Mycoplasma conjunctivae infection is self-maintained in the Swiss domestic sheep population

Bacteriological and serological investigations were performed to assess whether the domestic sheep population is a reservoir of Mycoplasma conjunctivae in Switzerland. Among a sample of 69 sheep showing clinical signs of infectious keratoconjunctivitis (IKC) in three Swiss cantons, M. conjunctivae was identified 53 times (76.8%). A commercially prepared indirect ELISA was used to detect M. conjunctivae antibodies in 674 sera of adult sheep. We analysed a stratified random sample of 123 sheep herds from 25 out of the 26 Swiss cantons. At least one positive animal was detected in 89.4% of the herds. In positive herds (n=110), 57.1% of the individual animals tested positive. To assess the importance of sheep's age in the spread of M. conjunctivae, 209 sera of adult sheep and 93 lamb sera among eight sheep herds were analysed using the indirect ELISA. Seroprevalence in 2-6-month-old lambs was 50.5%, indicating that the IKC agent is spread in sheep flocks during raising. Lambs experimentally infected with M. conjunctivae carried the agent for 8 and 23 weeks, respectively, depending on the strain used for challenge. We conclude that the M. conjunctivae-infection is endemic and self-maintained in the domestic sheep population in Switzerland.     

Detection of Mycoplasma conjunctivae in sheep affected with conjunctivitis and infectious keratoconjunctivitis

AIM: To determine the aetiology of a recurring and severe form of infectious keratoconjunctivitis (IKC) in sheep. METHODS: Five sheep flocks that had experienced a severe form of IKC were examined. Clinical history, conjunctival swabs and blood samples were collected from affected animals. Culture for bacteria, and also specifically for Mycoplasma and Chlamydophila spp, and detection of Mycoplasma conjunctivae DNA by polymerase chain reaction (PCR) were attempted. Serum samples were tested for antibodies to M. agalactiae, M. capricolum, M. conjunctivae and Chlamydophila spp. RESULTS: Mycoplasma conjunctivae DNA was detected using PCR in 3/5 flocks, and in all flocks antibodies to M. conjunctivae were detected in sera. A pure growth of Branhamella ovis was cultured from conjunctival swabs from a small proportion of sheep in two flocks. No other pathogens were detected. CONCLUSIONS: This investigation demonstrated that M. conjunctivae was a primary pathogen causing severe IKC in sheep, and is the first report of detection of this organism in sheep in New Zealand. Introduction of clinically normal carrier sheep appeared to have caused the outbreaks. KEYWORDS: Infectious keratoconjunctivitis, Mycoplasma conjunctivae, Chlamydophila pecorum, Branhamella ovis, polymerase chain reaction, ELISA, complement fixation test.     

Ovine keratoconjunctivitis experimentally induced by instillation of Mycoplasma conjunctivae

Five sheep, free from Mycoplasma conjunctivae and ocular Chlamydia infection, were experimentally inoculated with M. conjunctivae and five more sheep were exposed to the infection by contact. Keratoconjunctivitis developed in all ten sheep. As in natural outbreaks of infectious keratoconjunctivitis (IKC), clinical signs were generally moderate and transient, and recurred in some sheep. M. conjunctivae was detected throughout the 53-day observation period. Clinical diagnosis was confirmed by histopathologic examination of three sheep. Moraxella ovis, found in six of the ten sheep before the start of the experiment, appeared to play no etiologic role in the development of IKC.     

Detection of Mycoplasma conjunctivae in the eyes of healthy, free-ranging Alpine ibex: possible involvement of Alpine ibex as carriers for the main causing agent of infectious keratoconjunctivitis in wild Caprinae

Mycoplasma conjunctivae is considered the major cause of infectious keratoconjunctivitis (IKC) in Alpine ibex (Capra i. ibex) and chamois (Rupicapra r. rupicapra). While it is known that domestic sheep can act as healthy carriers for M. conjunctivae, this question has not been addressed in wild ungulates so far. In this study, bacteriological investigations and field observations were performed to assess whether free-ranging Alpine ibex can be healthy carriers of M. conjunctivae. Among 136 ibex without clinical signs of IKC, M. conjunctivae was identified 26 times (19.1%) by TaqMan PCR. To assess the potential pathogenicity of M. conjunctivae strains isolated from asymptomatic eyes, strains from three healthy ibex and from 15 IKC-ibex and IKC-chamois were analysed genetically by DNA sequence analysis of the variable part of the lppS gene. No significant differences were observed between strains from asymptomatic and clinically affected animals, reflecting the assumption that healthy ibex may act as carriers for M. conjunctivae strains that may be pathogenic for other individuals. Our results further indicate that development of IKC is associated with M. conjunctivae load in the eyes. In addition, a questionnaire survey revealed that IKC is generally less common in ibex than chamois and that infection in wild ungulates is not necessarily linked to the presence of sheep. These data support the hypothesis that apparently healthy ibex may be important in the epizootiology of IKC and indicate that host predilection may play a role in IKC development.     

