J. COMP.
PATH.
1976.
EARLY
VOL.
86.
59
PATHOGENESIS
AND
TRITRICHOMONAS
IN
VIRGIN
I. M. PARSONSON, CSIRO
Division
of Animal
PATHOLOGY INFECTION HEIFERS
OF
FOETUS
B. L.
CLARK
Health, Animal Health Parkville, Victoria,
and J. H. DUFTY
Research Laboratory, 3052, Australia
Private
Ba: nb. I, P.O..
INTRODUCTIOX
Trichomoniasis due to Tritrichomonas foetus infection in Australian cattle was first confirmed in 1946 by Dumaresq (1948)) and interest in this disease has increased in recent years when reports indicated a high incidence of infected herds in northern Australia (Donaldson, Lucas, Johnston and Ritson, 1967 ; Rogers, Flanagan and Hill, 1972; Ladds, Dennett and Glazebrook, 1973; Dennett, Reece, Barasa and Johnson, 1974). The clinical features and epizootiology of T. foetus infection in the female has been adequately described (Morgan, 1944; Bartlett, 1947; Laing, 1956), but studies on the pathology and pathogenesis are limited. This paper records the results of an experiment designed to study early pathogenesis and pathology of trichomoniasis in virgin heifers mated to a bull infected with T. foetus. MATERIALS
AND
METHODS
Experimental animals. Twenty-two nulliparous Hereford females aged 2 years that had been reared on CSIRO field stations were maintained as a group. Two animals from this group were kept as unmated controls. A Hereford bull aged 3 years was infected with a culture of T. j&us var. brisbane (Elder, 1964) introduced into the preputial cavity (Clark, Parsonson and Dufty, 1974) 10 months prior to mating with the heifers. Cervico-vaginal mucus (CVM) samples from the heifers were examined for T. foetus and Campylobacter ,fetus subsp. fetus and were free of both organisms. Experimental methods. During oestrus and immediately before mating a cervicovaginal mucus (CVM) sample was collected from each animal; following this cofiection each cbw wa$ allowed a single service to the bull. Cervico-vaginal mucus samples from the mated cows and a preputial sample from the bull were collected at weekly intervals until the termination of the experiment. The CVM samples were inoculated directly into modified Plastridge medium (Sutherland, Simmonds and Bell, 1953) incubated at 37 “C. and examined after 4 to 10 days for T. foetus. Preputial samples were treated as described by Clark, White and Banfield (1971). After T. foetus infection was confirmed in the first few cows following mating, 2 cows were killed at an abattoir at approximately 2-week intervals in the order of joining to the bull, irrespective of whether they were found to be infected or not. The genitalia were collected intact and taken to the laboratory where they were examined within 2 h. of death. Each tract was laid out and the dorsal surface swabbed with 70 per cent. ethyl alcohol. Beginning at the dorsal surface of the vagina, an incision was made through the cervix and along each uterine horn.
60
I. M.
et al.
PARSONSON
The oviducts were dissected from the mesosalphinx and severed at the utero-tubal junction and flushed from the infundibulum to the isthmus with 1 to 2 ml. of sterile isotonic saline. Swabs were taken from each of the following areas: vagina, cervix, body of uterus, right and left caudal, and right and left rostra1 uterine horns. If a foetus was present f&al liquids were collected. The resulting swabs and liquids were inoculated into Plastridge medium. All tracts were examined macroscopically and if present the foetus and foetal membranes were placed in 10 per cent. buffered form01 saline (BFS). Tissue samples were taken from the vagina, cervix, uterine body, both uterine horns, oviducts and ovaries and fixed in Bouin’s solution. Fixed tissues were embedded in paraffin and sections stained with HE., Grocotts’ methenamine silver (GMS) (Luna, 1970) and periodic acid Schiff (PAS) stains. RESULTS
Mating Results Seventeen cows were mated with the bull within 3 during the following 30 days. The first 2 cows mating foIlowing confirmatidn of infection with 1. not ascertained if these 2 animals were pregnant, animals 11 (61 per cent.) were pregnant (Table 1).
