Large Colony Type Research Articles

​Caprine Respiratory Mycoplasmosis (Contagious Caprine Pleuropneumonia CCPP)- A Global Perspective of the Disease, Epidemiology, Diagnosis, Chemotherapy and Immunization: A Review

Mycoplasma infection of the respiratory tract of goats is prevalent worldwide including the South Asian sub-continent. Owing to intensive and large scale goat farming, the incidence of the disease is on an increase. Among various species of mycoplasma, Mycoplasma capricolum subspecies Capri pneumoniae is increasingly incriminated in Contagious Caprine Pleurapneumonia (CCPP) in goat populations with considerable economic fallout in the form of high morbidity and mortality. The disease manifestations in caprines are recorded as anorexia, high febrile reaction and respiratory embarrassment in the shape of clinical dyspnoea, polypnea, paroxysmal cough and sero-purulent nasal discharges. The disease is thus contracted by the healthy animals through aerosol, contaminated feed and water sources in the herd premises, without a protective immunity and that the conferred immunity in recovered cases being short-lived. The true lesions of CCPP are confined to the lung alveolar tissues of infected goats, which distinguish it from other respiratory diseases of small ruminants caused by the members of the Mycoplasma mycoides cluster. Atypical pneumonia caused by the mycoplasma infection of goats, also known as Contagious Caprine Pleuropneumonia (CCPP) has been more often reported from Africa and Asia than Europe. Classical, acute CCPP attributed to Mycoplasma capricolum subsp. Capri pneumoniae, originally known asthe F38 biotype (World Organisation for Animal Health, 2008) causes heavy kid mortality. Two other organisms in this group, M. mycoides subsp. capri and M. mycoides subsp. mycoides large-colony type, can cause disease in small ruminants that clinico-pathologically mimics CCPP but may have extra pulmonary signs and lesions, sometimes. Mycoplasma Capri pneumoniae and other members of the M. mycoides cluster cross-react in serological tests and share biochemical and genetic similarities. The most favourable epidemiological scenario in the Sub-continent is the hot humid climate during monsoons. The diversity and multi-etiological subspecies involved in the disease is detrimental in the development of an effective vaccine even though in some places a liquid vaccine is presently in use. At other places, anti-mycoplasmal antibiotics of aminoglycoside and fluoroquinolone and perhaps the macrolide groups remain to be the main option in preventing flock mortalities.

Species of Mycoplasma causing contagious agalactia in small ruminants in Northwest Iran.

Contagious agalactia (CA) is a serious disease of small ruminants that occurs in many countries, and is usually characterized by mastitis, arthritis, keratoconjunctivitis, pleuropneumonia, and septicemia. Mycoplasma agalactiae (Ma) is the main causative agent in sheep and goats but other pathogens including Mycoplasma mycoides subsp. capri (Mmc, which incorporates the former M. mycoides subsp. mycoides Large Colony type), Mycoplasma capricolum subsp. capricolum (Mcc), and Mycoplasma putrefaciens (Mp) might be involved. They are all usually associated with infections in goats and may cause similar clinical signs. A total of 116 sheep and 16 goats suffering from the acute form of the disease were included in this study. They were recruited following a number of outbreaks suspected to be CA in the Ardebil province of Iran. Milk, lachrymal or synovial fluid were collected exclusively from the affected animals in order to identify the pathogen involved. Of the 132 collected samples, 33 (25%) were positive for Mycoplasma species by culture in PPLO broth and agar. The polymerase chain reaction followed by denaturing gradient gel electrophoresis (PCR/DGGE) method identified 18 (12 sheep and 6 goats) of the 33 Mycoplasma positive samples with mixed Mycoplasma population. In particular, 25 Ma (47.2%), 23 Mp (43.4%), 4 Mcc (7.5%), and 1 Mmc (1.9%) were identified. This confirms that the several Mycoplasma species rather than the Ma only are in circulation, and are able to cause CA in sheep and goats in Iran. This is the first report on the isolation and identification of Mp, Mmc and Mcc in infected small ruminant flocks in Iran.

