BRUCELLA ABORTUS INFECTION IN THE BULL
J. W. PLANT,B.V.Sc., P. D. CLAXTON,B.V.Sc., Dip. Bact., M.A.C.V.Sc., M.B. (Marburg), and W. DE SARAM,M.V.Sc., Ph.D. D. JAKOVLJEVIC, Veterinary Research Station, Glenfield, New South Wales, 21 67 Introduction
Hughes and Claxton (1968) except that 5 doubling
dilutions (1/5 to 1/80) and a 1/5 serum conFew reports are available on natural infection serum trol without antigen were used. of the bull with Brucellu abortus (Thomson Semen was also tested in the tube agglutination and 1943; Bendixen and Blom 1947; Christensen CFT following treatment with sodium azide (Morgan 1948; Manthei et a1 1950; Lambert et a1 1963; 1967). Rankin 1965) and fewer still relating to experi- Bacteriological Procedures mental infection (Seddon 1919; Lubbehusen and Semen was collected for bacteriological and serological examination using a modification of the techFitch 1926; Christensen 1948). niique described by Parsonson ef a1 (1971). The bulls Although infection in bulls may not be a major were tranquillised using approximately 8 mg acepromproblem in eradication programmes, difficulty is mine maleate per 100 kg given intramuscularly. Semen frequently experienced in diagnosing the disease was collected into 30 ml sterile McCartney bottles. Semen smears were stained by the modified Ziehl in bulls. Christie et a1 (1968) comment that the Neilsen (MZN) technique (Stamp et a1 1950). Specimajority of infected bulls have low serum mens for bacteriological examination were inoculated agglutination test (SAT) titres. They also sug- onto 10% sheep blood agar with and without 10 iu gest that infected bulls may be negative to the penicillidml. All plates were incubated in 10% COZ in air at 37°C for 10 days. complement fixation test (CFT) . More recently, andTesticular biopsy was performed using a sterile 18G McCaughey and Purcell (1973) isolated Br. needle inserted into the affected testicle after the scrotal abortus from semen of a bull with low SAT and skin had been shaved. high serum CFT titres, and with high semen Results agglutination test and low semen CFT titres. On day 0, bull 1 recorded a reaction in the Bendixen and Blom (1947), Cedro et a1 (1967, SAT (24 iu) and on day 21 a reaction to both 1968) failed to recover Br. abortus from some SAT (27 iu) and CFT (1/5). It was bled at bulls showing low SAT titres. However, Cedro approximately 14 day intervals and SAT reacet a1 (1967, 1968) recovered the organism from tions varied from 0-53 iu and CFT titres flucanimals showing ncgative SAT titres. tuated between 5 and 20. On day 141, the SAT This paper describes observations made on two reaction was 40 iu and the CFT titre was 5, and bulls ovcr a prolonged period and also discusses the bull was returned to the property of origin. problems in the diagnosis of brucellosis in the Br. abortus was never demonstrated in semen bull. samples collected on 4 separate occasions. The Materials and Methods bull showed no clinical abnormalities during the Animals period of observation. The observations were made on two 24-month-old Bull 2 was negative to both SAT and CFT on Charolais x Murray Grey bulls from the same property days 0 and 21, but on day 66, it gave positive in Victoria which were undergoing routine testing at the reactions in both SAT (106 iu) and CFT (80). Veterinary Research Station, Glenfield, before proceeding to an artificial insemination centre. The bulls On day 83, palpation of the scrotal contents arrived a t Glenfield on 6th June, 1972 (day 0) having revealed a hard swelling approximately 5 cm in previously been tested on the property for brucellosis with a negative result in the SAT. At Glenfield the bulls diameter involving the head of the right epididywere housed in individual yards and fed on lucerne and mis. The swelling increased in size and on day cereal hay. Observations continued on Bull 1 for 141 117 it was considerably larger. On day 122, the days and on Bull 2 for 363 days. bull stopped eating and on day 125 was febrile Serological Procedlcres and the right half of the scrotum was grossly The SAT was performed as a tube test using stan- enlarged, hot and painful. No treatment was dardised Br. abortus antigen* and standardised agglu- given and on day 131 the bull commenced eattinating serum* (Alton and Jones 1967). The results were expressed as international units/ml (iu/ml). The ing: The right testicle and epididymis were 2 to CFT was performed as a 6 tube test using Br. o b o r t u 3 tmes normal size and firm. Between days 131 antigen*. The test was similar to that described by and 353 the lesion regressed with atrophy and fibrosis of the epididymis and testicle. Wornrnonwealth Serum Laboratory, Parkville, Victoria. Australia. Australim Veterinary Journal, Vol. 52, January, 1976
