Detection of Antibodies against Mycoplasma mycoides subsp . capri in Goats with the Complement Fixation Test

Mycoplasma mycoides subsp. capri is the causative agent of severe and acute respiratory problems in goats, which spreads rapidly and represents high mortality. The serological profile of the goat population, from nine regions in seven states of Mexico, was screened by the Complement Fixation test (CF) in sera from asymptomatic goats and animals with mild respiratory symptoms. Sera and nasal swabs of 827 goats were collected for the isolation of the organism. An antiserum was prepared against a previously isolated field strain of Mycoplasma mycoides subsp. capri. CF Antibody titers were associated with the results of the isolates to determine the cutoff point. The CF was considered as positive if its result was ≥1/16. The CF registered 251 positive goats (30.35%) and 576 (69.65%) negative; the test showed high sensitivity (93.33%) and specificity (72.27%). In the specific case of diagnosis for mycoplasmosis associated with respiratory problems in goats, the CF proved to be a good diagnosis test, this study determined that 30% of the goat population showed antibody titers against Mycoplasma mycoides subsp. capri and revealed those animals who have had contact with this microorganism during their lives regardless of the presence or absence of respiratory symptoms.


Introduction
The caprine respiratory disease is regarded as a multifactorial disease in which virus and bacteria including mycoplasmas are involved.Mycoplasma species are phylogenetically tightly related and have been jointly clustered into the mycoides cluster [1].The most frequently involved mycoplasma is M. mycoides subsp.capri, and M. capricolum subsp.capripneumoniae is the etiological agent of the caprine contagious pleuropneumonia (CCPA), which can submit as an acute, subacute or chronic disease.Mortality rate due to CCPA ranges from 60% to 80% among goat herds in Africa [2] [3].In CCPA free areas, M. mycoides subsp.capri and M. capricolum subsp.capricolum turn to be the predominant pathogens involved in caprine diseases [4] [5].
In Mexico, although there is a scanty serological profile of the caprine herd, the mycoides cluster has been detected as well as isolations of members of the mycoides cluster [6], and M. mycoides subsp.capri [7] from goat lungs with acute and severe respiratory problems.In this study, the usefulness of the complement fixation assay was assessed to determine the serological status of nine regions.

Animals and Samples
A sample size of 692 sera samples was estimated with a 0.90 confidence.A total of 827 dairy goats from all ages and breeds from nine regions in the northwest, southeast and central Mexico were aseptically bled (Table 1).Sera were conserved at −20˚C until use.

Antiserum
The immunization methodology described by Tully, et al. 1983 [8] was followed to obtain a rabbit antiserum against a strain of M. mycoides subsp.capri isolated from the lung of a goat with pneumonia.This polyclonal antiserum was evaluated by double immunodiffusion test (DIT) and counter-immune electrophoresis (CIE) against Mycoplasma bovis, Acholeplasma laidlawii and Ureaplasma diversum.Furthermore, to rule out cross reaction during complement fixation (CF), it was also performed with polyclonal serum against mycoides subsp.capri, and a polyclonal antiserum to M. bovis and M. hyopneumoniae.

Complement Fixation Assay (CF)
CF was performed in a 96 wells microplate according to the described by Cunningham, 1986 [9].Briefly, whole cells of M. mycoides subsp.capri were used as antigen (Ag).
100% hemolysis was regarded with two units of complement (UC), and two hemolysis units in Triethanolamine Buffer Solution (TBS), pH 7.2.Field samples and control serums (CS); positive and negative, were serially diluted (from 1/2 to 1/256) in a final  The threshold for the cut point was established according to the relation between the sample titers from which Mycoplasma spp. was isolated, and those from the milk samples and nasal swabs from herds included in the study.Finally, the CF sensitivity and specificity was determined by the Bayes theorem and the positive and negative predictive values were estimated.

Antiserum against Mycoplasma mycoides subsp. capri
Rabbits were bled to white at day 25 post-inoculation.No cross reaction was detected against Mycoplasma bovis, A. laidlawii, and U. diversum in the CF.To assess the assay homogeneity Xi-square was used to compare whole, sonicated or soluble fraction of M. mycoides subsp.capri as antigen.No statistical significance (P = 0.1580, P < 0 -5) among them was observed, therefore whole antigen was used for further experiments.