In vitro inflammatory responses elicited by isolates of Alloiococcus otitidis obtained from children with otitis media with effusion

Alloiococcus otitidis is usually detected in children with otitis media (OM) by PCR as it is not often detected by routine culture. Our improved method for its isolation obtained A. otitidis from nearly 50% of 78 children with OM with effusion. The role of A. otitidis in pathogenesis of OM is unclear. This study tested two hypothesis: (1) that fresh isolates of A. otitidis would elicit pro-inflammatory cytokines from THP-1 monocytic cells equivalent to those induced by Streptococcus pneumoniae; (2) priming THP-1 cells with interferon-gamma (IFN-γ) a surrogate for virus infection, would enhance pro-inflammatory responses. Recent clinical isolates of A. otitidis, S. pneumoniae (ATCC 49619) and a blood culture isolate of S. pneumoniae (SP2) were used in the assays. Cytokines were quantified by BioRad bead assay and Luminex 200. IFN-γ priming enhanced cytokine responses. S. pneumoniae ATCC 49619 induced lower responses than SP2 for IL-1β, IL-6, TNF-α. A. otitidis LW 27 elicited higher IL-1β and TNF-α responses than either pneumococcal isolate. Small green colony types of A. otitidis induced higher responses than large white colony types for IL-8 and IL-1β. The hypothesis that A. otitidis elicits cytokines observed in middle ear effusions was supported; the need to use recent clinical isolates in studies of pathogenesis was highlighted.

Impact of identification of Streptococcus dysgalactiae subspecies equisimilis from throat cultures in an adult population

Streptococcus dysgalactiae subspecies equisimilis (SDSE) are isolated from the throat of patients with pharyngitis, although the clinical significance remains debated. We sought to determine the incidence and association with pharyngitis of SDSE in an adult veteran population. Organisms were phenotypically identified to subspecies and Lancefield group, with selective 16S rRNA gene sequencing. From 833 throat cultures, the overall frequency of SDSE was 3.4% (64% group C and 36% group G) as compared to 8.6% for S. pyogenes (GAS). SDSE was described as a large colony in only 29% of the original culture evaluations by bench technologists, and clinical symptoms were similar for GAS and SDSE. Laboratory algorithms that are limited to identification of only GAS or are based on Lancefield group or visual identification of “large-colony type” β hemolytic Lancefield group C and G streptococci may be missing or misidentifying SDSE along with Anginosus group streptococci.

Further evidence to justify reassignment of Mycoplasma mycoides subspecies mycoides Large Colony type to Mycoplasma mycoides subspecies capri

Analysis, using the polymerase chain reaction (PCR), restriction enzyme endonuclease analysis (REA), protein profile patterns, random amplification of polymorphic DNA (RAPD) fingerprinting, 16S rRNA gene sequencing and antisera growth inhibition tests, of 22 strains of Mycoplasma mycoides subsp. mycoides Large Colony type (MmmLC) and eight strains of M. mycoides subsp. capri (Mmc) are presented, along with a summary of comparative data from the literature for over 100 strains, all of which supports the reclassification of the MmmLC and Mmc strains into the single subspecies, M. mycoides subspecies capri.

Kinetics of substrate oxidation and hydrogen peroxide production by Mycoplasma mycoides subsp. mycoides Large Colony (LC) type and Mycoplasma mycoides subsp. capri

Mycoplasma mycoides subsp. mycoides Large Colony (LC) type is a pathogen of goats causing contagious agalactia and respiratory disease, found on all continents where small ruminants are kept. It shares close genetic characteristics with M. mycoides subsp. capri. Substrate oxidation by 22 strains of M. mycoides subsp. mycoides LC from nine countries was compared with that of eight strains of M. mycoides subsp. capri from five countries. There was considerable similarity in the substrates used, but substrate saturation coefficients ( K s) varied for different substrates. Substrate utilization patterns and K s values did not (1) significantly differentiate the LC strains from each other, (2) show any correlation with geographical origin, or (3) distinguish the LC strains from the capri strains. These results support previous studies justifying the reclassification of these subspecies as a single species.

Analysis of the immunoproteome of Mycoplasma mycoides subsp. mycoides small colony type reveals immunogenic homologues to other known virulence traits in related Mycoplasma species

Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small colony type ( MmmSC) has been eradicated in the developed world, but it is still present in many countries of sub-Saharan Africa. After initially successful control measures in the 1960s it has been spreading due to a lack of money, fragmentation of veterinary services, uncontrolled cattle movement, insufficient vaccine efficacy and sensitivity of current diagnostic tests. In this study we used two-dimensional polyacrylamide gel electrophoresis followed by immunoblot with sera from MmmSC-infected animals and MALDI-ToF mass spectrometry to identify novel immunogenic proteins as candidate molecules for improved diagnostics and vaccines. We identified 24 immunogens recognized by pooled sera from experimentally infected cattle. Furthermore, a serum from an animal with acute clinical disease as well as severe pathomorphological lesions recognized 13 additional immunogens indicating variation in the antibody responses to CBPP amongst cattle. Most immunogens showed compelling similarity to protein/gene sequences in the two ruminant pathogens M. capricolum subsp. capricolum and M. mycoides subsp. mycoides large colony type both belonging to the mycoides cluster. Three of these proteins, namely glycerol-3-phosphate oxidase, adenylosuccinate synthase, and glyceraldehyde-3-phosphate dehydrogenase, had no compelling homologue in the other distantly related bovine pathogen M. agalactiae. In addition, translation elongation factor Tu, heat shock protein 70, pyruvate dehydrogenase, and FKBP-type peptidyl-prolyl isomerase, which have been found to mediate adhesion to host tissue in other mycoplasmas were shown to be expressed and recognized by sera. These proteins have potential for the development of improved diagnostic tests and possibly vaccines.

Survey of antibodies to Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (large colony type) in dromedaries ( Camelus dromedarius) in close contact with an infected goat population

Given recent evidence of a possible role of camelids in the epidemiology of diseases caused by mycoplasma species in other ruminants, we examined the prevalence of the mycoplasmas most commonly responsible for contagious agalactia (CA) in small ruminants among the dromedaries ( Camelus dromedarius) of Lanzarote. On this island, one of the most arid regions of Spain in which CA is considered endemic, dromedaries and infected small ruminants such as goats and sheep, live in close contact. Specific IgG antibodies against Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (large colony biotype) were determined using two indirect enzyme immunoassays in 28% of the island's dromedary population. None of the serum samples tested were found to have antibodies against the two mycoplasma species, suggesting that the camels are not potential carriers of the main causative agents of CA.

Sugar uptake byMycoplasma mycoides strain PT994 andMycoplasma canis strain 85 B97

A pathogenic strain of each ofMycoplasma mycoides subsp.mycoides (Large Colony type) (strain PT994) andMycoplasma canis (strain 85 B97) showed different patterns of sugar uptake when provided with glucose and a disaccharide. Maltose had no effect on the uptake of glucose or of α-methylglucoside byM. mycoides, whereas glucose inhibited the uptake of maltose. Maltose uptake by growing cultures ofM. mycoides recommenced immediately after glucose exhaustion. There was thus an interaction between the two sugars for uptake, with glucose inhibiting the uptake of maltose either by competing for a common transporter or by suppression of a specific maltose-binding system. In contrast, uptake of glucose and sucrose byM. canis showed no interaction, with both sugars being used concurrently, and were thus transported by independent systems. Sugar uptake by both species was inhibited by fluoride, consistent with the operation of glycolysis, and possibly phosphoenolpyruvate-dependent sugar uptake systems.

Strains of Mycoplasma mycoides subsp . mycoides small colony type isolated in China between 1953 and 1960 show close similarity to strains of the Africa/Australia cluster

In this study, six Chinese strains of Mycoplasma mycoides subsp. mycoides small colony type (MmmSC) isolated between 1953–1960 were analysed and their molecular characteristics compared to those of the African PG1 and Afade strains, the European C305 and 138/5 strains and the closely related caprine M. mycoides subsp . mycoides large colony type Y-goat strain. PCR amplification of long DNA fragments showed that the six Chinese strains, the PG1 strain and the Y-goat strain, just like Afade, did not have the 8.84 kb deletion characteristic of the European strains C305 and 138/5. In comparison, the lppB gene sequence of the six MmmSC Chinese strains was found to be 99% homologous to that of PG1and Afade, but <93% homologous to the Y-goat sequence. The anti-rLppB antiserum reacted with PG1, Y-goat and the six Chinese strains at 67 kDa sites in Western blot, indicating that the lppB gene and its encoding protein exist in the Chinese strains. Multilocus sequence analysis (MLSA) of MmmSC strains from various regions confirmed that the Chinese strains were identical to the African and Australian cluster. This finding was further supported by the outcome of selective primer amplification. Based on these results, it is suggested that CBPP in China may have originated from Australia.

