Introduction

Mycoplasma agalactiae is an important pathogen causing contagious agalactia in small ruminants such as sheep, goats and wild Caprinae. Contagious agalactia is a notifiable disease listed by the World Organisation for Animal Health (OIE) due to the important economic losses to the dairy industry and to small ruminants holders [1]. The contagious agalactia caused by M. agalactiae includes clinical symptoms of mastitis, arthritis and kerato-conjunctivitis, but M. agalactiae infections occasionally also cause abortions, pneumonia and septicaemia in goat and sheep [2, 3]. M. agalactiae is highly contagious and transmitted through oral, respiratory, and mammary routes. Since the end of the nineteenth century, the disease had been reported to be enzootic in many parts of the world [1, 4, 5]. Outbreaks of M. agalactiae infections occur notably in the Mediterranean basin, India, Australia, Turkey, Iran, Mongolia, Nigeria, Senegal and Iraq [4]. The spread of contagious agalactia within a population is multifactorial including inadequate herding practices, inefficiency of antimicrobial therapies, and adoption of very few prophylactic measures. In addition, the high potential of M. agalactiae to persist in the host and to some extent in the environment often renders difficult to eradicate the infections. The disease is mostly reported from sheep where it causes highest economic losses mainly due to high morbidity rather than high mortality resulting in losses of milk production throughout the world, but goats are equally affected by M. agalactiae infections [4]. Mycoplasma agalactiae possess a family of related surface lipoproteins, named variable protein of M.agalactiae (Vpma) that are prone to high-frequency variation in expression and structure thus modulating the mycoplasma surface structure and accessibility to immune components of the host. This provides M. agalactiae the capability to rapidly adapt to various environmental changes and to evade the host’s immune defence. Phase and size variations have been shown to occur in vitro with frequencies estimated at 10E-2 to 10E-5 events/cell/generation [6]. M. agalactiae, type strain PG2 which was used for most molecular studies, carries a genetic locus containing a cluster of 6 vpma genes (vpmaU though Z), while other M. agalactiae strains such as strain 5632 show up to 18 different vpma genes occurring as two clusters on two different loci of the chromosome. In a given cell, only one vpma gene per locus is expressed, while in the overall population the different Vpma antigens occur at different rates [6,7,8]. The intra-clonal variation is due to the presence of a unique promoter at the vpma loci that is alternatingly placed by a DNA recombination mechanism in front of silent vpma genes, resulting in ON and OFF switching of the various Vpma expression. Vpma phase variation allows M. agalactiae to survive in immuno-competent hosts and to establish persistent infections and cause debilitating disease [9, 10]. The phase variation, which is important for the host immune evasion and the in-host survival of the pathogen is controlled to a major extent by the Xer1 recombinase, also named DNA invertase Mar, that is encoded by the vpma loci [11, 12]. Recently a novel role of Vpmas as major adhesins of M. agalactiae mediating differential adhesion and invasion of host cells has been unravelled by the use of phase locked mutants expressing specific well-characterized Vpma lipoproteins [13].

In order to better understand the origin of M. agalactiae infections and their mode of propagation within and among herds with the aim of controlling the disease, molecular epidemiological tools including variable-number tandem repeat (VNTR) and multi-locus sequence typing (MLST) have been developed. They have been used successfully to trace M. agalactiae outbreaks and emergence or re-emergence after eradication campaigns [2, 5, 14,15,16,17]. While certain European countries such as Spain seem to have endemic clones, in particular ST5, others such as Greece have to deal with several different clones including ST2, ST4, ST6, and ST8 [5, 17,18,19]. It was also shown that M. agalactiae strains circulating in the ibex (Capra ibex) population in the French Alps were highly interrelated and most likely originated from a single parental clone that has also spread to chamois (Rupicapra rupicapra), another wild ungulate species in the same geographical area [2]. The clone contains some genetic loci closely related to Mycoplasma conjunctivae, a pathogen that is frequently found in wild ungulates with infectious kerato-conjunctivitis (IKC) [2, 20,21,22]. However, the molecular basis of the potential specificity or predilection of the clone to the wild Caprinae ibex and chamois is still unknown. The current manuscript describes a particular strain of M. agalactiae of a previously undetected sequence type, ST35 that caused an uncommon outbreak of contagious agalactia affecting specifically goats. The outbreak strain had a particularly small genome size and lacked several characteristic genes involved in host adhesion and variable antigenicity.

