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This article was published in 1977
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Blood Testing of Rams - Results and Problems Encountered
John C. Thomas, B.V.Sc., Veterinary Inspector, Mudgee
INTRODUCTION
An extension programme for 1976-77 was to survey and test rams to determine the incidence of Ovine Brucellosis (0.B.) in the Mudgee Pastures Protection Board (P.P.B.) area.
The results and discussion that follow are those obtained from the initial test of 2,328 rams on 78 properties in the survey.
METHOD AND MATERIALS
A Survey letter on 0.B. was sent to all ratepayers who had 1,000 sheep or more. This was determined from the P.P.B. stock Returns. Those interested then contacted the P.P.B.
Before bleeding, all rams were identified and clinical examination carried out on the scrotal contents.
A blood sample was obtained using Vacutainer method and then forwarded to Glenfield Veterinary Research Station. The serum was subjected to the Complement Fixation (C.F.) test for the detection of Brucella ovis (Br. ovis) and Actinobacillus seminis (A. seminis).
Rams with C.F. titres of 20 or greater were classified as serologically positive for Br. ovis and C.F. titres of 10 were serologically doubtful.
The criteria for lesions were palpable chronic or acute conditions of the epididymus or testes. During the survey, no other pathological condition of the scrotal contents were observed.
No ewes were tested or examined during the survey.
RESULTS
A). Breeds. The only breeds of rams encountered throughout the survey were Dorset Horns (D.H.), Border Leicesters (B.L.) and Merinos (M.)
TABLE I shows the breed composition of the rams tested:—
Table 1. BREED COMPOSITION OF RAMS TESTED
| D.H. | B.L. | M. | TOTAL | |
|---|---|---|---|---|
| Number Bled | 507 | 602 | 1,219 | 2,328 |
| Percent Bled | 21.7% | 25.8% | 52.5% | 100% |
B. Properties. The number of properties visited was 78. TABLE 2 shows the distribution of the ram types among the properties:—
Table 2. RAM TYPE ON PROPERTIES SURVEYED
| D.H. | B.L. | M. | D.H.+ B.L. | D.H. + M. | B.L. + M. | D.H.+ B.L. + M. | TOTAL | |
|---|---|---|---|---|---|---|---|---|
| No. Rams | 251 | 279 | 792 | 268 | 99 | 365 | 271 | 2328 |
| Properties | 14 | 7 | 18 | 7 | 7 | 11 | 14 | 78 |
| % | 17.9 | 8.9 | 23.2 | 8.9 | 8.9 | 14.2 | 17.9 | 100 |
From TABLE 2, 50% of the properties had only one ram type while they accounted for 56.3% of the rams tested.
C). Incidence of Palpable Lesions
Of the total rams surveyed, 6.4% had palpable lesions. TABLE 3. shows the incidence of clinical lesions in the three breeds.
Table 3. COMPARISON BETWEEN BREEDS AND NUMBER OF RAMS WITH PALPABLE LESIONS
| Breed | ||||
|---|---|---|---|---|
| Rams with Lesions | D.H. | B.L. | M. | TOTAL |
| No. | 109 | 24 | 17 | 150 |
| % | 72.6 | 16 | 11.3 | 99.9 |
D. Incidence of Ovine Brucellosis
of the 2,328 rams involved, 13.9% were serologically positive for 0.B. TABLE 4 shows the three breeds and serological results obtained:—
Table 4. RESULT OF C.F.T. AND A COMPARISON BETWEEN BREEDS (No. of rams)
| RESULT OF C.F. TEST | BREED | NUMBER | TOTAL % | ||
|---|---|---|---|---|---|
| D.H. | B.L. | M. | |||
| Sero. positive | 204 | 93 | 26 | 323 | 13.9 |
| Sero. doubtful | 23 | 4 | 8 | 35 | 1.5 |
| Sero. negative | 277 | 504 | 1159 | 1940 | 83.3 |
| Anticomplementary | 2 | 1 | 26 | 30 | 1.3 |
| TOTAL | 507 | 602 | 1219 | 2328 | 100 |
The distribution of serologically positive rams between breeds and within breeds is shown as percentage in TABLE 5.
Table 5. COMPARISON OF SERO. POSITIVE RAMS BETWEEN BREEDS AND WITHIN BREEDS
| BREED | |||
|---|---|---|---|
| D.H. | B.L. | M. | |
| % Sero. positive between breeds | 63.1 | 28.7 | 8.1 |
| % Sero. positive within breeds | 40.2 | 15.5 | 2.1 |
The influence of the breed type and the occurrence of serologically positive rams is shown in TABLE 6.
