Molecular characterization and phylogenetic analysis of the causative agent of hemoplasma infection in small Indian Mongoose (Herpestes Javanicus)

Hemoplasmas are the trivial name for a group of erythrocyte-parasitizing bacteria of the genus Mycoplasma. This study is the first report of hemoplasma infection in Small Indian Mongoose (Herpestes Javanicus) based on molecular analysis of 16S rDNA. Whole blood samples were collected by sterile methods, from 14 live captured mongooses, in the south of Iran.     

Characterization of the in vitro core surface proteome of Mycoplasma mycoides subsp. mycoides, the causative agent of contagious bovine pleuropneumonia

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides (Mmm) is a severe cattle disease, present in many countries in sub-Saharan Africa. The development of improved diagnostic tests and vaccines for CBPP control remains a research priority. Polyacrylamide gel electrophoresis and mass spectrometry were used to characterize the Triton X-114 soluble proteome of nine Mmm strains isolated from Europe or Africa. Of a total of 250 proteins detected, 67 were present in all strains investigated. Of these, 44 were predicted to be lipoproteins or cytoplasmic membrane-associated proteins and are thus likely to be members of the core in vitro surface membrane-associated proteome of Mmm. Moreover, the presence of all identified proteins in other ruminant Mycoplasma pathogens were investigated. Two proteins of the core proteome were identified only in other cattle pathogens of the genus Mycoplasma pointing towards a role in host–pathogen interactions. The data generated will facilitate the identification and prioritization of candidate Mycoplasma antigens for improved control measures, as it is likely that surface-exposed membrane proteins will include those that are involved in host–pathogen interactions.     

Phylogenetic analysis of the genus Anaplasma in Southwestern China based on 16S rRNA sequence

To identify the species within the genus Anaplasma circulating among ruminants in the Southwest of China, we performed the phylogenetic analysis of the 16S rRNA gene of two Anaplasma isolates from cattle and seven from goats. The two sequences obtained from cattle strains belonged to the A. marginale cluster, whereas the other seven sequences from caprine strains formed two Anaplasma spp. clusters, which diverged earlier than the clusters of A. marginale, A. centrale and A. ovis. These results indicate that there are at least two Anaplasma species circulating among ruminants in Southwestern China.     

Detection and prevalence of four different hemotropic Mycoplasma spp. in Eastern North Carolina American black bears (Ursus americanus)

Hemotropic Mycoplasma spp. are globally emerging, obligate parasitic, epierythrocytic bacteria that infect many vertebrates, including humans. Hemoplasma infection can cause acute life-threatening symptoms or lead to a chronic sub-clinical carrier state. Hemotropic Mycoplasma spp. transmission, prevalence, and host specificity are uncertain. The purpose of this study was to determine the molecular prevalence of Mycoplasma species in blood from 68 free-ranging black bears from the eastern coast of North Carolina. DNA amplification of Mycoplasma 16S rRNA gene identified four distinct species infecting 34/68 (50%) of the black bear blood samples, including Candidatus M. haematoparvum. The high prevalence of hemotropic Mycoplasma infection in this wildlife species highlights the importance of understanding intra and inter species transmission. Black bears may play a role in the transmission of hemotropic Mycoplasma spp. between animals, arthropod vectors, and humans. Further studies are needed to elucidate black bears as a potential reservoir for hemotropic Mycoplasma infections.     

Novel hemotropic Mycoplasma species in white-tailed deer (Odocoileus virginianus)

Globally, hemotropic Mycoplasma spp. are emerging or re-emerging zoonotic pathogens that affect livestock, wildlife, companion animals, and humans, potentially causing serious and economically important disease problems. Little is known about hemotropic Mycoplasma spp. prevalence, host-specificity, or route of transmission in most species, including wildlife. DNA amplification by PCR targeting the 16SrRNA and the RNaseP genes was used to establish the presence and prevalence of hemotropic Mycoplasma spp. in a white-tailed deer (O. virginianus) population in eastern North Carolina. Sixty-five deer (89%) tested positive for hemotropic Mycoplasma spp. where sequence analysis of the 16SsRNA and the RNaseP genes indicated the presence of at least three distinct species. This study represents the first detection of three distinct hemotropic Mycoplasma species in white-tailed deer and the first report of two novel hemotropic Mycoplasma species.     

Comparison of seven isolates of Mycoplasma meleagridis

A comparative study of seven isolates of Mycoplasma meleagridis indicated that they were indistinguishable morphologically. Two isolates, E₂ and 8M92, induced hemagglutination of red blood cells of several different species while the others did not. Metabolic inhibition, growth inhibition and growth precipitation tests revealed minor differences among the seven isolates. According to these differences, isolates were divided into three groups: antiserum-sensitive isolate 1 466, less sensitive isolates N, 8M92, RY₃, 529 and E₂ and insensitive isolate 1940. One dimensional polyacrylamide gel electrophoresis of cell proteins revealed that all isolates of M. meleagridis had virtually identical patterns and that they were electrophoretically distinct from Mycoplasma gallisepticum and Mycoplasma synoviae. When nonhemagglutinating isolate N, and hemagglutinating isolate E₂ were examined by simple immunoelectrophoresis, no differences were detected. However, minor antigenic differences were detected between the two strains by means of two dimensional immunoelectrophoresis.     

