Risk factors associated with Mycoplasma agalactiae infection of small ruminants in northern Jordan

Serological detection of Mycoplasma agalactiae was carried out in 104 small ruminants flocks consisting of 18 sheep, 27 goat and 59 flocks containing both sheep and goats in northern Jordan between 2002 and 2003. At least 5 serum samples per flock were tested using an indirect ELISA for antibodies to M. agalactiae. To increase the chances of detecting this mycoplasma, sick or older animals were sampled. A high seropositivity to M. agalactiae was found in small ruminants suggesting a major role for M. agalactiae in contagious agalactia in northern Jordan. There was no significant difference in the seroprevalence of M. agalactiae in sheep and goats at flock level (X(2)=0.14, d.f.=1, p=0.7). A total of 31 variables including production and health management practices were tested as risk factors for seropositive flocks and analyzed using logistic regression analysis. Increasing risk factors for M. agalactiae seropositive flocks were: using outsider rams, improper cleaning of the milking utensils and separating young from dam, with odds ratios of 5, 3, 4.2, respectively; having mastitis problems in the flock was negatively associated (p=0.04) with M. agalactiae seropositivity. Educating small ruminant farmers to avoid the use of outsider rams, ensuring adequate cleaning of milking utensils and separating the young from dams would enhance the health of small ruminants.     

Changes in peripheral blood leucocytes of sheep experimentally infected with Mycoplasma agalactiae

Contagious agalactia is a serious disease of small ruminants affecting mainly mammary glands, joints and eyes. In sheep, the main aetiological agent is Mycoplasma agalactiae (Ma) whose abilities to persist in the target organs are known. Since there is no information on the effect of acute and chronic Ma infection on circulating leucocytes, the present study was designed to monitor granulocytes, monocytes, T and B lymphocytes, by flow cytometry, in female lactating sheep nasally infected with Ma. A profound depletion of leucocytes was observed from day 5 to day 34 post infection (p.i.). In particular, while the granulocytes returned to baseline levels by day 12 p.i., the monocytes remained significantly low until day 20 p.i. The infection caused a prolonged depletion of peripheral T lymphocytes (both CD4⁺ and CD8⁺) while B lymphocytes remained unaltered throughout the study. Mycoplasma agalactiae was detected by real-time PCR in several anatomical sites (ear, nose and milk) from day 2–5 p.i. until the end of the study (i.e., day 50 p.i.) while a transient bacteraemia was observed from day 5 to day 12 p.i. The leucopenia observed following intranasal Ma infection is likely due to leucocyte infiltration within the target organs.     

Comparison of two diagnostic tests for canine atopy using monoclonal anti-IgE antibodies

In 66 dogs of various races consulting the veterinary clinic for skin problems suspicious of atopic dermatitis, parallel skin tests with two commercial brands of allergens and serological tests with the CMG IMMUNODOT system and with an ELISA assay using monoclonal anti-IgE antibodies were performed. The most frequent sensitivities found were towards house dust (44%) and storage mites (50%). Depending upon the cut-off point chosen, the CMG IMMUNODOT test was shown to have sensitivity, in respect to the skin test, varying from 54 to 100%, in an inverse relationship to specificity. The ELISA assay was found to be slightly less sensitive. These investigations also revealed some major discrepancies in skin test results among allergen extracts of different origins, confirming that skin test results should no longer be considered as the sole 'gold standard' in diagnosis of IgE-mediated allergy. The experience gained confirms that modern serological tests using monoclonal anti-IgE antibodies for detection of allergen-specific IgEs in the dog are useful in the diagnosis of dog allergy.     

Comparison of four serologic tests for the detection of antibodies to bovine leukemia virus

