Comparison of different NAT assays for the detection of microorganisms belonging to the class Mollicutes.

BACKGROUND: Mollicutes detection can be cumbersome due to their slow growth in vitro. For this reason, the use of DNA based on generic molecular tests represents an alternative for rapid, sensitive and specific detection of these microorganism. For this reason, six previously described nucleic acid testing assays were compared to evaluate their ability to detect microorganisms belonging to the class Mollicutes. METHODS: A panel of 61 mollicutes, including representatives from the Mycoplasma, Acholeplasma, Mesoplasma, Spiroplasma and Ureaplasma genus, were selected to evaluate the sensitivity and specificity of these assays. A total of 21 non-mollicutes, including closely related non-mollicutes species, were used to evaluate specificity. Limits of detection were calculated to determine the analytical sensitivity of the assays. The two best performing assays were subsequently adapted into real-time PCR format, followed by melting curve analysis. RESULTS: Both assays performed satisfactorily, with a 100% specificity described for both assays. The detection limits were found to be between 10-4 and 10-5 dilutions, equivalent to 15 to 150 genome copies approximately. Based on our work, both van Kuppeveld and Botes real-time PCR assays were found to be the best performing tests in terms of sensitivity and specificity. Furthermore, Botes real-time PCR assay could detect phytoplasmas as well. CONCLUSIONS: These assays can be very useful for the rapid, specific and sensitive screening cell line contaminants, clinical samples as well as detecting non-culturable, unknown species of mollicutes or mollicutes whose growth is slow or difficult.

Two strains of Mycoplasma synoviae from chicken flocks on the same layer farm differ in their ability to produce eggshell apex abnormality.

Mycoplasma synoviae (Ms) is considered to be an economically important poultry pathogen. Although the full economic costs of infection in layer chickens are still under debate, the prevalence of Ms is known to be high in some countries and earlier reports have shown a correlation between infection and Eggshell Apex Abnormality (EAA). This work is a continuation of an earlier study of a clinical case of EAA on a layer hen farm where the presence of two different strains of Ms, based on the sequence of the 5' end of the vlhA gene, was demonstrated. Both strains could be detected in the trachea but only one (designated strain PASC8) appeared able to colonize the oviduct, while the other (designated TRACH) was not found in the oviduct and has not been related to EAA. The PASC8 partial vlhA gene sequence differs from that of the TRACH in having a 39 nucleotide deletion in the proline rich region and three point mutations in the RIII region. Based on this information an experimental infection was performed in SPF chickens using groups infected with either the PASC8 or the TRACH strain and a non-infected control group. Both Ms strains were detected in the trachea of infected birds, but only the PASC8 strain was found in the oviduct. Furthermore, EAA developed only in the group infected with PASC8 strain. Compared to the control group, both strains produced an adverse impact on egg production: a decrease in the numbers laid and in their average weight (P<0.05) This work demonstrates a difference in oviduct tropism between two Ms strains and a possible relationship to the production of EAA in experimental conditions.

Development and evaluation of an improved diagnostic PCR for Mycoplasma synoviae using primers located in the haemagglutinin encoding gene vlhA and its value for strain typing.

Using published primers, detection of Mycoplasma synoviae and strain identification using the vlhA gene sequence was attempted. However, of 21 M. synoviae strains examined, three could not be amplified, so a new reverse primer was designed with a target in the conserved region of the vlhA gene. This allowed all 21 M. synoviae strains, a further nine strains and also material from 11 swab samples from M. synoviae-positive birds, to produce a PCR product, suggesting that the method could also be suitable for clinical specimens. The protocol was then tested on the type strains of M. synoviae and the other 22 recognised avian Mycoplasma species, with amplification of M. synoviae only. Further testing demonstrated that this PCR was equally or more sensitive than other PCR tests used to detect M. synoviae. Subsequent DNA sequence analysis of the PCR product based on percent similarity and evolutionary relationship appeared to be a useful tool for strain differentiation.