Table 6. THE INFLUENCE OF BREED TYPE ON THE OCCURRENCE OF SEROLOGICALLY POSITIVE RAMS
Table 6a. ONLY ONE BREED TYPE
| BREED | NUMBER BLED | NUMBER SERO. POSITIVE |
|---|---|---|
| D.H. | 251 | 77 (30.6%) |
| B.L. | 279 | 11 (3.9%) |
| M. | 792 | 3 (0.37%) |
Table 6b. MERINO RUN WITH DORSET HORNS ONLY
| BREED | NUMBER BLED | NUMBER SERO. POSITIVE |
|---|---|---|
| D.H. | 46 | 25 (54.3%) |
| M. | 53 | 10 (18.8%) |
Table бс. MERINO RUN WITH BCRDER LEICESTER
| BREED | NUMBER BLED | NUMBER SERO. POSITIVE |
|---|---|---|
| B.L. | 109 | 40 (36.6%) |
| M. | 259 | 9 (3.4%) |
Table 6d. DORSET HORNS RUN WITH BORDER LEICESTER
| BREED | NUMBER BLED | NUMBER SERO. POSITIVE |
|---|---|---|
| D.H. | 130 | 70 (53.8%) |
| B.L. | 138 | 18 (13.1%) |
Table 6e. THREE TYPES TOGETHER
| BREED | NUMBER BLED | NUMBER SERO. POSITIVE |
|---|---|---|
| D.H. | 80 | 32 (40%) |
| B.L. | 76 | 24 (31.5%) |
| M. | 115 | 4 ( 3.4%) |
From TABLES 4, 5 and 6 it appears that the Dorset Horn is the breed with the greatest incidence of serologically positive rams and this breed has the greatest influence on the occurrence of positive titres in the other two breeds.
Note:In Table 6a. the 3 sero. positive Merino rams were Stud rams on 3 separate properties.
TABLE 7 shows the comparison of scrotal lesions and positive titres and their distribution between the three breeds.
Table 7. DISTRIBUTION OF SCROTAL LESIONS BETWEEN RAM TYPES AND CORRELATION WITH POSITIVE C.F. TITRES (Number of Rams)
| Breed | ||||
|---|---|---|---|---|
| D.H. | B.L. | M. | TOTAL | |
| Scrotal Lesions with sero. Positive titres | 100 | 24 | 3 | 127 |
| Scrotal lesions without C.F. titres | 9 | 0 | 14 | 23* |
The percentage of rams with serologically positive titres and palpable lesions is 39%. This agrees with Hughes et al. 1968, who quotes 39.7%. But in a literature search the figures can vary, such as Murray, 1969, found 23.4% of rams with positive C.F. titres having palpable lesions.
*Within this number is 7 ex 9 Merino rams having scrotal lesions, but negative C.F. titres for Br. ovis and A. seminis, on two bled. This was the same for 4 ex 14 D.H. While on one property 3 D.H. had palpable scrotal lesions, but only one was serologically positive for Br. ovis.
Only one ram with palpable lesions had a positive titre for A. seminis.
The number of properties which had titres to Br. ovis antibodies was 50. The details are in TABLE 8.
Table 8. PROPERTIES WITH BR. ovis TITRES
| C.F. TITRE | POSITIVE | DOUBTFUL | POSITIVE & DOUBTFUL | ZERO | TOTAL |
|---|---|---|---|---|---|
| No. of Properties | 31 | 6 | 13 | 28 | 78 |
The incidence of C.F. positive ranged from NIL to 100%. The incidence of C.F. positives is in TABLE 9.
The
Table 9. INCIDENCE OF C.F. POSITIVES ON PROPERTIES
| % SERO. POSITIVE | 0 | 0-10 | 10-20 | 20-30 | 30-40 | 40-50 | 50-60 | 60-70 | 70-80 | 80-90 | 90-100 |
|---|---|---|---|---|---|---|---|---|---|---|---|
| No. of properties | 34 | 12 | 7 | 3 | 2 | 3 | 5 | 2 | 3 | 3 | 4 |
NOTE The difference between TABLE 8 and TABLE 9 in regards to zero titres and zero serologically positives is that the 6 doubtful in TABLE 8 were regarded as zero sero. positives.
In respect to the situation in regards to Studs, of the 78 properties visited, 14 were Studs. They accounted for 48.6% of the rams examined. TABLE 10 gives the incidence of Brucella ovis antibodies in the 14 Studs examined.
Table 10. DISTRIBUTION OF BR. ovis ANTIBODIES IN THE 14 STUDS VISITED
| STUD NUMBER | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 M | 2 M | 3 M | 4 M | 5 M | 6 DH | 7 BL DH | 8 DH | 9 M | 10 BL | 11 DH | 12 BL | 13 DH | 14 DH | |
| No. Infected | 1? | 1+ | 1? | 0 | 1+ | 0 | 1? | 0 | 0 | 1+ | 0 | 1+ 1? | 42+ | 20+ |
| Total bled | 51 | 140 | 17 | 37 | 306 | 53 | 117 | 17 | 118 | 80 | 13 | 175 | 53 | 46 |
| % with titres | 1.9 | 0.7 | 5.8 | 0 | 0.3 | 0 | 0.8 | 0 | 0 | 1.25 | 0 | 1.1 | 79.2 | 43.4 |
Key: + = positive sero. titres. ? = doubtful titres.
