Mycoplasma agalactiae ST35: a new sequence type with a minimal accessory genome primarily affecting goats

Background Mycoplasma agalactiae, causing agent of contagious agalactia, infects domestic small ruminants such as sheep and goats but also wild Caprinae. M. agalactiae is highly contagious and transmitted through oral, respiratory, and mammary routes spreading rapidly in an infected herd. Results In an outbreak of contagious agalactia in a mixed herd of sheep and goats, 80% of the goats were affected displaying swollen udders and loss of milk production but no other symptom such as kerato-conjunctivitis, arthritis or pulmonary distress commonly associated to contagious agalactia. Surprisingly, none of the sheep grazing on a common pasture and belonging to the same farm as the goats were affected. Whole genome sequencing and analysis of M. agalactiae strain GrTh01 isolated from the outbreak, revealed a previously unknown sequence type, ST35, and a particularly small, genome size of 841′635 bp when compared to others available in public databases. Overall, GrTh01 displayed a reduced accessory genome, with repertoires of gene families encoding variable surface proteins involved in host-adhesion and variable antigenicity being scaled down. GrTh01 was also deprived of Integrative Conjugative Element or prophage, and had a single IS element, suggesting that GrTh01 has a limited capacity to adapt and evolve. Conclusions The lack of most of the variable antigens and the Integrative Conjugative Element, both major virulence- and host specificity factors of a M. agalactiae strain isolated from an outbreak affecting particularly goats, indicates the implication of these factors in host specificity. Whole genome sequencing and full assembly of bacterial pathogens provides a most valuable tool for epidemiological and virulence studies of M. agalactiae without experimental infections.

Vaccines for Mycoplasma Diseases of Small Ruminants: A Neglected Area of Research

Mycoplasmas cause some of the most economically important diseases of sheep and goats, including diseases listed by the World Organisation for Animal Health (OIE) such as contagious caprine pleuropneumonia (CCPP) and contagious agalactia (CA). Other important mycoplasma diseases include chronic respiratory and arthritic syndrome (CRAS) and atypical pneumonia, both present on all continents where small ruminants are farmed. Unfortunately, owing to a lack of investment, most commercial vaccines for these diseases are of poor quality, being mostly composed of killed bacteriocins of dubious or unknown efficacy. Several Mediterranean laboratories produce autogenous vaccines, but these can only be used on farms where outbreaks have been officially declared, and consequently have limited impact on disease nationally. Effective live vaccines are available, but their use is often restricted because of safety concerns. With the necessary safeguards in place, we argue for their greater use. This review examines reported vaccines for mycoplasma diseases of small ruminants and attempts to identify new candidate antigens that may enable the development of improved products. Vaccines for CCPP are covered elsewhere.

An Investigation of Contagious Agalactia Disease of Sheep and Goats in Isparta and Afyonkarahisar in Turkey

Background: Contagious agalactia causes significant economic losses. The aim of this study is to investigate the presence of contagious agalactia disease in cities of Isparta and Afyonkarahisar, Turkey. Methods: The study includes 45.500 animals in 220 ovine enterprises and samples were taken from those suspected of contagious agalactia disease. 202 animals in the 21 ovine enterprises comprised of 139 goats, 56 sheep, 3 kid goats, 2 goats, 2 lambs in total suspected of the disease were sampled. A total of 289 samples were collected, including 91 milk samples, 28 nasal swabs, 101 eye swabs, 8 joint fluids and 61 ear swabs. The isolates obtained after incubation were identified with polymerase chain reaction by using specific primers to assess film and spot formation, glucose fermentation, growth inhibition tests. Result: Three Mycoplasma spp. isolates obtained from 28 nasal swabs turned out to be negative for M. agalactiae after PCR analysis. Colony morphology, biochemical tests and growth inhibition tests revealed that one agent was M. arginine and the two factors were identified as M. ovipneumoniae with centerless colony morphology. The obtained results were confirmed with the polymerase chain reaction. None of the four factors causing contagious agalactia were isolated and identified.

Efflux Might Participate in Decreased Susceptibility to Oxytetracycline in Contagious Agalactia-Causative Mycoplasma spp.