19 days and the remaining were killed 15 days after foe&s. At necropsy it was but of the remaining 18
TABLE 1 PATHOGENICITY
or Tritrichomonas
j&us
FOR
Proportion of CVM Animal number
A23 S78 s77 S85 A44 S86 S66 s93 A28
Time from mating to necropsy
from-which T. foetus was cultured
Pregnancv status
P P
w
p’
516 g/g
;
919
cows
MATED
T O AN INFECTED
BULL
Examination
ND ND NP
3j3 516 717
VIRGIN
of cenital tracts at necropsy Sites from Pathological which T. foetus Jindings was cultured
cv; cv; cv; cv; cv;
u; u u; u; u
0 0
CV cv;o cv;u cv; u; 0 Foetal fluids CV
A6 A14 A36 A32 s92
lO/lO Ojll S/l1 ll/ll
ii::
cv;
:: NP
None None cv;u;o
S88 s99 s74 S64
12112 13/13 12/13 13/14
NP NP NP P
S67 s79
l/14 l/15
P P
0
0
None CV CV cv;u;o Foetal fluids None
* CVM samples were collected from each cow at weekly intervals throughout Abbreviations: ND-not determined; P-pregnant; NP-not pregnant; U-uterus; O-oviducts; Vag-vaginitis; Cerv-cervicitis; End-ndometritis; Plac-placentitis; None-no abnormalities found.
None None None None None None None None Vag; Cerv; Endo; Salp; Plac Endo; Salp Endo; Salp None None Vag ; Endo ; Salp; Pyometra Vag Vag; Endo; Salp None Vag ; Endo ; Cerv ; Salp; Plac None None the experiment. CV-cervix vagina; Salp-salpingitis;
1. foetus
INFECTION
IN VIRGIN
HEIFERS
61
CulturuZ Examination for T. foetus Prior to service T. foetus was not isolated from any CVM sample collected at oestrus. However, from 19 of the 20 cows (95 per cent.) T. foetus was cultured from either the first or second CVM sample collected after mating to the infected bull. The other cow was not infected. In 2 cows (S67, S79) T. foetus was isolated from the second CVM sample, but was not demonstrated in any further samples obtained. 2: foetus was cultured from the second to ninth CVM samples from A32, but not from the last 2 CVM collections or from the genital tract at slaughter. In the remaining 16 cows T. foetus was isolated from either the first or second CVM sample and from subsequent samples up to slaughter (Table 1). At necropsy 7: foe&s was found in the genital tract in 15 of the 20 cows (Table 1). All had infections of the vagina or cervix and 11 also had infections in either the uterus or oviducts. In 2 cows (A28 and S64) T. foetus was present in the foetal liquids and the foetus. Fourteen preputial samples were collected from the bull during the mating period. T. foetus was cultured from all except the third sample.
Examination
of Cows at Necropg
No macroscopic changes were noted in the genital tracts of the 2 unmated animals. Two of the 11 pregnant animals (A28 and S64) had evidence of impending abortion. The foetal liquids were a pale yellow and contained a fine colloidal-like suspension. Some of the placentomes were haemorrhagic and from these the cotyledons were separating. The remaining 9 foetuses appeared normal. The genital tracts of 8 of the 9 non-pregnant animals appeared normal. One animal (S92) had pyometra, and the uterine horns, cervix and oviducts contained a pale yellow material of semi-solid consistency.
Microscopic Examination Focal areas of lymphoid cells and a mild to moderate infiltration of macrophages, plasma cells and neutrophils occurred in the sub-epithelial tissues of the vagina in all cows examined. This appearance was regarded as normal and was distinguished from that seen in animals with a severe vaginitis. Microscopic lesions were present in the genital tract of 7 of the 12 cows killed between 60 and 100 days after mating. 1. foetus was present at necropsy in the genital tracts of 6 of the 7 cows (Table 1). No lesions were found in the genital tracts of the 8 cows killed less than 60 days after mating, although r. foetus was present in most areas sampled. In 2 animals that were pregnant at 60 (A28) and at 95 days (S64) respectively, T. foetus was present in the uterus and oviducts, and in foetal liquids and placentomes. Inflammatory cells were present between the cotyledon and caruncle and the majority of these cells were neutrophils with some macrophages present (Fig. 1). In sections stained by GMS, T. foetus was present in large numbers in the placentome, in inflammatory material lining the lumen of the uterus (Fig. 2) and packed into the lumen of endometrial glands (Fig. 3). The endometrium in the interplacentomal areas was eroded and large numbers
62
I. M.
PARSONSON
et c-21.
of inflammatory cells, mostly neutrophils and macrophages, were present throughout the stratum compacturn (Fig. 4). A concurrent salpingitis and cervicitis was also present in these animals.
Fig. 1. Cellular exudate.
infiltration of placentome HE. x 215.
Fig g. 2. Surface of endometrium numerous (arrow).