17
The results of SAT and CFT are shown in Fig. 1. The SAT reaction reached a peak on day 100 (212 iu) and had dropped to < 17 units on day 269. The CFT titre fluctuated between 40 and 80 between days 66 to 190 and had dropped to < 5 on day 276. Semen was collected on 12 occasions between days 90 and 212. Brucellae were not demonstrable either microscopically or culturally in any sample. Inflammatory cells were invariably absent, though spermatozoa were plentiful. The semen agglutination test was positive on one occasion (20 iu on day 111) but CF antibody was never demonstrated. Corynebacterium bovis was the only organism isolated from a testicular biopsy sample taken on day 218. Autopsy Findings The bull was slaughtered on day 363 and lesions were found only in the right testicle and epididymis. The parietal and visceral layers of the tunica vaginalis were thickened (3-4 mm) and completely adherent. The testicle was atrophied and fibrosed, the parenchyma pale and scattered small (1-2 mm) creamy-orange lesions were localised in one small area of the testicle. The head and tail of the epididymis were fibrosed with loss of tubular definition, and the epididymis appeared occluded. Bacteriological Findings Specimens were collected from the testicles, head, body and tail of the epididymides, the seminal vesicles and ampullae of vasu deferentiu and lymph nodes associated with the genitalia. No organisms resembling brucellae were observed in smears. Br. abortus was isolated only from the ampulla of the right vas deferens and from the right seminal vesicle. The isolate was
Figure 1. Results
18
of SAT and CFT carried out on Bull 2.
COz dependent and penicillin resistant. Delayed, fine agglutination occurred in the slide agglutination test using an unabsorbed anti-Br. abortus serum. A sub-culture of the isolate was subsequently identified at the W.H.O. Brucellosis Reference Laboratory* as Br. abortus biotype 1 (rough).
Histopathological Findings Severe longstanding progressive fibrosis was present in the right testicle with destruction of seminiferous tubules. The remaining seminiferous tubules showed severe epithelial degeneration with no evidence of mitotic activity or spermatogenesis. A mild scattered focal predominantly mononuclear cellular reaction was seen generally associated with degenerating tubules. Occasional small unencapsulated granulomatous foci were also present which showed early calcification. The right epididymis showed severe longstanding interstitial fibrosis. The epididymal duct was generally empty. A mild cellular reaction was associated with segments of the duct where the lumen contained a small quantity of cellular detritus. The right seminal vesicle was normal. The left testicle showed severe degenerative changes. A few spermatozoa were seen within tubules. No changes were observed in the left epididymis and seminal vesicles. Discussion
There are few reports of detailed studies on Br. abortus infection in the bull. Seddon (1919) produced testicular lesions in a 3-month-old bull after intravenous inoculation with Br. abortus. Buck et al (1919) and King (1932) isolated Br abortus from serologically (SAT) positive bulls with seminal vesiculitis and/or orchitis. The former authors suggested that high SAT titres are more likely to be associated with infection than low titres. Christie et al (1968) commented that the majority of infected bulls have low SAT titres. Bendixen and Blom (1947) carried out more detailed investigations on naturally infected bulls. Among 3 0 bulls which had agglutinins in the sperm plasma as well as in serum, brucellae were demonstrated in the semen of 15. In another two bulls whose semen was bacteriologically negative, brucellae were isolated from infected organs at slaughter. The SAT titres of these 17 bulls ranged from 10 to 2560 and the semen agglutination titres from 10 to 10,000. However, Br. abortus was not recovered either from semen or from organs of four bulls with vesiculitis or *Commonwealth Serum Laboratory, Parkville, Victoria. Australia.
