Molecular investigation of transplacental and vector-borne transmission of bovine haemoplasmas

The present study was carried out in a herd with concurrent infections of Mycoplasma wenyonii and 'Candidatus M. haemobos', to investigate if transplacental and/or vector-borne transmission is possible for one or both bovine haemoplasma species. For this purpose blood samples were collected from 38 mother animals and their newborn calves; as well as from 17 uninseminated cows twice three months apart. In addition, 311 mosquitoes and blood-sucking flies (Diptera: Culicidae, Tabanidae, Muscidae) were cought near the animals. DNA was extracted from all samples, followed by real-time PCR analysis. In 10.5% of neonate calves, that were born to cows harbouring both haemoplasmas, M. wenyonii and/or 'Candidatus M. haemobos' positivity was detected. Copy numbers in positive samples from cows and their calves indicated that - in comparison with M. wenyonii - 'Candidatus M. haemobos'-bacteraemia had usually lower levels. In samples of uninseminated cows the rate of infection with the latter species decreased. These findings may explain why M. wenyonii was significantly more frequently detected in blood-sucking flies, than 'Candidatus M. haemobos'. In conclusion, molecular evidence is provided for the first time on the transplacental transmission of bovine haemoplasmas. Regarding their spread by blood-sucking arthropods, new potential vectors were identified, i.e. the horn fly (Haematobia irritans), the stable fly (Stomoxys calcitrans) and two species of horse flies (Tabanus bovinus, T. bromius).     

Occurrence of 'Candidatus Mycoplasma turicensis' infection in domestic cats in Japan

We have examined the presence of hemoplasmas, hemotropic mycoplasmas, among domestic cats (Felis catus) in Japan by using a species specific PCR, and found 'Candidatus Mycoplasma turicensis', a recently recognized hemoplasma species. A total of 60 feline blood samples collected in 2004 and 2005 were subjected to PCR amplification for the detection of Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum' and 'Candidatus Mycoplasma turicensis'. Six blood samples collected from domestic cats were found infected with the 'Candidatus M. turicensis'. All of them are also infected with other species of hemoplasmas, M. haemofelis and/or 'Candidatus M. haemominutum'. This is the first to demonstrate 'Candidatus M. turicensis' infections among cat population in Japan.     

利用套式PCR检测牛羊乏质体

为同时检测和鉴定牛边缘乏质体、中央乏质体及绵羊乏质体,根据这3种病原体的msp4基因核苷酸序列,自行设计、合成了针对3种乏质体的2对通用引物,及分别针对三者的特异引物,通过PCR条件优化,建立了检测乏质体及分别鉴定3种乏质体的套式PCR方法,并与OIE推荐的msp5半套式PCR比较.结果显示:该方法对牛巴贝斯虫、双芽巴贝斯虫、羊莫氏巴贝斯虫、山羊泰勒虫、温氏附红细胞体、东方巴贝斯虫、刚地弓形虫、伊氏锥虫均未扩增出特异性片段.套式PCR检测乏质体DNA量为0.2 pg(相当于6个感染红细胞).检测l 119份来自6个不同地区的奶牛、肉牛、水牛及羊的临床样品,阳性106份,经鉴定边缘乏质体46份,中央乏质体15份,绵羊乏质体35份,混合感染中央乏质体和绵羊乏质体4份,混合感染边缘乏质体和绵羊乏质体3份,混合感染边缘乏质体和中央乏质体3份.首次在分子生物学水平证明中央乏质体存在于中国.同时,证明牛可以混合感染边缘乏质体和中央乏质体或绵羊乏质体,以及混合感染中央乏质体和绵羊乏质体.上述848份样品用OIE推荐的msp5半套式PCR同时检测,两者符合率为98.5％(835/848).检测结果表明,msp4套式PCR特异、敏感,可用于边缘乏质体、中央乏质体、绵羊乏质体的检测和鉴定.     

Molecular detection of Mycoplasma wenyonii and 'Candidatus Mycoplasma haemobos' in cattle in Hokkaido, Japan

Blood samples from 78 cattle were tested for hemoplasma infection using molecular methods. PCR and sequence analysis revealed that 17 cattle were infected with Mycoplasma wenyonii, while 13 were infected with 'Candidatus Mycoplasma haemobos'. Four animals were infected with both species. This is the first study to report hemoplasma species infection among cattle in Japan.     

