o
o
5
27
5
2
o
SALMONELLA TYPHIMURIUM IN
PULLETS
95
separated. The shells and yolks were placed in individual flasks contammg 100 ml selenite F medium and the yolks ruptured by means of a sterile applicator stick. The swabs, shells and yolks were cultured in a similar manner to the soft tissues . RESULTS
Clinical Depression, inappetence and diarrhoea were observed in all infected birds three to five days after infection, followed by an improvement in their clinical condition during the next few days. The majority of the point-of-Iay birds were more severely affected than the in-lay group and were a day or two slower in showing an improvement. Approximately 50% of the point-of-Iay and 25% of the in-lay birds had persistent diarrhoea during the second week ; those most severely affected and not in lay were killed 10 to 12 days after infection. A few birds in both groups continued to show evidence of diarrhoea until slaughtered at the end of the experiment. One point-of-Iay bird died and one of the in-lay group was slaughtered in a comatos.e condition six and seven days after infection respectively. The control birds showed no evidence of disease. T he patterns of lay and percentage egg production are given in Tables I and II. Egg production showed a varied response to infection which was less marked in the in-lay gro up, despite the presence of clinical disease in all birds and prolonged diarrhoea in a few. Nine of the in-lay group continued to lay well until the end of the experiment, five returned to lay after a short break in production seven to 12 days after infection and one after a break in production one to eight days after infection . Ten of this group went off lay o ne to three days after infection and did not resume production and two did not co me into lay during the experiment. A mo re severe effect on the point-of-Iay birds was noted, 14 of which did not come into lay before slaughter or death. Four birds came into production during the first week but two of these laid only one egg each while two birds started to lay near the end
TABLE II EGG PRODUCTION EXPRESSED AS A PERCENTAGE FO R SIX·DAY PERIODS F ROM 12 DAYS BEFORE TO 18 DAYS AFTER INOCULATION
In -lay birds
Point- oj-lay birds
Before inoculation 12-7 days 6- 1 days After inoculation 1-6 days 7- 12 days 13- 18 days
Infected
Control
Infected
. Control
5 12
0 3
40 61
37 47
13
13 37 47
43 38 60
47 47 63
II
37
96
BRITISH VETERINARY JOURNAL, 134, 2
of the experiment. Ten birds had begun to lay before infection , one of which continued to lay well throughout the experiment and one laid rather irregularly; the other eight birds went ofT lay during the first week after infection and four of these resumed production during the second week. The five in-lay and one of the point-of-lay control birds had come into production before the start of the experiment; of the four remaining control point-of- lay birds, three started to lay within the first week and the fourth by the 17th day. Egg production of all birds from 12 days before until 18 days after inoculation is shown in Table II. A gradual rise was noted throughout the experiment in both control groups, compared to a fall in infected birds where the maximum decrease occurred seven to 12 days after infection, followed by a rise in production over the next six days.
Gross patholof:Y The most marked changes were observed, six to 10 days after infection, in the caeca. The walls were thickened, the linings congested and there was narrowing of the lumina which were either empty or contained pale or blood-stained necrotic cores. Congestion of the lining of the duodenum and upper small intestine was also noted at this time. Similar but less marked changes were observed in birds killed 12 days after infection, and in a few of those killed at 17 to 19 days there was congestion of the lining of the large intestine. Enlargement of the spleen was a feature of infection in most birds examined up to 12 days after infection and in many killed at the end of the experiment. Abnormality of the ovarian follicles was evident in six of the point-of-lay and six of the in-lay birds. The contents of some ovules were inspissated, in others the capsule was congested and one bird had elongated follicular stalks.
Bacteriolof:Y Eggs. Bacteriological examination of the shells and contents of the 25 7 eggs laid by infected birds during the experiment was negative for the presence of S. typhimurium. Tissues . The results of bacteriological examination of tissues post mortem are given in Fig. 1. S. typhimurium was recovered from the spleen, ovary, caeca and liver of 84, 32, 28 and 25% respectively of the 57 birds. There was a higher frequency of recovery early in infection with those tissues being positive in 96, 57, 65 and 52% respectively of the 27 birds examined six to 12 days after infection, compared to 76, 15, 3 and 6% respectively of the survivors 17 to 19 days after infection. All tissues of control birds were negative for the presence of salmonellae. The organism was recovered from the ovaries of 18 birds. With one exception, this involved birds with either apparently inactive ovaries or in which only regressed ovules were positive. In the one exception, a point-of-lay bird, the ovary was becoming active and the organism was recovered from one of two samples of apparently normal ovules. The majority of birds with positive regressed ovules, had gone ofT or were going ofT lay, but one point-of-lay bird was coming back into production and two were in lay; there was no recovery of the organism from other ovule samples cultured from these birds. In no case was the organism recovered from an active ovule of a bird in lay.