Australian Veterinary Journal, Vol. 52, January, 1976
epididymitis and with SAT titres 80 to 640 and semen agglutination titres of 10 to 2560. Cedro et a1 (1967, 1968) in extensive examinations of abattoir samples, recovered Br. abortus from 6/69 and 7/108 SAT negative bulls respectively, which came from herds in which brucellosis had been confirmed. These workers failed to isolate Br. abortus from some serologically positive bulls with SAT titres as high as 800. In bulls with epididymitis, Bendixen and Blom (1947) found that the serum agglutinin titre had a tendency to be higher than that of sperm plasma especially in bulls with orchitis. The presence of agglutinins in seminal plasma was considered to be of diagnostic value. In 26 bulls in which agglutinins were demonstrated in seminal plasma, inflammatory changes were observed in the seminal vesicles, ampullae, epididymides or testicles. A review of the literature suggests that two clinical syndromes are associated with Br. abortus infection in the bull and that these may or may not occur in the same animal. The first, involving the testicle and epididymis, is often characterised clinically by orchitis (Buck et a1 1919; King 1932; Bendixen and Blom 1947; Rankin 1965). The second and apparently more common syndrome involves the seminal vesicles and ampullae (Buck et a1 1919; Bendixen and Blom 1947; Rankin 1965). In the present investigation, Br. abortus was not recovered from the semen of bull 2 and the serological response was of relatively short duration, even though infection was demonstrated at slaughter. This bull resembled the serologically negative bulls reported by Cedro et a1 (1967) in that Br. abortus was recovered only at slaughter and the animal came from an infected herd. The incubation period of Br. abortus infection in the bull is not known. Lambert et al (1964) observed orchitis due to Br abortus strain 19 10 days after vaccination and Danks (1943) reported orchitis due to Br. abortus strain 19 some 7 months after vaccination. Lubbehusen and Fitch (1926) observed no clinical changes in young bulls challenged on 3 occasions over a 6month period even though serological responses were produced. In the present case, epididymitis was present at least 39 days prior to the development of orchitis and, if it is assumed that the bull was infected on the property of origin, then the time from infection to the development of orchitis was at least 122 days. The isolation of a rough strain of Br. abortus from this bull may be significant in view of the Australian Veterinary Journal, Vol. 52, January, 1976
relatively short duration of the serological response. It could be that the peculiar conditions in the local lesion (perhaps causing a fall in oxygen tension) resulted in the selection of a rough mutant, regression of the lesion and disappearance of the serological titre. The isolate produced no detectable serological response (CFT and SAT) in 2 guinea pigs which, were tested 3 and 6 weeks after inoculation and Brucella could not be isolated from their spleens on autopsy at 6 weeks. These findings, together with evidence in the literature indicate major problems in the diagnosis of brucellosis in bulls. Reliance on serological tests alone will result in some infected bulls being regarded as uninfected. Infected bulls may be serologically positive or negative, and may or may not be excreting Br. abortus in the semen. Antibody titres both in serum and semen may be negative or weakly positive in infected bulls and similar titres may be found in animals from which Br. abortus cannot be isolated. Lesions may be confined to the seminal vesicle and/or ampullae and may not be readily detectable except at autopsy. These facts suggest that ali bulls from known infected herds should be viewed with suspicion irrespective of their serological status. It is further suggested that in such herds it may be reasonable to consider as infected any bull with palpable lesions in the ampullae, seminal vesicles, epididymides or testicles. Diagnosis should be based on serological examination, clinical examination (including palpation of seminal vesicles and ampullae), examination of semen, both bacteriological and for antibody, and herd history. As brucellosis can be transmitted to cows from infected semen used for artificial insemination (Bendixen and Blom 1947; Manthei et a1 1950) we further suggest that bulls from infected herds should not be used in artificial insemination programmes, nor should their semen be stored for use by the owner in his own herd, particularly when eradication programmes are in progress. Summary
Observations on 2 bulls from a brucellainfected property are reported. Bull 1 gave serological reactions to Br. abortus in both the SAT and CFT from day 0 to day 141. Br. abortus was not recovered from semen and the bull remained clinically normal. The serological status of bull 2 changed from negative to positive to negative over a 203 day 19
period and remained negative for a further 74 days. Semen agglutinins were only detected on one occasion (20 iu). The first clinical sign observed was epididymitis followed by orchitis, which became apparent on day 122. Br. abortus was not recovered from semen but at autopsy, on day 363, Br. abortus biotype 1 was isolated from the right seminal vesicle and ampulla. The histology of the lesion is described. The literature relating to Br. abortus infection in the bull is discussed. Recommendations are made regarding the diagnosis of Br. abortus infection in bulls. Acknowledgments
We thank Mr R. Larnach and Mr B. Cairns for their assistance in the collection of samples, Mr D. W. Dredge, Dip. M.T., A.A.I.M.T., for performing the serological tests and Mr B. Porter, Dip. M.T., A.A.I.M.T., for assistance with the bacteriological examinations. References Alton G. G. and Jones, Lois M. (1967)--'Laboratory techniques in Brucellosis'. W.H.O. Monograph series No. 55. Bendixen, H. C. and Blom, E. (1947)-Br. vet. J . 103: 337.