High inter-species and low intra-species variation in 16S-23S rDNA spacer sequences of pathogenic avian mycoplasmas offers potential use as a diagnostic tool.

In order to investigate its value for phylogenetic analysis, species characterisation and diagnosis, the 16S-23S rDNA intergenic spacer regions (ISRs) of the type strain of 23 avian Mycoplasma species were amplified and the sequences determined. Also sequenced were the reference strains of Mycoplasma iowae serotypes J, K, N, Q and R and a number of field strains of Mycoplasma synoviae, Mycoplasma gallisepticum, Mycoplasma meleagridis and M. iowae. The ISRs demonstrated a high level of size variation (178-2488bp) between species and did not include tRNA genes. Phylogenetic analysis performed using the information conflicted with that based on the 16S rDNA and was therefore not helpful for phylogenetic studies. However, the ISR did appear to be of value for determining species since there was high inter-species variation between all 23 avian Mycoplasma species, and in addition there was low intra-species variation, at least in the four pathogenic species. It could also be very useful as additional information in the description of a new species and as a target for species-specific PCRs.

Survey of campylobacter, salmonella and mycoplasmas in house crows (Corvus splendens) in Malaysia.

House crows (Corvus splendens) in Selangor, Malaysia were examined for the presence of Campylobacter species, Salmonella species, Mycoplasma gallisepticum and Mycoplasma synoviae by serology, culture and pcr. For the detection of Campylobacter and Salmonella species swabs were taken either from the intestine or cloaca. For the detection of mycoplasmas, swabs were taken either from the choanal cleft or trachea for culture and pcr and serum samples were tested by the rapid serum agglutination (rsa) and monoclonal antibody-blocking elisa (mbelisa) for antibodies to M gallisepticum and M synoviae. For campylobacter, 25.3 per cent of the crows were positive by culture, and the species identified were Campylobacter jejuni and Campylobacter coli. No Salmonella species were isolated. Four of 24 swabs were positive for M gallisepticum dna but none gave positive results for M synoviae dna. No M gallisepticum or M synoviae antibodies were detected by rsa but 60 per cent of the sera gave positive reactions for M gallisepticum and 13 per cent gave positive reactions for M synoviae by mbelisa.

Mycoplasma amphoriforme sp. nov., isolated from a patient with chronic bronchopneumonia.

A mycoplasma was isolated from the sputum of an immunodeficient patient with recurrent bronchitis. The isolate designated strain A39T was very fastidious and atypical for a mycoplasma in its colonial appearance. Classical biochemical tests for mycoplasma speciation could not differentiate the isolate from the pathogens Mycoplasma pneumoniae and Mycoplasma genitalium and serological identification as a recognized Mycoplasma species was lacking. Specific PCR detection for these two species was negative. Subsequently, other strains were isolated from human patients that appeared to be similar to strain A39T in their physiological and genetic characteristics. Analysis of the 16S rRNA gene placed strain A39T and other isolates in the pneumoniae group of mycoplasmas, with the highest sequence similarity to Mycoplasma testudinis (96.8 %), but with only 93.0 % similarity to M. pneumoniae and M. genitalium. Examination of the 16S-23S rRNA internally transcribed spacer sequence, protein electrophoresis profile, genome size and serological reactions indicated that this organism represents a novel species, for which the name Mycoplasma amphoriforme sp. nov. is proposed, with strain A39T (=NCTC 11740T=ATCC BAA-992T) as the type strain.

Gordon Memorial Lecture. Poultry mycoplasmas: sophisticated pathogens in simple guise.