Materials and methods

Observation farm

The observation farm was located in Aridea (northwestern Greece), a lowland at 120 m above sea level. The farm harboured a mixed herd of 850 Lacaune milking sheep (Ovis aries) and 320 Anglo-nubian milking goats (Capra aegagrus hircus) of the age of 1-12 years. About one third (106) of the animals were aged 15 – 17 months and were the first time in the milking season. Sheep and goats were kept in separate barns but shared meadows and were milked at the same milking place. Generally, all adult animals were vaccinated twice a year against contagious agalactia with a commercial vaccine (Agalax Uno; Laboratorios Syva, Leon, Spain) based on inactivated bacteria of Mycoplasma agalactiae. However, the year of the observed outbreak, no vaccination was recorded for both sheep and goats.

Clinical examinations

A veterinarian conducted a general clinical routine examination inspecting eye, ears, mouth, body, skin, hair, rectum vagina, udder, legs, hooves and records of appetence as recommended by Nagy and Pugh [23] giving special attention to udders as described for previous studies [24].

Bacteriological diagnosis

Composite milk samples (CMS) were collected from seven randomly selected goats that suffered from clinical mastitis, before any treatment was administered. Briefly, following proper disinfection of the teats and the withdrawal of the initial 3 to 4 squirts of milk, CMS (ca. 30 mL each), were collected in sterile falcon tubes. The samples were refrigerated and submitted in insulated boxes containing coolants to the laboratory of microbiology and infectious diseases at the Faculty of Veterinary Medicine of Aristotle University, Thessaloniki within 12 h of collection. Aliquots of 10 μL milk were cultured on Columbia Agar supplemented with 5% sheep blood (Oxoid Ltd., Cambridge, United Kingdom), MacConkey Agar (Biolife Italiana S.r.l., Milan, Italy) and Sabouraud Dextrose Agar (Oxoid Ltd., Cambridge, United Kingdom) aerobically and in an atmosphere that contained 5% CO2 at 37o C.

Detection of mycoplasmas, isolation, culture, extraction of genomic DNA, genome sequencing and annotation

In order to detect the presence of mycoplasmas 0.5 mL of each CMS was inoculated in 5 mL of Heyflicks broth and incubated at 37 °C under an atmosphere of 5% CO2. Additionally 50 μL milk volume was cultured on Heyflicks agar and incubated at 37 °C under an atmosphere of 5% CO2. Identification of Mycoplasma colonies was performed using quick lysis DNA extraction and polymerase chain reaction (PCR) using primers, specific for M. agalactiae and the Mycoplasma mycoides cluster [25, 26].

A strain (GrTh01) of M. agalactiae was obtained from one of the M. agalactiae isolates by filter cloning through a 0.2 μm filter and sub-culture of a single colony as described [27]. For preparing genomic DNA the strain was grown in Heyflicks broth under aerobic atmosphere for 72 h. Mycoplasma DNA was extracted using the Guanidine thiocyanate extraction method [28] with two subsequent phenol extractions and two ethanol precipitations.

The genome sequence of M. agalactiae strain GrTh01 was generated using a combination of Pacific Biosciences RSII and Illumina HiSeq sequencing. After sequencing and circularization using Pacific Biosciences (PacBio) RSII (Swiss Institute of Bioinformatics, Lausanne, Switzerland), the sequence was corrected with Illumina HiSeq data for the substitutions due to sequencing errors and for the frameshifts, using the Celera Assembler (CA) PacBio correction module PBcR (version 7.0) [29]. Illumina reads were obtained using the Nextera XT DNA protocol for the library preparation and the V3 chemistry to produce 2 × 150 bp paired-end reads that resulted in 9.1 million read pairs (GATC Biotech, Konstanz, Germany). The resulting full genome was annotated using the Prokka pipeline [30] and the NCBI Prokaryotic Genome Annotation Pipeline PGAP https://www.ncbi.nlm.nih.gov/genome/annotation_prok/ and deposited at NIH genetic sequence database GenBank. The GenBank/EMBL accession number of the full genome sequence of M. agalactiae strain GrTh01 is CP039447. Detailed analysis of Vpma loci, potential virulence genes and comparisons of genomes of M. agalactiae strains were performed using the software package Geneious® software package 9.1.8 (Biomatters Ltd., Auckland 1141, New Zealand), Virulencefinder https://cge.cbs.dtu.dk/services/VirulenceFinder/ [31] and Artemis [32]. Multi locus sequence typing (MLST) was performed using the M. agalactiae MLST Databases (https://pubmlst.org/magalactiae/) extracting the gene-loci of dnaA, gltX, gyrB, metS and tufA from the genome sequence [33].

