CASE NOTES

Review of a prolonged Brucella ovis eradication program

Michaela Woolford, CSU veterinary student and Libby Read, district veterinarian North West LHPA

Posted Flock & Herd March 2012

Introduction

Brucella ovis infection is a major cause of epididymitis and infertility in rams, resulting in reproductive failure and significant economic losses within Australia and worldwide. This report discusses a recent eradication program undertaken in an extensive Merino flock. The property is 18,049 Ha in size and located west of Carinda in north western NSW. B. ovis was eradicated after a 12 month intensive test and cull eradication program. The report focuses on what may have caused the prolonged course of the eradication, and questions the validity of the complement fixation test as the gold standard serological marker of disease.

REVIEW OF THE SYNDROME

Brucella ovis is most commonly carried by mature, sexually active rams. Transmission occurs via direct contact or passive venereal infection (Radostits et al., 2007). By the time B. ovis has been identified within a flock, as many as 80% of rams may already be infected (Dan Salmon, SDV Riverina LHPA, pers. comm.). Bacteraemia develops initially accompanied by a mild systemic response, however this soon resolves as the organism localises in the epididymides. Epididymitis can be profound, with sperm stasis and secondary spermatocele formation resulting in infertility. This acute phase is characterised by poor semen quality in the presence of scrotal oedema and inflammation. Palpable lesions in the epididymis and tunicae are often the primary clinical finding, however lesions may not develop until after the acute syndrome has resolved and the latent period has elapsed (Radostits et al., 2007). Lesions may also be confined to the internal genitalia. Thus, affected rams may be actively excreting Brucella ovis in the absence of palpable testicular abnormalities.

The organism has a low prevalence and low pathogenicity in ewes, the primary loci for infection being the placenta. This can, uncommonly, induce foetal death however low birth weight lambs are most often observed (Plant et al., 2007).

Poor reproductive performance in the flock is usually the principal factor that prompts the launch of an investigation and subsequent eradication program.

As infected rams are the source of infection within the flock, eradication programs are based upon identification and removal of infected rams and improving biosecurity measures to ensure the disease is not introduced by stray or purchased stock. In order for this program to be effective, testing protocols must be reliable, culling policies stringent and biosecurity tight.

CASE STUDY

A test and cull policy was implemented at 3-12 week intervals from September 2010 until the property was deemed free of Brucellosis in October 2011. The intertest period is demonstrated in Figure 1. The three peaks in the graph represent extended intertest periods due to flooding, joining and outside obligations of the producer. The rams were not tested for 4 weeks following joining.

Figure 1: Intertest periods shown as number of weeks between tests

Serological testing utilising a Complement Fixation Test (CFT) was conducted at the Elizabeth MacArthur Agricultural Institute (EMAI) Diagnostic and Analytical Services Laboratory. Thirty-four positive rams were culled after the initial serological test (CFT; 10/9/2010). The CFT results from the infected ('dirty') ram flock are outlined in Figure 2, demonstrating a low prevalence of infection persisting within the flock after the initial cull of 34 positive rams. The eradication was considered successful when all rams tested were CFT negative 11 weeks after removal of the last infected ram on 2 August 2011.

Figure 2: CFT results over time

A consignment of 43 replacement rams was purchased from an Ovine Brucellosis Accredited stud in October 2010. These rams were also serologically tested upon arrival. All new rams were CFT negative and were maintained as a separate 'clean' mob.

Clean musters of the dirty ram flock were not always achieved during the eradication program. A total of 6 rams failed to be mustered at different testing cycles during the 12 month eradication period. None of these rams appeared in subsequent musters and they were presumed dead.

Compared to eradication programs on properties with similar management procedures in the North West Livestock Health and Pest Authority (NWLHPA), the program on this property was prolonged. It was expected that eradication would be achieved within four or five test and cull cycles. Factors that may have been involved are explored below.

Discussion

Flock Management

The importance of flock management during an eradication program should not be underestimated. Failure to segregate clean from infected and/or potentially infected (dirty) stock can impede success of an otherwise well structured program. Other flock management failures include; failure to muster all rams for testing, failure to cull positive rams and straying rams which can all perpetuate a persistent infection within the flock.

Flock segregation

The maintenance of strict segregation between clean and dirty rams is essential during an eradication program. This may be more difficult during the joining period as rams are more likely to stray between mobs. When a clean mob is exposed to a possibly infected ram from the dirty mob, it must be assumed that all rams (and potentially ewes) in the clean mob have been exposed and possibly infected. These animals should then be included in ensuing tests, reclassified and culled if necessary.

This principle also applies to straying rams of unknown B. ovis infection status. It is pertinent to test these rams prior to removal to determine if they may have caused a breach in flock segregation.

In this case study, a ram from the dirty mob strayed into a joining paddock with 14 clean rams. These rams were tested after joining, and all were found to be negative on CFT.

Ryan (1964) reported evidence to suggest that a clean ram may become infected by joining with a ewe which had failed to conceive to an infected rams (4 months previously in this report). This suggests that the classification and segregation of clean and dirty ewe mobs may be beneficial in addition to segregation of rams, further complicating flock management during eradication.