Buck, J. M., Creech, G. T. and Ladson, H. H. (1915) -.I. agric. Res. 17: 239.
26
Cedro, V. C. F., Cisale, H. 0..de Menaldo, L. R. A. F., and Alvarez, N. 0. (1967)-Revta lnvesrnes Agropee. Ser. 4. (Patol. anim.). 4 ( 1 ) : 1. Cedro, V. C. F., Cisale, H. O., de Menaldo, L. R. A. F.. Bernadelli, A. A., Tome, J. S. G. and Alvarez, N. (1968)-Revta Znvestnes Agropec. Ser. 4 (Patol anim.) 5 ( 2 ) : 9. Christensen, N. 0. (1948)-Acta path. microbiol. Scand. 25: 202. Christie, T . E., Kerr, W. A. and McCaughey, W. J. (1968)--Vet. Rec. 82: 176. Danks, A. G. (1943)--CorneZl Vet. 33: 381. Hughes, K. L. and Claxton, P. D. (1968)-Aust. vet. J. 44: 41. King, R. 0. C. (1932)-Aust. vet. J . 8 : 226. Lambert, G., Manthei, C. A. and Deyoe, B. L. (1963) -Am. J. vet. Res. 24: 1152. Lambert, G., Deyoe, B. L. and Painter, G. M. (1964) -4.Am. vet. med. Ass. 145: 909. Lubbehusen, R. E. and Fitch, C. P. (1926)-J. Am. vet. med. Ass. 68: 467. McCaughey, W. J. and Purcell, D. A. (1973)--Vet. Rec. 92: 336. Manthei. C. A.. de Trav. D. E. and Goode. E. R. (195Oj-Proc: 87th Meeting Am. vet. med. Ass. 87: 177. Morgan, W. J . B. (1967)-Vet. Rec. 80: 612. Parsonson, I . M., Hall, C. E. and Settergren, I. (1971) -J. Am. vet. med. Ass. 158: 175. Rankin, J. E. F. (1965)-Vet. Rec. 77: 132. Seddon, H. R. ( 1 9 1 9 ) - J . comp. Path. Ther. 32: 1. Stamp, J. T., McEwan, A. D., Watt, J. A. A. and Nisbet, D. I. (1950)-Vet. Rec. 62: 251. Thomson, A. (1943)-J. comp. Parh. Ther. 53: 199. (Received for publication 27 September 1974)
Austbalian Veterinary Journal, VoL 52, Januapy, 1976
J. W. PLANT,B.V.Sc., P. D. CLAXTON,B.V.Sc., Dip. Bact., M.A.C.V.Sc., M.B. (Marburg), and W. DE SARAM,M.V.Sc., Ph.D. D. JAKOVLJEVIC, Veterinary Research Station, Glenfield, New South Wales, 21 67 Introduction
Hughes and Claxton (1968) except that 5 doubling
dilutions (1/5 to 1/80) and a 1/5 serum conFew reports are available on natural infection serum trol without antigen were used. of the bull with Brucellu abortus (Thomson Semen was also tested in the tube agglutination and 1943; Bendixen and Blom 1947; Christensen CFT following treatment with sodium azide (Morgan 1948; Manthei et a1 1950; Lambert et a1 1963; 1967). Rankin 1965) and fewer still relating to experi- Bacteriological Procedures mental infection (Seddon 1919; Lubbehusen and Semen was collected for bacteriological and serological examination using a modification of the techFitch 1926; Christensen 1948). niique described by Parsonson ef a1 (1971). The bulls Although infection in bulls may not be a major were tranquillised using approximately 8 mg acepromproblem in eradication programmes, difficulty is mine maleate per 100 kg given intramuscularly. Semen frequently experienced in diagnosing the disease was collected into 30 ml sterile McCartney bottles. Semen smears were stained by the modified Ziehl in bulls. Christie et a1 (1968) comment that the Neilsen (MZN) technique (Stamp et a1 1950). Specimajority of infected bulls have low serum mens for bacteriological examination were inoculated agglutination test (SAT) titres. They also sug- onto 10% sheep blood agar with and without 10 iu gest that infected bulls may be negative to the penicillidml. All plates were incubated in 10% COZ in air at 37°C for 10 days. complement fixation test (CFT) . More recently, andTesticular biopsy was performed using a sterile 18G McCaughey and Purcell (1973) isolated Br. needle inserted into the affected testicle after the scrotal abortus from semen of a bull with low SAT and skin had been shaved. high serum CFT titres, and with high semen Results agglutination test and low semen CFT titres. On day 0, bull 1 recorded a reaction in the Bendixen and Blom (1947), Cedro et a1 (1967, SAT (24 iu) and on day 21 a reaction to both 1968) failed to recover Br. abortus from some SAT (27 iu) and CFT (1/5). It was bled at bulls showing low SAT titres. However, Cedro approximately 14 day intervals and SAT reacet a1 (1967, 1968) recovered the organism