Detection of specific Mycoplasma conjunctivae antibodies in the sera of sheep with infectious keratoconjunctivitis

The serological cross reactions between Mycoplasma conjunctivae, the etiological agent of infectious keratoconjunctivitis (IKC), and the antigenetically and phylogenetically closely related Mycoplasma ovipneumoniae, which is often found in sheep, were analysed. Cross reacting antigens were identified using sera from sheep with IKC and from sheep of herds known to be free of IKC, as well as rabbit hyperimmune serum specific to the two Mycoplasma species. Cross reactions were predominantly due to the strongly antigenic proteins of 42 kDa and 83 kDa. Serospecific antigens of M. conjunctivae could be separated from cross-reacting antigens by the extraction of Tween 20-soluble membrane proteins. The Tween 20-extracted proteins of the M. conjunctivae strain HRC/581T were used for the development of an indirect ELISA test. This ELISA test was shown to be a useful serological method for the diagnosis of M. conjunctivae infections and to identify infected sheep herds.     

牛附红细胞体单管套式PCR检测方法的建立

为快速、准确地检测出牛附红细胞体,根据GenBank上发表的牛温氏附红细胞体(Mycoplasma wenyonii)l6S rRNA基因序列(登录号AF016546)设计合成内外2对引物,建立了牛附红细胞体单管套式PCR检测方法,并进行了特异性、敏感性及应用试验。结果:建立的牛附红细胞体单管套式PCR检测方法扩增的片段大小为361 bp,与GenBank中相应序列同源性为98%,该方法扩增不出牛瑟氏泰勒虫、新孢子虫、布氏杆菌及牛无乳链球菌等基因片段,检测出标准模板DNA的最小量为1.22 fgμ/L。通过对吉林省延边地区66份血液样本的临床检测显示,单管套式PCR检出率28.8%,高于常规PCR的21.2%和鲜血压片镜检的12.1%,具有准确、特异、敏感等优点,是检测牛附红细胞体的一种新型、可靠的诊断技术。     

奶牛温氏支原体16SrRNA基因的PCR扩增、克隆及分析

目的对奶牛温氏支原体16SrRNA基因进行PCR扩增及克隆分析。方法从自然感染体的广西奶牛无菌采集血液,分离温氏支原体并提取病原基因组,用血营养菌的16SrRNA基因的通用引物进行PCR扩增,将扩增产物克隆到PGEM-Teasy载体后进行溺,I序和分析,并与Genebank上搜索的温氏支原体相应序列进行比较,建立系统发育树。结果PCR扩增得到长约1．5kb的扩增片段,测序结果显示该片段全长为1453bp,同源性分析表明该序列与Neimark公布的温氏支原体（前称温氏附红细胞体）（AF016546）的16SrRNA基因序列同源性达到97．4％,与系统发育进化树表明本株温氏支原体同本地株的关系较近,而与国外株的新缘关系较远。国内公布的广西株同源性为99．8％。结论结果表明证实该病原为温氏支原体,从分子生物学水平证实了温氏支原体在广西的存在。由于本试验分离得到的牛温氏支原体与国外发表的牛温氏支原体核苷酸序列相差2．6％,因此两者的基因型存在一定的差异,这对该病的分子流行病学分析具有一定的意义。     

猪支原体不同靶基因PCR检测方法的比较

为筛选出检测猪支原体更为特异、敏感的PCR检测方法,本试验分别以16S rRNA、50S rRNA和膜蛋白OxaA为靶基因进行PCR检测,并从其敏感性、特异性和临床样本检出率等方面进行了比较。结果显示,以膜蛋白OxaA和16S rRNA为靶基因的PCR方法敏感性最高,最小检测DNA量为1.86 fg/μL,而以50S rRNA为靶基因的PCR方法最小检测DNA量为18.6 fg/μL;3种靶基因引物均扩增不出大肠杆菌、猪链球菌、猪肺炎支原体、牛附红细胞体等基因片段,具有较好的特异性;通过对临床60份血液样本的检测结果表明,以膜蛋白OxaA基因设计的引物检出率最高,为25%(15/60),明显高于16S rRNA基因的21.6%(13/60)和50S rRNA基因的18.3%(11/60)。本试验为猪支原体病的诊断及流行病学调查提供了更为敏感、特异的检测技术。     

猪附红细胞体50S核糖体基因PCR检测方法的建立及应用

根据GenBank上最新发布的猪附红细胞体基因组序列(NC-015155)设计一对引物,并以吉林省延边地区猪附红细胞体基因组DNA为模板,建立猪附红细胞体50 S核糖体基因PCR诊断方法,通过特异性、敏感性及临床应用试验验证,快速准确的检测出猪附红细胞体.试验结果显示,建立的猪附红细胞体PCR诊断方法扩增片段大小为10...     