Four tests for detection of antibodies to bovine leukemia virus (BLV) were compared. The sera that were tested came from cattle in naturally infected commercial dairy herds, cattle that were infected under experimental conditions, and cattle in an isolated BLV-free herd. The tests that were compared included a radioimmunoprecipitation assay (RIA) with p24 antigen, a RIA with glycoprotein (gp) antigen, an agar-gel immunodiffusion (AGID) test with gp antigen, and a virus-neutralization (VN) test that was based on inhibition of BLV-induced syncytia in cell culture. Results of the 4 serologic tests agreed for 96.8% of the sera from cattle in commercial herds. The gp RIA detected the greatest number of positive sera (188); it was followed in turn by the p24 RIA (187), the VN test (183), and the AGID test (176). The gpd RIA titers of the 12 sera that gave negative AGID results were 175 or less. In RIA, the percentage of precipitation of labeled antigen by positive sera was almost always higher with gp antigen than with p24 antigen. Satisfactory sensitivity in the p24 RIA required the acceptance of a low level of antigen precipitation, 15%, as a positive test. In the gp RIA, however, almost all positive sera precipitated at least 50% of the labeled antigen. Nonspecific precipitation of antigen in the RIA by sera from BLV-free cattle ranged from 4% to 10%. Examination of sequential serum samples from 17 experimentally infected cattle showed that BLV antibody was first detected 2 to 8 weeks after inoculation. In 9 cattle, seroconversion was detected simultaneously by all of the tests. Results from the other 8 cattle indicated that seroconversion could be detected first by p24 RIA, followed by the gp RIA and the VN test. The longest interval between RIA seroconversion and AGID seroconversion was 10 days. Monthly tests of sera from 10 laboratory cattle that were infected by contact exposure showed that 7 animals seroconverted in all tests at the same time. Two cattle were positive first in RIA, but the next month they were also positive in the VN and AGID tests. One animal was positive in the RIA and the VN test for 2 months before antibody was detected by AGID.     

Assessment of PCR for routine identification of species of the Mycoplasma mycoides cluster in ruminants

DNA amplification techniques offer considerable promise for the identification of Mycoplasma mycoides cluster members. They avoid antigenic cross-reactivity and variability that hamper serological methods. Many sets of primers, specific of these different members and of Mycoplasma putrefaciens, have been proposed. To assess the reliability of some of these PCR tests in routine laboratory diagnostic use, 230 field strains supposed to belong to this group were simultaneously identified by PCR and an antigenic method. The results were well correlated to antigenic identification for M. putrefaciens, but PCR failed to identify respectively 74% and 52% of M. mycoides subsp. mycoides Large Colony type and M. capricolum subsp. capricolum strains. Any identification of M. mycoides subsp. mycoides Small Colony type must be confirmed by two different tests. Difficulties in defining the M. species bovine serogroup 7 were also encountered with both the PCR and immunological methods. The occurrence of putative variable antigen(s) on the mycoplasma surface may explain part of the identification difficulties encountered with the immunological methods.     

Evaluation of PCR systems for the identification and differentiation of Mycoplasma agalactiae and Mycoplasma bovis: a collaborative trial

Diagnostic differentiation between the ruminant pathogens Mycoplasma agalactiae and Mycoplasma bovis is known to be problematic when only conventional serological and biochemical tests are used. The main reason for this is that both agents share a considerable number of related proteins and common epitopes. DNA-based detection methods offer advantages in terms of specificity and sensitivity. However, there is an urgent need to compare currently used PCR assays because they target different genomic regions and, therefore, may perform differently. In the present work, five laboratories, which use PCR routinely, evaluated the specificity of four different PCR systems for M. agalactiae and three systems for M. bovis on a total of 41 strains of the two Mycoplasma species including six previously unidentified strains. As the vast majority of PCR examinations (97.1% of all tests) correctly identified the strains the specificity of all seven detection systems appears to be high. In four cases, incorrect identification by conventional diagnostic methods was rectified by PCR. Isolates from non-typical hosts, i.e. three M. bovis strains from small ruminants and two M. agalactiae strains from cattle, were characterised by sequencing the 16S and part of the 23S ribosomal RNA genes.     

Anatomic location of Mycoplasma mycoides subsp. capri and Mycoplasma agalactiae in naturally infected goat male auricular carriers

