Molecular mechanisms of pathogenicity of Mycoplasma mycoides subsp. mycoides SC

Mycoplasma mycoides subsp. mycoides SC, the aetiological agent of contagious bovine pleuropneumonia (CBPP), is considered the most pathogenic of the Mycoplasma species. Its virulence is probably the result of a coordinated action of various components of an antigenically and functionally dynamic surface architecture. The different virulence attributes allow the pathogen to evade the host's immune defence, adhere tightly to the host cell surface, persist and disseminate in the host causing mycoplasmaemia, efficiently import energetically valuable nutrients present in the environment, and release and simultaneously translocate toxic metabolic pathway products to the host cell where they cause cytotoxic effects that are known to induce inflammatory processes and disease. This strategy enables the mycoplasma to exploit the minimal genetic information in its small genome, not only to fulfil the basic functions for its replication but also to damage host cells in intimate proximity thereby acquiring the necessary bio-molecules, such as amino acids and nucleic acid precursors, for its own biosynthesis and survival.     

Mycoplasma mycoides subsp. capri and Mycoplasma mycoides subsp. mycoides LC can be grouped into a single subspecies

Mycoplasma mycoides subsp. capri and Mycoplasma mycoides subsp. mycoides LC can be combined into one taxon on the basis of several contributions on both DNA sequence and protein analyses reported in the literature. Moreover, for the differentiation and identification of mycoplasmas of the "mycoides cluster", we investigated the rpoB gene, encoding the beta-subunit of the RNA polymerase. A segment of 527 bp of the rpoB gene was amplified from 31 strains of ruminant mycoplasmas by PCR. The nucleotide sequences were determined and aligned, and accurate genetic relationships were calculated. Cluster analysis of rpoB DNA allowed species differentiation within the "mycoides cluster" and confirmed that M. mycoides subsp. capri and M. mycoides subsp. mycoides LC cannot be distinguished from each other. "Mycoplasma mycoides subsp. capri" is proposed as a common name for both subspecies.     

Disease caused by Mycoplasma mycoides subspecies mycoides LC in Hungarian goat herds

The occurrence of a goat disease caused by Mycoplasma mycoides subsp. mycoides LC in Hungary is reported. The disease occurred in two goat herds in the spring of 1999. In one herd 25% of the 4-12 weeks old kids (10 animals) while in the other herd 33% of the 6-12 weeks old kids (20 animals) became affected. The goat kids developed polyarthritis. The most severe lesions developed in the carpal joints. All animals died after 3-8 days of disease. Four dead kids were necropsied. All of them had serofibrinous and purulent polyarthritis, and in two animals bronchopneumonia, fibrinous pleuritis and meningitis were also found. In the articular exudates the presence of mycoplasmas was detected by PCR using a general mycoplasma primer. Mycoplasmas were cultured from the joints of all animals, from the abdominal parenchymal organs of two kids and from the lungs of one animal. The cultured mycoplasmas grew in strikingly large colonies, proved to be glucose positive, arginine negative and phosphatase positive, and liquefied the coagulated serum. They survived incubation at 45 degrees C for more than 24 h. Based upon their biochemical properties, the results of the immunofluorescence (IF) and growth inhibition tests and the sequence analysis of the PCR product, the cultured strains were identified as M. mycoides subsp. mycoides LC. Animals purchased in the previous autumn had been introduced to both farms. The disease may have been introduced with asymptomatic carrier animals, as earlier no similar disease had been observed at either farm.     

Experimental infections of goats with Mycoplasma mycoides subspecies mycoides, LC type

In five experiments 29 goats were infected experimentally by five different routes with a strain of Mycoplasma mycoides subspecies mycoides, LC type, isolated from a contagious caprine pleuropneumonia-like outbreak on a farm in northern Sweden. All the goats were colonised except those inoculated subcutaneously with small doses. In its pattern of pathogenicity this strain was similar to other experimentally tested strains except that peroral infection in kids produced no clinical signs. A 'contact' goat was also colonised but the clinical signs seen in it were probably due to a concomitant infection with Pasteurella haemolytica.     