Mycoplasma sp. infection in captive Marcgrave's capuchin monkeys (Sapajus flavius)

Three species of hemoplasmas are known to infect monkeys, ‘Candidatus Mycoplasma kahanei’, ‘Candidatus Mycoplasma haemomacaque’ and ‘Candidatus Mycoplasma aoti’. Marcgrave’s capuchin monkey (Sapajus flavius) is a critically endangered neotropical primate species of the Brazilian Atlantic Forest, and thus, sustained efforts are needed to protect the health and well-being of these animals. This study has aimed to screen 12 captive S. flavius from the State of Paraíba, northeastern Brazil, for hemoplasma infection. Packed cell volume (PCV) was measured and a pan-hemoplasma PCR protocol performed. A total of 8/12 (66.6%) monkeys were positive for the 16S rDNA gene of Mycoplasma sp., showing an identity of 99% to Mycoplasma sp. previously detected in Sapajus apella and Sapajus sp. monkeys from the Brazilian Amazon. Further studies should be conducted in order to elucidate whether this potentially novel Mycoplasma sp. is able to cause anemia in Sapajus sp. monkeys from Brazil.     

Studies into the prevalence of Mycoplasma species in small ruminants in Benue State,North-central Nigeria

The indicative prevalence of respiratory Mycoplasma species in small ruminants (SR) was determined in North-central Nigeria. Nasal swabs from 172 sheep and 336 goats from the Northeast, Northwest and South Senatorial Districts of Benue State were examined. Initial Mycoplasma isolation used Mycoplasma culture techniques followed by digitonin sensitivity testing. Species identification was done using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). Overall, Mycoplasma organisms were isolated from 131 (25.8 %) of the 508 SR examined. Prevalence rates of 18.1 and 29.8 % were recorded for sheep and goats, respectively. A total of 135 isolates of Mycoplasma belonging to three different species were identified: Mycoplasma ovipneumoniae (127), Mycoplasma arginini (7) and Mycoplasma mycoides subspecies capri (1). More than one Mycoplasma species were detected in four (3.1 %) of the 131 confirmed Mycoplasma positive cultures. Mycoplasma was isolated from 16.2 and 29.1 % of animals with and without respiratory signs, respectively. The high isolation rate of mycoplasmas in apparently healthy and clinically sick sheep and goats in this study indicates a carrier status in these SR which may constitute a serious problem in disease control.     

Occurrence and genetic diversity of hemoplasmas in beef cattle from the Brazilian Pantanal,an endemic area for bovine trypanosomiasis in South America

Hemotropic mycoplasmas (hemoplasmas) are Gram-negative bacteria that parasitize the erythrocyte surface of a wide variety of mammals. The present study aimed at investigating the occurrence of hemoplasmas in beef cattle in the Brazilian Pantanal, an area endemic for bovine trypanosomiasis in South America. Additionally, the objective of this study was to characterize molecularly the genotypes of the found hemoplasmas. For this purpose, blood and serum samples of 400 beef cattle were collected from five properties in Corumbá, Nhecolândia sub-region, Mato Grosso do Sul, in Midwest Brazil. Blood samples underwent DNA extraction and standard 16S rRNA gene-based PCR assays for hemoplasmas. The sequences obtained were submitted to phylogenetic inferences, distance analysis, and genotype diversity. The Indirect Enzyme-Linked Immunoabsorbent Assay (iELISA) indicated the presence of anti-Trypanosoma vivax IgG antibodies in 89.75% of the animals sampled, confirming the endemicity of said agent in the studied region. Among the 400 bovine blood samples tested, 2.25% (9/400) were positive for hemoplasmas in cPCR. The phylogenetic analysis of the obtained sequences confirmed the presence of 'Candidatus Mycoplasma haemobos' and Mycoplasma wenyonii DNA in 0.5% (2/400) and 1.75% (7/400) animals, respectively. Five genotypes of M. wenyonii and one of 'Candidatus M. haemobos' were detected among the sequenced amplicons. The present study showed low molecular occurrence of haemoplasmas in beef cattle sampled in the Brazilian Pantanal, an area endemic for bovine trypanosomiasis. Despite of the conservation of the 16S rRNA gene, there was considerable diversity of hemoplasma genotypes infecting the sampled beef cattle.     