Contagious agalactia is associated with mastitis, keratoconjunctivitis, arthritis, pneumonia, and septicemia in small ruminants in countries with large dairy industries worldwide. The causative agents belong to four (sub)species of the Mycoplasma genus that have remained essentially susceptible to antimicrobials, including to the widely-used tetracycline family. However, some clinical isolates have been detected that show increased minimum inhibitory concentrations of tetracyclines, although they do not harbor the mutation in the 16SrRNA gene usually associated with resistance. The present work aimed to assess whether efflux pumps, infrequently described in mycoplasmas, could participate in the observed moderate loss of susceptibility. General efflux mechanisms were measured (i) using the fluorescence property of ethidium bromide when accumulated intracellularly and intercalated in the mycoplasma genomes, its active extrusion resulting in a temperature-dependent decrease in fluorescence and (ii) monitoring the growth inhibition of mycoplasmas by subinhibitory concentrations of tetracycline with or without reserpine, a known inhibitor of efflux in other bacteria. Both methods revealed non-specific efflux phenomena in most of the isolates tested, although their efficacy was difficult to quantify. This property could contribute to the acquisition of mutations conferring resistance by maintaining intracellular concentrations of tetracyclines at subinhibitory levels.

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.

PK/PD Analysis of Marbofloxacin by Monte Carlo Simulation against Mycoplasma agalactiae in Plasma and Milk of Lactating Goats after IV, SC and SC-Long Acting Formulations Administration

Contagious agalactia is a mycoplasmosis affecting small ruminants that have become an important issue in many countries. However, PK/PD studies of antibiotics to treat this problem in lactating goats affected by Mycoplasma (M.) agalactiae, the main CA-causing mycoplasma are almost non-existent. The aims of this study were to evaluate the plasma and milk disposition of marbofloxacin in lactating goats after intravenous (IV), subcutaneous (SC) and subcutaneous poloxamer P407 formulations with and without carboxy-methylcellulose (SC-P407-CMC and SC-P407) administration. Marbofloxacin concentrations were analysed by the High Performance Liquid Chromatography (HPLC) method. Minimum inhibitory concentrations (MIC) of M. agalactiae field isolates from mastitic goat’s milk were used to calculate surrogate markers of efficacy. Terminal half-lives of marbofloxacin after IV, SC, SC-P407 and SC-P407-CMC administration were 7.12, 6.57, 13.92 and 12.19 h in plasma, and the half-lives of elimination of marbofloxacin in milk were 7.22, 7.16, 9.30 and 7.74 h after IV, SC, SC-P407 and SC-P407-CMC administration, respectively. Marbofloxacin penetration from the blood into the milk was extensive, with Area Under the Curve (AUCmilk/AUCplasma) ratios ranged 1.04–1.23, and maximum concentrations (Cmax-milk/Cmax-plasma) ratios ranged 0.72–1.20. The PK/PD surrogate markers of efficacy fAUC24/MIC and the Monte Carlo simulation show that marbofloxacin ratio (fAUC24/MIC > 125) using a 90% of target attainment rate (TAR) need a dose regimen between 8.4 mg/kg (SC) and 11.57 mg/kg (P407CMC) and should be adequate to treat contagious agalactia in lactating goats.

Age and seasonal pattern of contagious agalactia in small ruminants in Ukraine

Introduction The aim of the study was to determine how the spread of contagious agalactia in sheep and goats in the Odesa region depended on the age of the animals and the season. Material and Methods From January 2016 to December 2018, 1,964 ewes and 1,484 nanny goats of different age groups were studied by ELISA for antibodies to Mycoplasma agalactiae. Results The highest incidence of contagious agalactia was registered in one-year-old animals and was 59.7‒83.0%, two-year-old ruminants showed 17.0‒40.3% prevalence, in livestock at the age of 3–4 years no serological evidence of the disease was registered and in ewes and nanny goats older than 5–6 years 1.5–3.6% were infected. The most susceptible were young animals at the age of one-month (11.6‒14.5%). The first peak of the disease was recorded in March‒April (21.0‒26.1%), in the lambing period, which coincided with the beginning of lactation and the suckling period, and the second peak occurred in June–July (28.9‒34.2%), the period of maximum lactation and of manual milking of sheep and goats. Conclusion The results of serological investigations indicate the circulation of M. agalactiae in small ruminants in the south of Ukraine. To avoid greater dissemination of the pathogen, appropriate measures should be applied and strategies for its control need to be drawn up.

Comparative analysis of the postvaccinal immune response in sheep in two farms after vaccination against infectious agalactia – a field study

The aim of the present study was to detect differences in immune response in sheep from 2 small extensive farms against contagious agalactia, after treatments against helminths and optimization of the diet (farm 2). The one-year experimental period was divided into 2 six-months subperiods. The first without intervention, the second after changes in some of the management conditions. Samples for every subperiods, obtained from the day of vaccination and 3, 7, 14, 30, 60, 90, 120, 180 days thereafter were examined. The samples were assayed by means of indirect ELISA. It was found that the antibody titer increased rapidly after administration of the vaccine, reached a maximum between 7 and 30 days, and decreased rapidly after the 90th day of the experimental period. Significantly higher (P <0.05) antibody titers were found in farm 2 after anti-parasite treatments compared to farm 1.