Induction of mycoplasmosis in goat kids by oral inoculation with Mycoplasma mycoides subspecies mycoides

A one-time, orally administered dose of greater than or equal to 1 X 10(6) colony-forming units of Mycoplasma mycoides subspecies mycoides was sufficient to induce clinical mycoplasmosis (n = 37) terminating in fatal mycoplasmemia in 73% (37 of 51) of the clinically affected kids. The pathogen was isolated from the blood samples as early as 24 hours after oral inoculation; hot, swollen joints frequently were evident by 4 or 5 days after exposure. Pyrexia (to 42.3 C) was detected in about 95% (35 of 37) clinically affected kids, although about 5% (2 of 35) died peracutely without fever or other premonitory signs. At necropsy, the cardinal lesions were a fibrinopurulent polyarthritis and red, patchy to diffuse areas of consolidation in 1 or more lung lobes. At death, usually within 4 to 16 days after oral inoculation, the concentration of M mycoides subspecies mycoides in the blood was 1 X 10(6) to 1 X 10(7) colony-forming units/ml. Histologically, the kids had diffuse fluid leakage into pulmonary alveoli and to a lesser extent into small vessels of various other organs. Fibrinocellular thrombi of terminal occurrence were occasionally present in various organs. The meningeal, pleural, and peritoneal surfaces had vascular leakage and a minimal perivascular accumulation of leukocytes. The disease was contagious. Of 14 noninoculated control kids in close confinement with affected kids, 8 (57%) developed mycoplasmosis in 7 to 15 days and died of mycoplasmemia. The remaining 5 noninoculated kids remained healthy, as did noninoculated kids that were kept isolated from affected kids.     

Development of real-time diagnostic assays specific for Mycoplasma mycoides subspecies mycoides Small Colony

Rapid and specific detection of Mycoplasma mycoides subsp. mycoides Small Colony (M. mycoides SC) is important for the effective control of contagious bovine pleuropneumonia. Although the United States has been free of this disease for over 100 years, it is necessary to develop modern diagnostic assays that are sensitive and specific for biological agents that would affect the US agricultural industry following accidental or intentional introduction into the US agricultural population. With this aim in mind, we have identified M. mycoides SC-specific genetic loci and developed TaqMan-based PCR assays for the detection of M. mycoides SC. The TaqMan assay allows for real-time detection of specific, amplified PCR products using portable equipment, enabling testing to be performed in the field. These assays are specific for M. mycoides SC, failing to amplify DNA from other organisms belonging to the M. mycoides cluster or two phylogenetically unrelated bovine mycoplasma species. Standard curves were drawn based on the linear relationships measured between the threshold fluorescence (C(T)) values and a measured quantity of genomic DNA. M. mycoides SC was successfully detected in bronchoalveolar lavage samples obtained from experimentally infected cattle. These TaqMan-based real-time PCR assays will allow for the rapid and specific detection of M. mycoides SC.     

Antigenic and genetic characterisation of lipoprotein lppC from Mycoplasma mycoides subsp. mycoides SC

Lipoprotein lppC, an immunodominant antigen, and its corresponding gene lppC were characterised in Mycoplasma mycoides subspecies mycoides small colony (SC) type, the etiological agent of contagious bovine pleuropneumonia (CBPP). The lppC gene was found in the type strain of M. mycoides subsp. mycoides SC and in field strains isolated in Europe, Africa, and Australia, as well as in vaccine strains. Southern blot analysis indicated the presence of at least four copies of lppC in the genome of M. mycoides subsp. mycoides SC, of which only one seems to be functional. Genes homologous to lppC have also been detected in closely related mycoplasmas such as M. mycoides subsp. mycoides large colony (LC) type and in M. sp. bovine group 7. lppC is encoded as a precursor with a consensus sequence for a prokaryotic signal peptidase II. The amino acid sequence of lppC and its precursor showed similarity to both LppB (at the N-terminal domain) and LppQ (at the C-terminal domain), two lipoproteins described previously in M. mycoides subsp. mycoides SC. The N-terminal domain of the mature lppC seems to be surface exposed. The C-terminal domain presented an integral membrane structure made up of five repeated units, rich in hydrophobic and aromatic amino acids, which may have pore forming potential in the mycoplasmal membrane. A recombinant peptide representing the N-terminal half of lppC was obtained following cloning in vector pETHIS-1 and expression in Escherichia coli hosts. The recombinant protein was used on immunoblots for serological analysis of sera from cattle that were naturally or experimentally infected with M. mycoides subsp. mycoides SC.     