Of the 14 Studs surveyed, 7 required further examination to determine the true serological O.B. status of the flock. In all Studs except 2, negative titres were had on retest.
The two exceptions were Studs 13 and 14 which were positive for Ovine Brucellosis in respect to both clinical evidence and laboratory testing. It is interesting to note that the only introductions Stud 14 were from Stud 13.
Two Studs had rams with palpable lesions of the epididymus and testes. Stud 8 had 4 ex 17 and Stud 4, 2 ex 37. Rebleeding have negative serology for both A. seminis and Br. ovis. Semen examination was not attempted, but culture and histopathology of ram from Stud 4 did not return any significant findings.
Studs 7 and 12 with doubtful titres for A. seminis - re-examination did not occur.
Table 11. NUMBER OF RAMS AND PROPERTIES WITH BR. ovis ANTIBODIES IN THE ABSENCE OF SCROTAL LESIONS IN ALL RAMS ON THE PROPERTY
| PROPERTY NUMBER | NUMBER OF RAMS | COMMENTS | ||
|---|---|---|---|---|
| Sero. Positive | Sero. Negative | Total Bled | ||
| 1 | 1 BL | 0 | 18 | On retest possible vaccinate |
| 2 | 0 | 2M | 10 | No retest |
| 3 | 0 | 1 DH | 5 | Retest - normal |
| 4 | 0 | 1 DH | 6 | Retest - normal |
| 5 | 0 | 1 BL | 8 | No retest |
| 6 | 0 | 2M | 10 | No retest |
| 7 | 1 M | 0 | 16 | CF 20 - no retest |
| 8 | 7 M, 18 BL | 0 | 47 | 23 M + 24 BL - retest - 16 BL and 9 M sero positive |
| 9 | 1 M | 0 | 34 | Retest normal |
| 10 | 3 M | 1 M | 13 | All M. - no retest |
| 11 | 1 BL | 1 M | 20 | Retest M. Normal BL still sero. positive |
| 12 | 1 BL, 2 M | 1 BL | 19 | Retest titres from 20 to 0 - possible vaccinate |
| 13 | 2 BL | 0 | 39 | Mixed flock - 1 CF 40, another CF 20 - vaccinate? |
| 14 | 3 M | 0 | 21 | No retest |
| 15 | 4 BL | 0 | 48 | 4 BL + 44 M - titres 40, - possible vaccinate |
| 16 | 2 DH, 4 M | 0 | 21 | No retest |
| 17 | 3 BL | 0 | 18 | 3 BL + 15 M - no retest - possible vaccinate |
| 18 | 1 BL | 1 BL | 13 | 5 BL + 8 M - retest results same |
| 19 | 1 | 0 | 51 | Stud |
| 20 | 1 | 0 | 140 | Stud |
| 21 | 0 | 1 | 17 | Stud |
| 22 | 1 | 0 | 80 | Retest normal - stud |
| 23 | 1 | 1 | 175 | Retest normal - stud |
| 24 | 1 | 0 | 306 | CF 20 - no retest |
Properties 1, 12, 13, 15, 17 and 18 had serologically positive titres from rams that may be due to vaccinations. On two properties semen culture was done but it did not help to clarify the results.
PROBLEMS
These refer to difficulties encountered in interpreting laboratory and clinical findings to the property concerned. With resultant perplexities in implementing control and eradication programmes for these places.
1. ANTI COMPLEMENTARY (A/C) ACTIVITY
This was experienced on 5 properties and involved 30 rans (1.3% of those bled - Table 4.). This meant that the true O.B. status of the flock could not be decided until these animals were retested.
The possible reasons for the A/C activity are:—
i. Climatic problems - especially in hot weather.
ii. slow bleeding, or
iii. deterioration of sera. These are operator induced and should be able to be avoided.
2. OCCURRENCE OF SMALL NUMBERS OF BR. ovis RAMS IN LARGE FLOCKS
This was especially evident in the Studs (Table 10). Three Studs had one serologically positive ram each, another three had a serologically doubtful ram each, while one stud had a serologically positive and doubtful ram (Stud 12). Where retesting occurred (Studs 3, 9 and 12) negative titres were obtained. This revisiting is needed to determine the true Br. ovis antibody status of the flock. This is strongly advised as reports have occurred where in similar cases Br. ovis has been recovered from these rams (cited Ryan, 1964).