from tions varied from 0-53 iu and CFT titres flucanimals showing ncgative SAT titres. tuated between 5 and 20. On day 141, the SAT This paper describes observations made on two reaction was 40 iu and the CFT titre was 5, and bulls ovcr a prolonged period and also discusses the bull was returned to the property of origin. problems in the diagnosis of brucellosis in the Br. abortus was never demonstrated in semen bull. samples collected on 4 separate occasions. The Materials and Methods bull showed no clinical abnormalities during the Animals period of observation. The observations were made on two 24-month-old Bull 2 was negative to both SAT and CFT on Charolais x Murray Grey bulls from the same property days 0 and 21, but on day 66, it gave positive in Victoria which were undergoing routine testing at the reactions in both SAT (106 iu) and CFT (80). Veterinary Research Station, Glenfield, before proceeding to an artificial insemination centre. The bulls On day 83, palpation of the scrotal contents arrived a t Glenfield on 6th June, 1972 (day 0) having revealed a hard swelling approximately 5 cm in previously been tested on the property for brucellosis with a negative result in the SAT. At Glenfield the bulls diameter involving the head of the right epididywere housed in individual yards and fed on lucerne and mis. The swelling increased in size and on day cereal hay. Observations continued on Bull 1 for 141 117 it was considerably larger. On day 122, the days and on Bull 2 for 363 days. bull stopped eating and on day 125 was febrile Serological Procedlcres and the right half of the scrotum was grossly The SAT was performed as a tube test using stan- enlarged, hot and painful. No treatment was dardised Br. abortus antigen* and standardised agglu- given and on day 131 the bull commenced eattinating serum* (Alton and Jones 1967). The results were expressed as international units/ml (iu/ml). The ing: The right testicle and epididymis were 2 to CFT was performed as a 6 tube test using Br. o b o r t u 3 tmes normal size and firm. Between days 131 antigen*. The test was similar to that described by and 353 the lesion regressed with atrophy and fibrosis of the epididymis and testicle. Wornrnonwealth Serum Laboratory, Parkville, Victoria. Australia. Australim Veterinary Journal, Vol. 52, January, 1976
17
The results of SAT and CFT are shown in Fig. 1. The SAT reaction reached a peak on day 100 (212 iu) and had dropped to < 17 units on day 269. The CFT titre fluctuated between 40 and 80 between days 66 to 190 and had dropped to < 5 on day 276. Semen was collected on 12 occasions between days 90 and 212. Brucellae were not demonstrable either microscopically or culturally in any sample. Inflammatory cells were invariably absent, though spermatozoa were plentiful. The semen agglutination test was positive on one occasion (20 iu on day 111) but CF antibody was never demonstrated. Corynebacterium bovis was the only organism isolated from a testicular biopsy sample taken on day 218. Autopsy Findings The bull was slaughtered on day 363 and lesions were found only in the right testicle and epididymis. The parietal and visceral layers of the tunica vaginalis were thickened (3-4 mm) and completely adherent. The testicle was atrophied and fibrosed, the parenchyma pale and scattered small (1-2 mm) creamy-orange lesions were localised in one small area of the testicle. The head and tail of the epididymis were fibrosed with loss of tubular definition, and the epididymis appeared occluded. Bacteriological Findings Specimens were collected from the testicles, head, body and tail of the epididymides, the seminal vesicles and ampullae of vasu deferentiu and lymph nodes associated with the genitalia. No organisms resembling brucellae were observed in smears. Br. abortus was isolated only from the ampulla of the right vas deferens and from the right seminal vesicle. The isolate was
Figure 1. Results
18
of SAT and CFT carried out on Bull 2.