Detection of mycoplasmas in goat milk by flow cytometry

The detection of mycoplasma in milk can be performed by either culture techniques or polymerase chain reaction (PCR) based methods. Although PCR can reduce the average diagnostic time to 5 h in comparison with the several days for the isolation of the agent, there is still a need to develop methods, which could give earlier results. For this purpose, we tested the ability of flow cytometry (FC) to detect mycoplasmas in milk samples. Milk samples inoculated with four different mycoplasmas, Mycoplasma agalactiae, Mycoplasma putrefaciens, Mycoplasma capricolum subsp. Capricolum, or Mycoplasma mycoides subsp. mycoides large-colony type, known to cause contagious agalactia in goats, were stained with the DNA stain SYBR Green I and analyzed by FC. Three goat milk samples, from which mycoplasmas have been isolated in broth medium were also analyzed. All mycoplasmas were easily distinguished from debris of milk samples, but it was not possible to distinguish between the different mycoplasma species. In our conditions, the detection limit of the technique was of the order of 10(3)-10(4) cells ml(-1). Furthermore, mycoplasmas were also distinguished from Staphylococcus aureus. FC together with SYBR Green I was able to distinguish between mycoplasma cells and debris present in milk samples and gave results in 20-30 min. This is an important first step in developing a robust, routine flow cytometric method for the detection of mycoplasmas in milk samples.

Field trial of a combined vaccine against caprine contagious agalactia: Humoral immune response in lactating goats

Two vaccines against Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (large colony type) were developed using inactivated strains selected in previous characterization studies. Formaldehyde and phenol were used as the inactivating agents for vaccines A and B, respectively. Aluminium hydroxide plus purified saponin (Quil-A) were added to both vaccines as adjuvant. The field trial was designed to evaluate the specific humoral immune response to the two mycoplasma species in lactating goats over a period of 7 months. The vaccines were tested on 120 goats randomly assigned to three groups of 40 animals each. Two groups received two injections of vaccine A or B respectively, and a third group remained in the herd as control. Antibody titres determined by ELISA indicated a significant difference between both vaccines and the control group over a 6-month period. Immunoblotting assays also revealed the production of antibodies against the two mycoplasma species. Further field trials are underway to evaluate the efficacy and protection conferred to the animals by these specific antibodies.

Field trial of two dual vaccines against Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (large colony type) in goats

Two vaccines against Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (LC type) were developed using inactivated strains selected in previous characterization studies. The vaccines differed in terms of the adjuvants used: aluminium hydroxide (vaccine A) or aluminium hydroxide plus purified saponin (vaccine B). These vaccines were tested on 60 pregnant goats and 60 seronegative kids that were challenged by placing in a herd with a history of caprine contagious agalactia (CCA). Our findings indicate the effectiveness of the vaccines in preventing the appearance of new clinical signs such as mastitis, abortion, pneumonia and polyarthritis in CCA affected herds.

In vitro susceptibilities of field isolates of Mycoplasma mycoides subsp. mycoides large colony type to 15 antimicrobials

In vitro susceptibilities of 16 Mycoplasma mycoides subsp. mycoides large colony type field isolates to 15 antimicrobial agents were determined using a broth microdilution method. The most effective antimicrobials were fluoroquinolones, tetracyclines and macrolides, with MIC values under 2 μg/ml. Resistance to nalidixic acid, gentamicin, streptomycin and spectinomycin was observed.

Identification and Characterization of an Aeromonas salmonicida (syn Haemophilus piscium) Strain That Reduces Selenite to Elemental Red Selenium

A bacterium that reduces toxic and mobile selenite to insoluble elemental selenium (Se0) was isolated from a laboratory scale permeable reactive biobarrier. Biochemical tests and 16S rRNA gene sequence alignment identified the isolate as Aeromonas salmonicida. Two colony types were isolated, one more resistant to selenite than the other. Both grew on agar plates containing 16 mM: selenite, although the colony diameter was reduced to 8% of controls with the small colony type and to 18% with the large colony type. Further study was done with the large colony type. In anaerobic culture, this bacterium was able to use nitrate as a term electron acceptor but not selenate or selenite. In aerobic culture, when no nitrate was present, early log phase cells removed selenite at a rate of 2.6 +/- 0.42 micromol SeO3 (-2)/mg protein/day. Reduction was retarded by 25 mM: nitrate. Mutants with a diminished ability to reduce selenite to Se0 also had a reduced ability to reduce nitrate to nitrous oxide. This bacterium, or perhaps its enzymes or DNA, might be used to remove selenite from contaminated groundwaters.