3. PROBLEMS OF PALPABLE SCROTAL LESIONS BUT NEGATIVE SEROLOGY
This was especially notable in a flock of 9 Merinos which had 7 rams with palpable scrotal lesions. 2 rams had bilateral lesions and the others varied from chronic epididymitis to acute testicular swelling. Serology on two consecutive bleeds was negative for both Br. ovis and A. seminis. Autopsy and semen culture is to be attempted.
A flock of 14 Dorset Horns had 4 rams with chronic scrotal lesions. Serology was negative for Br. ovis and A. seminis on two consecutive bleeds. No significant findings were had on autopsy or culture of testicular tissue and histopathology is not yet available.
In a flock of 10 mixed rams, 3 exhibited scrotal lesions. One ram was sero. positive for Br. ovis - the other two rams negative titres for Br. ovis and A. seminis.
The consequences of these are:—
i. The examination of other causes of scrotal lesions (as defined) - either they be traumatic or infectious.
ii. The possibility of negative serology in cases of long standing O.B. with clinical lesions.
4. PROBLEMS OF VACCINATED ANIMALS
Six properties visited where the possibility of vaccinated rams existed. From TABLE 11, these were properties 1, 12, 13, 15, 17 and 18. Besides these six, another three were encountered where vaccinated rams maybe existed but clinical evidence of O.B. was present and these rams were deemed to be infected after retest.
Of the six properties retest of rams of three gave results that indicate the rams may have been vaccinates, due to a fall in C.F. titre on retest. These were properties 1, 12, and 18 (TABLE 11).
Semen culture was not attempted on any of the six properties but was on the three others mentioned. No significant findings were to be had.
The problems of vaccinated rams are:—
i. owners do not know if the rams have been vaccinated or not
I suspect vaccination if:—
a. on retest the C.F. titre falls, or
b. in cases like property 15 (TABLE 11) which had 4 serologically positive B.L. rams with 44 serologically negative Merino rams all running together.
ii. the occurrence of vaccinated rams in infected flocks, exeamples:-
Property A. incidence of Sero. positives was 93%, on retest 2 B.L. rams gave lowered titres - semen culture gave no significant findings, and the rams were culled as being infected, as clinical lesions existed in the flock.
Property B. - incidence of sero. positives 40%, clinical lesions present. 1 B.L. had a lowered titre on retest - semen examination did not help to clarify the position. Ram classed as being infected.
Thus the problem of culling normal rams exists with the presence of vaccinates.
5 . OCCURRENCE OF 'FALSE POSITIVES'
These occur when rams give positive C.F. titres and,
i. the numbers are small compared to the flock number and no lesions are present,
ii. negative C.F. titres are had on subsequent bleeds,
iii. possibility of vaccinates,
iv. large numbers of C.F. positives but no clinical evidence of the disease.
Discussion of i; ii; and iii appears above. In respect of iv, the most pronounced case of this was property 8, TABLE 11
Both wool and fat lamb production occurred. This property had 47 rams in total, comprising 23 M. and 24 B.L. Lambing percentage varied 95-97%. Joining was at 2%. The property was first visited 29/10/1976 - On this visit all rams were clinically normal. The laboratory report on the sera gave 7 M. with positive titres and 18 B.L. On the second visit 23/12/1976, again all rams were clinically normal. This time the laboratory report gave 26 B.L. and 9 M. serologically positive. No autopsy or semen collection has yet been carried out.
The following may occur:—
i. the sero.logically positive rams may have been sensitised by Br. ovis. Thus clinical disease may not occur, due to termination of the infection.
ii. the possibility of cross reaction with other Brucella organisms giving rise to C.F. titres. (Meyer, 1976) states that 'forms of Br. abortus type 2 with revertant characteristics essentially indistinguishable in vitro from those of Br. ovis have been found'.
The Brucella abortus status of the property is one abattoir trace back with C.F. 256. Testing of cattle is anticipated.
iii. positive C.F. titres in a flock is not synonymous with clinical disease - on Property 8 the owner has been advised to keep the rams and have them retested on a two month basis.
As far as semen collection goes, on the three properties, it has been used - I have found that the method is time consuming and unrewarding unless frequent visits are made. This makes the procedure useless in control or eradication of Br. ovis on a wide scale.
CONCLUSION
From the survey conducted on the 78 properties described above and the 2,328 rams examined, the incidence of Br. ovis C.F. positive was 13.9%.
It would appear that Ovine Brucellosis is a serious source of economic loss in the sheep industry in the Mudgee P.P. Board district.
From the initial survey, efforts will be aimed at:—
i. controlling and eradicating the disease from commercial flocks, depending on which is the more suitable, and
ii. eradicating the disease from Studs in the P.P. area.