Failure to muster all rams for testing

The most significant threat posed by unaccounted rams is the possibility that they may be infected and evade culling or that they may become infected during the unaccounted period and renter the flock to reinstitute infection. In either case, re-entry of the ram into either the clean or dirty flock represents a considerable risk and must be accounted for by means of identification and testing of missing rams if they appear at subsequent tests. In this case, any rams that were not mustered did not appear in subsequent musters and thus the risk of perpetuating infection by missing rams was decreased but not eliminated.

Ewe infection

Although ewe infection with B. ovis is of low prevalence and often transient, Plant (2003) reported a small proportion of ewes (2/102) within an infected flock that were serologically positive at lambing. These ewes excreted B. ovis in foetal fluids and vaginal discharges. However, 8 weeks after lambing, these ewes and lambs were serologically negative. Given this finding, it is possible that the presence of clean rams with lambing ewes may perpetuate B. ovis infection within the ram mob. This may contribute to the failure of an eradication program. In this case however, no rams were present in any lambing flock, therefore the risk is negligible and cannot account for the persistent infection within the flock.

CFT positive ewes have been implicated in perpetuating brucellosis on three properties via perinatal infection of ram lambs (Walker and Walker, 2001). It is thought that the lambs were infected via B. ovis infected mammary tissue. As ram lambs are not retained on the case study property, it is unlikely that this was a source of persistent infection.

Testicular Palpation: Epididymitis and orchitis

Clinical examination and genital palpation have historically held a firm place as a screening process for B. ovis diagnosis. However, clinical methods alone are not adequate for detection of the disease. This is due to the variability in palpable testicular abnormalities which can be absent in the presence of active B. ovis excretion (especially in chronically excreted rams) (Plant et al., 2007; Worthington et al., 1985). In addition to this, 'hallmark' B. ovis lesions (epididymitis and orchitis) can often be confused with other conditions, including Histophilus somni (previously Haemophilus somnus), Histophilus ovis and Actinobacillus seminis reproductive tract infections. These organisms are commonly implicated in the reproductive tract of rams post-joining and may induce significant transient CFT titres, as observed during investigations at the Regional Veterinary Laboratory at Orange (M. Carrigan) (Plant et al., 2003).

In this case study, rams were not classified based on clinical lesions. Therefore alternative aetiologies for clinical lesions were not investigated.

Serological Testing

The complement fixation test (CFT) has been adopted in Australia as the standard test for the detection of Brucella ovis in sheep. It is generally accepted that rams will have a detectable titre on CFT between 2 and 7 weeks after exposure. For this reason, it is necessary to test and cull approximately every 4 weeks when utilising the CFT test to eradicate brucellosis in a ram flock. The flock is usually deemed brucellosis free when all rams test negative on CFT at least 7 weeks after the last positive ram was removed.

The specificity and sensitivity of the B. ovis CFT procedure used by EMAI has a reported 'sensitivity of 100% recorded for 59 serums from known infected rams and a specificity of 99.9% for 1593 serums from rams known or believed to be free of infection' (Searson JE, 1982).

However, problems with specificity and low sensitivity of CFT have been identified (Hicks et al., 1978). Culture positive rams with negative or fluctuating CFT titre are possible, especially in chronically infected animals (Worthington et al., 1985). False negative CFT results are most common in early and late infection (Burgess and Norris, 1982). Plant (2003) reported repeated testing in one flock that failed to eradicate the disease from the ram flock. A ram was found to be excreting B. ovis in the absence of clinical lesions whilst demonstrating a negative serological CFT. The ram remained serologically negative for over 12 months until a lesion was palpable. Four months following this the ram returned a serologically positive test, however at this time was semen culture negative (Plant et al., 2003).

This finding correlates with previous observations by Worthington and colleagues (1985), who investigated 37 chronically infected rams. Of these chronically infected rams, 11% of sera were CFT negative. When these CFT negative, actively excreting rams were tested using enzyme labelled immunosorbent assays (ELISA), 3 /4 were positive.

Furthermore, the CFT has limitations due to additional processing disadvantages such as the occurrence of anti-complementary and haemolysed sera (Vigliocco et al., 1997).

While it is unlikely, it is possible that an infected and excreting ram who was CFT negative was responsible (and may continue to be responsible) for brucellosis persisting in the flock in this case study.

Other serological tests include agar gel immunodiffusion (AGID) and ELISA. AGID has the lowest sensitivity (40.5%) relative to CFT (Estein et al., 2002) and is not recommended for use in an eradication program. Competitive ELISA (cELISA) has been introduced to replace the CFT for identification of Brucella melitensis in sheep and goats in the UK, due to its higher specificity and automation when compared to the previous CFT implemented (Perret et al., 2010). The sensitivity of cELISA (92.31%-100%) is more favourable than CFT (77.14%-100%) with no concurrent depreciation in specificity. This serological test is not available for B. ovis.

