Experimental pathogenicity of mollicutes from bovines with reproductive disorders in rabbit fallopian tube organ culture.

Mollicutes (10) belonging to Mycoplasma and Acholeplasma isolated from various reproductive disorders were tested in rabbit fallopian tube (FT) organ culture. Parameter for describing pathogenic status of Mollicutes in rabbit FT organ culture included multiplication of organisms, and its effect on ciliary activity along with histopathological changes in FT explants. M. mycoides (LC, Y-Goat), M. bovoculi, M. bovigenitalium, Mycoplasma sp. and A. oculi were categorized as pathogenic; A. axanthum and A. laidlawii as mildly pathogenic; and M. bovis, M. arginini. and A. granularum, as nonpathogenic to rabbit FT organ culture. Thus, rabbit FT organ culture is recommended for use as a suitable and economical in vitro model to assess the pathogenicity of Mollicutes of reproductive tract origin.     

The complete genome sequence of Mycoplasma bovis strain Hubei-1

Infection by Mycoplasma bovis (M. bovis) can induce diseases, such as pneumonia and otitis media in young calves and mastitis and arthritis in older animals. Here, we report the finished and annotated genome sequence of M. bovis strain Hubei-1, a strain isolated in 2008 that caused calf pneumonia on a Chinese farm. The genome of M. bovis strain Hubei-1 contains a single circular chromosome of 953,114 bp with a 29.37% GC content. We identified 803 open reading frames (ORFs) that occupy 89.5% of the genome. While 34 ORFs were Hubei-1 specific, 662 ORFs had orthologs in the M. bovis type strain PG45 genome. Genome analysis validated lateral gene transfer between M. bovis and the Mycoplasma mycoides subspecies mycoides, while phylogenetic analysis found that the closest M. bovis neighbor is Mycoplasma agalactiae. Glycerol may be the main carbon and energy source of M. bovis, and most of the biosynthesis pathways were incomplete. We report that 47 lipoproteins, 12 extracellular proteins and 18 transmembrane proteins are phase-variable and may help M. bovis escape the immune response. Besides lipoproteins and phase-variable proteins, genomic analysis found two possible pathogenicity islands, which consist of four genes and 11 genes each, and several other virulence factors including hemolysin, lipoate protein ligase, dihydrolipoamide dehydrogenase, extracellular cysteine protease and 5'-nucleotidase.     

Identification of Moraxella bovis by qualitative genetic transformation and nutritional assays.

Strains of Moraxella bovis were identified definitively through the combined use of a qualitative genetic transformation assay and determination of the ability of the organism under examination to grow in a defined medium (medium MB). Except for weak transformation by DNA from strains of M. lacunata, M. nonliquefaciens, and M. (Branhamella) ovis, DNA samples from all other members of the genus Moraxella failed to transform either of the two M. bovis auxotrophs used in this study.     

Identification of Moraxella bovis by qualitative genetic transformation and nutritional assays

Strains of Moraxella bovis were identified definitively through the combined use of a qualitative genetic transformation assay and determination of the ability of the organism under examination to grow in a defined medium (medium MB). Except for weak transformation by DNA from strains of M. lacunata, M. nonliquefaciens, and M. (Branhamella) ovis, DNA samples from all other members of the genus Moraxella failed to transform either of the two M. bovis auxotrophs used in this study.     

Mycoplasmas and bovine respiratory disease: studies related to pathogenicity and the immune response--a selective review

Three species of mycoplasma have been established as being of importance as causes of pneumonia in housed calves, based on pathogenicity studies and frequency of association with the disease. These three species are Mycoplasma bovis, M. dispar, and Ureaplasma diversum. M. bovis is the most pathogenic of these species but the disease outbreaks with which it is associated are sporadic. M. dispar is regularly isolated from pneumonic calves but is also found causing mild superficial and asymptomatic infections of the respiratory mucosa. The bovine ureaplasmas are serologically complex. They are distinct from ureaplasmas isolated from other non-ruminants by PAGE analysis, G + C content of DNA, and serology. A second species within the genus ureaplasma has been proposed to accommodate the bovine ureaplasmas, U. diversum. Control of mycoplasma respiratory infections of cattle based on immunization might be possible. Calves have been immunized against M. bovis and immunity has been related to antibody in the lung. M. dispar appears less immunogenic in calves than M. bovis and this may contribute to its pathogenicity.     

