Characterization of mycoplasma gallisepticum infection in captive house finches (Carpodacus mexicanus) in 1998

Since 1995, the epidemic of mycoplasmal conjunctivitis in eastern house finches has affected the Auburn, AL, house finch population. To better characterize the current status of this host-parasite interaction, we established a captive flock of 38 seronegative, healthy finches in fall 1998. After a minimum quarantine period of 4 wk, two Mycoplasma gallisepticum (MG)-infected house finches were introduced into this flock. Over a 12-wk period, the flock was captured every 2 wk and each bird was observed for conjunctivitis. Blood and choanal swabs were collected from each bird for serologic analysis and for the detection of MG by polymerase chain reaction. The infection spread rapidly through the flock just as it had in a similar study performed in 1996 at the height of the epidemic. Unlike the earlier study in which birds remained chronically infected, most of the birds in our study recovered rapidly, and only three of the birds died during the study. Two patterns of host response to infection with MG were observed. Twenty-seven birds (73%) experienced an acute conjunctivitis that resolved, and the birds appeared to clear the infection. Ten birds (27%) suffered prolonged clinical disease, and MG could be detected in these birds intermittently throughout the experiment. These results, in conjunction with our surveys of MG in the wild population, suggest an evolving host-parasite interaction.     

Mycoplasma gallisepticum in house finches (Carpodacus mexicanus) and other wild birds associated with poultry production facilities.

Since 1994, an epidemic of conjunctivitis caused by Mycoplasma gallisepticum (MG) has spread throughout the eastern population of house finches (Carpodacus mexicanus). The adaptation of MG to a free-flying avian species presents potential problems for the control of mycoplasmosis in commercial poultry. To evaluate risks associated with this emerging problem, a field survey was conducted to assess prevalence of MG infection in house finches and other passerine birds associated with poultry farms. Between November 1997 and March 1999, 1058 birds were captured by mist net or trap at 17 farms and at 10 feeder stations in northeast Georgia. Birds were bled and screened by serum plate agglutination (SPA) for antibodies to MG. Birds with negative or weak positive SPA results were released at capture sites, and those with strong positive SPA reactions were kept for further evaluation. Necropsies were performed on selected house finches and individuals of 11 other passerine species, and samples were collected for MG testing by culture, polymerase chain reaction (PCR), hemagglutination inhibition, and histopathology. Testing revealed 19.1% of 671 birds caught at farms and 11.6% of 387 birds caught at feeder sites were SPA positive for MG. Three house finches captured on farms were positive for MG by culture and PCR, whereas three from feeder sites were positive only by PCR. No MG isolates were made from tufted titmice (Baeolophus bicolor), but 40% were positive by PCR. Individuals from 10 additional species were SPA positive only. Results suggest that MG persists at low levels in house finches in northeast Georgia and that tufted titmice may be nonclinical carriers of MG or a related mycoplasma. Positive SPA reactions in other species may be caused by nonspecific reactions or contact exposure. Current biosecurity recommendations should be sufficient to minimize risks of transmission between wild and domestic birds.     

Field investigation of Mycoplasma gallisepticum infections in house finch (Carpodacus mexicanus) eggs and nestlings.

We conducted a field study to investigate the occurrence of Mycoplasma gallisepticum (MG) in eggs and nestlings from nests of house finches (Carpodacus mexicanus). Forty-three nests were located between the months of April and August 1998 and were followed with one to three sampling efforts. Vitelline membrane of fresh eggs, whole embryos, or swabs from the choanal cleft or conjunctiva of nestlings were inoculated into mycoplasma broth for MG isolation and polymerase chain reaction (PCR) testing. No isolation of MG was made from 39 eggs or 110 nestlings sampled during the study. Pooled choanal and conjunctival swab samples from two broods of nestlings, however, tested positive for MG by PCR. None of the nestlings examined showed clinical signs of conjunctivitis, and no nestling mortality could be linked to MG infection. Serologic tests from 37 older nestlings were negative for antibodies to MG. The results suggest transmission of MG is occurring between breeding adults and their dependent offspring (pseudovertical transmission). Evidence supporting transovarian transmission of MG was not found in these house finches.     