Mycoplasmas are a genus within the class Mollicutes (trivial name mollicutes), which are the smallest known prokaryotes capable of self-replication. They have a very small genome, and have evolved to this 'minimalist' status by losing non-essential genes, including those involved in cell wall synthesis. The mollicutes exploit their limited genetic material to the maximum and many are successful pathogens in man, animals, birds and plants. Most of those of veterinary importance are in the genus Mycoplasma and include 4 poultry pathogens of economic importance: Mycoplasma gallisepticum, Mycoplasma synoviae, Mycoplasma meleagridis and Mycoplasma iowae. The pathogenetic mechanisms of mycoplasmas are not fully understood, but they are successful pathogens because they can enter the host and multiply, evade the defence mechanisms, cause damage and escape to infect new hosts. M. gallisepticum is one of several motile species and possesses a terminal tip structure that mediates adherence to its target tissues. For some species, including M. gallisepticum, some of the organisms may become intracellular. Some Mycoplasma species, including the pathogenic poultry species, have a remarkable ability to vary their major surface antigens, a mechanism that is thought to help them to persist in their host by evading the immune response. The molecular and cellular events that lead to the development of lesions and clinical disease are still obscure. Some lesions appear to be the result of indirect damage from the host's inflammatory and cellular responses. Despite short survival times in the environment, mycoplasmas are able to transmit successfully to new hosts. In poultry flocks there is both horizontal and vertical transmission, the former being encouraged by intensive husbandry and stress factors. Establishing the pathways of transmission and the possible role of other birds, such as game and wild birds, as intermediate vectors between poultry flocks is now greatly aided by the availability of modern molecular methods for strain typing.

Effects of cyclosporin A on the immune responses and pathogenesis of a virulent strain of Mycoplasma gallisepticum in chickens.

Immune responses to the virulent S6 strain of Mycoplasma gallisepticum in immunocompetent and cyclosporin A (CsA)-treated specific pathogen free chickens were investigated, and pathogenesis of the M. gallisepticum strain was also examined. Ten-day-old specific pathogen free chickens were inoculated by eye-drop with M. gallisepticum, and a control uninfected group was inoculated with mycoplasma broth. Blood was collected weekly for 4 weeks from five birds in each group and whole blood lymphocyte transformation assayed against concanavalin A and lipopolysaccharide. Blood samples were also collected at intervals for serological assays. Live body weight, clinical signs and lesions were monitored, and recovery of M. gallisepticum was attempted from choanal cleft of live birds and also from various sites at necropsy. In parallel to the aforementioned groups, another set of two groups of chicks treated with CsA was infected with M. gallisepticum S6 or mycoplasma broth. These groups were subjected to the same experimental procedures. In the immunocompetent chickens, M. gallisepticum caused temporary T-cell suppression at 2 weeks post-infection. Comparison of the clinical signs and macroscopic lesions produced in immunocompetent and CsA-treated chickens indicated that T cells may not play an active role in disease development. The percentage of birds with mycoplasma isolation and the load of mycoplasmas suggested that T cells may have some role in resisting mycoplasma colonization or in the elimination of the infection.

The complex matter of DNA double-strand break detection.

To maintain genomic stability, despite constant exposure to agents that damage DNA, eukaryotic cells have developed elaborate and highly conserved pathways of DNA damage sensing, signalling and repair. In this review, we concentrate mainly on what we know about DNA damage sensing with particular reference to Lcd1p, a yeast protein that functions early in DNA damage signalling, and MDC1 (mediator of DNA damage checkpoint 1), a recently identified human protein that may be involved in recruiting the MRE11 complex to radiation-induced nuclear foci. We describe a model for the DNA damage response in which factors are recruited sequentially to sites of DNA damage to form complexes that can amplify the original signal and propagate it to the multitude of response pathways necessary for genome stability.

Infectious agents associated with respiratory disease in pheasants.

