Mycoplasma infection of ducks and geese.

Production of ducks and geese in certain parts of the world is very important. Mycoplasma diseases cause significant losses to the duck and goose industry. This review summarizes the epidemiological, clinical, and pathomorphological characteristics of mycoplasma diseases of ducks and geese and the involvement of the various mycoplasma species in their pathogenesis. The role of mycoplasma infections in the development of clinical signs, pathological lesions, and mortality of challenged birds is demonstrated in challenge experiments. Transmission of mycoplasma in the ovary and eggs resulting in the reduction of egg production and an increase of embryo mortality has been shown in challenge experiments as well as in field studies. The susceptibility of many mycoplasma isolates of the most important mycoplasma species of duck and goose origin were tested and showed relatively high average minimum inhibitory concentrations of lincomycin, tilosin, oxytetracycline, chlortetracycline, and enrofloxacin but not for tiamulin. The successful treatment of mycoplasma infections with antibiotics in ducks and geese should be selected based on the minimum inhibitory concentration values against the mycoplasmas isolated from the flock.

Pathologic lesions caused by coinfection of Mycoplasma gallisepticum and H3N8 low pathogenic avian influenza virus in chickens.

Chickens were infected under experimental conditions with Mycoplasma gallisepticum and low pathogenic avian influenza (LPAI) strain A/mallard/Hungary/19616/07 (H3N8). Two groups of chickens were aerosol challenged with M. gallisepticum strain 1226. Seven days later, one of these groups and one mycoplasma-free group was challenged with LPAI H3N8 virus; one group without challenge remained as negative control. Eight days later, the birds were euthanized and examined for gross pathologic and histologic lesions. The body weight was measured, and the presence of antimycoplasma and antiviral antibodies was tested before the mycoplasma challenge, before the virus challenge, and at the end of the study to confirm both infections. Chickens in the mycoplasma-infected group developed antibodies against M. gallisepticum but not against the influenza virus. Chickens of the group infected with the influenza virus became serologically positive only against the virus, while the birds in the coinfected group developed antibodies against both agents. The LPAI H3N8 virus strain did not cause decrease in body weight and clinical signs, and macroscopic pathological lesions were not present in the chickens. The M. gallisepticum infection caused respiratory signs, airsacculitis, and peritonitis characteristic of mycoplasma infection. However, the clinical signs and pathologic lesions and the reduction in weight gain were much more significant in the group challenged with both M. gallisepticum and LPAI H3N8 virus than in the group challenged with M. gallisepticum alone.

Prevalence of postweaning multisystemic wasting syndrome in pigs sired by 13 Hungarian Landrace boars.

Between February 20 and October 31, 2003, 2034 sows were inseminated with semen collected from 13 Hungarian Landrace boars. Altogether 16,013 piglets were born: 13,801 (86.2 per cent) alive, 796 (4.97 per cent) stillborn and 156 (0.97 per cent) mummified. A total of 1260 (7.87 per cent) of the pigs developed signs of postweaning multisystemic wasting syndrome (PMWS). In the groups of sows inseminated with semen from each of the 13 boars, the percentages of farrowings producing piglets with signs of PMWS, stillborn piglets or mummified piglets were very high (59.4 per cent, 57.6 per cent and 13.8 per cent, respectively). There were significant differences between the proportions of piglets with signs of PMWS, stillborn piglets and mummified piglets sired by the different boars: 3.06 to 15.6 per cent, 1.76 to 8.52 per cent and 0 to 3.22 per cent, respectively.

Detection of Mycoplasma bovis with an improved pcr assay.

A Mycoplasma bovis species-specific PCR assay has been developed with improvement of a previously described method (Ghadersohi et al., 1997). This test and its semi-nested version (Hayman and Hirst, 2003) did not function at all in our hands. A new reverse primer (Mbr2) was designed using previously published sequence data. For testing specificity, DNA was extracted from the most frequently occurring mycoplasma species and bacteria of bovine origin. The new PCR detected only Mycoplasma bovis. Moreover, no cross-reaction was observed with the genetically closest relative species, M. agalactiae. The target organism could be detected in a dose as low as 150 CFU ml(-1) in broth cultures using ethidium-bromide-stained agarose gels.

Differentiation of mycoplasma gallisepticum strains using molecular methods.