Results

Outbreak description

Between April and June 2016, 87 out of 106 young milking goats of the age of 15 to 17 months that were their first milking season showed a general malaise, absence of appetite, and signs of clinical mastitis. In most of the cases, both the right and the left udder were hot, swollen and tender while the milk became discolored and granular. No signs of respiratory complications, arthritis or keratitis were detected. Furthermore, the sheep population of the same farm remained unaffected, showing no clinical signs of contagious agalactia. The study of the medical records of the farm showed that, for an undefined reason, the whole farm (sheep and goats) was not vaccinated against contagious agalactia during the year preceding the outbreak. After the outbreak the goats of the farm were culled.

Bacteriological analyses

Milk samples of 7 of the 87 affected goats were analysed bacteriologically of which all 7 showed M. agalactiae by culture and subsequent species differentiation using PCR. No other bacterial species were diagnosed from the 7 milk samples. One M. agalactiae isolate was twice filter cloned, and the strain isolated named GrTh01 was used for full genome analysis.

Whole genome sequencing and analysis of strain GrTh01

The whole genome sequence (WGS) of M. agalactiae strain GrTh01was obtained by combining PacBio and Illumina sequencing and revealed a circular chromosome of 841′635 bp (GenBank full Genome accesion nr. CP039447). The Illumina HiSeq coverage was of 1895 x. As for the other M. agalactiae strains so far sequenced, no plasmid was identified in GrTh01. Annotation of the genome using the NCBI Prokaryotic Genome Annotation Pipeline PGAP revealed a total of 767 genes of which 623 encode proteins. Strain GrTh01 contains two full sets of rRNAs (5S, 16S, 23S), 34 tRNAs and 3 non coding RNAs (Table 1).

Table 1 General properties of M. agalactiae circularized genomes and corresponding strains. Note the full genome sequence of M. agalactiae, strain JF4428 is also available but not included in this table as JF4428 is a sample of strain PG2 that was propagated in vitro as internal diagnostic control for several decencies
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The GrTh01 MLST analysis revealed a new sequence type, namely ST35, having the following profile: dnaA: 21; gltX: 1; gyrB: 1, metS: 2; tufA: 1. ST35 stands apart because of its novel dnaA allele, which sequence was verified by additional Sanger sequencing using primers recommended by Mc Auliffe [33]. The MLST analyses also indicated that the ST35 GrTh01 strain clustered together with several strains isolated from Greece, with dnaA being the only discriminating allele and from the PG2 type strain isolated in Spain, with two discriminating alleles (Table 2). The dnaA allele in strain GrTh01 leads to a T/S change at a.a. position 91. While strain GrTh01 is currently the only strain among M. agalactiae and the phylogenetically closely related Mycoplasma bovis, the mutation is in a serine-rich area of DnaA and a serine residue is found at the analogous site of DnaA of Mycoplasma simbae (GenBank accession nr. WP 051630113).

Table 2 Allelic profiles and sequence types (ST) of M. agalactiae type strain PG2 and field strains isolated from Greece. Data taken from https://pubmlst.org/magalactiae/
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Of the currently publicly available M. agalactiae genomes, the GrTh01 genome is the smallest showing ca. 36 and 165 Kbp less than the two representative M. agalactiae strains with circularized genome, namely PG2 and 5632 [7, 34] (Table 1). More specifically, comparison of GrTh01 with these two strains revealed that GrTh01 lacks the 23-kb Integrative Conjugative Element (ICE) found in 5632 in three copies and the 20-kb ICE vestige of PG2 [34, 35] (Fig. 1). Interestingly, GrTh01 displayed 2 small ICE vestiges of ca 2.4 and 6.0 kb that are composed of only few CDSs and pseudogenes. The smallest is found in the three strains at the same locus, while the largest is only found in 5632 and GrTh01 (Fig. 1). Of note, PG2 and GrTh01 are deprived of IS elements, a situation that contrasts with 5632 having 15 belonging to the IS30 family [34].