空间位阻对丁胺侧链取代截短侧耳素衍生物鸡毒支原体抑制活性的影响

为给截短侧耳素类动物专用抗生素的研发提供试验依据,本试验合成了4种含丁胺侧链的截短侧耳素衍生物,研究了取代基的空间位阻对4种截短侧耳素衍生物鸡毒支原体抑制活性的影响。试验结果表明,化合物1、2的最小抑菌浓度均为0.0125 μg/mL,化合物3的最小抑菌浓度为0.25 μg/mL,化合物4的最小抑菌浓度为0.5 μg/mL。     

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 microg/ml. Resistance to nalidixic acid, gentamicin, streptomycin and spectinomycin was observed.     

In vitro susceptibilities of field isolates of Mycoplasma agalactiae

In order to determine how widespread antibiotic resistance has become to standard treatments, the in vitro susceptibilities of 28 Mycoplasma agalactiae Spanish field isolates to 16 antimicrobial agents were determined using a broth microdilution method. The most effective antimicrobials based on minimum inhibitory concentration (MIC)₉₀ values were fluoroquinolones, tetracyclines and macrolides. Two strains were tetracycline resistant. Streptomycin, erythromycin and nalidixic acid resistance was observed in all strains.     

Control of Mycoplasma hyopneumoniae infections in pigs

Mycoplasma hyopneumoniae, the primary pathogen of enzootic pneumonia, occurs worldwide and causes major economic losses to the pig industry. The organism adheres to and damages the ciliated epithelium of the respiratory tract. Affected pigs show chronic coughing, are more susceptible to other respiratory infections and have a reduced performance. Control of the disease can be accomplished in a number of ways. First, management practices and housing conditions in the herd should be optimized. These include all-in/all-out production, limiting factors that may destabilize herd immunity, maintaining optimal stocking densities, prevention of other respiratory diseases, and optimal housing and climatic conditions. Strategic medication with antimicrobials active against M. hyopneumoniae and, preferably, also against major secondary bacteria may be useful during periods when the pigs are at risk for respiratory disease. Finally, commercial bacterins are widely used to control M. hyopneumoniae infections. The main effects of vaccination include less clinical symptoms, lung lesions and medication use, and improved performance. However, bacterins provide only partial protection and do not prevent colonization of the organism. Different vaccination strategies (timing of vaccination, vaccination of sows, vaccination combined with antimicrobial medication) can be used, depending on the type of herd, the production system and management practices, the infection pattern and the preferences of the pig producer. Research on new vaccines is actively occurring, including aerosol and feed-based vaccines as well as subunit and DNA vaccines. Eradication of the infection at herd level based on age-segregation and medication is possible, but there is a permanent risk for re-infections.     

Pathogenicity of endophytic entomopathogenic fungi to Ornithodoros erraticus and Ornithodoros moubata (Acari: Argasidae)

The argasid ticks O. erraticus and O. moubata are of great medical and veterinary importance because they are vectors of the African swine fever virus and several species of human relapsing fever borreliae. Biocontrol of these ticks using entomopathogenic fungi has not been previously reported. We examined the pathogenicity to different developmental stages of these two argasids of six strains of the fungal species Beauveria bassiana (strains Bb1764 and Bb2157), Lecanicillium lecanii (strains Ll586, Ll618 and Ll3047) and Tolypocladium cylindrosporum (strain Tc3398). Three strains, Bb1764, Bb2157, and Tc3398, caused in Spanish O. erraticus mean mortality rates between 34.4% and 62% in 14-28 days post-inoculation. Additionally, Bb2157 also induced in African O. moubata mean mortality rates of 31.9%. The remaining strains caused lower mortality rates and were not considered effective. This is the first study in which some strains of entomopathogenic fungi are found to be effective against argasid ticks of the genus Ornithodoros, and its results might justify further efforts towards the application of entomopathogenic fungal strains as anti-argasid biocontrol agents.     