T.foetus
showing
neutrophils
and lumen of uterus GMS x 215.
and macrophages
showing
inflammatory
in the inflammatory
exudate
containi
w
r.
fot?t!LJ
INFECTION
IN VIRGIN
63
HEIFERS
The cow (S92) with pyometra had a marked endometritis with inflammatory cells present throughout the stratum compacturn. The cells, mostly neutrophils and macrophages, had infiltrated into the endometrial gland epithelium, and the endometrial lining of the uterus was eroded. A mixture of cells, cellular debris and amorphous eosinophilic material was present in the lumen of the endometrial glands and uterus. In deeper areas of the stratum compactum, more chronic inflammatory foci were present surrounding endometrial glands or distributed perivascularly. The cells in these areas were composed of neutrophils, macrophages and lymphocytes and a small number of plasmacytes. In the oviduct inflammatory cells were present in folds and sub-epithelial tissues in all regions (Fig. 5). Cells, cellular debris and eosinophilic amorphous material was present in the lumen and there was an infiltration of the epithelium by neutrophils and macrophages. Three other cows (A6, Al4 and S99) had lesions of chronic endometritis and salpingitis. In the uterus perivascular and periglandular foci were present in the stratum compacturn. The foci were mostly composed of lymphocytes with some macrophages and occasional small groups of plasma cells and there was an increase in the number of cells of the same type in the stroma of the stratum compacturn. In the oviducts there were areas of chronic salpingitis involving the stroma and epithelium. In some areas fusion of folds had occurred, forming small cysts. Small foci of inflammatory cells were present, usually consisting of lymphocytes, macrophages and occasionally plasma cells. Severe vaginitis was seen in several cows (A28, S64, S88, S92 and S99). The lesions consisted of large perivascular lymphoid foci and many cells of lymphocytic, plasma cell and macrophage types were present in the subepithelial tissues and infiltrating into the epithelium.
Fig. 3. Uterus;
endometrial
glands
containing
large
numbers
of T. foetus in the lumen.
GMS.
x 215.
64
Fig. 4. Uterus; erosion HE. x 120.
Fig. 5. Oviduct; Cellular
et al.
I. M. PARSONSON
of the endometrium
salpingitis, inflammatory
and
marked
cellular
inflammatory cellular infiltration exudate present in lumen. HE.
x
infiltration
through 120.
of stratum
the stroma
and
compactum
epithelium.
T.jJbetus INFECTION
IN VIRGIN HEIFERS
65
DISCUSSION
Within 2 weeks after mating to an infected bull, 19 of 20 susceptible cows were shown by culture of CVM samples to be infected with 1. foetus var. &bane. The high transmission rate of the organism confirms the findings of Bartlett (1947)) Kerr and Robertson (1943, 1946) and Pierce (1947). Bartlett (1947) suggested that the uterus is the definitive and persistent site of infection and that the vagina, which plays only a passive inconsequential role in the progress of the disease, provides a relatively unreliable source of T. foetus for diagnosis. In this study the persistence of 1. foetus in the vagina and/or cervix for periods up to 95 days was confirmed by the culture of CVM samples at approximately weekly intervals, and these samples provided a reliable and accurate method of diagnosis. T. foetus was cultured from the vagina, cervix and uterus of all 4 animals killed 15 and 28 days after mating and also from the oviducts of 3 of them. The presence of 1: foetus in the oviducts so early in the infection indicates that the oviducts are more readily infected than is usually believed. No macroscopic or microscopic lesions were seen in the genital tracts or foetuses of the 8 cows killed before 50 days, although T. foetus was cultured from several areas of the genital tract in all animals. At least 5 of the 8 cows were pregnant at necropsy. In the 9 cows killed between 60 and 95 days after infection and from which T. foetus had been consistently cultured from CVM samples 2 had impending abortions and 5 were not pregnant, 1 having pyometra. Seven cows had microscopic lesions ranging from a marked vaginitis, to generalized acute or chronic inflammation of the genital tract. The remaining 2 cows had no lesions of the genital tract. Seemingly, in this experiment T. foetus did not produce recognizable lesions in the genital tract or terminate pregnancy until 50 or more days after natural infection. In tissue sections where 1. &&us was seen on microscopic examination the organism was present in surface secretions, in the lumen of endometrial glands or between the maternal and foetal components of placentomes. The localization of I: foetus in the mucous secretions of the female genital tract and the resulting absorption of antigenic material through the epithelium would appear to be the stimulator of the inflammatory response seen in those tissues. SUMMARY
The mating of a bull infected with Tritrichomonas foetus var. brisbane to 20 susceptible nulliparous cows resulted in 19 of the animals becoming infected after a single service. After T. foetus infection was confirmed, 2 cows were killed at approximately 2 week intervals in the order of mating to the bull. All cows were killed within 100 days of the commencement of mating. Eleven of the 18 cows that were killed 28 days or more after mating were pregnant. 1. foetus was cultured from several areas of the genital tract in the 8 cows killed from 15 to 50 days after mating. The organism was found in the vagina and cervix of all cows, in the uterus in 6 and in the oviducts of 4. No macroscopic or microscopic lesions attributable to T. jbetus infection were seen in any of these animals. Of the 12 cows killed from 60 to 100 days after mating
66
I. M.
PARSONSON
et al.