Use of flow cytometry for enumeration of Mycoplasma mycoides subsp. mycoides large-colony type in broth medium

The potential of using flow cytometry (FC) in combination with a fluorescent dye (SYBR green-I) for rapidly estimating Mycoplasma mycoides subSPS. mycoides large-colony type (MmmLC) in broth culture was examined. The FC analysis was performed by staining the MmmLC cells with a fluorescent dye, SYBR green-I (SYBR), and the results were compared with plate count method (colony forming units, - CFUs). There was a good correlation (linear regression, r(2) = 0.93) between mycoplasma counts determined by FC (cells ml(-1)) and by traditional plate count method (CFU ml(-1)). The lowest bacterial concentration detected by FC and traditional plate count was of the order of 10(4) cells ml(-1) and 10(3) CFU ml(-1), respectively. FC method allowed results in 20-30 min, whereas at least 24 h were necessary to obtain results with the traditional plate count method (CFU). Growth rates of MmmLC in broth medium determined by FC were highly reproducible and correlated well with mycoplasma counts assessed by the plate count method. These findings suggest that FC could be a good alternative to replace other time-consuming techniques that are currently used to enumerate mycoplasma in broth medium, such as plate count method (CFU).

First isolation of Mycoplasma capricolum subsp. capricolum, one of the causal agents of caprine contagious agalactia, on the island of Lanzarote (Spain)

During an unusually long period of bad weather, several outbreaks of caprine contagious agalactia (CCA) were reported in a number of flocks on the island of Lanzarote (Canary Islands, Spain). Clinical and subclinical mastitis in lactating goats and some cases of arthritis and pneumonia in kids were observed in the affected flocks. Mycoplasma capricolum subsp. capricolum was isolated as the main causal agent of the outbreaks, associated with M. mycoides subsp. mycoides “large colony type” ( Mmm LC) in two flocks. This is the first report of an isolation of M. capricolum subsp. capricolum on the island of Lanzarote. The finding is of epidemiological importance and could complicate plans to control the disease. The significance of this mycoplasma species in association with CCA must now be studied in detail.

Isolation and molecular identification of small ruminant mycoplasmas in Jordan

Mycoplasma infections of small ruminants are known to exist in the Mediterranean region, Asia, Africa and cause significant economic impacts but little is known of the Mycoplasma spp. in sheep and goats in the Middle East. During the period of 2002–2003, 104 flocks of local sheep and goats (17 sheep, 27 goat and 60 mixed flocks) were surveyed for the occurrence of mycoplasma infections in Northern Jordan. The clinical signs seen in the studied flocks were, to varying degrees, mastitis in sheep and goats, arthritis, mainly in kids, and pneumonia in both sheep and goats of most age groups. Small ruminant farms were sampled and pooled milk samples and nasal swabs were collected for culture and isolation of mycoplasma. Mycoplasmas were isolated from 17 (26%) of the 62 milk samples and 12 (3.9%) of the 310 nasal swab samples collected from goats and from 8 (13%) of the 62 milk samples and 7 (2.3%) of the 310 nasal swabs collected from sheep. In all 44 isolates were fully identified using growth inhibition test (GI), denaturing gradient gel electrophoresis (DGGE) and specific polymerase chain reactions. These were: Mycoplasma mycoides subsp. mycoides large colony type ( Mmm LC) ( n = 9), Mycoplasma capricolum subsp. capricolum ( Mcc) ( n = 13), Mycoplasma putrefaciens ( n = 21) and Mycoplasma agalactiae ( n = 1); all were isolated from both sheep and goats. Several isolates could not be identified. This is the first report on the isolation and identification of small ruminant mycoplasmas in Jordan.

Assessment of PCR for routine identification of species of the Mycoplasma mycoides cluster in ruminants

DNA amplification techniques offer considerable promise for the identification of Mycoplasma mycoides cluster members. They avoid antigenic cross-reactivity and variability that hamper serological methods. Many sets of primers, specific of these different members and of Mycoplasma putrefaciens, have been proposed. To assess the reliability of some of these PCR tests in routine laboratory diagnostic use, 230 field strains supposed to belong to this group were simultaneously identified by PCR and an antigenic method. The results were well correlated to antigenic identification for M. putrefaciens, but PCR failed to identify respectively 74% and 52% of M. mycoides subsp. mycoides Large Colony type and M. capricolum subsp. capricolum strains. Any identification of M. mycoides subsp. mycoides Small Colony type must be confirmed by two different tests. Difficulties in defining the M. species bovine serogroup 7 were also encountered with both the PCR and immunological methods. The occurrence of putative variable antigen(s) on the mycoplasma surface may explain part of the identification difficulties encountered with the immunological methods.

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.