Quantitative real-time polymerase chain reaction for detecting Mycoplasma hyosynoviae and Mycoplasma hyorhinis in pen-based oral,tonsillar, and nasal fluids

Mycoplasma (M.) hyorhinis and M. hyosynoviae are pathogens known to cause disease in pigs post-weaning. Due to their fastidious nature, there is increased need for culture-independent diagnostic platforms to detect these microorganisms. Therefore, this study was performed to develop and optimize quantitative real-time PCR (qPCR) assays to rapidly detect M. hyorhinis and M. hyosynoviae in pen-based oral fluids as well as nasal and tonsillar fluids as proxies for samples used in swine herd surveillance. Two methods of genomic DNA extraction, automated versus manual, were used to compare diagnostic test performance. A wean-to-finish longitudinal study was also carried out to demonstrate the reproducibility of using pen-based oral fluids. Overall, pen-based oral and tonsillar fluids were more likely to be positive for both types of bacteria whereas only M. hyorhinis was detected in nasal fluids. DNA extraction protocols were shown to significantly influence test result. Although the initial detection time somewhat differed, both organisms were repeatedly detected in the longitudinal study. Overall, this study evaluated two qPCR methods for rapid and specific detection of either mycoplasma. Results from the present investigation can serve as a foundation for future studies to determine the prevalence of the two microorganisms, environmental load, and effectiveness of veterinary interventions for infection control.     

Production and characterization of recombinant transmembrane proteins from Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia (EP), a chronic respiratory disease which causes significant economic losses to the swine industry worldwide. More efficient strategies for controlling this disease are necessary. In this study, we cloned17 genes coding for transmembrane proteins from M. hyopneumoniae, among which six were successfully expressed in Escherichia coli and had their immunogenic and antigenic properties evaluated. All proteins were immunogenic in mice and sera from naturally infected pigs reacted with the recombinant proteins, suggesting that they are expressed during infection. These antigens may contribute for the development of new recombinant vaccines and diagnostic tests against EP.     

The effect of including anti-Ig sera in the haemagglutination inhibition test forMycoplasma gallisepticum

Addition of anti-immunoglobulin M (anti-IgM), G (anti-IgG) and A (anti-IgA) sera to the haemagglutination-inhibition (HI) test (anti-Ig HI test) forMycoplasma gallisepticum resulted in 2- to 8-fold increases in the HI titres. On investigating the anti-Ig HI reaction using IgM and IgG antibodies separated by affinity chromatography, it was confirmed that, in the enhanced HI titres, specificity existed between the chicken Ig classes having antibody activity and the antisera used in the test. Four days after inoculation ofM. gallisepticum, the anti-Ig HI reaction was markedly enhanced by anti-IgM antiserum in the intravenously inoculated chickens and by anti-IgA serum in the nasally inoculated chickens. Ten days after inoculation ofM. gallisepticum marked enhancement of the reaction was produced by anti-IgG serum in both intravenously and nasally inoculated chickens, but the enhancement of the anti-Ig HI reaction diminished from the second week after inoculation.     

Long-term immunogenicity and protection against Mycoplasma agalactiae induced by an oil adjuvant vaccine in sheep

The long-term protective immunity of an inactivated mineral-oil adjuvanted Mycoplasma agalactiae vaccine was evaluated in sheep. The antigen suspension was emulsified with a mixture of three mineral oils (Montanide ISA-563, Marcol-52, Montane-80 at the ratio of 30%, 63%, and 7%, respectively). Twenty-two animals were divided in 2 groups (A and B) and immunised with two doses of the vaccine (group A, n = 14) or used as unvaccinated control (group B, n = 8). Five months after the second vaccination, seven animals of group A and four animals of group B were challenged by nasal route with M. agalactiae. The remaining seven vaccinated and four control animals were challenged intranasally eight months after vaccination. The vaccine was able to induce a full-protective immunity preventing the clinical signs of contagious agalactia and the infection by M. agalactiae in all groups of animals irrespective of the time of challenge after booster administration.     

非结核性分支杆菌的致病性及其鉴定方法

非结核性分支杆菌属于放线菌目,分支杆菌科,分支杆菌属,其分布广泛,可传染家畜及人类。近年来随着越来越多的非结核性分支杆菌疾病被发现并报道出来,已经引起人们的广泛关注和学者的研究兴趣。由于非结核性分支杆菌其临床表现与结核性分支杆菌极为相似,诊断起来有一定的困难。根据国内外近几年的报道,对非结核性分支杆菌从易感人群、感染部位及长期慢性生长等角度综述其致病性,举例说明其临床症状,再总结比较了传统分析鉴定法、噬菌体分析法、聚合酶链式反应技术和核酸探针等几种非结核性分支杆菌的鉴定方法,建议在临床表征相似时,不仅仅采用传统方法进行分离鉴定,应该采用多种分离方法相结合来进行细菌的分离鉴定,减少时间,节约成本,同时继续研发新的分类方法。     