7 had acute or chronic microscopic lesions in many areas of the genital tract. Abortion was pending in 2 that were mated at 60 and 95 days previously, and a third cow had a pyometra. The remaining 5 cows had no macroscopic or microscopic lesions and T. foetus was cultured from the cervix and vagina of 1 animal only. T. foetus appeared to be confined to the surface secretions in the genital tract. ACKNOWLEDGMENTS
The able technical assistance of Messrs G. .J. Donovan and P. R. Staples, and the excellent histological preparations of Miss Marian Casan and Mr A. J. Rowlatt are gratefully acknowledged. The manager and staff of the C.S.I.R.O. field station at Werribee are thanked for their care and attention to the animals. REFERENCES
Bartlett, D. E. (1947). Bovine venereal trichomoniasis: its nature, recognition, intraherd eradication, and interherd control. Proceedings of the United States Livestock Sanitary Association, 5 1st Annual Meeting, pp. 170-l 8 1. Clark, B. L., Parsonson, I. M., and Dufty, J. H. (1974). Experimental infection of bulls with Tritrichomonus foe&s. Australian Veterinary Journal, 50, 189-l 91. Clark, B. L., White, M. B., and Banfield, J. C. (1971). Diagnosis of Trichomonas foetus infection in bulls. Australian Veterinary Journal, 47, 18 l-l 83. Dennett, D. P., Reece, R. L., Barasa, J. O., and Johnson, R. H. (1974). Observations on the incidence and distribution of serotypes of Tritrichomonus foetus in beef cattle in north-eastern Australia. Australian Veterinary Journal, 50, 427-43 1. Donaldson, L. E., Lucas, M. H., Johnston, L. A. Y., and Ritson, J. B. (1967). The reproductive efficiency of several North Queensland beef herds. 2. The influence of vibriosis, trichomoniasis and lesions of the reproductive tract. Australian Veterinary Journal, 43, 4 l-44. Dumaresq, J. A. (1948). A note recording the presence of Trichomonus foetus infection of cattle on King Island. Australian Veterinary Journal, 24, 282. Elder, J. K. (1964). Examination of twelve strains of Tritrichomonusfoetus (Riedmiiller) isolated in Queensland and the description of a new serotype, T. foetus var. Brisbane. Queensland Journal of Agricultural Science, 21, 193-203. Kerr, W. R., and Robertson, M. (1943). A study of the antibody response of cattle to Trichomonas foetus. Journal qf Comparative Pathology, 53, 280-297. Kerr, W. R., and Robertson, M. (1946). Experimental infections in virgin heifers with Trichomonas foetus in vaccinated and unvaccinated animals. Journal of Comparative Pathology, 56, 10 1-l 13. Ladds, P. W., Dennett, D. P., and Glazebrook, J. S. (1973). A survey of the genitalia of bulls in northern Australia. Australian Veterinary Journal, 49, 335-340. Laing, J. A. (1956). Trichomonas foetus infection of cattle. FAO Agricultural Studies No. 33, Food and Agriculture Organization of the United Nations, Rome. Luna, L. G. (Ed.) (1970). Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. 3rd edit. McGraw-Hill Book Co., New York. Morgan, B. B. (1944). Bovine Trichomoniusis. Burgess Publishing Company, Minneapolis. Pierce, A. E. (1947). The demonstration of an agglutinin to Trichomonus foetus in the vaginal discharge of infected heifers. Journal of Comparative Patholou, 57, 84-97. Rogers, R. J., Flanagan, M., and Hill, M. W. M. (1972). A survey of infectious causes of reproductive failure in beef cattle in north-eastern Australia. Australian Veterinary Journal, 48, 203-207. Sutherland, A. K., Simmonds, G. C., and Bell, A. T. (1953). An outbreak of bovine trichomoniasis. Part 2 : Diagnosis. Australian Veterinary Journal, 29, 67-69. [Received for publication,
June 11 th, 19751
PATH.