Increasing use of Mycoplasma gallisepticum (MG) live vaccines has led to a need for the differentiation of MG strains. The MG strains MK-7, MS-16, S6, FS-9 and R strains and the MG live vaccine strain F were compared by random amplification of polymorphic DNA (RAPD) in this study. Using RAPD, different patterns were found among the MG strains. In addition to this, we examined the differentiating potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) primers targeted at the crmA, crmB, crmC, gapA, mgc2 and pvpA genes encoding cytadherence-related surface proteins. These proteins may take part in the pathogenesis of MG-induced disease. Differentiation of strain F is based on the identification of restriction enzyme sites in the PCR amplicons. Using HphI enzyme, crmC PCR amplicons produced different RFLP patterns. Digestion of amplicons of gapA-specific PCR with MboI enzyme also produced distinct patterns. Differences were observed among strains R and F by digestion of mgc2 PCR amplicons with HaelIl and VspI enzymes and digestion of pvpA PCR amplicons with AccI, PvulI and ScrFI endonucleases. This method can be used for the rapid differentiation of vaccine strain from wild strains. Differentiation of MG strains is a great advantage for diagnosticians or practitioners and it is useful for epidemiological studies.

Binding of mycoplasmas to solid phase adsorbents.

The capture of mycoplasmas (M. hominis, M. buccale, M. fermentans, M. bovis, M. synoviae, M. gallisepticum and M. arthritidis) based on lipid structures and adhesion molecules present in the mycoplasmal membrane was tested using different chromatographic resins (ActiClean Etox, ClarEtox, Heparin-Actigel, Sulfated Hiflow and SulfEtox). All of the resins efficiently reduced mycoplasma concentrations in Phosphate Buffered Saline (PBS) and in Fetal Bovine Serum (FBS) by 3-8 logs in a few minutes. This technology could be used for removing mycoplasmas from tissue culture components such as serum, and for concentrating mycoplasmas in vaccine production.

Safety and efficacy of Mycoplasma gallisepticum TS-11 vaccine for the protection of layer pullets against challenge with virulent M. gallisepticum R-strain.

Mycoplasma gallisepticum TS-11 vaccine was studied for its safety and protective ability in 49-day-old M. gallisepticum-free and Mycoplasma synoviae-free commercial Tetra SL layer chickens. Sixty birds were distributed into four groups: 15 were unvaccinated but were challenged with M. gallisepticum R-strain, 15 were vaccinated by eye drop and then challenged with virulent M. gallisepticum R-strain 4 weeks post vaccination, 15 were designated as controls without vaccination and challenge, and 15 received TS-11 vaccine but no challenge. Based on the post-challenge clinical signs, body weight gain, gross pathological examination of air sacs and peritoneum, histological examination of the trachea, lung, spleen and liver, and reisolation of mycoplasmas from inner organs, the TS-11 vaccine is safe and does not produce clinical signs, a major decrease of body weight gain or pathological lesions. Vaccination induced a slight serological response to M. gallisepticum antigen in serum plate agglutination and blocking enzyme-linked immunosorbent assay tests and prevented development clinical signs of airsacculitis, peribronchitis and interstitial pneumonia on M. gallisepticum challenge.

The efficacy of valnemulin (Econor) in the control of disease caused by experimental infection of calves with Mycoplasma bovis.

Mycoplasma bovis infection was experimentally induced in groups of six young calves. A further group was uninfected and served as a control. Ten days after infection, medication with either enrofloxacin (Baytril, Bayer) or valnemulin (Econor, Novartis) was instituted via the milk replacer for a further 10 days, after which all calves were killed. Infection resulted in depression, pyrexia, inappetance and prominent respiratory signs. Arthritis occurred in two animals and two (unmedicated) animals died. At post-mortem examination extensive lesions were present in the lungs and M. bovis was re-isolated from infected unmedicated calves' lungs. Medication with either enrofloxacin or valnemulin resulted in a rapid diminution of clinical signs, restoration of appetite and reversal of weight loss. Isolation of Pasteurella multocida from the calves' lungs was suppressed by both medicaments. Valnemulin resulted in a more rapid reduction of clinical scores and eliminated M. bovis from the lungs more effectively than enrofloxacin.

Examination of the role of Mycoplasma bovis in bovine pneumonia and a mathematical model for its evaluation.

