Experimental evidence of indirect transmission of Mycoplasma synoviae

The aim of the study was to analyse experimental transmission of Mycoplasma synoviae, an avian pathogen. Three experiments using specific pathogen-free day-old chicks placed in isolators were conducted. In the first experiment, the birds were introduced in an isolator previously contaminated with a M. synoviae broth culture. After 34 days, these birds were eliminated and, for the second trial, the chicks were introduced in the same isolator without disinfecting. In the third assay, the chicks were placed in an isolator containing a mixture of food, feathers and dust collected less than an hour earlier from a M. synoviae infected laying hen flock. In the second and third experiments in order to exacerbate the M. synoviae infection, the birds were inoculated with infectious bronchitis (IB) virus. The presence of M. synoviae in the environment and in tracheal swabs was monitored by culture, a multiplex PCR (mPCR) detecting M. synoviae and Mycoplasma 16S rDNA and a multiplex RT-PCR (mRT-PCR) detecting the M. synoviae mRNA coding for a membrane protein and Mycoplasma 16S rRNA. In in vitro experimental conditions, M. synoviae mRNA and 16S rRNA were detected up to 20 min and 23 h respectively after mycoplasma death. In the first assay, the first infected bird was detected on the 13th day. In the second trial, culturable M. synoviae or viable M. synoviae were detected in the isolator for 3 or 4 to 5 days respectively after depopulation of the birds of the first assay whereas the first culture positive tracheal swabs were detected on the 33rd day, after IB inoculation. In the third experiment, the first infected birds were detected on the 54th day. Thus, the different assays showed that M. synoviae contaminated material (dust, feathers and food) can infect chicks, sometimes after remarkably long silent periods.     

The humoral immune response of chickens to Mycoplasma gallisepticum and Mycoplasma synoviae studied by immunoblotting

The humoral immune response over time of White Leghorn chickens experimentally infected with Mycoplasma gallisepticum or M. synoviae by an aerosol inoculation or a contact exposure were compared by immunoblotting. The response of chickens infected with M. gallisepticum were similar with respect to proteins recognized and intensity of response, regardless of mode of infection. On the other hand, chickens infected by aerosolization of M. synoviae responded to more proteins and with greater intensity than did M. synoviae contact-exposed birds. Chickens infected with M. gallisepticum responded with antibodies to over 20 proteins, while chickens infected with M. synoviae responded with antibodies to 12 proteins. Field sera from chickens naturally infected on commercial poultry farms with M. gallisepticum or M. synoviae were analyzed by immunoblotting and were found to react with a number of mycoplasma proteins. However, no correlation was seen when comparing intensity of immunoblot staining and hemagglutination-inhibition titer of the field sera. The experimental antisera were used to identify species-specific proteins of M. gallisepticum and M. synoviae. Six immunogenic species-specific proteins of M. gallisepticum with relative molecular masses of 82 (p82), 65-63 (p64), 56 (p56), 35 (p35), 26 (p26), and 24 (p24) kilodaltons (kDa) were identified. Two species-specific proteins of M. synoviae with relative molecular masses of 53 (p53) and 22 (p22) kDa were identified. Additionally, a highly immunogenic 41 (p41) kDa protein of M. synoviae was identified. Species-specific proteins identified in these mycoplasmas and the 41 kDa protein of M. synoviae were purified by preparative SDS-PAGE in amounts sufficient for further characterization and for use in serodiagnostic tests.     

Laboratory infection of house sparrows (Passer domesticus) with Mycoplasma gallisepticum and Mycoplasma synoviae

House sparrows were infected by aerosol with Mycoplasma gallisepticum (MG) or M. synoviae (MS). MG was reisolated from 5 to 11 sparrows 10 days postinfection, but infection appeared to be temporary. Mycoplasma-free chickens reared in the experimental house became infected with MG during the trial. MS was recovered from only one sparrow. Serological tests were unsatisfactory for diagnosing infected birds. The results suggest that house sparrows may be temporary biological carriers of MG.     