COz dependent and penicillin resistant. Delayed, fine agglutination occurred in the slide agglutination test using an unabsorbed anti-Br. abortus serum. A sub-culture of the isolate was subsequently identified at the W.H.O. Brucellosis Reference Laboratory* as Br. abortus biotype 1 (rough).
Histopathological Findings Severe longstanding progressive fibrosis was present in the right testicle with destruction of seminiferous tubules. The remaining seminiferous tubules showed severe epithelial degeneration with no evidence of mitotic activity or spermatogenesis. A mild scattered focal predominantly mononuclear cellular reaction was seen generally associated with degenerating tubules. Occasional small unencapsulated granulomatous foci were also present which showed early calcification. The right epididymis showed severe longstanding interstitial fibrosis. The epididymal duct was generally empty. A mild cellular reaction was associated with segments of the duct where the lumen contained a small quantity of cellular detritus. The right seminal vesicle was normal. The left testicle showed severe degenerative changes. A few spermatozoa were seen within tubules. No changes were observed in the left epididymis and seminal vesicles. Discussion
There are few reports of detailed studies on Br. abortus infection in the bull. Seddon (1919) produced testicular lesions in a 3-month-old bull after intravenous inoculation with Br. abortus. Buck et al (1919) and King (1932) isolated Br abortus from serologically (SAT) positive bulls with seminal vesiculitis and/or orchitis. The former authors suggested that high SAT titres are more likely to be associated with infection than low titres. Christie et al (1968) commented that the majority of infected bulls have low SAT titres. Bendixen and Blom (1947) carried out more detailed investigations on naturally infected bulls. Among 3 0 bulls which had agglutinins in the sperm plasma as well as in serum, brucellae were demonstrated in the semen of 15. In another two bulls whose semen was bacteriologically negative, brucellae were isolated from infected organs at slaughter. The SAT titres of these 17 bulls ranged from 10 to 2560 and the semen agglutination titres from 10 to 10,000. However, Br. abortus was not recovered either from semen or from organs of four bulls with vesiculitis or *Commonwealth Serum Laboratory, Parkville, Victoria. Australia.