In a case-control study of the infectious agents associated with natural outbreaks of respiratory disease in pheasants, 28 batches of birds from sites affected by disease and eight batches of birds from unaffected sites were examined by six veterinary laboratories in England, Wales and Scotland, and tested for mycoplasmas, other bacteria and viruses. Sinusitis was the commonest sign of disease and was associated with Mycoplasma gallisepticum as detected by PCR in the trachea (P < 0.05) and conjunctiva (P < 0.01). Sinusitis was also associated with pasteurella cultured from the sinus (P < 0.05), antibody to avian pneumovirus (APV) (P < 0.01) and avian coronaviruses as detected by reverse-transcriptase PCR (P < 0.05); there was no association between disease and APV as detected by PCR. Avian coronaviruses were the most common infectious agents detected. They were genetically close to infectious bronchitis virus (IBV) but differed in their gene sequence from all the serotypes of IBV previously identified in domestic fowl, and serological tests with six known IBV types showed little cross reactivity. Mycoplasma species other than M gallisepticum were cultured in 18 batches of pheasants but, with the exception of Mycoplasma gallinaceum, were not associated with disease.

Detection of Mycoplasma synoviae in clinically normal pheasants.

Mycoplasma synoviae was isolated from the tracheas of seven clinically normal pheasants found in the vicinity of a chicken farm infected with M synoviae, but not from 120 pheasants and partridges with respiratory disease. When specimens were examined by the polymerase chain reaction only two additional pheasants infected with M synoviae were identified, one healthy and one diseased.

Avian pneumovirus infection in broiler chicks inoculated with Escherichia coli at different time intervals.

The effects of dual infection of 1-day-old broiler chicks with a chicken isolate of avian pneumovirus (APV) and a pool of pathogenic Escherichia coli strains were studied by supraconjunctival application of the bacteria simultaneously with the virus, or at 4, 7 or 11 days afterwards. When the agents were given together, the clinical disease was significantly more severe than that caused by the virus alone, but when the bacterium was given later the signs were less severe. None of the infections resulted in swollen head syndrome by 32 days. All mixed infections caused moderate to severe congestion in the turbinates, when birds were examined at 32 days of age, at which time no such lesions were present in birds having been infected with APV alone. E. coli was isolated from almost 100% of birds with mixed infections, while rates of those given only E. coli isolation varied between 56 and 67%. Furthermore, E. coli colony counts were consistently higher from mixed infection groups. Virus persistence in the choanal cleft was slightly prolonged in birds with the simultaneous mixed infection. Although the pool of E. coli included O2, O78 and O18 serotypes, only those of the O2 serotype and a small number of untypable strains were re-isolated from selected mixed and single E. coli-infected groups. Mixed APV and E. coli infection did not affect APV enzyme-linked immunosorbent assay antibody titres at 21 or 32 days. Thus, experimental infection of broiler chicks with APV and E. coli, simultaneously or at intervals afterwards, demonstrated a synergistic effect between the two agents, but none of the infection protocols caused swollen head syndrome.

Mycoplasmas and respiratory disease in pheasants and partridges.

Pheasants and partridges with signs of upper respiratory disease were cultured for mycoplasmas and were also examined for Mycoplasma gallisepticum and Mycoplasma synoviae using commercial polymerase chain reaction (PCR) kits. Sixty-two incidents of disease were investigated in pheasants and 12 in partridges. M. gallisepticum was detected by culture in only four and three incidents in pheasants and partridges, respectively, but with PCR a further 15 M. gallisepticum-positive incidents were detected in pheasants and another five in partridges. Several fast-growing Mycoplasma species, in particular Mycoplasma glycophilum, Mycoplasma gallinaceum and Mycoplasma pullorum, were isolated frequently and were thought to be impeding the isolation of M. gallisepticum by outgrowing it. Samples yielding M. gallisepticum isolates contained significantly fewer "contaminating" species and were exclusively from specimens submitted as whole heads rather than as swabs or as cultures from other laboratories. M. synoviae was not isolated and was detected in only one specimen by PCR.

Dual infection of turkey poults with avian pneumovirus and Mycoplasma synoviae.