Influence of a 12.5 per cent rapeseed diet and an avian reovirus on the production of leg abnormalities in male broiler chickens

The incidence of different forms of leg abnormality were recorded in reovirus (S1133) infected and control male broiler chickens fed on a normal commercial diet or one of similar nutritive value containing 12.5 per cent rapeseed meal. Regular serological examination showed that birds remained free from Mycoplasma gallisepticum and M synoviae infection throughout the 10 week period of investigation. Precipitating antibodies to the reovirus were detected in 90 per cent of the infected birds between the third and 10th week after infection. Carotene levels in rapeseed fed groups showed no significant differences between reovirus infected and control birds or between birds with or without clinical signs of leg abnormality. The most frequent and severe leg abnormalities were present in the infected birds fed on the rapeseed diet, followed by those fed on the normal commercial diet. There was a highly significant difference (P less than 0.01) between the number of birds with leg abnormalities in each of these groups and their corresponding control groups. The lesions which were mainly responsible for these differences were tenosynovitis and enlarged hocks. Oral infection with reovirus did not appear to make the birds more susceptible to other types of leg abnormality, although the severest lesions of dyschondroplasia were seen in birds which had been exposed to the dual effects of reovirus and rapeseed diet.     

Isolation of Mycoplasma synoviae from infectious synovitis of chickens

This report describes the first isolations of Mycoplasma synoviae from the synovial sheaths and joints of commercial chickens affected with synovitis in Australia. Over 4 years 3 separate outbreaks were investigated in which up to 20% of birds exhibited clinical signs of poor growth and "hot foot" syndrome (swollen inflamed footpads). Once an outbreak occurred, chronic infection of the farm usually ensued. Grossly the hocks and footpads were swollen by a purulent exudate and associated inflammatory changes with histological features of a severe acute synovitis. Seroconversion of the flocks occurred at the time of the development of lesions. M. synoviae specific antibodies were demonstrated by ELISA in the joint fluid of affected birds. It is concluded that the cases described are similar to avian infectious synovitis syndrome caused by M. synoviae previously described overseas.     

Epidemiological study on Mycoplasma synoviae infection in layers

Mycoplasma synoviae infection occurs worldwide in commercial poultry flocks and may result in severe economic losses. The prevalence of this mycoplasma in standard layers older than 60 weeks was studied in a French department and the characteristics of infected or free flocks were compared. The genomic profiles of isolates from 36 infected flocks were studied by pulsed-field gel electrophoresis and random amplified polymorphic DNA methods in order to investigate possible routes of transmission. The minimum inhibitory concentrations of antibiotics were determined. Results showed that infection was more frequent in multi-age farms. Egg production and mortality of infected flocks were respectively lower and higher than in non-infected flocks but the differences were not statistically significant. The genomic profiles of isolates were quite homogeneous, a feature which does not facilitate the understanding of routes of transmission. All isolates were susceptible to tetracyclines, macrolides (except erythromycin), spectinomycin and fluoroquinolones.     

Serological survey of the infectious disease status of Old English Game fowl in the lower North Island, New Zealand

AIM: To investigate the serological status of Old English Game (OEG) cockerels for a range of infectious diseases of poultry. METHODS: Standard methods were used to screen serum collected from approximately 200 birds during routine dubbing operations, in 2004 and 2005. RESULTS: There was no serological evidence of infection with Newcastle disease, infectious bursal disease, or Salmonella Pullorum. Antibodies to infectious bronchitis virus, avian encephalomyelitis (AE) virus, Mycoplasma gallisepticum and Mycoplasma synoviae were detected. CONCLUSIONS AND CLINICAL RELEVANCE: The disease status of OEG birds is similar to that of commercial poultry.     