The identification of an organism isolated from maned sheep as Mycoplasma mycoides subsp. mycoides

The natural host-range of M. mycoides subsp. mycoides is generally believed to be restricted to cattle, although the production in experimental conditions of a generalized infection of sheep and goats has been reported on some occasions.     

Isolation of Mycoplasma mycoides subspecies mycoides from Polyarthritis and Mastitis of Goats in Canada

The clinical signs, pathomorphological changes, and microbiological findings in Canadian goats infected with Mycoplasma mycoides subspecies mycoides are discussed. The disease affected mainly young goats and was characterized by septicemia and polyarthritis. Mastitis followed by septicemia was seen in two mature goats. The diagnosis was made by culture and identification of the mycoplasma. Infected goats without clinical signs were identified by cultural and serological (complement fixation) techniques. Healthy carriers are presumably able to transmit the infection and may have brought the disease to Canada.     

Biological effects of sonicated suspension and phenol-water extract of Mycoplasma mycoides subspecies mycoides in goats.

Strain Y3343 isolated from a goat with septicemia and polyarthritis was studied. The strain was virulent and induced septicemia, polyarthritis and coagulopathy in two goats. Limulus amebocyte lysate active material was present in plasma, but not in higher titre in inoculated goats. Sonicated mycoplasma material induced a dramatic somatic cell response in the mammary gland of cows and goats and marked clotting of the cows' milk, but it did not clot limulus amebocyte lysate or kill chick embryos. Phenol-water extract clotted limulus amebocyte lysate and induced somatic cell response in cows but not in goats. The phenol-water extract did not kill chick embryos, was not pyrogenic in rabbits or goats, and did not induce generalized Shwartzman reaction or change the leukocyte kinetics in rabbits. It therfore appears that the virulence mechanisms of strain Y3343 can not be explained on the basis of factors with strong endotoxin activity.     

Genetic and antigenic characterisation of elongation factor Tu from Mycoplasma mycoides subsp. mycoides SC

Specific serodiagnosis of contagious bovine pleuropneumonia (CBPP) is hampered by the low antibody titers against Mycoplasma mycoides subsp. mycoides small-colony type (MmmSC) antigens in calf serum due to persistent infections and by the existence of cross-reactions among the members of the mycoides cluster. In order to identify potential diagnostic antigens, we have constructed a genomic library from MmmSC which was screened with antibodies from naturally-infected animals. Using this strategy, a genome fragment has been isolated and characterised. The complete nucleotide sequence of this fragment revealed the presence of several open reading frames, including that of translation elongation factor Tu (EF-Tu), whose product was responsible of the positive reaction observed when expressed in E. coli. The organisation of this MmmSC genome region differed from that of other Mycoplasma species whose complete genome sequences are known, but was similar, by PCR amplification analysis of genomic DNA, to other members of the mycoides cluster, such as Mycoplasma capricolum subsp. capricolum (Mcc). Nevertheless, the MmmSC and Mcc amplicons could be distinguished by digestion with restriction enzymes AseI or HindIII, strategy that could be used as a tool for differential diagnosis of infections caused by members of the mycoides cluster. The full recombinant EF-Tu was produced in E. coli, after correction of an unusual tryptophan codon by site-directed mutagenesis, and used to investigate anti-EF-Tu circulating antibodies in bovine sera.     

Studies on Mycoplasma mycoides subsp. mycoides (LC) in lambs and calves.

Six cesarean-derived lambs were inoculated either with 4.5 X 10(4), 4.5 X 10(6) or 4.5 X 10(8) Mycoplasma mycoides subsp. mycoides intratracheally. One animal receiving the intermediate dose died four days post-inoculation, the two receiving the high dose died six days postinoculation, while one receiving the low dose died eight days postinoculation. The two surviving lambs were challenged on day 20 postinoculation with 1 X 10(8) organisms subcutaneously and 2 X 10(9) organisms intravenously. One animal died eight days following this challenge while the other survived and was killed. Six conventionally reared lambs challenged with 90 to 8500 organisms by intranasal and intraocular instillation failed to become infected. Three conventionally reared calves were each inoculated with 1 X 10(8) organisms by each of intratracheal, subcutaneous and intravenous routes. They were killed 20 days post-inoculation without having shown any clinical signs.