1976.
EARLY
VOL.
86.
59
PATHOGENESIS
AND
TRITRICHOMONAS
IN
VIRGIN
I. M. PARSONSON, CSIRO
Division
of Animal
PATHOLOGY INFECTION HEIFERS
OF
FOETUS
B. L.
CLARK
Health, Animal Health Parkville, Victoria,
and J. H. DUFTY
Research Laboratory, 3052, Australia
Private
Ba: nb. I, P.O..
INTRODUCTIOX
Trichomoniasis due to Tritrichomonas foetus infection in Australian cattle was first confirmed in 1946 by Dumaresq (1948)) and interest in this disease has increased in recent years when reports indicated a high incidence of infected herds in northern Australia (Donaldson, Lucas, Johnston and Ritson, 1967 ; Rogers, Flanagan and Hill, 1972; Ladds, Dennett and Glazebrook, 1973; Dennett, Reece, Barasa and Johnson, 1974). The clinical features and epizootiology of T. foetus infection in the female has been adequately described (Morgan, 1944; Bartlett, 1947; Laing, 1956), but studies on the pathology and pathogenesis are limited. This paper records the results of an experiment designed to study early pathogenesis and pathology of trichomoniasis in virgin heifers mated to a bull infected with T. foetus. MATERIALS
AND
METHODS
Experimental animals. Twenty-two nulliparous Hereford females aged 2 years that had been reared on CSIRO field stations were maintained as a group. Two animals from this group were kept as unmated controls. A Hereford bull aged 3 years was infected with a culture of T. j&us var. brisbane (Elder, 1964) introduced into the preputial cavity (Clark, Parsonson and Dufty, 1974) 10 months prior to mating with the heifers. Cervico-vaginal mucus (CVM) samples from the heifers were examined for T. foetus and Campylobacter ,fetus subsp. fetus and were free of both organisms. Experimental methods. During oestrus and immediately before mating a cervicovaginal mucus (CVM) sample was collected from each animal; following this cofiection each cbw wa$ allowed a single service to the bull. Cervico-vaginal mucus samples from the mated cows and a preputial sample from the bull were collected at weekly intervals until the termination of the experiment. The CVM samples were inoculated directly into modified Plastridge medium (Sutherland, Simmonds and Bell, 1953) incubated at 37 “C. and examined after 4 to 10 days for T. foetus. Preputial samples were treated as described by Clark, White and Banfield (1971). After T. foetus infection was confirmed in the first few cows following mating, 2 cows were killed at an abattoir at approximately 2-week intervals in the order of joining to the bull, irrespective of whether they were found to be infected or not. The genitalia were collected intact and taken to the laboratory where they were examined within 2 h. of death. Each tract was laid out and the dorsal surface swabbed with 70 per cent. ethyl alcohol. Beginning at the dorsal surface of the vagina, an incision was made through the cervix and along each uterine horn.
60
I. M.
et al.
PARSONSON
The oviducts were dissected from the mesosalphinx and severed at the utero-tubal junction and flushed from the infundibulum to the isthmus with 1 to 2 ml. of sterile isotonic saline. Swabs were taken from each of the following areas: vagina, cervix, body of uterus, right and left caudal, and right and left rostra1 uterine horns. If a foetus was present f&al liquids were collected. The resulting swabs and liquids were inoculated into Plastridge medium. All tracts were examined macroscopically and if present the foetus and foetal membranes were placed in 10 per cent. buffered form01 saline (BFS). Tissue samples were taken from the vagina, cervix, uterine body, both uterine horns, oviducts and ovaries and fixed in Bouin’s solution. Fixed tissues were embedded in paraffin and sections stained with HE., Grocotts’ methenamine silver (GMS) (Luna, 1970) and periodic acid Schiff (PAS) stains. RESULTS
Mating Results Seventeen cows were mated with the bull within 3 during the following 30 days. The first 2 cows mating foIlowing confirmatidn of infection with 1. not ascertained if these 2 animals were pregnant, animals 11 (61 per cent.) were pregnant (Table 1).