Poor serologic response to upper respiratory infection with Mycoplasma synoviae in turkeys

Mycoplasma synoviae (MS) was isolated from a flock of commercial tom turkeys in which a small percentage of the birds exhibited clinical signs and lesions typical of MS synovitis. However, serologic testing of such flocks revealed poor to inconsistent reactivity by agglutination, enzyme-linked immunosorbent assay (ELISA) or hemagglutination inhibition; isolation of MS from such flocks proved to be very difficult. Turkeys were challenged with one of the isolates (K4463B) either by aerosol or systemically by a combination of intravenous, foot pad, and eyedrop routes. Turkeys challenged by the systemic route responded normally to all serologic tests, whereas those challenged by aerosol either responded very poorly on all serologic tests or were seronegative up to 6 wk postchallenge even though they were positive for MS by tracheal culture. These results suggest that turkeys may harbor an upper respiratory infection with MS while remaining serologically negative.     

鸡滑液囊支原体的分离鉴定及病理组织学观察

为了解宁夏及其周边不同地区蛋鸡场中的鸡滑液囊支原体的种类及其致病力,采用改良Frey氏鸡滑液囊支原体培养基,从疑似感染鸡滑液囊支原体的不同蛋鸡群中分离出病原株,设计鸡滑液囊支原体vlhA基因特异性引物,对分离到的病原进行基因序列扩增并测序,使用MEGA6.0软件中的邻接法(Neighbor-joining,NJ)构建分离株系统发育树并进行遗传进化分析,采用从临床症状最明显的鸡体分离的菌株进行动物感染试验,制作石蜡切片,HE染色,病理组织学观察。结果表明,所分离到的病原菌均为鸡滑液囊支原体,部分菌株之间极其相似;鸡滑液囊支原体不仅可以使鸡只关节肿胀、生长缓慢,也可引起鸡只的肝脏轻微肿大,肝细胞部分坏死、间质增生;脾脏结缔组织增生,出血。     

Persistence of Mycoplasma synoviae in hens after two enrofloxacin treatments and detection of mutations in the parC gene

The ability of Mycoplasma synoviae, an avian pathogen, to persist despite fluoroquinolone treatments was investigated in hens. Groups of Mycoplasma-free hens were experimentally infected with the M. synoviae 317 strain and treated twice with enrofloxacin at the therapeutic dose. The results show that the two treatments did not have any influence on this strain of M. synoviae recovery from tracheal swabs. Mycoplasmas were isolated from tracheal swab cultures, but not from inner organs such as the liver or spleen, suggesting that this strain of M. synoviae was not able to cross the mucosal barrier to disseminate throughout the host. A significant increase of the resistance level to enrofloxacin of five re-isolated mycoplasma clones, was observed after the second treatment. This increase was associated in two clones to a Ser81-->Pro substitution, found in the ParC quinolone-resistance determining region (QRDR) of DNA topoisomerase IV. This is the first time that a mutation in a gene coding for topoisomerase IV is described in M. synoviae after in vivo enrofloxacin treatments in experimentally infected hens.     

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.     

Infection of turkeys with Ornithobacterium rhinotracheale and Mycoplasma synoviae

Within 1 mo, two separate outbreaks of respiratory disease occurred in two flocks on the multiage market turkey farm in Slovenia. More severe dinical signs and higher mortality were observed in male birds. Ornithobacterium rhinotracheale (ORT) was isolated in pure culture from tracheas of the affected birds in both outbreaks. Commercial enzyme-linked immunosorbent assay test showed the presence of antibodies to ORT in sera of birds from both clinically affected flocks and also in two flocks of younger birds without clinical sings. Immunoblotting with ORT culture isolated during the outbreak as an antigen confirmed the presence of antibodies to ORT in sera of turkeys of all four flocks examined. In addition, three different serologic assays also detected antibodies to Mycoplasma synoviae (MS) in three out of four flocks. The concomitant infection with MS did not show an obvious effect on mortality rates nor on the antibody response against ORT. Younger birds appeared to be less susceptible to ORT pathogenicity because in those flocks the infection was subclinical.     

