Chapter 6
Surveillance of bovine brucellosis

This section provides a summary of identification, serological and other tests, vaccines and diagnostic biologicals drawn from Chapter 2.3.1 of the OIE Manual of Standards for Diagnostic Tests and Vaccines (OIE, 2000).

Bovine brucellosis is usually caused by Brucella abortus, less frequently by B. melitensis, and rarely by B. suis. It is usually manifested by abortion, with excretion of the organisms in uterine discharges and in milk. Diagnosis depends on the isolation of Brucella sp. from abortion material, udder secretion or from tissues removed at postmortem examination. Alternatively, specific cell-mediated or serological responses to Brucella antigens can be demonstrated.

Identification of the agent

The demonstration by modified acid-fast or immunospecific staining of organisms of Brucella morphology in abortion material or vaginal discharges provides presumptive evidence of brucellosis, especially if supported by serological tests. Whenever possible, the organism should be isolated, and the species and biovar should be isolated, and the species and biovar should be identified by phage lysis or oxidative metabolism tests, or both, and by cultural, biochemical and serological criteria. The recently developed polymerase chain reaction (PCR) and DNA-probe methods provide additional means of detection.

Serological tests

No serological test is appropriate for all epidemiological situations. The buffered Brucella antigen tests (Rose Bengal plate agglutination test and buffered plate agglutination test) are suitable for screening herds and individual animals. The reactivity of positive samples should be confirmed by the complement fixation test or by enzyme-linked immunosorbent assay (ELISA), both of which can also be used for both screening and confirmation. The serum agglutination test is inferior to other tests in specificity and sensitivity, and is not recommended if other procedures are available. The milk ring test and indirect ELISA performed on bulk milk samples are effective for screening and monitoring dairy cattle for brucellosis, but are less reliable in large herds and less sensitive with B. melitensis. Another immunological test is the brucellin skin test, which can be used for screening unvaccinated herds, provided that a standardized allergen preparation (e.g., brucellin INRA) is available.

Requirements for vaccines and diagnostic biologicals

B. abortus Strain 19 live vaccine should be prepared from USA-derived seed cultures^[1], and each batch must conform to minimum standards for viability, smoothness, pathogenicity and ability to immunize guinea pigs or mice against challenge with a virulent strain of B. abortus. Brucellin preparations for the intradermal test must be free of lipopolysaccharide and must not produce non-specific inflammatory reactions or interfere with serological tests. Diagnostic antigens must be prepared from an approved smooth strain of B. abortus and comply with minimum standards for identity, purity, sensitivity and specificity. Antigens for serological tests should be standardized against standard sera calibrated against the International Standard B. abortus serum.

Note: B. abortus infections, besides occurring in cattle (Bos taurus and Bos indicus), also occur in domestic buffalo (Bubalus bubalus), African Buffalo (Syncerus cafer), North American buffalo (Bison bison), as well as cervidae and camelidae. Occasional infections in horses and dogs have also been reported.

B. melitensis and B. suis infections have been reported in cattle, especially if in contact with infected small ruminants and swine, respectively. It is important to recognize that disease may not always occur with these two Brucella species.

The following surveillance techniques are appropriate for the four phases of bovine brucellosis control and eradication.

Where calving is seasonal, actual testing periods might best be implemented to coincide with herd management practices.

1. High or unknown prevalence phase with no control programmes

During this phase, the magnitude and distribution of the problem should be determined, as discussed earlier.

On-farm surveillance

• Voluntary investigation of abortion incidents and submission of tissues to a veterinary diagnostic laboratory for culture (passive, and frequently biased).

• Sero-surveillance (active).

Off-farm surveillance

• Percentage of abortion incidents confirmed by laboratory as brucellosis (passive and frequently biased).

• Bacteriological and serological examination of tissues and blood from cattle of breeding age at markets or slaughter (active).

2. Mass vaccination phase

B. abortus strain 19 is still considered the standard vaccine, although some countries are now using strain RB51 (rough) vaccines. Strain RB51 apparently does not result in antibodies detectable by current serological tests. The dose, route of inoculation, and age or sex of vaccination may vary from country to country.

On-farm surveillance

• Testing random samples of animals or herds within 2-3 weeks after vaccination using buffered Brucella antigen tests to evaluate strain 19 vaccine coverage. Over 80% of vaccinated animals should be seropositive. Also check the identification of vaccinated animals.

• Monitor abortion incidents as in the previous phase, or carry out surveillance on selected or sentinel herds to monitor abortion rates (active).