Detection frequency of haemoplasma infections of the domestic cat in Germany

We present epidemiological data on the frequency of infections with haemotrophic Mycoplasma spp. (feline haemoplasmas) in domestic cats in Germany. From November 2004 to October 2006 135 blood samples of anaemic patients and cats without clinical symptoms were examined with conventional and real-time PCR methods. In 15,6 % of the samples DNA of one or more haemoplasma species could be detected. 8,9 % of the samples (12 cats) were infected with "Candidatus Mycoplasma haemominutum, whereas 7,4 % (10 cats) were infected with Mycoplasma haemofelis. Out of these, one cat harboured both species.The recently described species "Candidatus Mycoplasma turicensis" was found in 2.2 % of all samples (3 cats) and was restricted to animals coinfected with M. haemofelis. No correlation could be detected between the infection with haemotrophic Mycoplasma spp. and clinical signs of anaemia or disease. Infections were significantly correlated with age, male gender or coinfections with retroviruses (FIV, FeLV). Our data indicate, that chronically infected carriers without clinical symptoms are frequent in the investigated cat populations in Germany and that the screening of blood-donors for the presence of Mycoplasma spp. infections is advisable before clinical use.     

A multiplex PCR to identify porcine mycoplasmas present in broth cultures

Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and Mycoplasma flocculare can be present in the lungs of pigs at the same time. These three mycoplasma species all require similar growth conditions and can be recovered from clinical samples using the same media. We have developed a multiplex PCR as a helpful tool for rapid differentiation of these three species in the course of isolation. Based on the 16S ribosomal DNA sequences, three different forward primers and a single reverse primer were selected. Each forward primer was compared to available mycoplasma sequences, showing the primers to be specific. The three amplification products observed of 1129 bp (M. hyorhinis), 1000 bp (M. hyopneumoniae) and 754 bp (M. flocculare) were clearly distinguishable on a 1% agarose gel. In addition, no cross-reaction with Mycoplasma hyosynoviae, another porcine mycoplasma, was noted. This multiplex PCR using the proposed set of primers is the first reported assay that allows the simultaneous identification of the different Mycoplasma species isolated from the lungs of pigs.     

Severe anemia associated with Mycoplasma wenyonii infection in a mature cow

The clinical findings, diagnostic tests, and treatment of clinical anemia in a mature Angus cow infected with the hemoplasma Mycoplasma wenyonii are described. Mycoplasma wenyonii has been previously reported to cause clinical anemia in young or splenectomized cattle; however, infection has not been associated with severe anemia in mature animals.     

Prevalence of hemoplasma infection among cattle in the western part of Japan

We have examined for hemoplasma infection among cattle in the Hiroshima and Miyazaki prefectures by using a sensitive real-time PCR, with SYBR Green I and with melting curve analysis, which allow to distinguish the two bovine hemoplasma species, Mycoplasma wenyonii and 'Candidatus M. haemobos'. We found 69.4% of 36 cattle in Hiroshima and 93.8% of 32 cattle in Miyazaki infected with either of these two hemoplasma species. High morbidity in western part of Japan may reflect the activity of arthropod vectors for hemoplasma transmission. We also demonstrated neonatal calves less than three months old affected with hemoplasmas without grazing in summer, suggesting a possibility of vertical transmission.     