Antimicrobial resistant Escherichia coli isolates in cattle and house sparrows on two Czech dairy farms

Rectal smears of calves, cows and young bulls, as well as cloacal smears of house sparrows (Passer domesticus), from farms at the villages of Sumice and Troskotovice, Czech Republic, were examined for E. coli resistant to 12 antimicrobials. The resistant isolates were tested for antimicrobial-resistance genes and integrons. Totals of 40% (n=183), 3% (n=95), 0% (n=33), and 9% (n=54) of Escherichia coli isolates from calves, cows, young bulls and house sparrows, respectively, were antimicrobial resistant. The following genes were identified in cattle E. coli isolates: tetA, tetB (isolates resistant to tetracycline), bla(TEM) (beta-lactams), strA, aadA (streptomycin), sul1, sul2 (sulphonamides), and cat, floR (chloramphenicol). Seven of 16 antimicrobial-resistant calf isolates from the Sumice farm possessed class 1 integrons with the aadA1 gene cassette integrated, 1 kb in size. On the Troskotovice farm, eight of 57 antimicrobial-resistant calf isolates possessed class 1 integrons. Integrons of 1.5kb with the dhfr1- aadA1 gene cassette were found in four isolates, followed by a 1kb integron with the aadA1 gene found in three isolates, and a 1.7kb integron with the dhfr17-aadA5 gene cassette and the phenotype ASSuTSxtNaCipCCfG. The prevalence of resistant E. coli in calves compared to adult cattle was much higher and probably was influenced by oral antimicrobial usage in calves, feeding with milk and colostrum from treated cows, as well as mechanisms unrelated to antimicrobial drug selection. Although house sparrows lived together with the cattle and came into contact with cattle waste on the farm, they were not infected by resistant E. coli isolates with the same characteristics as those found in cattle.     

4种佐剂对绵羊肺炎支原体的免疫辅佐效力研究

不同种类的佐剂对同种抗原的免疫辅佐效力不同。为筛选绵羊肺炎支原体(Mycoplasma ovipneumoniae,MO)灭活疫苗的理想佐剂,本研究制备了4种不同佐剂的MO灭活疫苗,分别免疫成年家兔,用MO间接血凝试剂盒测定免疫后1～9周血清抗体效价。检测结果显示,4种疫苗在免疫后都能迅速产生抗体,其中用MO Buonavoglial's和ISA-760佐剂疫苗免疫家兔,产生抗体均于免疫后第3周达到高峰((5.67±0.70) log2,6.00 log2),到第9周时维持抗体高峰(6.00 log2,(6.00±1.00) log2);MO ISA-206和白油佐剂疫苗免疫抗体均于免疫后第4周达到高峰((4.33±0.57) log2,5.00 log2),至第9周下降约1个滴度。统计学分析结果显示,ISA-760、Buonavoglial's佐剂对MO的免疫增强效力显著优于ISA-206和白油佐剂(P<0.05),是MO灭活疫苗的理想候选佐剂,为高效MO灭活疫苗的研发提供了科学依据。     

Characterisation of protein and antigen variability among Mycoplasma mycoides subsp. mycoides (LC) and Mycoplasma agalactiae field strains by SDS-PAGE and immunoblotting

Mycoplasma mycoides subsp. mycoides (LC) (Mmm LC) and Mycoplasma agalactiae are the most important mycoplasma species involved in the contagious agalactia syndrome. A total of 25 field strains from Spain and the two type strains were analysed by SDS-PAGE and immunoblotting. Two polyclonal antisera (PAbs) raised against a pool of strains of each mycoplasma species were used. The results revealed a high degree of protein variability among the field strains. The type strain of Mmm LC appeared to be representative of the field strains of this species, whereas this was not the case with the M. agalactiae type strain. Whereas M. agalactiae is known to possess a gene family regulating surface antigen diversity, there is a need to study the mechanisms used byMmm LC to generate antigenic variability in more detail.     