• Monitor randomly selected herds periodically using tests capable of distinguishing between serological responses due to vaccination and those due to infection, such as the complement fixation, immunodiffusion and competitive ELISA tests (Active).

Off-farm surveillance

Active surveillance of tissues and blood samples from randomly selected slaughter animals of breeding age, using bacteriological and serological tests as above.

3. Test and removal, segregation or slaughter phase

Note that, as the incidence or prevalence of infected animals and herds decreases to low levels, vaccination (especially with strain 19) may be phased out to reduce the problem of false-positive reactions. However, the premature ban of strain 19 vaccination is a common source of problems in the later stages. Combined conjunctival vaccination of 3-4-month-old replacement animals and eradication by test and slaughter are compatible.

On-farm surveillance

• Dairy cattle. The milk ring test (MRT) and the indirect ELISA tests are recognized as low cost and efficient methods to identify herds with infected animals, even if the within-herd prevalence is low. If positive results are detected, then all animals in the herd should be serologically tested (see below). Note that in large herds the MRT test should be modified by either increasing the volume of milk tested or by taking segmented samples throughout the milking period. The milk ring test is usually carried out at intervals of 4-6 months, except in known infected herds, where monthly testing is advised. The MRT test may yield false-negative results in the early stages of B. melitensis-infected herds.

• Beef cattle. The brucellin test is a low cost alternative to other screening tests, especially where the herd rate of infection is low. Although not recommended in vaccinated herds, it has been successfully used in New Zealand. The test is carried out by intradermal injection either in the cervical or caudal fold regions. It is examined by palpation 72 hours after injection, and a swelling of 2 mm or greater considered to be positive.

The sensitivity of the test is approximately 60-75% and the specificity 95-100%. An important practical advantage of this test is that it can be carried out concurrently with the tuberculin test. It may also be used to distinguish false-positive brucellosis serological reactions. The table gives the probability that a Brucellin skin test will identify a herd as infected with brucellos

The prevalence of infection (P) in a herd may be estimated from the following formula:

P = (t + S_p -1) × (S_e + S_p -1)

where t = positive test frequency, S_p = test specificity, and S_e = test sensitivity.

• Investigation of abortions If abortion is made a notifiable event, this can be used as a supplementary tool, especially in herds that calve seasonally before the MRT is used. However, it may not be cost effective as a programme progresses.

A specific set of samples from each abortion (vaginal swabs, foetal membranes, foetal lung, and stomach contents, and serum from each animal) must be submitted to a diagnostic laboratory.

• Movement controls If there are controls on the movement of cattle during a control programme, testing of eligible animals for brucellosis may be undertaken both before and after movement and quarantine on the farm of destination. The second test is particularly critical, especially given the variable incubation period of brucellosis and if the tested animals originate from a herd of unknown status. Movement testing can be a very useful surveillance tool, especially for those owners or dealers who regularly buy and sell cattle.

• Epidemiological investigations When infected herds are identified and subsequently investigated (see section on surveillance), this may lead to testing of adjacent herds and the source herds of recent introductions.

• Individual animal tests Once an infected herd has been identified, all test-eligible animals (sexually intact animals 18 months of age and over, or, if vaccinated as calves with strain 19, this may be increased to 20-24 months), are bled at intervals, usually 3-6 months, and reactor animals identified and removed from the herd.

The sequence of testing most often used has been:

1. Use a buffered Brucella antigen test, such as the Rose Bengal or Brucella plate agglutination test. All positives are then examined with the complement fixation or ELISA tests and the positive animals removed.
2. If these tests can be automated it may be more economical to test all samples initially with the complement fixation or indirect ELISA tests. There are many advantages to centralizing these two tests to ensure quality control and consistency.

Serum agglutination tests are not recommended for routine testing and surveillance because they are inferior to complement fixation and ELISA tests in terms of sensitivity, specificity and practicability.

Off-farm surveillance

As the incidence or prevalence of infected herds decreases, it may be more cost effective to commence testing at markets and slaughterhouses if a reliable system can be developed of trace-back to the herd of origin. This may be accomplished by using permanent identifiers on the farm, such as ear or tail tags, or supplementing these with temporary identifiers such as bar-coded ‘back tags’ applied at entry to the market or slaughterhouse.

The probability of detecting infection in an individual herd is influenced by the herd size, rate of culling (removal) from the herd, and the prevalence of infection in the herd. In general, larger herds have a better chance of being located by market or slaughter surveillance than smaller herds. The percentage of ‘successful’ trace-backs to the herd of origin should be carefully monitored during this phase. If it falls below 50% then this is an indication that problems exist and should be corrected. In general, the MRT is more efficient in detecting infected herds than market or slaughter testing, but a combination of strategies is ideal.