Fig. 1
figure 1

Genome comparison of M. agalactiae strains PG2 (Type strain) (upper), GrTh01 (mddle) and 5632 (lower) whose full circularized genomes are available. Orange circles: bpsA. Green, vpma loci. Red blocks show homologous genome stretches. Magneta: spma locus. Purple blocks: ICE or ICE vestiges (smaller blocks). : IS elements

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Other smaller deletions, detected in GrTh01 when compared to PG2 or 5632, mostly affect gene families encoding surface proteins. More specifically, the repertoire of the Bacteroides like surface protein A (BspA) gene family is the smallest with 4 entire and 4 pseudo-genes in GrTh01 for 9 and 12 full copies in PG2 and 5632, respectively [34]. Of note, this family was previously described and annotated as drp genes by Nouvel et al. [34] or as PARCEL in M. bovis by Wise et al. [36] and were characterized by the presence of the motif DUF285, also present in BspA. In this study, we refer to all this CDS as bspA. The function of the proteins carrying a DUF285 domain is not known but these occurred across a large number of mycoplasma species and bacteria. In strain GrTh01, the spma locus that encodes a family of surface lipoprotein with unknown function is 13,475 bp and similar to PG2. Finally, as shown in Fig. 1, strain GrTh01 displayed two vpma loci as in 5632, a situation that contrast with the single locus of PG2 [34].

One striking feature of the GrTh01 loci is that they are highly degenerated, with only four genes encoding a signal peptide, a conserved N-terminal and a vpma block of amino acids (Fig. 2). Multiple copies of sequences encoding either a signal peptide and a few amino acids or a Vpma block without a signal sequence were identified on both loci (Fig. 2). These loci also contained multiple “vis” motifs, a highly conserved sequence of 21 nt that is recognized by the Xer recombinase, which one is responsible for DNA rearrangements [37] (Fig. 2). As in 5632, each of the two vpma loci contains a highly conserved xer1 gene (100% nucleotide identity in between the two copies). In PG2 and 5632, each vpma locus possesses a single conserved promoter placed upstream of the expressed vpma gene, so that a maximum of 1 and 2 Vpma are expressed in PG2 and 5632, respectively. Here, the conserved promotor sequence was detected, but no entire vpma gene was found directly behind raising the question of whether the sequenced GrTh01 strain expressed a Vpma or whether another promoter, yet unidentified, is used by this strain for Vpma expression at the surface of the cell. Remarkably, GrTh01 locus II is flanked by two short direct repeats also found in 5632 in which they were generated upon IS integration (Fig. 2). Full identical copies including promoter and ribosome binding site were found in both strains PG2 and GrTh01 for the virulence genes nifS – nifU [38], the gene encoding the adhesin P40 (Fleury et al., 2002), and the lipoprotein P80 [39] that is involved in neutrophil extracellular trap (NET) formation [40].

Fig. 2
figure 2

Genetic loci of vpma genes in M. agalactiae strain GrTh01. Arrowhead-Boxes represent open reading frames (ORF). Green arrowheads: vpma. Hatched green arrowheads partial or degenerated vpma genes. Black arrowheads: promoter sequences. Red arrowheads: 21 nt ‘vis’ motif encoding the Xer1 recombinase recognition site. Grey boxes labelled with ‘S’: signal sequences. HP: hypothetical protein