Study of infection with an Iranian field-isolated H9N2 avian influenza virus in vaccinated and unvaccinated Japanese quail

In the present study, we examined the mortality rate, egg production, and clinical signs of quail experimentally infected with a field isolate of A/Chicken/Iran/339/02 (H9N2) avian influenza virus obtained from an infected commercial layer farm with severe morbidity and mortality. A total of 120 quail at 14 days old were randomly divided into four groups of vaccinated (B and C) and unvaccinated (A and D) birds. Vaccination was done on days 20 and 32, and viral inoculation of birds in groups C and D was then carried out on day 43. For evaluation of viral transmission, at 24 hr postinoculation additional unvaccinated birds were placed in direct contact with challenged birds. All the birds were evaluated for clinical signs, egg production, antibody production, viral titration in lung homogenates, and viral transmission following inoculation. All unvaccinated-challenged birds were infected and showed clinical signs, whereas the infection rate along with clinical signs of vaccinated-challenged birds reached 30%-40%. Although vaccination induced high antibody titers, reduction in food and water consumption was evident in this vaccinated-challenged group compared with the unchallenged control group. These results could indicate that inactivated vaccine did not fully prevent the infection, although it was capable of protecting birds against clinical signs and significantly decreased viral titers in lungs after intranasal challenge.     

Serological detection of Mycoplasma synoviae infection in turkeys.

The antibody response of turkeys experimentally infected with Mycoplasma synoviae was determined by the serum plate agglutination (SPA), hemagglutination-inhibition (HI), and microagglutination (MA) tests and the enzyme-linked immunosorbent assay (ELISA). No antibody response was detected until 2 weeks postinfection (PI) with the MA test (17% positive), 3 weeks PI with the SPA test (11% positive), 4 weeks with the HI test (21% positive), and 5 weeks PI with the ELISA, and even then, only 16% of the birds were positive. Although at least 89% of the birds were positive by culture, only 58% of the turkeys developed a detectable antibody response.     

Pathogenesis in call ducks inoculated intranasally with H5N1 highly pathogenic avian influenza virus and transmission by oral inoculation of infective feathers from an infected call duck

To evaluate the possibility of virus transmission through feathers of call ducks, we performed two experiments, intranasal infection study and transmission study, using the Japanese H5N1 highly pathogenic avian influenza virus (HPAIV) A/chicken/Yamaguchi/7/2004 (Ck/Yama/7/04). In Experiment 1, 1-day-old, 2-wk-old, and 4-wk-old birds were inoculated intranasally with Ck/Yama/7/04. Birds in all age groups exhibited necrosis and/or viral antigens in the feather epithelium. Nonpurulent encephalitis and focal necrosis of the pancreas and heart also were common to inoculated birds. In Experiment 2, nine 2-wk-old birds that were orally inoculated with feathers of an infected call duck exhibited the nonpurulent encephalitis, necrosis of the feather epithelium, and focal necrosis of the pancreas and heart, accompanied by viral antigens. These lesions were similar to those in intranasal infection. Some birds were positive for the virus isolation from cloacal swabs and hemagglutination inhibition antibody. The infection was confirmed in seven of nine birds. This study confirmed that the Japanese HPAIV can replicate in the feather epithelium, causing necrosis in call ducks through the natural infection route. It also suggests that feathers of call ducks infected with Ck/Yama/7/04 can be a potential source of infection for unaffected birds in nature.     