Detection of Mycoplasma conjunctivae in sheep affected with conjunctivitis and infectious keratoconjunctivitis

AIM: To determine the aetiolog y of a recurring and severe form of infectious keratoconjunctivitis (IKC) in sheep.

METHODS: Five sheep flocks that had experienced a severe form of IKC were examined. Clinical history, conjunctival swabs and blood samples were collected from affected animals. Culture for bacteria, and also specifically for Mycoplasma and Chlamydophila spp, and detection of Mycoplasma conjunctivae DNA by polymerase chain reaction (PCR) were attempted. Serum samples were tested for antibodies to M. agalactiae, M. capricolum, M. conjunctivae and Chlamydophila spp.

RESULTS: Mycoplasma conjunctivae DNA was detected using PCR in 3/5 flocks, and in all flocks antibodies to M. conjunctivae were detected in sera. A pure growth of Branhamella ovis was cultured from conjunctival swabs from a small proportion of sheep in two flocks. No other pathogens were detected.

CONCLUSIONS: This investigation demonstrated that M. conjunctivae was a primary pathogen causing severe IKC in sheep, and is the first report of detection of this organism in sheep in New Zealand. Introduction of clinically normal carrier sheep appeared to have caused the outbreaks.     

Serological responses to two serovar‐independent ELISA antigens of Actinobacillus pleuropneumoniae in Australian commercial pig herds

Objective To compare serological responses in pig herds classified as low or high risk for disease caused by Actinobacillus pleuropneumoniae, using two ELISA tests based on serovar‐independent antigens. Procedure Cross‐sectional sampling was undertaken in 13 commercial herds, the clinical and slaughter histories of which indicated either freedom from (n = 5) or prior confirmed cases of A. pleuropneumoniae (n = 8). In nine herds, approximately 40 pigs each were sampled at 4, 8, 12, 16 and 20 weeks. Three of the remaining four herds were sampled between 6 and 30 weeks of age, and the last was sampled only prior to slaughter, at approximately 24 weeks. Sera were tested in ELISA based on two antigens common among A. pleuropneumoniae serovars: a 39‐kDa outer membrane protein and a recombinant ApxIVA‐N terminus protein. Results Sampling of 1 and 5 to 6‐month‐old pigs provided the most useful information on herd status. The 39‐kDa ELISA was sensitive in detecting infected herds, but had evidence of cross‐reactivity with high seroreactivity rates in older pigs in some low‐risk herds. The ApxIVA‐N ELISA was less seroreactive in high‐risk herds and had higher specificity in low‐risk herds. Conclusion ELISA based on the 39‐kDa subunit are of limited use, because of possible cross‐reactivity, but a high negative predictive value may be useful for risk assessment in suspect herds. Maternal antibody to ApxIVA‐N may be of value in detecting high‐risk herds, but 5% of 4‐week‐old pigs in low‐risk herds were also seropositive in this assay.     

Detection and identification of Mycoplasma conjunctivae in infectious keratoconjunctivitis by PCR based on the 16S rRNA gene.