Prevalence of mycoplasma antibodies in lesser prairie-chicken sera

Serologic testing by the serum plate agglutination (SPA) procedure was performed to detect the presence of cross-reacting antibodies to Mycoplasma meleagridis, Mycoplasma synoviae, and Mycoplasma gallisepticum in lesser prairie-chickens (Tympanuchus pallidicinctus) trapped over a 2-yr period in Finney and Kearny counties of southwestern Kansas. Sera examined from birds (n = 50) obtained in March-April 2000 tested positive for M meleagridis, M. synoviae, and M. gallisepticum at levels of 6%, 10%, and 10%, respectively, for the population examined. Mycoplasma meleagridis antibodies were detected in 3 samples (2.7%), M. synoviae antibodies in 2 samples (1.7%), and M. gallisepticum antibodies in 3 samples (2.7%) from birds (n = 112) collected in March-April 2001. Data obtained by SPA can result in false positives and should be verified by additional procedures such as the hemagglutination-inhibition test. Low amounts of sera prohibited this additional testing. Thus, the positive SPA results should be considered presumptive for the presence of Mycoplasma antibodies. Although Mycoplasma antibodies have been detected in wild turkeys (Meleagris gallopavo) from Kingman and Butler counties in Kansas, this report is the first of possible mycoplasmosis in Finney and Kearny counties, Kansas. All birds testing positive by this procedure should be considered as potential carriers of Mycoplasma and should not be used in relocation efforts.     

Diagnosis of feline haemoplasma infection using a real-time PCR assay

Haemobartonella felis has been reclassified within the genus Mycoplasma as Mycoplasma haemofelis and 'Candidatus Mycoplasma haemominutum', collectively referred to as the feline haemoplasmas. A total of 78 cats from the Johannesburg area that had blood samples submitted to a private veterinary laboratory were tested using a real-time polymerase chain reaction (PCR) assay able to detect and distinguish the two feline haemoplasma (basonym Haemobartonella) species. All samples had been diagnosed with haemoplasma infection by cytological examination of blood smears. Statistical analysis was performed to evaluate associations between haemoplasma status, age, and haematological and biochemical parameters. On PCR assay 43 cats (55%) were haemoplasma negative, 25 (32.1%) positive for 'Candidatus Mycoplasma haemominutum', 5 (6.4%) positive for Mycoplasma haemofelis and 5 (6.4%) positive for both species. Significant inverse correlation was found between the amount of M. haemofelis DNA present in the blood and the haematocrit value. Cats that were positive for M. haemofelis showed macrocytic regenerative anaemia, monocytosis and thrombocytopaenia. This report documents the existence of both haemoplasma species in cats in South Africa.     

Identification by culture, PCR, and immunohistochemistry of mycoplasmas and their molecular typing in sheep and lamb lungs with pneumonia in Eastern Turkey

This study used cultures, polymerase chain reaction (PCR), and immunoperoxidase to examine samples from 216 lungs from sheep and lambs with macroscopic pneumonia lesions for the presence of Mycoplasma species. DNA was extracted from lung tissue samples and broth cultures with the help of a DNA extraction kit and replicated using genus-specific and species-specific primers for mycoplasma. The lung samples were examined by the immunoperoxidase method using hyperimmune Mycoplasma ovipneumoniae serum. The randomly amplified polymorphic DNA (RAPD) test was used for the molecular typing of M. ovipneumoniae isolates. Mycoplasma was isolated in the cultures of 80 (37.03 %) of a total of 216 lung samples. Genus-specific mycoplasma DNA was identified by PCR in 96 (44.44 %) samples in broth cultures and 36 (16.66 %) directly in the lung tissue. Of these 96 cases in which genus-specific identification was made, 57 (59.37 %) were positive for reaction with species-specific primers for M. ovipneumoniae and 31 (32.29 %) for Mycoplasma arginini. The DNA of neither of the latter two species could be identified in the remaining eight samples (8.33 %) where mycoplasma had been identified. As for the immunoperoxidase method, it identified M. ovipneumoniae in 61 of 216 lung samples (28 %). Positive staining was concentrated in the bronchial epithelium cell cytoplasm and cell surface. RAPD analysis resulted in 15 different profiles. Our results suggest that PCR methods could be successfully used in the diagnosis of mycoplasma infections as an alternative to culture method and identifying this agent at the species level.     