The authors screened 34 large cattle herds for the presence of Mycoplasma bovis infection by examining slaughtered cattle for macroscopic lung lesions, by culturing M. bovis from lung lesions and at the same time by testing sera for the presence of antibodies against M. bovis. Among the 595 cattle examined, 33.9% had pneumonic lesions, mycoplasmas were isolated from 59.9% of pneumonic lung samples, and 10.9% of sera from those animals contained antibodies to M. bovis. In 25.2% of the cases M. bovis was isolated from lungs with no macroscopic lesions. The proportion of seropositive herds was 64.7%. The average seropositivity rate of individuals was 11.3% but in certain herds it exceeded 50%. A probability model was developed for examining the relationship among the occurrence of pneumonia, the isolation of M. bovis from the lungs and the presence of M. bovis specific antibodies in sera.

Testing the efficacy of fermented wheat germ extract against Mycoplasma gallisepticum infection of chickens.

The effect of fermented wheat germ extract (FWGE, Immunovet-HBM) was studied in chickens challenged with Mycoplasma gallisepticum. Ninety M. gallisepticum- and M. synoviae-free 3-wk-old chickens were exposed to aerosol infection of M. gallisepticum. One group (30 birds) was treated with FWGE, a second group with tiamulin, and a third group was untreated. The fourth group was exposed to PBS aerosol as a negative control. On d 9, all chickens were slaughtered and examined for the presence of gross and histological lesions, the presence of the challenge strain in the organs and specific antibodies in the serum. Body weight gains and feed conversion rates were recorded. In the groups treated with FWGE and with tiamulin, the chickens remained clinically healthy: their BW gains were 441.7 g and 446.8 g, respectively. Feed conversion ratios were 1.72 and 1.71 for FWGE- and tiamulin-treated birds, respectively. Control birds had BW gain of 480.8 g, and feed conversion ratio of 1.78. The numbers of birds with gross lesions (15 and 11, respectively) and lesion scores (25 and 25, respectively) of the FWGE- and tiamulin-treated groups were significantly lower than in the infected untreated group (25 birds, lesion score of 190). No mycoplasma was reisolated from brain, liver, spleen, heart, or kidneys of the FWGE-treated birds, and the number of mycoplasma isolations from the respiratory tract samples was less frequent (10) than from the infected untreated group (64). In addition, 35 samples from other internal organs were also positive. Twenty percent of the birds treated with FWGE showed serological response with a 5.0% reaction score, whereas in the infected untreated group, 83.3% of birds were reactors, with a 62.5% reaction score.

Reduction of economic losses caused by mycoplasmal pneumonia of pigs by vaccination with Respisure and by Tiamutin treatment.

The possibilities and economic benefits of controlling mycoplasmal pneumonia of pigs caused by Mycoplasma hyopneumoniae by immunisation with Respisure and by Tiamutin treatment were studied. The experiment was carried out in a herd comprising 1000 sows which was free of PRRS, Aujeszky's disease, swine dysentery and leptospirosis, and the prevalence of mycoplasmal pneumonia was low because the farm had recently been restocked. Groups C1 and C2 served as untreated controls, while Groups R1 and R2 received a prestarter diet containing 100 ppm Tiamutin from the time of weaning. Piglets of Group R1 were vaccinated with Respisure vaccine once on day 69, while those of Group R2 twice, on days 65 and 80. Piglets of Groups ST1 and ST2 were fed 100 ppm Tiamutin in the diet for 7 days at the time of weaning and then at 4 months of age, while pigs of Group ST2 received such treatment also in the 6th month of life. The efficacy of treatment was analysed on the basis of the number of animals that died, were emergency slaughtered or were retarded in growth in the different groups, the body weight of animals at weaning, at 94 and 148 days of age and at the time of slaughter, their daily body weight gain, the lung lesions found in animals slaughtered from the different groups, the costs of medication and vaccination, and the cost-benefit calculations of the results. The mortality and emergency slaughter rate was 2.88% and 4.62% in Groups ST2 and ST1, respectively, 4.23% and 4.62% in Groups R2 and R1, respectively, and 8.39% and 9.44% in the control groups (C2 and C1, respectively). The rate of growth retardation was 0.48% and 2.12% in Groups R1 and R2, respectively, 1.59% and 3.46% in Groups ST1 and ST2, respectively, as compared to 8.03% and 6.55% in the control groups (C1 and C2, respectively). The severity score of lung lesions was 1.82 and 1.46 in Groups R1 and R2, 2.18 and 2.93 in Groups ST1 and ST2, and 3.83 and 4.02 in the control groups C1 and C2, respectively. The mean finishing weight of pigs was 102.4-107.8 kg and 95.2-106.6 kg in the treated groups and 94.5-98.6 kg in the control groups. The classification of pigs according to the EUROP categories showed a shift to the E and U categories in the treated groups. The average feed cost per one kg of liveweight was 77.89-82.64 Forints in the treated groups and 85.66 Forints in the control groups.