This study sought to determine whether male goat auricular carriers of mycoplasmas known to cause contagious agalactia could harbour these microorganisms at anatomical sites other than the ears. A microbiological study was conducted in 6 naturally infected bucks that had been diagnosed as chronic auricular asymptomatic carriers of Mycoplasma (M.) mycoides subsp. capri (Mmc) more than one year previously. To detect mycoplasmas, cultures and PCR were performed on 46 samples taken from each goat from the cardio-respiratory, digestive, nervous, lymph and genitourinary systems and several joints. Of a total of 274 samples analyzed, 28 were positive for mycoplasmas (10.1%): Mmc was detected in 17 (6.1%), Mycoplasma (M.) agalactiae in 12 (4.3%) and both microorganisms were identified in one of the samples. In all 6 goats, mixed infection was observed despite none being auricular carriers of M. agalactiae. Mycoplasma spp. were identified at 15 different sites; the most frequent sites being the joints (31.2%, 5 positive samples), lymph nodes (25%, 4 positive samples) and respiratory tract (25%, 4 positive samples). Positive results were also obtained in three brain tissue (18.7%), two cardiac tissue (12.5%) and one ileum, urethra, testicle and bulbourethral gland (6.25%) samples. The histopathological findings may suggest the presence of mild chronic conditions in some of the organs where the bacteria were found. Our findings reveal for the first time the capacity of Mmc and M. agalactiae to colonize several other organ systems in chronically naturally infected auricular carriers, possibly representing an added risk factor for the spread of these microorganisms. In the case of M. agalactiae, colonization seemed to be independent of the animal's auricular carrier state.     

Recovery of Mycoplasma agalactiae from the ears of goats experimentally infected by the intramammary route

The role of inapparent carriers of Mycoplasma agalactiae and the strategies used to colonise the external ear canal in goats remain unclear. This study examined the ability of M. agalactiae to colonise the ears of goats infected experimentally by the intramammary route. The right mammary glands of 15 lactating goats were inoculated with 10(10) colony forming units (cfu) of M. agalactiae. The goats were randomly assigned to three groups of five animals each and sampled at slaughter at 5, 15 or 45 days post-infection (dpi). A further four goats served as uninfected controls. Right and left ear swabs were collected for detection of M. agalactiae by culture before and after sacrifice. M. agalactiae was detected in 19/20 (95%) ear swabs from goats sampled at 15 and 45dpi, whereas all ear swabs collected before inoculation, ear swabs collected from the group sampled at 5dpi and ear swabs from control goats at the time of sacrifice were negative for M. agalactiae. Blood samples collected at 6, 12, 24, 48 and 72h post-infection for detection of M. agalactiae by culture were also negative. There were differences in the antigenic profiles of isolates recovered from the ears compared to the 7MAG strain used to inoculate the animals and most isolates from the mammary gland, milk and supramammary lymph nodes.     

Comparison and evaluation of four serological tests for detection of antibodies to Aujeszky's disease virus

Four methods used for the detection of antibodies to Aujeszky's disease virus in porcine sera were compared. The serum neutralisation test, countercurrent immunoelectrophoresis test, micro-immunodiffusion test and the enzyme-linked immunosorbent assay (Elisa) were assessed with particular regard to their use in large scale screening of porcine serum samples. The Elisa test was found to be the most suitable on the grounds of sensitivity, speed and cost.     

Evaluation of two herd-level diagnostic tests for Streptococcus agalactiae using a latent class approach

Streptococcus agalactiae mastitis persists as a significant economic problem for the dairy industry in many countries. In Denmark, the annual surveillance programme for this mastitis pathogen initially based only on bacteriological culture of bulk tank milk (BTM) samples, has recently incorporated the use of the real-time PathoProof Mastitis PCR assay with the goal of improving detection of infected herds. The objective of our study was to estimate the herd sensitivity (Se) and specificity (Sp) of both tests of BTM samples using latent class models in a Bayesian analysis while evaluating the effect of herd-level covariates on the Se and Sp of the tests. BTM samples were collected from all 4258 Danish dairy herds in 2009 and screened for the presence of S. agalactiae using both tests. The highest Se of PCR was realized at a cycle threshold (Ct) cut-off value of 40. At this cut-off, the Se of the PCR was significantly higher (95.2; 95% posterior credibility interval [PCI] [88.2; 99.8]) than that of bacteriological culture (68.0; 95% PCI [55.1; 90.0]). However, culture had higher Sp (99.7; 95% PCI [99.3; 100.0]) compared to PCR (98.8; 95% PCI [97.2; 99.9]). The accuracy of the tests was unaffected by the herd-level covariates. We propose that screenings of BTM samples for S. agalactiae be based on the PCR assay with Ct readings of <40 considered as positive. However, for higher Ct values, confirmation of PCR test positive herds by bacteriological culture is advisable especially when the between-herd prevalence of S. agalactiae is low.     