19 days and the remaining were killed 15 days after foe&s. At necropsy it was but of the remaining 18
TABLE 1 PATHOGENICITY
or Tritrichomonas
j&us
FOR
Proportion of CVM Animal number
A23 S78 s77 S85 A44 S86 S66 s93 A28
Time from mating to necropsy
from-which T. foetus was cultured
Pregnancv status
P P
w
p’
516 g/g
;
919
cows
MATED
T O AN INFECTED
BULL
Examination
ND ND NP
3j3 516 717
VIRGIN
of cenital tracts at necropsy Sites from Pathological which T. foetus Jindings was cultured
cv; cv; cv; cv; cv;
u; u u; u; u
0 0
CV cv;o cv;u cv; u; 0 Foetal fluids CV
A6 A14 A36 A32 s92
lO/lO Ojll S/l1 ll/ll
ii::
cv;
:: NP
None None cv;u;o
S88 s99 s74 S64
12112 13/13 12/13 13/14
NP NP NP P
S67 s79
l/14 l/15
P P
0
0
None CV CV cv;u;o Foetal fluids None
* CVM samples were collected from each cow at weekly intervals throughout Abbreviations: ND-not determined; P-pregnant; NP-not pregnant; U-uterus; O-oviducts; Vag-vaginitis; Cerv-cervicitis; End-ndometritis; Plac-placentitis; None-no abnormalities found.
None None None None None None None None Vag; Cerv; Endo; Salp; Plac Endo; Salp Endo; Salp None None Vag ; Endo ; Salp; Pyometra Vag Vag; Endo; Salp None Vag ; Endo ; Cerv ; Salp; Plac None None the experiment. CV-cervix vagina; Salp-salpingitis;
1. foetus
INFECTION
IN VIRGIN
HEIFERS
61
CulturuZ Examination for T. foetus Prior to service T. foetus was not isolated from any CVM sample collected at oestrus. However, from 19 of the 20 cows (95 per cent.) T. foetus was cultured from either the first or second CVM sample collected after mating to the infected bull. The other cow was not infected. In 2 cows (S67, S79) T. foetus was isolated from the second CVM sample, but was not demonstrated in any further samples obtained. 2: foetus was cultured from the second to ninth CVM samples from A32, but not from the last 2 CVM collections or from the genital tract at slaughter. In the remaining 16 cows T. foetus was isolated from either the first or second CVM sample and from subsequent samples up to slaughter (Table 1). At necropsy 7: foe&s was found in the genital tract in 15 of the 20 cows (Table 1). All had infections of the vagina or cervix and 11 also had infections in either the uterus or oviducts. In 2 cows (A28 and S64) T. foetus was present in the foetal liquids and the foetus. Fourteen preputial samples were collected from the bull during the mating period. T. foetus was cultured from all except the third sample.
Examination
of Cows at Necropg
No macroscopic changes were noted in the genital tracts of the 2 unmated animals. Two of the 11 pregnant animals (A28 and S64) had evidence of impending abortion. The foetal liquids were a pale yellow and contained a fine colloidal-like suspension. Some of the placentomes were haemorrhagic and from these the cotyledons were separating. The remaining 9 foetuses appeared normal. The genital tracts of 8 of the 9 non-pregnant animals appeared normal. One animal (S92) had pyometra, and the uterine horns, cervix and oviducts contained a pale yellow material of semi-solid consistency.
Microscopic Examination Focal areas of lymphoid cells and a mild to moderate infiltration of macrophages, plasma cells and neutrophils occurred in the sub-epithelial tissues of the vagina in all cows examined. This appearance was regarded as normal and was distinguished from that seen in animals with a severe vaginitis. Microscopic lesions were present in the genital tract of 7 of the 12 cows killed between 60 and 100 days after mating. 1. foetus was present at necropsy in the genital tracts of 6 of the 7 cows (Table 1). No lesions were found in the genital tracts of the 8 cows killed less than 60 days after mating, although r. foetus was present in most areas sampled. In 2 animals that were pregnant at 60 (A28) and at 95 days (S64) respectively, T. foetus was present in the uterus and oviducts, and in foetal liquids and placentomes. Inflammatory cells were present between the cotyledon and caruncle and the majority of these cells were neutrophils with some macrophages present (Fig. 1). In sections stained by GMS, T. foetus was present in large numbers in the placentome, in inflammatory material lining the lumen of the uterus (Fig. 2) and packed into the lumen of endometrial glands (Fig. 3). The endometrium in the interplacentomal areas was eroded and large numbers
62
I. M.
PARSONSON
et c-21.
of inflammatory cells, mostly neutrophils and macrophages, were present throughout the stratum compacturn (Fig. 4). A concurrent salpingitis and cervicitis was also present in these animals.
Fig. 1. Cellular exudate.
infiltration of placentome HE. x 215.
Fig g. 2. Surface of endometrium numerous (arrow).