Mycoplasmas: Use of Polyvalent Antisera for Identification by Indirect Immunofluorescence

It would be an advantage, under many circumstances, to be able to make use of polyvalent antisera in the process of identifying mycoplasmas. As the indirect immunofluorescence test is sufficiently sensitive and also generally accepted as being rather specific, this technique was chosen to investigate whether polyvalent antisera are applicable in routine identification of mycoplasmas. Three polyvalent sera were used, each consisting of 9 or 10 rabbit antisera raised against 29 of the more common species of the genus Mycoplasma. Twenty-six field strains were examined. One strain did not react with any of the 3 polyvalent antisera although it was later identified as M. bovigenitalium. The remaining 25 strains reacted with 1 and only 1 of the polyvalent antisera and were subsequently identified by immunofluorescence utilizing monospecific antisera. Strains of the following species were identified: M. arginini, M. bovigenitalium, M. bovis, M. bovoculi, M. canis, M. capricolum, M. cynos, M. edwardii, M. hominis, M. hyorhinis, M. molare, M. mycoides subsp. mycoides and M. opalescens. It is concluded that polyvalent antisera may be used in identification procedures and thereby permit the use of a limited number of monospecific antisera without preceding biochemical testing.     

Interaction of Mycoplasma bovis and Mycoplasma bovigenitalium with preimplantation bovine embryos

In vitro exposures of bovine embryos to Mycoplasma bovis and Mycoplasma bovigenitalium were conducted to determine if these organisms adhered to the zona pellucida-intact (ZP-I) bovine embryo, and standard procedures for washing and treating embryos were evaluated to determine their effectiveness for removing or killing mycoplasmas. Mycoplasma bovis and M. bovigenitalium were isolated from 19 of 19 and 24 of 24 ZP-I embryos, respectively, after in vitro exposure and subsequent washing, thus demonstrating adherence of the two species of Mycoplasma to the ZP. Additionally, M. bovis was isolated from 20 of 20 and 23 of 23 embryos, while M. bovigenitalium was isolated from 25 of 25 and 22 of 22 embryos after antibiotic and trypsin treatment, respectively. It was concluded that neither of the standard procedures currently used for cleansing embryos should be relied upon for insuring freedom from mycoplasmas.     

Pulmonary mycoplasmosis in farmed white-tailed deer (Odocoileus virginianus)

An outbreak of respiratory disease at a farmed cervid facility resulted in isolation and identification of Mycoplasma boris in four affected white-tailed deer (Odocoileus virginianus) fawns. Microscopically, pulmonary lesions similar to those associated with M. bovis infections in calves, inclulding lymphoplasmacytic peribronchiolar cuffing and caseonecrotic bronchiectasis, were present. Arcanobacterium pyogenes was recovered from lung tissue as well. This report indicates that M. bovis can be associated with respiratory disease in white-tailed deer.     

Vsp antigens and vsp-related DNA sequences in field isolates of Mycoplasma bovis

The expression of the 1E5 epitope which is common to the three characterized variable lipoproteins VspA, VspB and VspC of Mycoplasma bovis type strain PG45 and the presence of vsp gene DNA sequences were assessed in field isolates randomly collected from cattle showing clinical manifestations due to M. bovis infection. Among 250 isolates tested, only four failed to react with mAb 1E5. Southern blot analysis of these four isolates and of 20 isolates expressing the 1E5 epitope were performed using synthetic oligonucleotide probes corresponding to a sequence located in the Vsp signal peptide coding region common to all known Vsp products or to selected regions of previously characterized vsp genes, vspA, vspE and vspF. The results demonstrate the presence of multiple vsp-related DNA sequences in all M. bovis field isolates tested and indicate that the vsp repertoire varies in size and composition among isolates.     

