Development of Immunochromatographic Assay for the Rapid Detection of Mycoplasma capricolum subsp. capripneumoniae Antibodies

Mycoplasma capricolum subsp. capripneumoniae (Mccp) is the cause of contagious caprine pleuropneumonia (CCPP), which is a highly significant respiratory disease in goats leading to significant economic losses in Africa and Asia. Currently available procedures for the diagnosis of CCPP have some limitations in sensitivity, specificity, operation time, requirement of sophisticated equipment or skilled personnel, and cost. In this study, we developed a rapid, sensitive, and specific colloidal gold-based immunochromatographic assay (GICA) strip for the efficient on-site detection of antibodies against Mccp in the serum within 10 min. For the preparation of this colloidal GICA strip, recombinant P20 protein, the membrane protein of Mccp, was expressed by Escherichia coli prokaryotic expression system after purification was used as the binding antigen in the test. The rabbit anti-goat immunoglobulin G labeled with the colloidal gold was used as the detection probe, whereas the goat anti-rabbit immunoglobulin G was coated on the nitrocellulose membrane as the control line. The concentration of the coating antibody was optimized, and the effectiveness of this colloidal GICA strip was evaluated. Our results proved that the detection limit of the test strip was up to 1:64 dilutions for the Mccp antibody-positive serum samples with no cross-reactivity with other pathogens commonly infecting small ruminants,including goat pox virus, peste des petits ruminants virus, foot-and-mouth disease virus type A, or other mycoplasmas. Moreover, the colloidal GICA strip was more sensitive and specific than the indirect hemagglutination assay for the detection of Mccp antibodies. The 106 clinical serum samples were detected by the colloidal GICA strip compared with the complement fixation test, demonstrating an 87.74% concordance with the complement fixation test. This novel colloidal GICA strip would be an effective tool for the cost-effective and rapid diagnosis of CCPP in the field.

​Caprine Respiratory Mycoplasmosis (Contagious Caprine Pleuropneumonia CCPP)- A Global Perspective of the Disease, Epidemiology, Diagnosis, Chemotherapy and Immunization: A Review

Mycoplasma infection of the respiratory tract of goats is prevalent worldwide including the South Asian sub-continent. Owing to intensive and large scale goat farming, the incidence of the disease is on an increase. Among various species of mycoplasma, Mycoplasma capricolum subspecies Capri pneumoniae is increasingly incriminated in Contagious Caprine Pleurapneumonia (CCPP) in goat populations with considerable economic fallout in the form of high morbidity and mortality. The disease manifestations in caprines are recorded as anorexia, high febrile reaction and respiratory embarrassment in the shape of clinical dyspnoea, polypnea, paroxysmal cough and sero-purulent nasal discharges. The disease is thus contracted by the healthy animals through aerosol, contaminated feed and water sources in the herd premises, without a protective immunity and that the conferred immunity in recovered cases being short-lived. The true lesions of CCPP are confined to the lung alveolar tissues of infected goats, which distinguish it from other respiratory diseases of small ruminants caused by the members of the Mycoplasma mycoides cluster. Atypical pneumonia caused by the mycoplasma infection of goats, also known as Contagious Caprine Pleuropneumonia (CCPP) has been more often reported from Africa and Asia than Europe. Classical, acute CCPP attributed to Mycoplasma capricolum subsp. Capri pneumoniae, originally known asthe F38 biotype (World Organisation for Animal Health, 2008) causes heavy kid mortality. Two other organisms in this group, M. mycoides subsp. capri and M. mycoides subsp. mycoides large-colony type, can cause disease in small ruminants that clinico-pathologically mimics CCPP but may have extra pulmonary signs and lesions, sometimes. Mycoplasma Capri pneumoniae and other members of the M. mycoides cluster cross-react in serological tests and share biochemical and genetic similarities. The most favourable epidemiological scenario in the Sub-continent is the hot humid climate during monsoons. The diversity and multi-etiological subspecies involved in the disease is detrimental in the development of an effective vaccine even though in some places a liquid vaccine is presently in use. At other places, anti-mycoplasmal antibiotics of aminoglycoside and fluoroquinolone and perhaps the macrolide groups remain to be the main option in preventing flock mortalities.

