This study compared two commercial ELISA kits widely used in Europe to detect antibodies against M. agalactiae, the primary agent of CA. Kits were tested on a large number of sera collected from 5900 animals from 211 farms. The constitution of representative samples of farms with well known infection status was possible for several reasons: first, the epidemiological situation of CA in France is well surveyed , second, a large number of farms and animals had been monitored for many years as part of several distinct eradication campaigns and third, animals had not been vaccinated against this agent. This large sample size guaranteed the statistical reliability and accuracy of the study. Nevertheless, the average performance values should be regarded with caution because a sampling bias may still have occurred, and furthermore, the values varied according to the region, host species or M. agalactiae strain.
Accurate determination of the infection status of individual animals regarding M. agalactiae is difficult in the absence of a gold standard diagnostic technique. Thus, the criteria used to define animals as “infected reference” differ from one study to another and may be clinical, bacteriological or serological [5–8]. The OIE recommends that in the absence of a gold standard for classifying an animal as infected, isolation of the disease agent can legitimately be considered the method of choice . Classification of an animal as infected may be biased because animals may shed the organism early in the course of infection even if the humoral immune response is still low. Furthermore, animals with latent infection that have developed an immune response may intermittently or no longer shed the organism and are therefore difficult to detect.
Here, the “infected reference” status of an animal was based on bacteriological identification of M. agalactiae in individual milk samples. The M. agalactiae detection protocol used in this study is highly sensitive and specific. Sensitivity was provided by the first step of culture: an inter-laboratory assay including 32 French diagnosis laboratories demonstrated that 70% of them detected M. agalactiae by culture when test aliquots contained only 2 to 25 CFU and 100% when test aliquots contained 25 to 250 CFU [unpublished data]. Specificity was provided by using two sets of PCR primers for the identification of M. agalactiae, and was confirmed by dot-immunobinding. Finally, bias associated with intermittent shedding and with early infection were limited by i) repeating bacteriological examinations of individual milk samples at regular intervals and ii) not taking into account milk samples performed during the period of 45 days prior to blood sampling.
The uninfected reference animals were recruited from geographical areas where M. agalactiae-related CA had never been reported or where routine monitoring had been in place for over 20 years and where no case had occurred in the last 10 years. Different independent populations and animal species (goats, sheep) were included to ensure diversity. Nevertheless, a number of animals, sometimes from the same farm, gave positive results with the TA-ELISA kit at the threshold recommended by the manufacturer. These positive results could have resulted from insufficient test specificity or from individual infections that escaped prior detection. A further technique was needed to unequivocally determine the status of these animals.
IBT is used for the serological diagnosis of contagious bovine pleuropneumonia (CBPP), another mycoplasma disease of ruminants, when routine tests are insufficiently accurate or ambiguous . Sera from infected animals give five specific IBT antigenic bands at 110, 98, 95, 60 and 48 kDa simultaneously (http://www.oie.int). IBT has been used in several studies to identify M. agalactiae infection in cases of CA [6, 7, 19, 20]. These authors described five stable immunogenic proteins with specific molecular masses of 80, 55, 48, 40, and 30 kDa, which presumably could be used as a specific signature of M. agalactiae infection much like with CBPP. However, three proteins (55, 30 and 40 kDa) are not expressed by all strains [6, 12, 21]. Thus an M. agalactiae-IBT should only be interpreted in the light of the antigenic profile of local strains.
A thorough analysis of 63 isolates collected between 1977 and 2007 in the P.A. showed that a single strain, very similar to the reference strain PG2, had been present for 30 years in the P.A . This strain expressed the 80, 48, 40, and 30 kDa proteins but not the 55 kDa one identified in Sardinian strains . A posteriori, the 55 kDa protein appeared to be antigenically similar to a variable protein of M. agalactiae known as Vpma U . On the PG2 antigenical phenotype used in IBT, Vpma U is apparent at 27 kDa. Understandably, if a single M. agalactiae strain, similar to PG2 reference strain, was circulating in the P.A., it seems reasonable to suspect that all serum samples from infected animals would express the same IBT profile with four major bands corresponding to stable proteins and a 27 kDa band, as confirmed by analysis of the 22 sera from infected reference animals. In contrast, the profiles of uninfected animals were heterogeneous and bands at 80, 48, 40, 30 kDa never occurred simultaneously. One recently infected flock that had escaped monitoring was detected by IBT. So IBT proved to be useful in determining the status of P.A. flocks with regard to M. agalactiae when the ELISA test results were ambiguous.
Several sera from the uninfected population gave positive results with the TA-ELISA kit. Even in the most doubtful cases (several positives in one farm), the IBT profiles were never similar to those of infected animals. Presumably, these were false positives and the wide distribution of non-negative responses to the TA-ELISA kit on several farms would support this hypothesis.
Variability within M. agalactiae strains can affect test performances, and test sensitivity may be improved by using a local strain as antigen . In the present study, the kinetics of antibody response were followed after experimental inoculations of goats with three different M. agalactiae strains. Detection of the immune response to one strain was poor with both ELISA kits. The variability of M. agalactiae is expressed in two ways. First, not all strains express certain specific immunogenic stable proteins [12–21]. Second, the expression and size of several surface proteins, such as the Vpma family, can show rapid and random fluctuations, thereby generating multiple antigenic surface configurations and broad intra-clonal variability . These two concomitant phenomena may alter the host’s immune response, thereby affecting the sensitivity of diagnostic tests based on the degree of proximity between the phenotype of the test antigen and the phenotypes of the infecting strain. The consequences of antigenic variability on test performance are difficult to predict from strain characteristics. For example, both ELISA kits detect an immune response to strain 5632 even though this strain is genetically very different from the reference strain PG2 .
Test performance is also modified by geographic region and host species (sheep or goats). Regional differences may be due to antigenic variations between locally circulating strains. The TA-ELISA kit is less specific when used to detect M. agalactiae antibodies in sheep than in goats. This may be because it was developed with a goat strain that is possibly “adapted” to this species. Test specificity is not affected when goats are infected concurrently with other mycoplasma(s), even though these organisms may have many genes in common with M. agalactiae. However, given the genetic and antigenic variability of M. mycoides subsp. capri[15, 26], certain strains may well express atypical cross antigens, as is the case with CBPP .
The main source of CA contagion is by M. agalactiae shedding in milk [1, 2]. This study showed that the correlation between milk shedding and a detectable serological IgG response in blood was poor, implying that serology should not be used as an indicator of infectiousness. The percentage of animals that shed and tested negative with the TA-ELISA kit varied from 0 to 48% (average 16%) between farms, and from 4 to 62% (average 31%) with the P48-ELISA kit. Reports in the literature concerning the duration of the humoral response to mammary infection show considerable divergence, ranging from a rapid, systematic and persistent sero-conversion of at least 320 days  to a highly transient sero-conversion of less than 32 days . Further studies with reliable tests will be required to see whether the negativity in some infected animals results from the absence of serological response or from the rapid decrease of antibodies.