Testing of a hyperimmune serum in calves infected intranasally with Mycoplasma bovis

Mycoplasma (M.) bovis hyperimmune serum was subcutaneously injected to 16 of 26 calves repeatedly intranasally infected with M. bovis, during and/or after experimental infection. Antibody titres between 1:32 and 1:256 were recorded by means of indirect haemagglutination from the calves treated. Transmission of film-inhibitory antibodies failed to work. Neither clinical manifestations nor pathologico-anatomic alterations to the lungs of experimentally infected animals were mitigated by hyperimmune serum treatment. M. bovis, in high germ counts, was re-isolated from nasal swabs, trachea, pulmonary lymph nodes, and inflammatory lung tissue of both treated and untreated calves.     

Risk factors associated with Mycoplasma capricolum subspecies capripneumoniae and morbillivirus infection in small ruminants in Tanzania

Tropical Animal Health and Production - Mortality of domestic small ruminants caused by contagious caprine pleuropneumonia (CCPP) and Peste des petits ruminants (PPR) is frequently reported in...     

Immunohistochemical detection of Mycoplasma agalactiae in formalin-fixed,paraffin-embedded tissues from naturally and experimentally infected goats

Samples from the mammary tissue of 14 lactating goats (12 naturally infected and two experimentally infected) were examined for the presence of Mycoplasma agalactiae. A monoclonal antibody (5G12) was applied to formalin-fixed, paraffin-wax-embedded sections and labelled by the avidin-biotin peroxidase complex (ABC) method. Histological examination of tissue sections revealed strong immunoreactivity in all animals included in the study. Mycoplasma agalactiae antigen was mainly detected in the cellular debris at the periphery of purulent exudates present within lactiferous sinuses, and lactiferous and interlobular ducts. In addition, M. agalactiae organisms appeared in the cytoplasm of the epithelium of ducts, and in infiltrating macrophages and neutrophils within the ducts, alveoli, interstitial tissue and regional lymph node sinuses. It is concluded that this monoclonal antibody-based immunohistochemical technique is an efficient and specific method for the post-mortem detection of M. agalactiae in cases of clinical mastitis as well as being a useful tool for the study of the route of infection and cellular types involved during mastitis caused by this organism.     

A coagglutination assay with monoclonal antibodies for rapid laboratory identification of Mycoplasma synoviae.

An agglutinating monoclonal antibody (MAb S2) specific for a 55,000-molecular-weight surface protein of Mycoplasma synoviae was developed by fusion of spleen cells from immunized BALB/c mice with P3X63 Ag8.653 myeloma cells. Immunogold labeling experiments confirmed the cell surface location of the MAb-recognized antigen. MAb S2-coated Staphylococcus aureus was used in a rapid slide coagglutination assay. Eleven M. synoviae strains, including the type strain WVU 1853, coagglutinated with MAb-coated S. aureus, but five M. gallisepticum strains (PG31, S6, R, F, and A5969) did not.     

Comparison of the enzyme-linked immunosorbent assay and the indirect hemagglutination and complement fixation tests for detecting antibodies to Mycoplasma hyopneumoniae.

Caesarean-derived, colostrum-deprived swine were exposed to a broth culture of a low passage field isolate of Mycoplasma hyopneumoniae by intranasal inoculation. The intranasal-inoculated swine subsequently were commingled with their litter-mates to effect transmission via contact-exposure. Sera were collected from the swine at two to four week intervals for approximately one year postexposure and evaluated by the enzyme-linked immunosorbent assay (ELISA), indirect hemagglutination and complement fixation tests. The intranasal-exposed swine seroconverted earlier, developed higher titers and remained indirect hemagglutination and complement fixation positive longer than the contact-exposed swine. It was concluded that the antibody response of intranasal-exposed swine was artificially high and that sera from such swine were not suitable for evaluating the sensitivity of mycoplasmal pneumonia of swine serodiagnostic tests. The indirect hemagglutination test was relatively insensitive and technically cumbersome and the least promising as a practical field test. The complement fixation test appeared to be slightly more sensitive in detecting early antibody production (especially in contact-exposed swine) but it was the least sensitive in detecting late antibodies. The ELISA was generally the most sensitive procedure. Individual high ELISA titers were from ten to 32 times greater than maximum complement fixation and indirect hemagglutination titers. The most striking difference among the three tests was the persistence of high ELISA titers late in the study. All swine were ELISA positive at necropsy approximately one year postexposure despite the fact that lungs were devoid of lesions and culturally and immunofluorescent negative for M. hyopneumoniae.