Synthesis, cloning, and expression of Mycoplasma suis inorganic pyrophosphatase gene using PCR-based accurate synthesis and overlap-extension PCR, and its immunogenicity analysis

Mycoplasma suis (M. suis), a hemotrophic pathogen of pigs, causes economic losses in swine production throughout the world. Inorganic pyrophosphatase (ppa) is a very important gene in M. suis. The ppa gene of M. suis was synthesized by PCR-based accurate synthesis (PAS) and overlapextension PCR, inserted into vector pMD18-T, and then subcloned to the prokaryotic expression vector pET28c.The recombinant plasmid pET28c_ppa was transformed to E. coli BL21 for expression under induction of isopropyl thiogalactoside. The expressed product was identified by SDS–PAGE and Western blot, which suggested that the recombinant protein has good antigenicity. Piglets were immunised with purified recombinant protein, and specific antibodies to the recombinant protein were detected in piglet serum. The results show that the ppa gene can be efficiently expressed in E. coli and that the expressed recombinant protein can elicit a specific serum antibody response in piglets. PAS and overlap-extension PCR were first used to synthesize the ppa of M. suis. They provide simple, rapid, reliable and relatively inexpensive methods to synthesize, clone, and express genes. The experiment conducted in this paper will enable future research into the role and function of the ppa gene.     

Prevalence of Calodium hepaticum (Syn. Capillaria hepatica) in house mice (Mus musculus) in the Azores archipelago

Calodium hepaticum (Syn. Capillaria hepatica) is a zoonotic liver nematode of mammals distributed worldwide. Rodents are believed to be the main reservoirs of this nematode. In this paper, prevalence of the parasite was analyzed in liver histological sections from 51 house mice (Mus musculus) caught in human-inhabited houses, from two localities (Furnas and Rabo de Peixe) on São Miguel island from the Azores archipelago (Portugal). Mean prevalence of infection was 19.6%, with 33.3% prevalence in Furnas and 4.1% in Rabo de Peixe (P = 0.07). No significant differences were found between the prevalence of infection and the age, body weight and the sex of mice. Hepatic lesions found were either acute and/or chronic stage and consisted of moderate to severe multifocal pyogranulomatous hepatitis with encapsulated eggs with typical bipolar plugs and moderate to severe necrotizing hepatitis consistent with larva tracks. Periportal inflammatory infiltration, hepatocyte regeneration and bile duct hyperplasia were also noted. In most cases, hepatic lesions occupied more than 50% of the liver, but despite severe lesions, in some mice, no signs of hepatic failure were noticed. The high rate of infection found in the present study suggests that house mice are an important reservoir for this parasite in the Azores and could have a role in human transmission.     

Molecular cloning of a Mycoplasma meleagridis-specific antigenic domain endowed with a serodiagnostic potential

A recombinant phage library harbouring Mycoplasma meleagridis (MM) genomic DNA fragments was generated in the bacteriophage lambda gt11 expression vector. The library was screened for expression of MM specific antigens with a polyclonal antiserum that had been preadsorbed with antigens of the most common unrelated avian mycoplasma species. A 49-amino acid antigenic domain unique to MM was isolated, expressed in Escherichia coli, and its serodiagnostic potential was demonstrated. An antiserum raised against this MM-specific antigenic domain recognized a cluster of seven membrane-associated MM proteins with molecular masses ranging from 34 to 75 kDa. Overall, this study resulted in the identification of a potent serodiagnostic tool and revealed the complex antigenic nature of MM.     

Optimization of culture media of pathogenic Mycoplasma hyopneumoniae by a response surface methodology

Composition of culture medium for mass production of Mycoplasma hyopneumoniae was optimized using a response surface methodology (RSM). Initially, the influence of glucose, thallium acetate, fresh yeast extract, horse serum, and porcine serum on the production of mycoplasmal protein was assessed using a ''one factor at a time'' technique. Next, factors with a significant effect, including fresh yeast extract, and horse and porcine sera, were selected for further optimization using a central composite design (CCD) of RSM. The experimental results were fitted into a second order polynomial model equation. Estimated optimal condition of the factors for maximum production of mycoplasmal protein (i.e., triple-fold increase from 0.8 mg/L produced by basal mycoplasma media to 2.5 mg/L) was 10.9% fresh yeast extract, 15% horse serum, and 31.5% porcine serum (v/v). For the optimized conditions, a 2.96 mg/L experimental result was observed, similar to the estimated optimal conditions result of the CCD.