The use of ELISA to improve serological diagnosis of B. ovis infection in rams has been thoroughly investigated. The higher sensitivity of the ELISA (stated above) reduces the risk of false negative serological results in chronically excreting rams (Vigliocco et al., 1997). The practicality and validity of replacing the CFT with ELISA for serological diagnosis of B. ovis in Australia is an area for future consideration.

Semen Culture

Historically, a combination of semen examination and genital palpation has been the foundation of definitive B. ovis diagnosis in rams. The limitation of these methods arise due to the intermittent nature of B. ovis excretion that occurs in chronically infected rams in the absence of testicular lesions (as discussed above).

PCR

A species specific PCR test has been developed for the detection of B.ovis in semen, urine, tissue samples and preputial wash (Xavier et al., 2010). This PCR has been shown to detect the organism 6 weeks post-infection and detect a higher number of positive samples when compared to conventional bacteriology. Importantly, the sample can be obtained from a standard preputial wash as in comparison to the semen sampled required for conventional culture techniques.

However, a considerable number of serologically positive rams may never excrete B. ovis. As such, the field sensitivity of semen PCR and culture declines significantly.

Conclusion

Twelve serological tests were required to eradicate B. ovis from the flock in this case study. Breakdowns in flock management were encountered during the program which may have contributed to the extended testing and eradication period. A breakdown in flock segregation occurred during joining, however all clean rams that came in contact with the dirty ram were retested at the subsequent test and found to be uninfected. Rams went missing during the program; however they did not return and were presumed dead. Palpation of rams was only completed once, towards the end of the eradication program. However of animals with identified abnormalities, none were serologically positive.

The intertest period was variable and extended at times. The prolonged intertest periods between tests 5 and 8 may have been crucial to the prolonged nature of this eradication program, especially when combined with the short (3 week) period between tests 4 and 5. This could have resulted in a ram to be incubating at test 5 and then spreading the disease in the 9 week period before test 6.

The serological test (CFT) utilised in this case is gold standard for the sheep industry in Australia. However false negatives are possible, and the protracted nature of eradication on this property suggests that presence of an infected, excreting ram in the mob is possible. This also represents a threat to the continued brucellosis free status of the ram flock.

This case highlights the importance of careful flock management and record keeping during an ovine brucellosis eradication program. It also emphasises the need for consistent testing intervals. Testicular palpation at the initial test, and repeated throughout the program may have decreased the time taken to eradicate the disease.

During prolonged eradications (say beyond 5 tests) it may also be pertinent to consider incorporating compounding tests to increase the overall testing sensitivity and reduce the likelihood of false negatives. This might allow for more rapid identification and removal of chronic shedders and decrease the amount of testing required. Such procedures might include a combination of serological (ELISA - CFT) and auxiliary tests (genital palpation and semen culture), especially on suspect chronically infected rams. In the future, PCR on preputial washings may become available.

References

Burgess G & Norris M (1982) Evaluation of the cold complement fixation test for diagnosis of ovine brucellosis Australian Veterinary Journal 59:23-25
Estein SM, Baldi PC & Bowden RA (2002) Comparison of serological tests based on outer membrane or internal antigens for detecting antibodies to Brucella ovis in infected flocks Journal of Veterinary Diagnostic Investigation 14:407
Hicks JD, Burr GR, Marshall DR & Vidler BM (1978) CFT inaccurate for epididymitis New Zealand Veterinary Journal 26:135
Perret LL, McGiven JA, Brew SD & Stack JA (2010) Evaluation of Competitive ELISA for Detection of Antibodies to Brucella Infection in Domestic Animals Croatian Medical Journal 51:314-319
Plant J (2003) Breakdowns in Brucella ovis eradication programs ASV Conference Proceedings pp 199-201
Plant J & Seaman JT (2007) Ovine Brucellosis Primefact 472 NSW DPI
Radostits OM, Gay CC, Hinchcliff KW & Constable PD Veterinary Medicine: A textbook of the diseases of cattle, horses, sheep, pigs and goats Saunders, London, 10^th Edition, 2007
Searson, JE (1982) Sensitivity and specificity of two microtitre complement fixation tests for the diagnosis of Brucella ovis infection in rams Australian Veterinary Journal 58:5-7
Ryan, F (1964) Eradication of ovine brucellosis Australian Veterinary Journal 40:162-165
Vigliocco AM, Silva Paulo PS, Mestre J, Briones GC, Draghi G, Tossi M & Nielsen K (1997) Development and validation of an indirect enzyme immunoassay for detection of ovine antibody to Brucella ovis Veterinary Microbiology 54:357-368
Walker R & Walker B (2001) Ovine brucellosis in ewes In: Proceedings of the Australian Sheep Veterinary Society
Worthington R, Stevensont B & De Lisle G (1985) Serology and semen culture for the diagnosis of Brucella ovis infection in chronically infected rams New Zealand Veterinary Journal 33:84-86
Xavier MN, Silva TMA, Costa A, Paixio TA, Moustacas VS, Carvalho Junior CA, SantaAnna FM, Robles CA, Gouveia AMG, Lage AP, Tsolis RM & Santos RL (2010) Development and evaluation of a species-specific PCR assay for the detection of Brucella ovis infection in rams Veterinary Microbiology 145:158-164