4. Freedom phase: herds, regions and countries

According to the OIE International Animal Health Code (see Chapter 2.3.1, Bovine Brucellosis), for a country or region to be considered as officially free from bovine brucellosis it must satisfy the following requirements:

1. Bovine brucellosis or any suspicion thereof is compulsorily notifiable in the country.
2. The entire cattle population of a country or part of the territory of a country is under official veterinary control and it has been ascertained that the rate of brucellosis infection does not exceed 0.2% of the cattle herds in the country or area under consideration.
3. The serological tests for bovine brucellosis are periodically conducted in each herd, with or without the milk ring test.
4. No animal has been vaccinated against bovine brucellosis for at least the past three years.
5. All reactors are slaughtered.
6. Animals introduced into a free country of part of the territory of a country shall only come from herds officially free from bovine brucellosis.

In a country where all herds of cattle have qualified as officially free from bovine brucellosis and where no reactor has been found for the past five years, the system for further control may be decided by the country concerned.

For herds to be considered officially free, they must satisfy the following requirements:

1. Be under official veterinary control.
2. Contain no animal that has been vaccinated against bovine brucellosis during at least the past three years.
3. Only contain animals that have not showed evidence of bovine brucellosis infection during the past six months. Some authorities would argue that a longer period of several years is necessary to reduce the risk still further. All suspect cases (such as animals that have prematurely calved) should have been subjected to the necessary laboratory investigations.
4. All cattle over the age of one year were subjected to serological tests, with negative results, performed twice at an interval of 12 months. This requirement is maintained even if the entire herd is normally tested every year or testing is conducted in accordance with other requirements established by the veterinary administration of the country concerned.
5. Additions to the herd shall only come from herds officially free from bovine brucellosis. This condition may be waived for animals that have not been vaccinated, but come from a herd free from bovine brucellosis, provided negative results were shown following a buffered Brucella antigen test and the complement fixation test during 30 days prior to entry into the herd. Any recently calved or calving animal should be retested after 14 days, as tests are not considered valid in female animals that have calved during the past 14 days.

For herds that may still be under a vaccination programme, a separate category of herds freefrom brucellosis is recognized, namely:

1. Be under official veterinary control.
2. Be subjected to either a vaccination or a non-vaccination regime.
3. If a live vaccine is used in female cattle, vaccination must be carried out between three and six months of age, in which case these female cattle must be identified with a permanent mark.
4. All cattle over the age of one year are controlled as provided in paragraph 4) of the definition of a herd of cattle officially free from bovine brucellosis; however, cattle under 30 months of age that have been vaccinated using a live vaccine before reaching six months of age, may be subjected to a buffered Brucella antigen test with a positive result, with the complement fixation test giving a negative result.
5. All cattle introduced into the herd come from a herd officially free from bovine brucellosis, or from a country or part of the territory of a country free from bovine brucellosis. This condition may be waived for animals which have been isolated and which, prior to entry into the herd, were subjected to the serological tests for bovine brucellosis with negative results on two occasions, with an interval of 30 days between each test. These tests are not considered valid in female animals that have calved during the past 14 days.
6. Risk assessment procedures have been utilized to evaluate the appropriateness of changes in surveillance in the transition from test-and-removal to maintaining freedom from brucellosis.

The current OIE Animal Health Code (2002) does not recognize off-farm surveillance, such as market or slaughter testing. The probability of locating infected herds by market or slaughter testing is dependent on the cull rate from the herd, the prevalence of infection in the herds, as well as the probability that an infected animal will pass through the surveillance system and the herd of origin is located. This in turn will depend on the percentage of identified animals, the percentage of samples collected at slaughter and the diligence of tracing activity.

The MRT and indirect ELISA are primary methods of surveillance for dairy herds once freedom has been established. Their efficiency depends upon the percentage of herds from which samples are taken, the number of times each herd is tested within a year, and the accuracy of identification of the herd.

Other surveillance activities in Brucella-free countries or regions should include:

• Testing prior to movement.

• Testing after movement and quarantine.

• Testing areas adjacent to borders where illegal introductions may occur.

• Testing imported animals of breeding age.

• Testing imported semen, embryos and ova.

Finally, there have been a number of instances recorded where a human case or outbreak of brucellosis has lead to a trace-back to an infected herd in a region hitherto considered free of brucellosis.