Complement Fixation Assay
The cut off point for CF was established as positive above titers ≥1/16 and negative up to ≤1/8.According to this cut off point 576 (69.65%) samples were negative and 251 (30.35%) were positive (Table 2).Noteworthy, the CF detected around 10% of positive sera in regions 9 and 2, in which no previous events of respiratory problems had been reported.In regions 3, 5, 1 and 7, with respiratory problems history the positive percentage was up to 20%.On the other hand, those herds in which the samples were drawn during a respiratory event in region 4, 6 and 8, the positive percentage was over 60% (Table 2).Statistically significant difference was determined to CF (P < 0.01) by Xi-square.Furthermore, the sensitivity and specificity of CF were determined by the Bayes Theorem, as shown in Table 3.

Discussion
Mexico is not an endemic area; hence the CF threshold is fixed at titers ≥1/16.In contrast, threshold of ≥1/32 is used in Kenia, in which micoplasmosis due to Mycoplasma capricolum subsp.capripneumoniae is endemic [10] [11].On the other hand, in Oman a threshold for immunoagglutination has been established at 1/40 for M. capricolum subsp.capripneumoniae [12].In these countries, vaccination against mycoplasma is widely accepted.In Mexico, the mycoplasmosis vaccination in goats is not practiced yet; therefore a tighter criterion for results interpretation of the CF was applied.In the mycoplasmosis free herds, ten goats were CF positive with titers of 1/16 -1/32.These results may be explained due to herd mobilization among adjacent regions.In herds with micoplasmosis history, 66 goats were diagnosed as positive by CF (titers 1/16 -1/128).These results suggest previous contact with the antigen [13].According to experimental assays with M. capricolum subsp.capripneumoniae, CF may detect previous exposure to Mycoplasma spp. as long as four months post inoculation [14].In the herds included in this study, micoplasmosis was diagnosed by CF in 69.46% in region 8 and up to 100% in regions 4 and 6.These results are in accordance with the disease morbidity, which may range from 60% to 100%.
In Mexico, Mycoplasma has been reported since the 60's.Solana et al. (1966) used the CF and/or Immunoagglutination for M. mycoides subsp.capri to screen 16 regions including regions 4, 5, 6 and 8 [15].In region 8 they reported 90% of positive sera, and in regions 4, 5 and 6, 50% of positive results.However, no relation among the Mycoplasma spp.isolation, the disease symptoms and the CF results was depicted.Besides, Mycoplasma spp.has been isolated in the South from pneumonia outbreaks [16].
In this work, we report enhancement of the sensitivity and specificity of the CF by using the whole antigen of Mycoplasma mycoides subsp.capri.It turns to be quick and reliable for the serological diagnosis of the caprine mycoplasmosis.This assay is able to detect antibodies against Mycoplasma spp. up to a two years period post-exposure.We were able to establish that the goat population had been exposed during their productive life to Mycoplasma mycoides subsp.capri with an overall seroprevalence of 30.35%.
Additionally, the serological profile by this sensible and specific serological test allows discerning which animals have been in contact with the mycoides cluster members in the short and long term and guides the preventive and control measures for goats' mycoplasmosis.
volume of 25 µl.Twenty-five µl of the M. mycoides subsp.capri 1/700 were added to each serum dilution, and to each control.Each anticomplement control (AntiC) was graduated to 25 µl with TBS, and 25 µl complement (C) with two UC were added to the serum dilutions, positive and negative controls, antigen complement (Ag-C) and C.The plate was incubated at 37˚C in a laboratory bath for 60 min.The hemolytic system (HS) was incubated at 37˚C for 30 min, then 50 µl were added to each well and again incubated at 37˚C for 30 min.Periodical observations were done since the first 5 min of incubation to determine the serum titers, and were established at the highest dilution in which a positive reaction of sedimentation of the HS was observed.

Table 1 .
Geographic location of the herds included in this study.
a Isolation based diagnosis, b Histopathology based diagnosis.

Table 3 .
Sensitivity and specificity of the CF.