POINT- OF- LAY GROUP
~
t--
:s;
()
~
t--
[;: ...., ~ ::t: IN - L AY GROUP
-;2:'~----
t-I
r __ .. _
~
c:::
::t> .....
c::: :s; Z
'" c: •
o
Positive
0- died
r r
Nega tive
C - comatose when killed
Vl
~
~ No sample Fig. I. Bacteriol ogica l exam ination of tissue post mortem.
98
BRITISH VETERINARY JO U RNAL, 134,2
DISC USSION
Experimental oral infection with 1 x 10 10 viable S. typhimurium org\lnisms resulted in severe clinical disease in all birds three to five days after infection, with one dea th a nd one killed in a comatose condition during the first week. The point-of-lay birds were more severely affected than the in-lay group and were one to two days longer in showing recovery. The clinical disease was more severe than previously observed with experimental infection of six-month-old Brown Leghorn cockerels after administration of a similar inoculum prepared from the same strain of S. typhimurium (Brown, Ross & Smith, 1975), but was comparable to that observed in hens naturally infected with S. typhimurium in which depression, inappe tence, diarrhoea, but no mortality was reported (Kashiwazaki et al., 1966). Severe clinical disease was also reported by Mundt & Tugwell (1958 ) up to four days after the intravenous inoculation of adult layers with other motile salmonellae. In co ntrast with the controls which laid well during the investigation, infection had a varied effect on egg production in both groups (Table I) . Apart from two of the inlay birds which did not come into production, the remainder of this group showed three main types of response; those which continued to lay well despite the presence of clinical disease, others which went off lay completely and those which had a break in production during the second week . A more severe effect was observed in the pointof- lay birds, half of which did not come into lay; those which were in lay before inoculation showed a varied response to infection (Table I). Because of the small number of controls and the removal of some non-laying infected birds during the second week for bacteriological examination, only a tentative comparison is made of egg production figures. Both control groups showed a gradual rise in production throughout the experiment, while a reduction was noted in the infected groups, this being most marked during the seven to 12-day period after infection, when a 23% fall was recorded from the in-lay birds (Table 11). Surviving infected birds showed a subseq uent rise in production during the 13th to 18th days . A gradual drop in production was reported by Kashiwazaki et al. (1966) in a flock showing evidence of clinical disease associated with natural S. typhimurium infection . Mundt & Tugwell ( 1958 ), following intravenous inoculation of layers with other motile salmonellae, reported variation in egg production ranging from normal lay to a maximum fall of 2 1% depending on the seroLype. The most marked gross pathological changes were seen in birds which were killed or had died six to 12 days after infection and involved the caecum, duodenum and spleen, although splenic enlargement was still a feature of infection in birds killed 17 to 19 days after inoculation. These findings are consistent with those previously observed in adult cockerels slaughtered three to nine days after oral administration of a similar inoculum (Brown et al., 1975). Distorted ovules were noted in birds in varying stages of lay and similar observations on fowls naturally infected with S. typhimurium are reported by Wilson ( 1947 ) and Kashiwazaki et al. ( 1966). Systemic infection was confirmed post mortem in 49 of the 57 birds (84%). The spleen was the most frequent site of recovery, being positive in 84% of cases, in contrast with previous experimental infection of cockerels with S. typhimurium (Brown et al. , 1975 ) when caecal recovery was predominant and more frequent recovery from the liver
SALMONELLA TYPHIMURIUM IN P ULLETS
99
reported by Kas hiwazaki et al. (1966). After oral inoculation of layers with other motile salmonellae, Turnbull & Snoeyenbos (1974) reported recovery from the spleen at nine, but not at 18 days, although frequent recoveries were obtained from various levels of the intesti nal tract. In the present experiment the organism was recovered from the ovary of 18 birds. This involved birds either with apparently inactive ovaries or in wh ich only regressed ovules were positive; most of those showing regression of ovules ha d go ne off lay, but two were in lay and one was coming in to lay. On one occasion on ly, the organism was recovered from one of two samples of apparently normal ovules of a bird which was coming in to lay. Isolation of S. typhimurium from regressed ovules of fowls has also been reported by Wilson (1947 ) and other reports