The phylogeny of Mycoplasma bovis as determined by sequence analysis of the 16S rRNA gene

Abstract The nucleotide sequence of the 16S rRNA gene of Mycoplasma bovis has been determined. Comparisons with other 16S rRNA sequences of mycoplasmas showed that Mycoplasma agalactiae is phylogenetically the closet relative. In total, only eight nucleotides differed between the M. bovis and M. agalactiae 16S rRNA sequences. The phylogenetic position of M. bovis with respect to other mycoplasmas was determined by sequence comparisons and from features in the secondary structure of 16S rRNA.     

Quantification of Moraxella bovis haemagglutinating adhesins with monoclonal antibodies

Six monoclonal antibodies (MAbs) against Moraxella bovis GF 9 were used to quantify haemagglutinating adhesins of 16 strains of this organism. The amount of each MAb necessary to inhibit one haemagglutinating unit of each strain varied between 4 and 0.007 times that required by strain GF 9. Five strains reacted with six MAbs, one with five, two with four, one with three, two with two and three with none. The procedures used enabled to detect dominant strains candidates for vaccines.     

Mycoplasma bovis shares insertion sequences with Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides SC: Evolutionary and developmental aspects

Three new insertion elements, ISMbov1, ISMbov2 and ISMbov3, which are closely related to ISMag1 (Mycoplasma agalactiae), ISMmy1 and IS1634 (both Mycoplasma mycoides subsp. mycoides SC), respectively, have been discovered in Mycoplasma bovis, an important pathogen of cattle. Southern blotting showed that the genome of M. bovis harbours 6-12 copies of ISMbov1, 11-15 copies of ISMbov2 and 4-10 copies of ISMbov3, depending on the strain. A fourth insertion element, the IS30-like element, is present in 4-8 copies. This high number of IS elements in M. bovis, which represent a substantial part of its genome, and their relatedness with IS elements of both M. agalactiae and M. mycoides subsp. mycoides SC suggest the occurrence of two evolutionary events: (i) a divergent evolution into M. agalactiae and M. bovis upon infection of different hosts; (ii) a horizontal transfer of IS elements during co-infection with M. mycoides subsp. mycoides SC and M. bovis of a same bovine host.     

Preliminary studies on the etiology of keratoconjunctivitis in reindeer (Rangifer tarandus tarandus) calves in Alaska

Keratoconjunctivitis outbreaks occur each summer in reindeer (Rangifer tarandus tarandus) herds in western Alaska, USA. This condition has not been well characterized nor has a definitive primary etiologic agent been identified. We evaluated the eyes of 660 calves near Nome, Alaska, between 29 June and 14 July 2005. Clinical signs of keratoconjunctivitis were observed in 26/660 calves (3.9%). Samples were collected from the conjunctival sac of both affected (n=22) and unaffected (n=24) animals for bacterial culture, enzyme-linked immunosorbent assay testing for Chlamydophila psittaci, and for polymerase chain reaction assays for Mycoplasma and Moraxella spp. No primary bacterial or viral etiologic agent(s) were isolated or identified. The cause of keratoconjunctivitis among reindeer calves was not determined, but it could involve an anaerobic bacteria, a difficult-to-isolate viral agent, stress associated with repeated handling, ocular foreign bodies, exposure to corral dust or arthropods, or a combination.     

Cloning and characterization of the fur gene from Moraxella bovis

A homologue of the ferric uptake regulator gene (fur) was isolated from Moraxella bovis by degenerate polymerase chain reaction and cloning. Fur protein of M. bovis exhibited 72.1% amino acid identity with Acinetobacter calcoaceticus Fur. Western blot analysis showed a decrease of Fur expression in response to sufficient-iron conditions compared with deficient-iron conditions. An electrophoretic mobility-shift assay indicated that Fur protein binds to DNA fragments containing a putative Fur-box derived from the upstream region of the M. bovis fur gene. Fur of M. bovis may regulate the expression of iron transport systems in response to iron limitation in the environment.     

A haemolytic cell-free preparation of Moraxella bovis confers protection against infectious bovine keratoconjunctivitis

Abstract Protection conferred by a cell-free preparation from a haemolytic Moraxella bovis isolate, UQV 148NF, was compared to an equivalent fraction from a non-haemolytic M. bovis isolate, Gordon 26L3, and to a recombinant DNA-derived pili vaccine. Three groups of ten calves were vaccinated twice with one of the three preparations and, together with ten non-vaccinated calves, challenged with virulent M. bovis isolate Dal 2d. Compared to the control group, significant protection was observed in the group receiving the pili vaccine and the group receiving the preparation from haemolytic isolate, UQV 148NF.     