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Discussion

A particular strain of M. agalactiae, strain GrTh01, was isolated from an outbreak of caprine contagious agalactia that was restricted to Carpinae and did not affect sheep that were grazing on the same meadows and kept in the same farm as the affected goats. Also rather uncommon was the profile disease symptoms that was restricted to mastitis and did not reveal arthritis, conjunctivitis or respiratory distress. Full genome analysis revealed that the chromosome of GrTh01 was significantly shorter than type strain PG2 and other fully sequenced M. agalactiae strains. Type strain of M. agalactiae PG2 was originally isolated from a goat but shown in several infection experiments to efficiently infect sheep [38]. There are no differences between PG2 and GrTh01 in the essential housekeeping genes, the virulence associated genes nifS and nifU that are required for host invasion, protein P40 involved in cell adhesion or the lipoprotein P80 involved in NET formation. However, strain GrTh01 lacks a locus of 20 kbp including conjugal transfer genes such as traE and DDE transposase and genes belonging to a potential type IV secretion system whose function in M. agalactiae is unknown. Furthermore, GrTh01 fully lacks genes encoding transposases such as the IS30 transposase gene that is present in type strain PG2. Most interestingly, strain GrTh01 shows deletions in two groups of genes that both are potentially involved in adhesion: i) genes encoding the Bacteroides-like surface protein A (BspA) and ii) the vpma genes of the variable proteins of M. agalactiae. All currently sequenced strains of M. agalactiae possess several gene copies coding BspA family leucine-rich repeat surface proteins. While strain PG2 contains 9 full copies and 3 truncated bspA genes, and strain 5632 has 12 full, and one truncated copy, strain GrTh01 only contains 4 full copies and 4 truncated versions of bspA. The BspA protein includes a domain that seems to have regulatory function by suppressing the σ54 dependent transcription and was found to have adhesin functions. In Tannerella forsythia the BspA-like protein is essential in pathogenesis [41]. In Entamoeba histolytica the BspA-like protein binds to human TNF receptor 1 (TNFR1) and is essential for invasion of the human colonic mucosa possibly via a fibronectin/fibrinogen integrin binding process [42]. Hence, the various BspA proteins in M. agalactiae can be assumed to play a role in adhesion in host cell adhesion and tropism.

The vpma loci of M. agalactiae are considered as pathogenicity island-like loci possessing key features of pathogenicity islands such as the tRNA-lys gene, the invertase-recombinase xer1 and the virulence associated vpma genes [37]. Furthermore, the Vpma proteins have been shown to be involved in virulence features such as immune evasion and immunomodulation and colonization of M. agalactiae in particular and Mycoplasmas in general [2, 6, 8, 43]. More recently, their involvement in differential cell adhesion and invasion was shown by the analysis of phase locked mutants that steadily express single well-characterized Vpma antigens [11, 13]. Co-infections of sheep with phase locked mutants stably expressing VpmaY and VpmaU revealed a dominance in the host of VpmaY expressors in particular during early stages of conjunctival and intra-mammary infections compared to VpmaU expressers, indicating differential roles of the antigenic variants in infection. Their data also revealed the importance of antigenic variation for survival and persistence inside the immunocompetent host as Xer1 independent antigenic variation was detected in the sheep infection model with Xer1 phase locked mutants [11]. While the surface antigenic variation caused by the Vpma proteins was mostly shown to be implicated as important mechanisms for better survival and persistence of M. agalactiae inside the host, a recent study using phase-locked mutants expressing the various Vpmas of strain PG2 revealed the role of Vpmas in adhesion [13]. Using sheep primary mammary epithelial cells and mammary stromal cells Hegde and collaborators showed that VpmaV exhibited the highest adhesion rate of the various Vpmas. Furthermore, they also could show a strong correlation between functions of Vpma proteins in cell adhesion and invasion. This strongly indicates that the variability of these lipoproteins not only causes evasion of the host’s immune response, but also changes the adhesion characteristics of the pathogen to allow it to adapt and spread in different host niches. Interestingly strain GrTh01 reveals two vpma loci similar to M. agalactiae strain 5632 [44], one located at the tRNA-lys gene (locus I) and the other at the 16S rRNA gene (locus II) (Fig. 2). Both loci contain a full-length xer1 gene but many strongly truncated vpma genes respectively residues with locus I having full length copies of vpmaW, vpmaX and vpmaZ and locus II showing solely vpmaY in full length. VpmaV, which was shown to have highest adhesion properties to sheep cells, is absent in strain GrTh01. The reduced set of available vpma genes and in particular the lack of vpmaV could explain the reduced host range and a predilection of the outbreak strain GrTh01 for Caprinae and for the mammary gland as revealed from the clinical and epidemiological data of the outbreak. Our clinical and molecular genetic data on strain GrTh01 show certain correlations to the observations by Chopra-Dewasthaly and collaborators [13] that the various Vpma lipoproteins are involved in host and tissue specificity. However, additional analysis of expressions of Vpma lipoproteins in strain GrTh01 under in vitro and under in vivo conditions would be necessary to confirm these correlations. We assume that the antigenic variation gives M. agalactiae the opportunity to infect and invade various hosts and tissues, hence generally causing multiple symptoms in various small ruminants unless certain adhesins are missing thus reducing host range and tissue tropism.