illustrate the occurrence of ovarian infection in the fowl with this serotype and other motile salmonellae (Clarenburg & Romijn, 1954; Mundt & Tugwell, 1958; Kashiwazaki et al., 1966; Snoeyenbos et ai., 1969 ; Slavkov & Vaptsarova, 1970 ; Savov et at., 1974), but none o f these workers relate ovule infection with direct ovarian transmission. Some reports do not state if infection involved quiescent, active or regressed o'vules, factorsof potential significance in considering this mode of transmission. Desp ite severe clinical disease, systemic infection and the recovery of the organism post mortem from the ovaries of birds which had laid, on no occasion was the organism recovered from an active ovule of a bird which was in lay, nor from any of the 257 wh ole eggs examined . Following inoculation of caged layers with other motile salmonellae, Mellor & Banwart (1965 ) and Brown, Ross & Smith (1976 ) also failed to recover the organisms from the shell or contents of eggs laid during the experiment. The presence of S. typhimurium and other motile salmonellae in egg contents has been reported (Wilson, 1945, 1950; Gordon & Tucker, 1965 ; Smyser et ai., 1966; Forsythe, Ross & Ayres, 1967; Olesiuk et ai., 1969; Slavkov & Vaptsarova, 1970; Cox et ai. , 1973 ; Savoy et at., 1974). As some of these workers have also recorded isolation of the organisms from the shell, the possibility of shell penetration must be considered as a potential r oute of infection of egg contents in such cases. Wilson (1950) reported yo lk infection in three eggs examined within minutes of lay from a fowl naturally infected with S. typhimurium and concluded that direct ovarian transmission had occurred . The literature, however, suggests that this is a rare occurrence in the fowl with motile salmonellae (Gordon, 1971; Smith, 1971; Williams, 1972). Cox et ai. (1973) found no evidence to support this mode of transmission with S. typhimurium and similar findings with other motile salmonellae are reported (Gibbons & Moore, 1946 ; Buxton & Gordon, 1947; Clarenburg & Romijn, 1954; Mundt & Tugwell, 1958; Mellor & Banwart, 1965). Whereas it is accepted that the challenge of experimental and natural field infection cannot be equated, the findings of the present in~estigation, obtained under conditions designed to preclude the possibility of shell contamination and subsequent shell penetration, produced no evidence of direct ovarian transmission of S. typhimurium and suggest the alternative of early regression of infected ovules. ACKNOWLEDGEMENTS
We wish to thank Dr J. G. Ross and Mr R. D. Lapraik for their helpful criticism during the preparation of the paper and the staff of the Bacteriology Section for their technical assistance.
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BROWN , D. D ., ROSS,]. G. & SMITH, A. F. G. (1975 ). Res. vet. Sci. 18, 165. BROWN, D. D., ROSS,]. G. &SMITH, A. F. G. (1976). Res. vet. Sci . 20, 237. BUXTON, A. & GORDON, R. F. ( 1947).]. Hyg. , Camb. 45, 265 . CLARENB URG, A. & ROMIJN, C . (1954 ). Proc. 10th Wld Poultry Congr. p. 214 . Cox, N. A., DAVIS, B. H., WATTS, A. B. & COLMER, A. R . (1973 ). Poultry Sci. 52, 661. FORSYTHE, R . H., Ross, W.]. & AYRES,]. c. (1967 ). Poultry Sci. 46, 849. GIBBONS, N . E. & MOORE, R . L. (1946 ). Poultry Sci. 2,115. GORDON, R . F. (1971). Vet. A. p. 95 . Bristol:]. Wright & Sons Ltd. GORDON, R . F. & TUCKER,]. F. (1965 ). Br. Poultry Sci. 6, 251. JORDAN, F. T. W. (1956 ). Poultry Sci. 35,1019. KASHIWAZAKI, M., AOKI, S., HORI UCH I, T., SHOYA, S. & NAM IOKA, S. (1966 ). Natn 1mt. Anim. Hlth Q" Tokyo 6, 144. MELLOR, D . B. & BANWART, G. j. ( 1965). ]. Food Sci. 30, 333. M UNDT,]. 0. & T UGWELL, R. L. (1958 ). Poultry Sci. 37, 415. OLESI UK, 0. M., CARLSON, V. L. , SNOEYENBOS, G. H. &SMYSER, c. F. ( 1969 ). Avian Dis. 13,500. SAVOY, D. , VRIGAZOV, A., DIMITROV, N. & GROMKOV, N. (1974 ). In Salmonelite i salmoneloz.ite v Bulgariya p. 265. Sofia, Bulgaria: Academy of Sciences. SLAVKOV, I. & VAPTSAROVA , M. (1970 ). Vet . Med. Nauki, Sofia 7,103 . SMI TH, H. WILLIAMS ( 1971 ). Poultry Disease and World Economy, ed. R. F. Gordon and B. M. Freemen, p. 37. Edinburgh: Longman for British Poultry Science . SMYSER, C. F., ADINARAYANAN, N., RO EKEL, H . VAN & SNOEYENBOS, G. H. (1960). Avian Dis. 10, 314 . SNOEYENBOS, G. H., SMYSER, C. F. & RO EKEL, H. VAN ( 1969 ). Avian Dis. 13,668. TURNBULL, P. C. B. & SNOEYENBOS, G. H. ( 1974). Avian Dis. 18, 153. WILLIAMS,]. E. (1972). Diseases of Poultry, 6th edn, p. 135, ed. M . S . Hofstad and others. Ames , Iowa: State University Press. WILSON,]. E . (1945 ). Vet . Rec. 57,411. WILSO N, ]. E. ( 1947). Br. vet.}. 103, 101. WILSON,]. E. (1950). Vet. Rec. 62,449. WILSON, j. E. ( 1958). Agric. Rev., Lond. 3, 23.
(AcceptedfOT publication 22 March 1977)