Prevalence of mycoplasmas in the semen and vaginal swabs of Danish stallions and mares

The reproduction rate of horses is one of the lowest within domestic livestock despite advances the veterinary medicine. Infertility in horses may be due mainly to the lack of suitable selection criteria in the breeding of horses. However, acquired infertility due to genital, bacterial infections may occur. Mycoplasmas have been implicated in genital disorders and infertility of many species including humans and horses. However, their role as commensals or pathogens of the genital tract of horses is still not determined. Bacteriological examinations made on the fossa glandis, urethra, penis and semen of stallions, showed the presence of different Mycoplasma species. Therefore our study aimed to find the prevalence of Mycoplasma species and a possible association with fertility problems in Danish riding horses. Eighty semen samples from stallions and 19 vaginal swab samples from mares were tested by PCR for presence of mycoplasmal DNA. The vaginal swab samples were also cultured in the Mycoplasma specific medium. None of the samples were positive for presence of genital mycoplasmas during the screen. The lack of genital mycoplasmas observed in this study may be due to a very extensive use of artificial insemination of modern sport horses.     

Fatal bovine anaplasmosis in a herd with new genotypes of Anaplasma marginale, Anaplasma ovis and concurrent haemoplasmosis

Haematological and molecular analysis of blood samples was carried out during an outbreak of bovine anaplasmosis in Hungary. Acute disease was observed in five animals, two of which died. Anaplasma-carrier state was diagnosed in 69 (92%) of cattle. Further evaluation of 24 blood samples revealed concurrent infections with Mycoplasma wenyonii and 'CandidatusM. haemobos' in 22 and 21 animals, respectively. In addition, two cows were identified with rickettsaemia. Regarding molecular investigation of potential hard tick vectors, Haemaphysalis inermis and Dermacentor marginatus males collected from the animals were PCR-negative. However, in one pool (out of 18) of Ixodesricinus males, and in six pools (out of 18) of D. reticulatus males the msp4 gene of Anaplasma marginale was detected. In the same I. ricinus pool Anaplasma ovis was also identified. All ticks were negative for haemoplasmas. Anaplasma sequences yielded 97-99% homology to sequences deposited in the Genbank. This is the first report of fatal bovine anaplasmosis associated with divergent A. marginale genotypes and concurrent 'CandidatusM. haemobos' infection, as well as of an A. ovis strain in ticks collected from cattle.     

Rapid Identification of Mycoplasma pulmonis Isolated from Laboratory Mice and Rats Using Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

Mycoplasma species identification is based on biochemical, immunological, and molecular methods that require several days for accurate identification. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a novel method for identification of bacteria and has recently been introduced into the clinical microbiology laboratory as a rapid and accurate technique. This method allows a characteristic mass spectral fingerprint to be obtained from whole inactivated mycoplasmal cells. In this study, we evaluated the performance of the MALDI-TOF MS for the identification of Mycoplasma by comparison with standard sequence analysis of 16S rRNA. We developed the first database of MALDI-TOF MS profiles of Mycoplasma species, containing Mycoplasma pulmonis, M. arthritidis, and M. neurolyticum, which are the most common pathogens in mice and/or rats, and species-specific spectra were recorded. Using the database, 6 clinical isolates were identified. Six tracheal swabs from 4 mice and 2 rats were cultured on PPLO agar for 4 to 7 days, and the colonies were directly applied to analyze the protein profiles. Five strains were identified as M. pulmonis, and 1 strain from a mouse was identified as M. neurolyticum (spectral scores were >2.00); the results were consistent with the results of the 16S rRNA gene sequence analysis (homologies>97.0%). These data indicate that MALDI-TOF MS can be used as a clearly rapid, accurate, and cost-effective method for the identification of M. pulmonis isolates, and this system may represent a serious alternative for clinical laboratories to identify Mycoplasma species.     

An avidin-biotin enhanced dot-immunobinding assay for the detection of Mycoplasma gallisepticum and M. synoviae serum antibodies in chickens

A dot-immunobinding assay was enhanced by the incorporation of avidin and biotin reagents into the test system (DAB assay). This assay was used to detect serum antibodies to Mycoplasma gallisepticum (MG) and M. synoviae (MS) from chickens. Serum samples were tested by rapid serum plate (RSP), hemagglutination-inhibition (HI), and DAB assay methods. These results were compared. The DAB assay was at least 20 times more sensitive in detecting antibodies for MS and at least 75 times more sensitive in detecting antibodies for MG than the HI test. The DAB assay was as specific as the HI test. The DAB assay was also more sensitive and specific than the RSP test. Some cross-reactions occurred when low dilutions of high-titer sera were used in the DAB assay. Parameters for determining negative, suspicious, and positive samples were established. The DAB assay for MG and MS may have several applications, including use as a screening test and a confirmatory test.