Poults were infected with avian pneumovirus (APV) at 1-day-old, followed by Mycoplasma synoviae (Ms) 3 days later. Dual infection did not cause an increase in severity of clinical disease, or gross and microscopic lesions due to APV. The patterns of virus and Ms isolations from tracheal swabs or tissues from single and dual infected groups were similar. Ms infection did not induce Ms antibodies, nor did it affect seroconversion to APV in the dual infection.

Pathogenicity of Mycoplasma imitans in mixed infection with infectious bronchitis virus in chickens.

Mycoplasma imitans (Mim) has been isolated from ducks, geese and partridges, and is closely related to Mycoplasma gallisepticum (Mg). The pathogenicity of Mim for chicks was investigated in single and mixed infections with infectious bronchitis virus (IBV) by giving IBV strain M41 at 1-day-old and Mim 2 days later. Single infections with IBV or Mim were also performed. No clinical signs or gross lesions were seen in chicks infected with Mim or uninfected control chicks, but they were seen in the other two groups. Clinical scores were consistently higher in birds with mixed infections than those infected with IBV alone, and were significantly higher (P < 0.05) between days 7 and 14. More birds developed sinusitis, tracheitis and airsacculitis (with greater severity) in the mixed than the single IBV infections. Mim was recovered more frequently and in greater numbers from the respiratory tract of birds with mixed than single infections. It was recovered from the lower trachea, air sacs and lungs only in mixed infections. Seroconversion to Mim occurred by day 14 in mixed infections, but not until day 21 in single infections. It appears that Mim can act synergistically with IBV in young chickens in a similar manner to Mg, although Mg may act as a primary pathogen under some circumstances.

Pathogenicity of in vivo-passaged Mycoplasma imitans in turkey poults in single infection and in dual infection with rhinotracheitis virus.

Mycoplasma imitans (Mim) is a close relative of Mycoplasma gallisepticum (Mg), but has been isolated from ducks, geese and partridge. In order to investigate its potential pathogenicity for turkeys a UK isolate of Mim from a partridge with sinusitis was first passaged through turkey poults and then assessed for pathogenicity in turkey embryo tracheal organ cultures (TOCs) and in one-day-old turkey poults with or without turkey rhinotracheitis virus (TRTV). Mim appeared to gain virulence on passage through poults and this was confirmed by an increased ciliostatic effect in TOCs. In single infection in poults the organism caused mild upper respiratory disease but in dual infection with TRTV there was a significant increase in clinical signs and lesions. The mycoplasma was only isolated from upper respiratory tract in single infection but was recovered also from lung and airsacs in the presence of the virus. There was also a higher humoral immune response in the dual infection than the single Mim infection and there were some cross-reactions with commercial Mg stained antigen in the rapid serum agglutination test.

Pathogenicity of Mycoplasma gallisepticum and Mycoplasma imitans in red-legged partridges (Alectoris rufa).

Groups of 3-day-oId red-legged partridges were infected intranasally either with the S6 strain of M. gallisepticum or with an M. imitans strain from a partridge with sinusitis. Starting 6-8 days post-infection (p.i.) birds in both groups developed signs of depression, nasal exudation, tracheal rales, sneezing, gasping, head shaking, watery eyes and eye scratching. The most outstanding feature was bilateral swelling of the infraorbital sinuses. Morbidity reached 100% in the M. gallisepticum infection and 80% in the M. imitans infection and mean clinical scores in the former were significantly greater than those of the latter group on days 11 and 14 p.i. There was also slower recovery in the M. gallisepticum infection. Necropsies at weekly intervals for 5 weeks revealed nasal and sinus exudate in both groups but tracheal exudate and cloudy airsacs were seen only in M. gallisepticum infection. M. gallisepticum was isolated from both upper and lower respiratory tract throughout the experiment while M. imitans was recovered less frequently from the upper respiratory tract and from the lungs and air sacs only at 7 days p.i. The numbers of isolations from eyes, tracheas, lungs and thoracic air sacs of the M. gallisepticum group were significantly greater than those from the M. imitans group. Seroconversion occurred in both groups using homologous antigen.