A specific PCR assay based on unique sequences of the rrs genes (16S rRNA) of Mycoplasma conjunctivae was developed for direct detection and identification of this pathogen from clinical material. DNA from eye swabs was amplified after a simple lysis step by either a single PCR with the M. conjunctivae specific primer pair McoR1 and McoF1, or by a nested PCR with the Mycoplasma genus specific primer pair MOLIGEN1-L and 16UNI-R in the first step and McoR1 and McoF1 in the second step. The specificity of the primer pair McoR1 and McoF1 was verified with purified DNA from the type strain, from 17 field isolates of M. conjunctivae and from several Mollicutes which are phylogenetically related to M. conjunctivae or which can be isolated from the same host animals. This method identified mycoplasma isolates from goat, sheep, ibex and chamois originating from different countries as M. conjunctivae. No cross amplifications with other mycoplasmas which are related to M. conjunctivae were observed. Eye swab samples containing known numbers of M. conjunctivae cells were analysed after direct lysis of the material. The detection level was estimated to be 20 cells per swab when the nested PCR procedure was used and 2 x 10(5) by the single PCR method. In an experimental infection model of sheep, the nested PCR method for detection of M. conjunctivae gave results which were comparable to mycoplasmal culture. These are the implications for diagnostic purposes: M. conjunctivae isolates can be identified by the one-step PCR method, whereas for detection and identification of M. conjunctivae in clinical material the two-step method should be used (higher sensitivity).     

Use of a novel serum ELISA method and the tonsil-carrier state for evaluation of Mycoplasma hyosynoviae distributions in pig herds with or without clinical arthritis

A novel indirect ELISA test using deoxycholate extracted antigens coated onto a hydrophobic polystyrene surface for measurement of serum antibodies specific for Mycoplasma hyosynoviae was developed. Sensitivity and specificity of the test were found to be superior to previous ELISAs as tested on porcine serum following experimental Mycoplasma infections as well as by analysis of samples from one Danish herd known to be free of M. hyosynoviae and samples from two Norwegian herds without clinical suspicion of M. hyosynoviae infections since their establishment. The epidemiology of M. hyosynoviae infection was then investigated in Danish pig herds with evidence of clinical M. hyosynoviae arthritis (MhA herds, n = 4) and in herds with M. hyosynoviae-carriers among slaughter pigs, but with limited clinical lameness (MhC herds, n = 4). M. hyosynoviae bacteriaemia and tonsil-carrier state were determined by culture of cross-sectional samples of whole-blood (n = 238) and tonsil scrapings (n = 322), respectively. Levels of serum antibodies (n = 396) were measured by the novel indirect ELISA test. There was no significant difference in the ELISA results between the MhA and the MhC herds. Pigs that were tonsil-carriers had a significantly higher response in the ELISA test (P < 0.001) than non-carriers. Slaughter pigs showed higher ELISA values (P < 0.001) and they were more prone to be tonsil-carriers (P < 0.001). The most critical period for spread of the infection seems to be the nursery period (4-12 weeks). The results indicate that M. hyosynoviae infection progresses similarly in herds irrespective of the presence of clinical arthritis. Thus, clinical arthritis due to M. hyosynoviae is probably triggered by other host or herd factors than low levels of serum antibodies or by differences in virulence factors between M. hyosynoviae strains.     

Studies of the origin of false positive reactions in the serodiagnosis of contagious bovine pleuropneumonia

Four groups of cattle were experimentally immunised by four mycoplasma species of "mycoides-like" group, Mycoplasma (M) capricolum, M. mycoides subsp. mycoides (LC), M. mycoides subsp. capri and M. species group 7 of LEACH (PG50). They were then bled weekly during 2 months to establish antibodies kinetics against homologous and heterologous antigens. The standard method of complement fixation test (CFT) used in Europe and a new ELISA test for diagnosis of contagious bovine pleuropneumonia were performed in comparison with passive haemagglutination test (PHA) against antigens used for experimental immunisation. Cross reactions obtained are rather equal to the degree of similitude between these mycoplasma species. With CFT-cross reactions are transitory and occur only while homologous titers are very high, particularly with "PG50" and the two caprine mycoides strains. ELISA results using a threshold of positivity of optical density of 0.20, were similar to that obtained with CFT except ELISA specificity is not so different from CFT one. This experimental model could explain some natural situations.     

Experience with simple ELISA test systems for Brucella serology in cattle, sheep and goats

The objective of this work was to use the ELISA technique for the serological surveillance for freedom of brucellosis of cattle, sheep and goats. By comparing 28 cattle sera taken after a brucellosis outbreak, 15 bovine sera supplied by the Federal Institute for Health Protection of Consumers and Veterinary Medicine (BgVV) and 497 serum slow agglutination test (SSAT) and complement fixation test (CFT) negative bovine sera from herds officially declared free of brucellosis, the ELISA technique not only shows higher sensitivity as compared to SSAT and CFT but also distinguishes clearly between positive and negative reactions. The serological comparison by SSAT, CFT and ELISA of 615 cattle, 624 sheep and 630 goat sera from herds acknowledged as brucellosis free showed equivalent specificities for both CFT and ELISA. The specificity of the SSAT was much lower, 81.1% in cattle and 96.2% in goat sera. The examination of 5796 cattle, 1337 calf, 5031 sheep and 1796 goat sera demonstrates the advantage of the ELISA technique as routine method. The possible application of the ELISA technique as a screening method for serological brucellosis tests in sheep, goats and possibly also in pigs is discussed.     