Rapid detection of Mycoplasma mycoides subsp. capri and Mycoplasma capricolum subsp. capripneumoniae using high-resolution melting curve analysis

Mycoplasma capricolumsubsp.subsp. capripneumonia (Mccp) and Mycoplasma mycoidessubsp.sbusp. capri (Mmc) cause caprine pleuropneumonia (CCPP) and mycoplasmal pneumonia in goats and sheep (MPGS), respectively. These diseases cannot be identified on clinical symptoms alone and it is laborious to distinguish them using biochemical methods. It is therefore important to establish a simple, rapid identification method for Mccp and Mmc. Here, we report a high-resolution melting (HRM) curve analysis using specific primers based on the Mmc 95010 strain MLC_0560 and Mccp F38 strain MCCPF38_00984 gene sequences. The method was highly specific with intra- and inter-batch coefficients of variation < 1%. The lower limit of detection for Mccp and Mmc was 55 copies/μL and 58 copies/μL, respectively. HRM and fluorescence qPCR results were compared using 106 nasal swabs and 47 lung tissue samples from goats (HRM-qPCR coincidence rate 94.8%; 145/153). Mycoplasma isolation and identification was performed on 30 lung tissue samples and 16 nasal swabs (HRM-culturing coincidence rate 87.0%; 40/46). HRM analysis was more sensitive than fluorescence qPCR and Mycoplasma isolation, indicating the practicality of HRM for accurate and rapid identification of Mccp and Mmc, and diagnosis and epidemiology of CCPP and MPGS.

Mycoplasma agalactiae: The Sole Cause of Classical Contagious Agalactia?

Contagious agalactia (CA) is suspected when small ruminants show all or several of the following clinical signs: mastitis, arthritis, keratoconjunctivitis and occasionally abortion. It is confirmed following mycoplasma isolation or detection. The historical and major cause is Mycoplasma agalactiae which was first isolated from sheep in 1923. Over the last thirty years, three other mycoplasmas (Mycoplasma mycoides subsp. capri, Mycoplasma capricolum subsp. capricolum and Mycoplasma putrefaciens) have been added to the etiology of CA because they can occasionally cause clinically similar outcomes though nearly always in goats. However, only M. agalactiae is subject to animal disease regulations nationally and internationally. Consequently, it makes little sense to list mycoplasmas other than M. agalactiae as causes of the OIE-listed CA when they are not officially reported by the veterinary authorities and unlikely to be so in the future. Indeed, encouraging countries just to report M. agalactiae may bring about a better understanding of the importance of CA. In conclusion, we recommend that CA should only be diagnosed and confirmed when M. agalactiae is detected either by isolation or molecular methods, and that the other three mycoplasmas be removed from the OIE Manual of Diagnostic Tests and Vaccines in Terrestrial Animals and associated sources.

Seroprevalence and risk factors of Mycoplasma capricolum subsp. capricolum among small ruminants in northern Jordan

During 2002-2003 the seroprevalence of Mycoplasma capricolum subsp. capricolum was studied among 104 small ruminant’s flocks (18 sheep, 27 goat and 59 mixed flocks) in northern Jordan. At least 5 serum samples/flock were tested using iELISA test. The true flock-level seroprevalences of Mycoplasma capricolum subsp. capricolum were 56%, 39%, 28% in small ruminant (sheep and goats), sheep and goats respectively. There was no significant difference (X2 = 2, d.f. =1, p = 0.15) between seroprevalences in sheep and goats at the flock level. A total of 29 variables including production and health management practices were tested as risk factors for seropositive flocks and analyzed using logistic regression analysis. The use of communal grazing was found to be a risk factor for Mycoplasma capricolum subsp. capricolum seropositivity with odds ratio of 5.2 and drinking the animals with spring water was a protective factor with odds ratio of 0.27. More than half of the examined flocks were seropositive to Mycoplasma capricolum subsp. capricolum indicating a role for Mycoplasma capricolum subsp. capricolum in contagious agalactiae in small ruminants. The education of farmers regarding the use of communal grazing and housing the newborn in separate barns is expected to help reducing the Mycoplasma capricolum subsp. capricolum infections in their flocks.

Structure of a tRNA-specific deaminase with compromised deamination activity

Nucleotide 34 in tRNA is extensively modified to ensure translational fidelity and efficacy in cells. The deamination of adenosine at this site catalyzed by the enzyme TadA gives rise to inosine (I), which serves as a typical example of the wobble hypothesis due to its diverse basepairing capability. However, recent studies have shown that tRNAArgACG in Mycoplasma capricolum contains unmodified adenosine, in order to decode the CGG codon. The structural basis behind the poorly performing enzyme M. capricolum TadA (McTadA) is largely unclear. Here we present the structures of the WT and a mutant form of McTadA determined at high resolutions. Through structural comparison between McTadA and other active TadA enzymes as well as modeling efforts, we found that McTadA presents multiple structural conflicts with RNA substrates and thus offered support to previous studies from a structural perspective. These clashes would potentially lead to reduced substrate binding affinity of McTadA, consistent with our in vitro deamination activity and binding assays. To rescue the deamination activity of McTadA, we carried out two rounds of protein engineering through structure-guided design. The unsuccessful attempts of the activity restoration could be attributed to the altered dimer interface and stereo hindrance from the non-catalytic subunit of McTadA, which could be the inevitable outcome of the natural evolution. Our study provides structural insight into an alternative decoding and evolutionary strategy by a compromised TadA enzyme at a molecular level.