Confirmation of the presence of Mycoplasma bovis in Hungarian cattle with pneumonia resembling pleuropneumonia.

Cattle from several farms in Hungary were investigated for the presence of mycoplasmal infections after the discovery of pulmonary lesions in some animals at slaughter. The pneumonic lesions, which resembled those of contagious bovine pleuropneumonia (CBPP) macroscopically and histologically were found to be caused by Mycoplasma bovis and not Mycoplasma mycoides subspecies mycoides (MmmSC) which is the causative agent of CBPP. No other bacterial pathogens were isolated. Negative results in complement fixation tests also showed that there was no serological evidence of CBPP. PCR tests for the detection of the M mycoides cluster and specifically for MmmSC were also negative. However, PCR and bacteriological culture detected cases of M bovis and the pneumonias may therefore be attributed to this mycoplasma.

Use of valnemulin in the control of Mycoplasma bovis infection under field conditions.

In a blind trial, alternate calves in six consecutive production batches of calves (total 70), on a farm with a high incidence of respiratory and reproductive disease, were allocated to treatment with either valnemulin or a placebo premix added to the milk from four days of age. The calves were weighed at the beginning and end of a 21-day period of medication. Blood samples and nasal swabs were taken and examined for the presence of Mycoplasma and Pasteurella species, and antibodies to viral agents. Clinical condition, rectal temperature, respiratory and other signs and refusals of milk were recorded daily. Dead calves were examined postmortem. The calves medicated with valnemulin gained weight more quickly, had fewer cases of Mycoplasma infection and fewer respiratory signs, and required fewer treatments with antibiotics than those in the placebo group.

Treatment of pigs experimentally infected with Mycoplasma hyopneumoniae, Pasteurella multocida, and Actinobacillus pleuropneumoniae with various antibiotics.

The authors have performed a comparative study of the efficacy of various in-feed medications for the treatment of 5- to 6-week-old specific pathogen-free (SPF) piglets experimentally infected on day 1 with Mycoplasma hyopneumoniae, on day 8 with Pasteurella multocida (serotype A), and on day 15 with Actinobacillus pleuropneumoniae (serotype 2). The treatment started on day 9 and continued for 12 consecutive days, then the piglets were euthanized for examination of macroscopic, histologic, and pathologic lesions and for the presence of mycoplasmas and bacteria in the lungs. Based on the results of clinical observations (respiratory signs, rectal temperature, body weight gain, and feed conversion efficiency), macroscopic and histologic lesions of the lungs, and microbiologic findings, the best results were obtained by treatment of pigs with Econor + chlortetracycline, followed by Tetramutin, Pulmotil, Cyfac, and lincomycin + chlortetracycline.

Screening of Hungarian cattle herds for Mycoplasma mycoides subspecies mycoides small colony infection with negative results.

At abattoirs and farms, 1248 sera were collected from animals representing 121 farms, and examined by complement fixation test using Mycoplasma mycoides subspecies mycoides small colony type (MmmSC) antigen. All sera were negative except seven from four farms, giving ++ reactions in the serum dilution of 1:10. On retesting, these sera and additional 30 sera collected repeatedly in both farms gave negative results. In isolation attempts, 953 lung samples collected from slaughtered cattle at the same abattoirs, and 326 nasal swabs collected from 11 herds proved to be negative for the presence of MmmSC, but M. bovis was isolated frequently. In the small farms 23.95% of the animals had pleurisy and/or pneumonia while in the large herds 34.69% had lesions. DNA extracted from 50 nasal swabs and 430 lung samples was examined by polymerase chain reaction (PCR) using M. mycoides cluster-specific primers. DNA from further 325 lung samples was tested by the more specific M. mycoides subspecies mycoides small colony/large colony/capri specific primers and 196 samples by nested PCR specific for MmmSC. All gave negative results. The detection level of cluster-specific primers and the more specific primers was 33.4 pg of DNA, whereas that of nested PCR was 0.33 pg.

Clinical study of the disease of calves associated with Mycoplasma bovis infection.