Mycoplasma species and related organisms isolated from ruminants in Britain between 1990 and 2000

Between 1990 and 2000, more than 1600 mycoplasmas and the related acholeplasmas were identified from ruminant animals by the Mycoplasma Group at the Veterinary Laboratories Agency--Weybridge. Mycoplasma bovis was the most commonly identified pathogen, mostly from pneumonic calves but occasionally from cattle with mastitis and arthritis. Mycoplasma canis was first isolated in Britain in 1995 from pneumonic calves and the number of isolates increased to 18 per cent of the total mycoplasmas isolated from cattle in 1999. The ELISA for antibodies to M. bovis detected 1971 positive samples (22 per cent) among 8959 serum samples, mainly from pneumonic cattle. Other mycoplasmas identified included Mycoplasma dispar from the lungs of cattle with respiratory disease, and Mycoplasma bovigenitalium from the reproductive tract of cows with vulvovaginitis and infertility. Mycoplasma bovirhinis and Acholeplasma species were found commonly but are thought to be more opportunistic than pathogenic. In sheep and goats, the majority of Mycoplasma species isolated were identified as Mycoplasma ovipneumoniae from pneumonic sheep, Mycoplasma conjunctivae from sheep with keratoconjunctivitis, and the ubiquitous Mycoplasma arginini.     

Evaluation of serovar-independent ELISA antigens of Actinobacillus pleuropneumoniae in pigs, following experimental challenge with A. pleuropneumoniae, Mycoplasma hyopneumoniae and Pasteurella multocida

Objective To compare the sensitivity and specificity of six serological enzyme‐linked immunosorbent assays (ELISAs) based on serovar‐independent antigens of Actinobacillus pleuropneumoniae (App) and investigate cross‐reactivity in disease‐free pigs challenged with Mycoplasma hyopneumoniae and Pasteurella multocida. Design Five experimental pig trials using direct challenge with App serovars 1, 7 or 15 or direct challenge with M. hyopneumoniae and/or various dose rates of P. multocida. Procedure A 39‐kDa outer membrane protein antigen and five recombinant antigens from the apxIVA gene of App were evaluated. The latter were derived from the ApxIVA N‐terminus (ApxIVA‐N, ApxIVA‐NP, ApxIVA‐NPS) or C‐terminus (ApxIVA‐C, ApxIVA‐CP). Pigs were sampled after challenge and clinical and necropsy findings evaluated. Results The 39‐kDa ELISA had high sensitivity but lacked specificity, with significantly increased cross‐reactivity following P. multocida challenge. ELISAs based on ApxIVA N‐terminus antigens were significantly more sensitive than C‐terminus antigens for the detection of App‐induced disease. Although ApxIVA‐N and ApxIVA‐NP ELISAs had increased reactivity following P. multocida challenge, they retained high specificity for App‐induced disease (90–93%). Affinity purified ApxIVA‐NP antigen had marginally better specificity than ApxIVA‐N, without reduced sensitivity. Mycoplasma hyopneumoniae did not affect serological cross‐reactivity. In disease‐free pigs, the specificity of the ApxIVA‐NPS ELISA may be adversely affected by nasal carriage of apparently low‐virulence App strains. Conclusions ApxIVA‐N‐based ELISAs can be used for evaluating App status in commercial herds, but some appear limited by high carriage rates of low‐virulence App. The 39‐kDa antigen is only of merit in exclusion of App disease by negative serology.     

Use of an ELISA for differential diagnosis of Mycoplasma agalactiae and M mycoides subspecies mycoides (LC) in naturally infected goat herds.