Scientometrics Approach to Research in Ovine Mastitis from 1970 to 2019 (with a Complete List of Relevant Literature References)

The present study is a scientometrics evaluation of refereed publications on bacterial mastitis in sheep; the objectives were the evaluation of the relevant papers and the presentation of quantitative characteristics regarding their scientific content and bibliometric details. The Web of Science platform was used with search terms: [mastitis OR *mammary infection*] AND [sheep OR ewe* OR ovine] for papers from 1970 tο 2019; only ‘articles’, ‘reviews’, ‘proceedings papers’, or ‘data papers’ were evaluated, whilst documents related solely to contagious agalactia, mammary aspects of lentiviral infections, or infections of the teats and the udder skin were excluded. Finally, 580 papers were considered in detail. The number of published papers increased from 8 during the 1970s to 273 during the 2010s. These papers originated from 43 countries (most from Greece or Spain, n = 87 from each) and 240 institutions (145 universities and 95 other establishments), of which 35 produced ≥ 5 papers each. Most papers present original studies (n = 539) with a few reviews (n = 41). The original papers refer to dairy (n = 428), meat (n = 113), or wool (n = 1) production systems and present field (n = 329), laboratory (n = 163), or experimental (n = 67) work; the papers report aetiology (n = 146), risk factors (n = 100), pathogenesis (n = 92), diagnosis (n = 88), effects (n = 66), treatment (n = 50), control (n = 36), or descriptive epidemiology (n = 32) of the disease. Papers related to dairy production present more field and fewer experimental work than papers related to meat production; also, in papers describing work performed in dairy sheep, studies about aetiology, risk factors, and diagnosis of the disease predominate, whilst in papers performed in meat sheep, studies about aetiology, pathogenesis, and effects/diagnosis are reported more often. The papers were published in 175 scientific journals (most in Small Ruminant Research, n = 90, or Journal of Dairy Science, n = 54). On average, the papers received 16.8 total citations and 1.6 yearly citations (h-index = 47). Most papers were published in Scimago classification Q1 (n = 240) or Q2 (n = 230) journals and received 23.4 or 15.4 total citations, respectively. Reviews received more citations than original papers; among the latter, papers with work referring to dairy production received more yearly citations than papers referring to meat production; no differences in citations were seen according to type of work or mastitis aspect covered. Most citations were received by papers from France. Papers published in Journal of Dairy Science or Small Ruminant Research received the most citations. In total, there were 1558 individual authors of the papers, with 24 authors having co-authored > 10 papers each (max: 73 papers); on average, there were 5.2 co-authors per paper (min–max: 1–25). Average number of co-authors progressively increased from 2.1 in the 1970s to 6.3 in the 2010s, with original papers having a higher number of co-authors than reviews: 5.3 and 3.7, respectively. Papers from France had highers number of co-authors (7.9). The findings of this first ever scientometrics study into ovine mastitis indicate that the disease has not been studied as other sheep diseases and that future studies in it should be directed to its control.

Determination of Contagious Agalactia in Sheep and Goats and Investigation of Antibiotic Susceptibility of Mycoplasma spp. Positive Isolates

Antibiotic resistance is one of the most important issues encountered globally in managing infectious diseases and is a potential problem in the treatment of Mycoplasma infections. The aims of this study were to: determine carrier rates in sick and healthy herds of sheep and goats; determine the presence in herds of carrier animals that were clinically asymptomatic for contagious agalactia and use antibiogram tests to investigate the susceptibility to antibiotics of Mycoplasma spp. positive isolates derived from sheep and goat ear swabs. The presence of contagious agalactia was diagnosed by analyzing ear swabs (n = 300, bacterial method, Mycoplasma spp.) and blood serum samples (n = 300, serological method [ELISA], Mycoplasma agalactiae) taken from sheep and goat herds located in Elazýð and Malatya provinces in eastern Turkey. The ELISA tests revealed seropositivity in 10 (3.33%) of 300 samples. In order to determine the most effective antibiotic for disease treatment, antibiogram testing was performed on 87 (29%) positive isolates that had been isolated from swab cultures. We determined tulathromycin (MIC 16 µg/mL) and tiamulin (MIC 16 µg/mL) to be the most effective antibiotics, whereas disease agents were resistant to trimethoprim/sulfamethoxazole and neomycin.