Identification of a capsular polysaccharide from Moraxella bovis

The bacterium Moraxella bovis is the causative agent of an economically important disease of cattle: Infectious Bovine Keratoconjunctivitis (IBK), otherwise known as pinkeye. Little is known regarding the structure of the carbohydrates produced by M. bovis. The structure of a capsular polysaccharide from M. bovis (strain Mb25) has been determined using NMR and MS analysis. From these data it is concluded that the polysaccharide is composed of the unmodified chondroitin disaccharide repeat unit.     

Potential pathogens carried by Spanish ibex (Capra pyrenaica hispanica) in southern Spain

The Spanish ibex (Capra pyrenaica hispanica) population of southern Spain was surveyed for potential pathogens associated with the conjunctiva, external ear canal, as well as reproductive and upper respiratory tracts. We sampled 321 ibex (131 adult males, 100 adult females, and 90 yearlings); these included 271 apparently healthy animals and 50 that were naturally infected with Sarcoptes scabiei. A total of 688 bacterial isolates were identified (377 gram-negatives, 225 gram-positives, and 86 Mycoplasma spp.); sex, age, location, infection with S. scabiei, and disposition of the animal (free-ranging versus captive) were evaluated as risk factors for infection. Infections with Mycoplasma agalactiae and Mycoplasma arginini were associated with age, having a higher frequency of isolation in young animals. With Escherichia coli, Mannheimia haemolytica, Pasteurella multocida biotype A, and Staphylococcus aureus, significantly higher isolation rates were associated with adults. The isolation frequency for E. coli was higher in females, whereas Moraxella bovis isolations were mostly associated with males. The presence of mange increased the risk of infection with both Streptococcus equi subsp. zooepidemicus and M. haemolytica. The geographic origin of sampled animals was related to the isolation of Branhamella ovis, M. agalactiae, and all Pasteurella sp. Isolations of M. haemolytica, P. multocida biotype A, E. coli, and B. ovis were more prevalent in samples from free-ranging rather than captive animals. Of the gram-positive bacteria, S. aureus represented the predominant species isolated from nasal, vaginal, and ocular samples. Mycoplasma agalactiae and M. arginini were the predominant Mycoplasma spp., and both were associated most often with the external ear canal. The most frequently isolated gram-negative bacteria included E. coli, M. haemolytica, P. multocida biotype A, and B. ovis. Isolation rates of gram-negative species varied by source. In nasal samples, M. haemolytica and P. multocida biotype A were isolated most frequently, whereas in ocular and vaginal samples, B. ovis and E. coli, respectively, were most frequently isolated.     

α-Enolase, an adhesion-related factor of Mycoplasma bovis

Mycoplasma bovis is the causative agent of Mycoplasma bovis-associated disease (MbAD). Although the mechanisms underlying M. bovis adherence to host cells is not clear, recent studies have shown that the cell surface protein α-enolase facilitates bacterial invasion and dissemination in the infected host. In this study, we cloned, expressed and purified recombinant M. bovis α-enolase and induced polyclonal anti-α-enolase antibodies in rabbits. M. bovis α-enolase was detected in the cytoplasmic and membrane protein fractions by these antibodies. Triple immunofluorescence labeling combined with confocal laser scanning microscopy (CLSM) revealed that the plasminogen (Plg) enhanced the adherence of M. bovis to embryonic bovine lung (EBL) cells; the values obtained for adherence and inhibition are consistent with this finding. Interestingly, we found that trace amounts of trypsin acted as a more effective enhancer of cell adherence than Plg. Hence, our data indicate that surface-associated M. bovis α-enolase is an adhesion-related factor of M. bovis that contributes to adherence by binding Plg.     

Inhibition of Autoagglutination of Moraxella bovis by 10% Magnesium Chloride

A 10% solution of magnesium chloride was used to uniformly disperse Moraxella bovis, an organism that autoagglutinates in most liquid media. The magnesium chloride did not affect the viability or morphology of this organism.