Contagious agalactia is an ovine and caprine mycoplasmosis which manifests as mastitis, arthritis and keratoconjunctivitis. Mycoplasma agalactiae is recognised as a causal agent but M mycoides subspecies mycoides (LC), and M capricolum may also be responsible for this syndrome in goats. The clinical signs are not pathognomonic; diagnostic procedures are based on isolation of the organism from diseased animals or by detection of seroconversion. An ELISA specific for M agalactiae and M m mycoides (LC) is described. The specificity of the antigens was demonstrated by immunoblotting and by ELISA using monospecific hyperimmune rabbit sera. A correlation of ELISA activity with other serological tests and isolation of mycoplasmas was carried out in two goat herds under field conditions. Results indicate the ability to detect subclinical mycoplasma infection and individual carrier goats on the basis of ELISA, a finding which will assist control procedures.     

Bacteriological investigation of infectious keratoconjunctivitis in Norwegian sheep

Contagious keratoconjunctivitis is a rather common disease in Norwegian sheep. Since the knowledge of its aetiology is limited, the present study was performed to determine the microorganisms involved. Local veterinarians throughout the country collected conjunctival swabs from both sick (n = 43) and healthy (n = 42) sheep on 15 farms with outbreaks of ovine keratoconjunctivitis, and further from healthy sheep (n = 50) on 17 farms not showing any signs of conjunctival disease. All samples were cultivated for bacteria and mycoplasma. Listeria monocytogenes was isolated from 3 cases (1%) in one single herd. Staphylococcus aureus (5%), Corynebacterium spp. (2%) and Escherichia coli (4%) were isolated only in herds with keratoconjunctivitis, but from both sick and healthy animals. Moraxella (Branhamella) ovis was isolated from 28% of sampled animals in affected herds and from 10% of sampled animals in healthy herds. The corresponding numbers for Moraxella spp. were 9%/12%, for Pseudomonas spp. 7%/8%, for Staphylococcus spp. 22//22%, for Bacillus spp. 12%/14%, for Micrococcus spp. 6%/2% and for Streptococcus/Enterococcus spp. 2%/2%. Mycoplasma conjunctivae was isolated from 16 animals with keratoconjunctivitis (37%) and from 3 animals without clinical signs (7%) in farms with keratoconjunctivitis. In farms without clinical signs of keratoconjunctivitis, M. conjunctivae was isolated in 4 animals (8%). To our knowledge, this is the first time M. conjunctivae has been isolated in Norway. Other predisposing agents found were Moraxella (Branhamella) ovis and Listeria monocytogenes. The etiological importance of different microorganisms in ovine keratoconjunctivitis seems to vary; some are probably only present as secondary invaders. Other possible causes of ovine keratoconjunctivitis in Norway, such as Chlamydia psittaci, remain to be investigated.     

The indirect hemagglutination test for the detection of antibodies in cattle naturally infected mycoplasmas.

Stable mycoplasma antigens for the indirect hemagglutination test (IHA) were prepared employing glutaraldehyde treated sheep erythrocytes sensitized with Mycoplasma agalactiae subsp. bovis and Mycoplasma bovigenitalium antigens. Employing these antigens mycoplasma antibodies were detected in sera from cattle which had mastitic symptoms due to natural infection with either M. agalactiae subsp. bovis or M. bovigenitalium. A total of 200 cows from four herds were examined at varying intervals for the presence of M. agalactiae subsp. bovis and for the detection of antibody using growth inhibition and IHA tests. Mycoplasmas were isolated from 37 animals. Growth inhibiting antibody was detected from 56 of the 200 animals. In the IHA tests, antibody titer greater than or equal to 1:80 were detected in 148 animals, 76 of these having antibody titers greater than or equal to 1:160, while sera of 116 normal control animals had no growth inhibiting antibody and none had IHA antibody titers greater than 1:40. M. bovigenitalium was isolated from the milk of three of 26 animals in a fifth herd during an outbreak of mastitis. Growth inhibiting antibodies were demonstrated in the sera of ten of the 26 animals. However, the IHA test detected antibody titers of greater than or equal to 1:160 in 13 animals and of 1:80 in one of the 26 animals. To determine the specificity of the IHA tests, M. agalactiae subsp. bovis and M. bovigenitalium antigens were reacted with rabbit hyperimmune typing sera produced against 12 species of bovine mycoplasmatales. Homologous antisera showed IHA antibody titers of 1:1280 and 1:2560 against M. agalactiae subsp. bovis and M. bovigenitalium respectively, whereas heterologous antisera showed IHA antibody titers of less than or equal to 1:20. Also eight type-specific bovine antisera were reacted with M agalactiae subsp. bovis and M. bovigenitalium antigens in homologous and heterologous tests. Homoogous reactions showed IHA antibody titers greater than or equal to 1:320, whereas heterologous reactions showed IHA titers of less than or equal to 1:20. This IHA test promises to be useful for the detection of bovine mycoplasma antibodies in sera from cattle infected with M. agalactiae subsp. bovis or M. bovigenitalium. Thes test is sensitive, reproducible and specific and the technique is relatively simple and rapid. The antigens were stable for at least seven months.     