Australian Veterinary Journal, Vol. 52, January, 1976
epididymitis and with SAT titres 80 to 640 and semen agglutination titres of 10 to 2560. Cedro et a1 (1967, 1968) in extensive examinations of abattoir samples, recovered Br. abortus from 6/69 and 7/108 SAT negative bulls respectively, which came from herds in which brucellosis had been confirmed. These workers failed to isolate Br. abortus from some serologically positive bulls with SAT titres as high as 800. In bulls with epididymitis, Bendixen and Blom (1947) found that the serum agglutinin titre had a tendency to be higher than that of sperm plasma especially in bulls with orchitis. The presence of agglutinins in seminal plasma was considered to be of diagnostic value. In 26 bulls in which agglutinins were demonstrated in seminal plasma, inflammatory changes were observed in the seminal vesicles, ampullae, epididymides or testicles. A review of the literature suggests that two clinical syndromes are associated with Br. abortus infection in the bull and that these may or may not occur in the same animal. The first, involving the testicle and epididymis, is often characterised clinically by orchitis (Buck et a1 1919; King 1932; Bendixen and Blom 1947; Rankin 1965). The second and apparently more common syndrome involves the seminal vesicles and ampullae (Buck et a1 1919; Bendixen and Blom 1947; Rankin 1965). In the present investigation, Br. abortus was not recovered from the semen of bull 2 and the serological response was of relatively short duration, even though infection was demonstrated at slaughter. This bull resembled the serologically negative bulls reported by Cedro et a1 (1967) in that Br. abortus was recovered only at slaughter and the animal came from an infected herd. The incubation period of Br. abortus infection in the bull is not known. Lambert et al (1964) observed orchitis due to Br abortus strain 19 10 days after vaccination and Danks (1943) reported orchitis due to Br. abortus strain 19 some 7 months after vaccination. Lubbehusen and Fitch (1926) observed no clinical changes in young bulls challenged on 3 occasions over a 6month period even though serological responses were produced. In the present case, epididymitis was present at least 39 days prior to the development of orchitis and, if it is assumed that the bull was infected on the property of origin, then the time from infection to the development of orchitis was at least 122 days. The isolation of a rough strain of Br. abortus from this bull may be significant in view of the Australian Veterinary Journal, Vol. 52, January, 1976
relatively short duration of the serological response. It could be that the peculiar conditions in the local lesion (perhaps causing a fall in oxygen tension) resulted in the selection of a rough mutant, regression of the lesion and disappearance of the serological titre. The isolate produced no detectable serological response (CFT and SAT) in 2 guinea pigs which, were tested 3 and 6 weeks after inoculation and Brucella could not be isolated from their spleens on autopsy at 6 weeks. These findings, together with evidence in the literature indicate major problems in the diagnosis of brucellosis in bulls. Reliance on serological tests alone will result in some infected bulls being regarded as uninfected. Infected bulls may be serologically positive or negative, and may or may not be excreting Br. abortus in the semen. Antibody titres both in serum and semen may be negative or weakly positive in infected bulls and similar titres may be found in animals from which Br. abortus cannot be isolated. Lesions may be confined to the seminal vesicle and/or ampullae and may not be readily detectable except at autopsy. These facts suggest that ali bulls from known infected herds should be viewed with suspicion irrespective of their serological status. It is further suggested that in such herds it may be reasonable to consider as infected any bull with palpable lesions in the ampullae, seminal vesicles, epididymides or testicles. Diagnosis should be based on serological examination, clinical examination (including palpation of seminal vesicles and ampullae), examination of semen, both bacteriological and for antibody, and herd history. As brucellosis can be transmitted to cows from infected semen used for artificial insemination (Bendixen and Blom 1947; Manthei et a1 1950) we further suggest that bulls from infected herds should not be used in artificial insemination programmes, nor should their semen be stored for use by the owner in his own herd, particularly when eradication programmes are in progress. Summary
Observations on 2 bulls from a brucellainfected property are reported. Bull 1 gave serological reactions to Br. abortus in both the SAT and CFT from day 0 to day 141. Br. abortus was not recovered from semen and the bull remained clinically normal. The serological status of bull 2 changed from negative to positive to negative over a 203 day 19
period and remained negative for a further 74 days. Semen agglutinins were only detected on one occasion (20 iu). The first clinical sign observed was epididymitis followed by orchitis, which became apparent on day 122. Br. abortus was not recovered from semen but at autopsy, on day 363, Br. abortus biotype 1 was isolated from the right seminal vesicle and ampulla. The histology of the lesion is described. The literature relating to Br. abortus infection in the bull is discussed. Recommendations are made regarding the diagnosis of Br. abortus infection in bulls. Acknowledgments
We thank Mr R. Larnach and Mr B. Cairns for their assistance in the collection of samples, Mr D. W. Dredge, Dip. M.T., A.A.I.M.T., for performing the serological tests and Mr B. Porter, Dip. M.T., A.A.I.M.T., for assistance with the bacteriological examinations. References Alton G. G. and Jones, Lois M. (1967)--'Laboratory techniques in Brucellosis'. W.H.O. Monograph series No. 55. Bendixen, H. C. and Blom, E. (1947)-Br. vet. J . 103: 337.
Buck, J. M., Creech, G. T. and Ladson, H. H. (1915) -.I. agric. Res. 17: 239.
26
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Austbalian Veterinary Journal, VoL 52, Januapy, 1976