T.foetus
showing
neutrophils
and lumen of uterus GMS x 215.
and macrophages
showing
inflammatory
in the inflammatory
exudate
containi
w
r.
fot?t!LJ
INFECTION
IN VIRGIN
63
HEIFERS
The cow (S92) with pyometra had a marked endometritis with inflammatory cells present throughout the stratum compacturn. The cells, mostly neutrophils and macrophages, had infiltrated into the endometrial gland epithelium, and the endometrial lining of the uterus was eroded. A mixture of cells, cellular debris and amorphous eosinophilic material was present in the lumen of the endometrial glands and uterus. In deeper areas of the stratum compactum, more chronic inflammatory foci were present surrounding endometrial glands or distributed perivascularly. The cells in these areas were composed of neutrophils, macrophages and lymphocytes and a small number of plasmacytes. In the oviduct inflammatory cells were present in folds and sub-epithelial tissues in all regions (Fig. 5). Cells, cellular debris and eosinophilic amorphous material was present in the lumen and there was an infiltration of the epithelium by neutrophils and macrophages. Three other cows (A6, Al4 and S99) had lesions of chronic endometritis and salpingitis. In the uterus perivascular and periglandular foci were present in the stratum compacturn. The foci were mostly composed of lymphocytes with some macrophages and occasional small groups of plasma cells and there was an increase in the number of cells of the same type in the stroma of the stratum compacturn. In the oviducts there were areas of chronic salpingitis involving the stroma and epithelium. In some areas fusion of folds had occurred, forming small cysts. Small foci of inflammatory cells were present, usually consisting of lymphocytes, macrophages and occasionally plasma cells. Severe vaginitis was seen in several cows (A28, S64, S88, S92 and S99). The lesions consisted of large perivascular lymphoid foci and many cells of lymphocytic, plasma cell and macrophage types were present in the subepithelial tissues and infiltrating into the epithelium.
Fig. 3. Uterus;
endometrial
glands
containing
large
numbers
of T. foetus in the lumen.
GMS.
x 215.
64
Fig. 4. Uterus; erosion HE. x 120.
Fig. 5. Oviduct; Cellular
et al.
I. M. PARSONSON
of the endometrium
salpingitis, inflammatory
and
marked
cellular
inflammatory cellular infiltration exudate present in lumen. HE.
x
infiltration
through 120.
of stratum
the stroma
and
compactum
epithelium.
T.jJbetus INFECTION
IN VIRGIN HEIFERS
65
DISCUSSION
Within 2 weeks after mating to an infected bull, 19 of 20 susceptible cows were shown by culture of CVM samples to be infected with 1. foetus var. &bane. The high transmission rate of the organism confirms the findings of Bartlett (1947)) Kerr and Robertson (1943, 1946) and Pierce (1947). Bartlett (1947) suggested that the uterus is the definitive and persistent site of infection and that the vagina, which plays only a passive inconsequential role in the progress of the disease, provides a relatively unreliable source of T. foetus for diagnosis. In this study the persistence of 1. foetus in the vagina and/or cervix for periods up to 95 days was confirmed by the culture of CVM samples at approximately weekly intervals, and these samples provided a reliable and accurate method of diagnosis. T. foetus was cultured from the vagina, cervix and uterus of all 4 animals killed 15 and 28 days after mating and also from the oviducts of 3 of them. The presence of 1: foetus in the oviducts so early in the infection indicates that the oviducts are more readily infected than is usually believed. No macroscopic or microscopic lesions were seen in the genital tracts or foetuses of the 8 cows killed before 50 days, although T. foetus was cultured from several areas of the genital tract in all animals. At least 5 of the 8 cows were pregnant at necropsy. In the 9 cows killed between 60 and 95 days after infection and from which T. foetus had been consistently cultured from CVM samples 2 had impending abortions and 5 were not pregnant, 1 having pyometra. Seven cows had microscopic lesions ranging from a marked vaginitis, to generalized acute or chronic inflammation of the genital tract. The remaining 2 cows had no lesions of the genital tract. Seemingly, in this experiment T. foetus did not produce recognizable lesions in the genital tract or terminate pregnancy until 50 or more days after natural infection. In tissue sections where 1. &&us was seen on microscopic examination the organism was present in surface secretions, in the lumen of endometrial glands or between the maternal and foetal components of placentomes. The localization of I: foetus in the mucous secretions of the female genital tract and the resulting absorption of antigenic material through the epithelium would appear to be the stimulator of the inflammatory response seen in those tissues. SUMMARY
The mating of a bull infected with Tritrichomonas foetus var. brisbane to 20 susceptible nulliparous cows resulted in 19 of the animals becoming infected after a single service. After T. foetus infection was confirmed, 2 cows were killed at approximately 2 week intervals in the order of mating to the bull. All cows were killed within 100 days of the commencement of mating. Eleven of the 18 cows that were killed 28 days or more after mating were pregnant. 1. foetus was cultured from several areas of the genital tract in the 8 cows killed from 15 to 50 days after mating. The organism was found in the vagina and cervix of all cows, in the uterus in 6 and in the oviducts of 4. No macroscopic or microscopic lesions attributable to T. jbetus infection were seen in any of these animals. Of the 12 cows killed from 60 to 100 days after mating
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et al.