Ovine keratoconjunctivitis experimentally induced by instillation of Mycoplasma conjunctivae

Summary

Five sheep, free from Mycoplasma conjunctivae and ocular Chlamydia infection, were experimentally inoculated with M. conjunctivae and five more sheep were exposed to the infection by contact. Keratoconjunctivitis developed in all ten sheep. As in natural outbreaks of infectious keratoconjunctivitis (IKC), clinical signs were generally moderate and transient, and recurred in some sheep. M. conjunctivae was detected throughout the 53‐day observation period. Clinical diagnosis was confirmed by histopathologic examination of three sheep. Moraxella ovis, found in six of the ten sheep before the start of the experiment, appeared to play no etiologic role in the development of IKC.     

Use of immunoblotting to detect antibodies to Mycoplasma crocodyli infection in the sera of crocodiles (Crocodylus niloticus)

An immunoblotting protocol for the detection of antibodies to Mycoplasma crocodyli was developed using sonicated antigen of the reference strain 266/93. Immunoblotting detected nine reacting antigens, of which the 33 and 40kDa antigens were immunodominant. There was no difference in reactivity of the antigens against sera obtained from vaccinated and infected crocodiles. Both antigens are candidates for other serological and molecular studies. This is the first report to develop and apply an immunoblotting test for detection of antibody to M. crocodyli infection in crocodiles.     

Serological reactivity to Mycobacterium bovis protein antigens in cattle

The serological response to 12 purified Mycobacterium bovis antigens were examined in an ELISA assay. These antigens included the majority of M. bovis protein antigens described to date and in most cases they were very similar to the M. tuberculosis antigens of the same molecular mass.The purified antigens were tested against sera from M. bovis infected cattle, M. bovis culture-negative cattle from infected herds and animals infected with related microorganisms, mainly other mycobacterial species. All the antigens gave strong reactions with at least some sera from the M. bovis infected group and showed cross-reactivity with some of the sera from the other two groups. The antigen with the highest specificity reacted strongly with only 60% of the M. bovis infected sera. Antigens that reacted with most or all of the M. bovis infected sera also gave the highest cross-reactivity with sera from the other two groups. These results indicate that a serological test based on any one or a combination of these antigens, without removal of the cross-reacting epitopes, would be unsatisfactory.     

Evaluation of the specificity and sensitivity of two commercial enzyme-linked immunosorbent assay kits, the serum plate agglutination test, and the hemagglutination-inhibition test for antibodies formed in response to Mycoplasma gallisepticum

Two commercial enzyme-linked immunosorbent assay (ELISA) kits, seven serum plate agglutination (SPA) antigens, and the hemagglutination-inhibition (HI) test for antibodies to Mycoplasma gallisepticum (MG) were compared for sensitivity and specificity using known MG-positive and MG-negative sera from leghorn chickens. All SPA antigens proved to be highly sensitive when testing MG-positive sera. Laboratory-prepared SPA antigens yielded fewer positive reactions when testing MG-negative sera than commercial SPA antigens. Both MG ELISA kits showed high rates of positive reactions when testing sera from birds given commercial M. synoviae bacterin, fowl coryza (Haemophilus paragallinarum) bacterin, inactivated infectious bursal disease virus vaccine, and to a lesser extent fowl cholera (Pasteurella multocida) bacterin. Immunization with Frey's medium with 12% swine serum-in-oil or Staphylococcus aureus-in-oil resulted in sera which yielded numerous positive ELISA reactions. During the first 1 to 3 weeks, antibodies induced by experimental infection with MG were better detected by the SPA test than by the ELISAs and the HI test, thus confirming the SPA test's importance in Mycoplasma diagnostic serology. The HI test can serve to confirm positive SPA results.