Serological responses of chickens to low challenge doses of Mycoplasma synoviae

Groups of eight chickens were challenged with 10-fold dilutions of one of two strains of Mycoplasma synoviae (MS); each challenge group contained two noninfected sentinels. Both strains were highly efficient in colonizing the respiratory tract with challenge doses as low as 76 and 24 color-changing units/bird. Infection spread rapidly (within 7 days) to sentinels, while uninfected control chickens separated from infected chickens by two empty pens remained uninfected for the 56-day experimental period. Although sentinels and birds challenged with the lowest doses had weaker or slightly slower antibody responses in some cases as measured by serum plate agglutination, enzyme-linked immunosorbent assay (ELISA), and hemagglutination inhibition (HI), they generally exhibited a typical antibody response. Agglutination reactions tended to be weak, but a high percentage of tests (generally >30% from day 14 postchallenge) were positive. ELISA results were variable, and in some cases reactor rates were low (generally <20%), even though the chickens were colonized in the upper respiratory tract. The HI test was reliable in detecting infected groups; usually >50% were positive from 14 days postchallenge. Mean HI titers were higher when using hemagglutination antigens prepared from the homologous MS strain as compared with antigen prepared from the heterologous strain or with standard antigen prepared from WVU 1853.     

Acquisition of immunity to Eimeria maxima in newly hatched chickens given 100 oocysts

The acquisition of immunity to Eimeria maxima by chicks infected 18 hr after hatch with a single dose of 100 oocysts was investigated. In the first experiment, birds were moved each day to clean cages in order to prevent the possibility of secondary infection resulting from ingestion of oocysts passed in their feces. Immunity was measured at 4 wk of age by calculation of oocyst production following challenge with 500 oocysts or weight gain following challenge with 100,000 oocysts. Large numbers of oocysts were produced by infected birds following challenge, although numbers were significantly less than those from birds that had been reared in the absence of infection (susceptible controls). The weight gain of infected birds following challenge was significantly greater than that of susceptible controls but less than that of unchallenged controls. Thus, only partial protection had been acquired, whether parasite replication or body weight gain was used to assess the extent of immunity development. In a second experiment, acquisition of immunity at 4 wk by chicks infected 18 hr after hatch with 100 oocysts of E. maxima and reared in floor pens in contact with their droppings was investigated. Infected birds produced no oocysts following challenge, and weight gains were not significantly different from the unchallenged controls, which indicates that full immunity had developed by 4 wk. It is concluded that if oocysts of Eimeria species are used to vaccinate day-old chicks, reinfection by oocysts present in the litter is necessary for the establishment of protective immunity.     

Serological examination and egg production of progeny of fowl experimentally infected with egg drop syndrome 1976 virus

Following EDS'76 virus (BC14 virus) infection of breeder chickens by the conjunctival route, vertical transmission occurred in the first week after infection. In the progeny which had been infected with EDS'76 virus by the vertical route, increasing haemagglutination inhibiting (HI) titres to BC14 virus and increasing numbers of birds with HI titres were observed from 3 weeks to 15 weeks of age. Sixty-one per cent of the hens and 77 per cent of the cocks had 2 log HI BC14 virus titres exceeding 4 at an age of 15 weeks. Some birds which han been serologically negative throughout the rearing period, seroconverted between 25 and 28 weeks of age. This phenomenon occurred in hens as well as in cocks. Simulation of stress twice during the laying period by injection of corticosteroid hormone did not increase the number of birds serologically positive to EDS'76 virus. EDS'76 was observed in the group of hens that was vertically infected, since egg production was significantly depressed between 28 and 34 weeks of age. Probably this was mainly the results of a production drop in the hens showing serconversion at 27 or 28 weeks of age. In this group of fowl vertically infected with EDs'76 virus, serologically positive birds appeared to be protected for the greater part to BC14 virus challenge at 50 weeks of age, while negative birds seemed to be fully susceptible. Chicks hatched from eggs collected in the third and fourth week after infection of the dams had maternal antibodies. Fertility and hatchability of apparently normally shelled eggs seemed not to be affected after BC14 virus infection of the dams. Intensive contact with contaminated faeces is probably an indispensable condition for lateral transmission of the virus.