7 had acute or chronic microscopic lesions in many areas of the genital tract. Abortion was pending in 2 that were mated at 60 and 95 days previously, and a third cow had a pyometra. The remaining 5 cows had no macroscopic or microscopic lesions and T. foetus was cultured from the cervix and vagina of 1 animal only. T. foetus appeared to be confined to the surface secretions in the genital tract. ACKNOWLEDGMENTS
The able technical assistance of Messrs G. .J. Donovan and P. R. Staples, and the excellent histological preparations of Miss Marian Casan and Mr A. J. Rowlatt are gratefully acknowledged. The manager and staff of the C.S.I.R.O. field station at Werribee are thanked for their care and attention to the animals. REFERENCES
Bartlett, D. E. (1947). Bovine venereal trichomoniasis: its nature, recognition, intraherd eradication, and interherd control. Proceedings of the United States Livestock Sanitary Association, 5 1st Annual Meeting, pp. 170-l 8 1. Clark, B. L., Parsonson, I. M., and Dufty, J. H. (1974). Experimental infection of bulls with Tritrichomonus foe&s. Australian Veterinary Journal, 50, 189-l 91. Clark, B. L., White, M. B., and Banfield, J. C. (1971). Diagnosis of Trichomonas foetus infection in bulls. Australian Veterinary Journal, 47, 18 l-l 83. Dennett, D. P., Reece, R. L., Barasa, J. O., and Johnson, R. H. (1974). Observations on the incidence and distribution of serotypes of Tritrichomonus foetus in beef cattle in north-eastern Australia. Australian Veterinary Journal, 50, 427-43 1. Donaldson, L. E., Lucas, M. H., Johnston, L. A. Y., and Ritson, J. B. (1967). The reproductive efficiency of several North Queensland beef herds. 2. The influence of vibriosis, trichomoniasis and lesions of the reproductive tract. Australian Veterinary Journal, 43, 4 l-44. Dumaresq, J. A. (1948). A note recording the presence of Trichomonus foetus infection of cattle on King Island. Australian Veterinary Journal, 24, 282. Elder, J. K. (1964). Examination of twelve strains of Tritrichomonusfoetus (Riedmiiller) isolated in Queensland and the description of a new serotype, T. foetus var. Brisbane. Queensland Journal of Agricultural Science, 21, 193-203. Kerr, W. R., and Robertson, M. (1943). A study of the antibody response of cattle to Trichomonas foetus. Journal qf Comparative Pathology, 53, 280-297. Kerr, W. R., and Robertson, M. (1946). Experimental infections in virgin heifers with Trichomonas foetus in vaccinated and unvaccinated animals. Journal of Comparative Pathology, 56, 10 1-l 13. Ladds, P. W., Dennett, D. P., and Glazebrook, J. S. (1973). A survey of the genitalia of bulls in northern Australia. Australian Veterinary Journal, 49, 335-340. Laing, J. A. (1956). Trichomonas foetus infection of cattle. FAO Agricultural Studies No. 33, Food and Agriculture Organization of the United Nations, Rome. Luna, L. G. (Ed.) (1970). Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. 3rd edit. McGraw-Hill Book Co., New York. Morgan, B. B. (1944). Bovine Trichomoniusis. Burgess Publishing Company, Minneapolis. Pierce, A. E. (1947). The demonstration of an agglutinin to Trichomonus foetus in the vaginal discharge of infected heifers. Journal of Comparative Patholou, 57, 84-97. Rogers, R. J., Flanagan, M., and Hill, M. W. M. (1972). A survey of infectious causes of reproductive failure in beef cattle in north-eastern Australia. Australian Veterinary Journal, 48, 203-207. Sutherland, A. K., Simmonds, G. C., and Bell, A. T. (1953). An outbreak of bovine trichomoniasis. Part 2 : Diagnosis. Australian Veterinary Journal, 29, 67-69. [Received for publication,
June 11 th, 19751