Clinical, bacteriological and serological examination of 35 calves from the age of 5 to 26 days was performed in a Holstein-Friesian dairy herd endemically infected with Mycoplasma bovis. M. bovis was isolated from 48.6% of nasal swabs taken from the calves at the age of 5 days, and from 91.4% of the same calves at the age of 26 days, indicating the gradual spread of infection. The isolation rate of Pasteurella multocida did not change much, and varied from 28.6 to 25.7%. No P. haemolytica could be detected. In addition to M. bovis and P. multocida, the herd was also infected with different viruses (including bovine viral diarrhoea virus, infectious bovine rhinotracheitis virus, bovine adenoviruses, parainfluenza-3 virus, and bovine respiratory syncytial virus) as a large proportion of the sera of newborn calves contained colostral antibodies against these viruses. In most of the newborn calves severe clinical signs (fever, depression, inappetence, hyperventilation, dyspnoea, nasal discharge and coughing) due to M. bovis infection developed. The clinical signs appeared already on the fifth day of life, and their incidence was the highest at the age of 10 to 15 days. Three calves (8.6%) died as a result of severe serofibrinous pneumonia. The surviving calves showed very poor weight gain (ranging from 1.5 to 3.5 kg) during the first two weeks of life.

Protective effect of two Mycoplasma gallisepticum protein fractions affinity purified with monoclonal antibodies.

Two protein fractions of Mycoplasma gallisepticum (Mg) were affinity purified with monoclonal antibodies A3 and B3, and tested for protective capacity in chickens. One fraction, designated MgP1, appeared as a doublet of 64 and 62kDa bands in sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels, while MgP2 consisted of five polypeptides (64, 56, 47, 45 and 43 kDa). The molecular mass, haemagglutination activity and matching amino acid sequence of MgP1 suggest that it is identical to pMGA1.2, the putative haemagglutinin of Mg. Groups of Mg-free chickens were immunized once or twice with 1 or 5 mug MgP1 or MgP2, or a combination of the two, and adjuvanted with immunostimulating complexes. Except for the group given 1 mug MgP1, all vaccinated groups showed a significant (P < 0.01) reduction in air sac lesions after challenge compared with unvaccinated controls. MgP2 appeared more protective than MgP1. Vaccination twice with MgP2 was the only regime that produced a detectable serum antibody response.

Testing the compatibility of a combination of tiamulin/chlortetracycline 1:3 premix (Tetramutin-Novartis) given in feed at different levels with salinomycin in chickens.

The compatibility of salinomycin (SA) (60 mg/kg feed) and Tetramutin (TM), a combination of tiamulin (TI) and chlortetracycline (CTC) in a 1:3 ratio, included in varying concentrations, was tested in broiler chickens. Assessment was based on clinical signs, body weight gain, feed conversion efficiency, gross pathological lesions and histological lesions in liver, heart and pectoral muscles, as well as analysis of blood for lactate dehydrogenase (LDH), aspartate dehydrogenase (AST) and creatine phosphokinase (CPK) activity. It was shown that chickens can be safely treated in feed for 7 days with TM containing TI concentrations of 33 or 55mg/kg feed together with SA at 60mg/kg. However, higher levels of TI (100 and 150 mg/kg feed) caused some adverse effects. There was no mortality but there was mild depression, 'leg weakness' accompanied by specific histological lesions in pectoral muscles (obliteration of cross-striation and swelling of muscle fibres, nuclei of muscle cells, hyalinization and destruction of sarcoplasm), a mild increase of AST activity, and a significant increase of CPK and LDH activity in blood. These latter two parameters can be used as an early indication of TI and SA incompatibility.

Antibody response detected by immunoblot in respiratory tract washings of chickens after infection with Mycoplasma gallisepticum.

Two experiments were conducted to test the sensitivity of Western blotting for detection of M. gallisepticum antibodies in respiratory washings and sera of infected chickens by mouse monoclonal antibodies to chicken IgG, IgM and IgA. In the first experiment, birds infected at 10 days of age were examined 2 weeks later. In the respiratory washings, IgA antibodies reacted with eight polypeptides of M. gallisepticum, while IgM and IgG reacted with three. In the serum IgA antibodies were not detected but IgM antibodies reacted with eight polypeptides and IgG with 16. In the second experiment birds were infected at 3 weeks of age and a subgroup was examined every week for 3 weeks post-infection. In the respiratory washings IgA was the principle immunoglobulin detected in the first week and it reacted to six major polypeptides of M. gallisepticum (p72, p64-67, p60, p56, p45, p40). In serum IgM was predominant in the first week and reacted to nine polypeptides. From the second week IgG antibodies were the most important as they reacted to 13 polypeptides in respiratory washings and to 11 polypeptides in the serum, while they reacted to nine polypeptides in respiratory washings and to 13 in the serum in the third week.