Impact of various preservation and storage methods on the viability of mycoplasma field strains isolated in Mali

The survival of five mycoplasma strains was studied in different storage media (Mycoplasma complet media without cryopreservative agent, Mycoplasma complete media with addition of horse serum, Mycoplasma complete media with addition of glycerol and lyophilized cultures without stabilizer) under different temperatures (+37°C, +4°C, -20°C, -85°C) during 24 months. Five Mycoplasma strains, Mycoplasma mycoides subsp mycoides (Mmm), Mycoplasma bovis (Mb), Mycoplasma agalactiae (Ma), Mycoplasma gallisepticum (Mg) and Mycoplasma synoviae (Ms) were isolated from various parts of the country. The initial titers of the strains determined by the agar plate count before storage were 42.4x10 7 UFC/ml (8.6 log UFC/ml) for Mmm strain; 32.4x10 8 UFC/ml (9.51 log UFC/ml) for M.bovis strain; 12.4x10 9 UFC/ml (10.09 log UFC/ml) for Ma strain; 2.4x10 9 UFC/ml (9.38 log UFC/ml) for Mg and 2.8x10 9 UFC/ml (9.45 log UFC/ml) for Ms strain. After 3 weeks of storage, no viable mycoplasmas were detected in all the conservation media at +37°C and after 3 months of storage at +4°C except for the lyophilized cultures in which an average viability rate of 17.81% was observed. Overall, the mycoplasma strains remained viable at freezing temperatures after 24 months regardless of the storage medium, but with decreasing titers, which was noticeable with mycoplasma complete media, and mycoplasma media with horse serum. Conversely, at -20°C the average viability rates after 24 months of storage were 84.36% (with glycerol) and 90.04% (lyophilized cultures). At -85°C after 24 months of storage, this was 87.98% (with glycerol) and 91.44% (lyophilized cultures). These findings suggest that, in the absence of the lysophylisation process, the addition of glycerol may be recommended for long-term storage of frozen mycoplasma isolates.

Serological survey and isolation of Mycoplasma mycoides subspecies mycoides from cattle slaughtered at Katsina state central abattoir, Nigeria

serological survey for the detection of antibodies to Mycoplasma mycoides subspecies mycoides (Mmm) from cattle slaughtered at Katsina Central Abattoir was conducted from March to October 2012 using competitive ELISA (CIRAD) version: P05410/04 antibody detection kit. A total of 300 cattle sera were screened for Mmm antibodies out of which 22 (7.3%) obtained from 15 (68.2%) cows and 7 (31.8%) bulls were positive. Age distribution indicated that 3 (13.6%) were less than 2 years, 11 (50%) were between 2 and 4 years of age, while 8 (36.4%) were above 4 years. The study confirmed the susceptibility of some cattle breeds from all ages and sexes to Mmm. However, middle-aged cows were shown to be more associated with the infections though not statistically significant (P˃0.01). Similarly, a total of 130 pneumonic lung tissues were used for isolation and biochemical characterization of Mmm out of which 2 (1.5%) samples one each from adult White Fulani and Red Bororo cows were found to be positive. The study established baseline information on the status of Mmm infection in Katsina State. Continuous disease surveillance and annual mass vaccination programme using effective vaccines were recommended.

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 of contagious bovine pleuropneumonia in three selected south-eastern states of Nigeria

Contagious bovine pleuropneumonia is a trans-boundary animal disease caused by Mycoplasma mycoides subsp. mycoides. This study was designed to determine the seroprevalence of contagious bovine pleuropneumonia (CBPP) in three selected south eastern states (Anambra, Enugu and Imo) of Nigeria using competitive enzyme-linked immunosorbent assay (c-ELISA). A total of 438 bovine sera samples were collected randomly for four months (December 2019 to March 2020) and screened for antibodies to Mycoplasma mycoides subsp. mycoides (Mmm) using IDEXX CBPP antibody ELISA kit (CIRAD, France). Results showed an overall prevalence of 59.4% for the three states screened. Antibodies to Mmm were detected in all the three states. Enugu state had the highest prevalence (64.3%) followed by Imo state (63%) and Anambra state (50.7%). Female animals had higher prevalence of CBPP than male. However, it was not statistically significant (P> 0.05). This study confirms the presence of CBPP in south eastern Nigeria, and could be used as a base line data for future studies in this region. It is recommended that active surveillance and vaccination protocol should be undertaken in the region for the control and prevention of this disease. Keywords: c-ELISA, Contagious bovine pleuropneumonia, Mycoplasma , Nigeria, Seroprevalence

Editorial. Biología sintética y SARS-Cov-2

La biología sintética, desde hace ya varios años —tal vez una década—, ha generado la idea de la creación de la vida desde cero. Los investigadores han diseñado experimentos para sintetizar proteínas en un papel de filtro. Se trasplantó el genoma bacteriano entre diferentes especies (Mycoplasma mycoides a Mycoplasma caplicolium), se creó un genoma sintético y se puso a funcionar en una bacteria (Mycoplasma laboratorium), cuyo material genético había sido previamente removido. Además, actualmente se están desarrollando microorganismos con un genoma mínimo, a los que podrían añadirse genes y de esta manera realizar funciones específicas, tales como la biodegradación de agentes tóxicos del ambiente, la reducción de CO2 en cantidades que permitan minimizar el calentamiento global y la producción de bioetanol o biodiésel (Hernández-Fernández, 2012).

Recent Developments in Vaccines for Bovine Mycoplasmoses Caused by Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides

Two of the most important diseases of cattle are caused by mycoplasmas. Mycoplasma bovis is a world-wide bovine pathogen that can cause pneumonia, mastitis and arthritis. It has now spread to most, if not all, cattle-rearing countries. Due to its increasing resistance to antimicrobial therapy, vaccination is the principal focus of the control of infection, but effective vaccines are currently lacking. Despite being eradicated from most parts of the world, Mycoplasma mycoides subsp. mycoides, the cause of contagious bovine pleuropneumonia (CBPP), continues to plague sub-Saharan Africa, affecting at least 25 countries. Numerous new experimental vaccines have been developed over the last 20 years to improve on protection afforded by the T1/44, a live vaccine in continuous use in Africa for over 60 years, but none so far have succeeded; indeed, many have exacerbated the disease. Tools for diagnosis and control are adequate for eradication but what is necessary are resources to improve vaccine coverage to levels last seen in the 1970s, when CBPP was restricted to a few countries in Africa. This paper summarizes the results of the main studies in the field of experimental mycoplasma vaccines, reviews data on commercially available bacterin vaccines and addresses issues relating to the search for new candidates for effective vaccines to reduce economic losses in the cattle industry caused by these two mycoplasmas.