Microscopic and molecular identification of hemotropic mycoplasmas in South American coatis (Nasua nasua)

Hemoplasmas were detected in two apparently healthy captive South American coatis (Nasua nasua) from southern Brazil during an investigation for vector-borne pathogens. Blood was subjected to packed cell volume (PCV) determination, a commercial real-time PCR panel for the detection of Anaplasma spp., Babesia spp., Bartonella spp., Hepatozoon spp., Leishmania spp., Mycoplasma haemofelis, ‘Candidatus Mycoplasma turicensis’, ‘Candidatus Mycoplasma haemominutum’, Neorickettsia risticii, Rickettsia rickettsii and Leptospira spp., and a pan-hemoplasma conventional PCR assay. PCV was normal, but both coatis tested positive for hemoplasmas and negative for all the remaining pathogens tested. Using different techniques for microscopy (light, confocal or SEM), structures compatible with hemoplasmas were identified. Sequencing of the 16S rRNA gene identified an organism resembling Mycoplasma haemofelis and another hemotropic Mycoplasma sp., with a sequence identity of 96.8% to a Mycoplasma sp. previously detected in capybaras.     

Diversity and molecular characterization of novel hemoplasmas infecting wild rodents from different Brazilian biomes

Although hemoplasma infection in domestic animals has been well documented, little is known about the prevalence and genetic diversity of these bacteria in wild rodents. The present work aimed to investigate the occurrence of hemotrophic mycoplasmas in wild rodents from five Brazilian biomes, assessing the 16S rRNA phylogenetic position of hemoplasma species by molecular approach. Spleen tissues were obtained from 500 rodents, comprising 52 different rodent species trapped between 2000 and 2011. DNA samples were submitted to previously described PCR protocols for amplifying Mycoplasma spp. based on 16S rRNA, followed by sequencing and phylogenetic inferences. Among 457 rodent spleen samples showing absence of inhibitors, 100 (21.9%) were PCR positive to Mycoplasma spp. The occurrence of hemotropic mycoplasmas among all sampled rodents was demonstrated in all five biomes and ranged from 9.3% (7/75) to 26.2% (38/145). The Blastn analysis showed that amplified sequences had a percentage of identity ranging from 86 to 99% with other murine hemoplasmas. The ML phylogenetic analysis of 16S rRNA gene of 24 positive randomly selected samples showed the presence of ten distinct groups, all clustering within the Mycoplasma haemofelis. The phylogenetic assessment suggests the circulation of novel hemoplasma species in rodents from different biomes in Brazil.     

Mycoplasma synoviae cell invasion: Elucidation of the Mycoplasma pathogenesis in chicken

Fluorochrome-labelled cells of two field isolates and Mycoplasma synoviae (Ms) were inoculated onto monolayer cultures of fluorochrome-labelled HEp-2 cells and monitored by confocal laser scanning microscopy (CLSM). Ms was detected initially adhered to and subsequently inside the host cells. Between 24 and 48 h of infection, Ms was detected in the perinuclear region, and after 72 h of infection was confirmed by gentamicin invasion assay. High and low passage Ms strains showed no differences in adherence or invasion. The morphology and the actin filaments of the infected HEp-2 cells were preserved throughout the study period. The observed invasion by Ms is consistent with the biology of Mollicutes, and could explain the difficulties in recovering field isolates of the mycoplasma and in controlling the infection in birds even after long-term antibiotic treatment.     

Detection and identification of avian mycoplasmas by polymerase chain reaction and restriction fragment length polymorphism assay

The polymerase chain reaction (PCR) with primers complementary to the 16S rRNA genes was used to detect avian mycoplasmas. A primer pair designed for the detection of human and rodent mycoplasmal species was examined for its ability to detect the most important avian mycoplasmas. After testing the respective reference strains, we found that Mycoplasma iowae, Mycoplasma meleagridis and Mycoplasma synoviae could be detected by PCR with this primer pair, and distinction could be made among them by restriction fragment length polymorphism (RFLP) assay with two restriction enzymes (BamHI and RsaI). For the detection of Mycoplasma gallisepticum by PCR, we needed species-specific primers. The results of the PCR- and RFLP-based identification procedures of 17 different field isolates agreed with those obtained by conventional methods.     

Identification of Arcanobacterium pyogenes isolated by post mortem examinations of a bearded dragon and a gecko by phenotypic and genotypic properties

The present study was designed to identify phenotypically and genotypically two Arcanobacterium (A.) pyogenes strains isolated by post mortem examinations of a bearded dragon and a gecko. The A. pyogenes strains showed the typical biochemical properties and displayed CAMP-like synergistic hemolytic activities with various indicator strains. The species identity could be confirmed genotypically by amplification and sequencing of the 16S rDNA gene and, as novel target gene, by sequencing of the beta subunit of RNA polymerase encoding gene rpoB, of both strains and of reference strains representing nine species of the genus Arcanobacterium. The species identity of the two A. pyogenes strains could additionally be confirmed by PCR mediated amplification of species specific parts of the 16S-23S rDNA intergenic spacer region, the pyolysin encoding gene plo and by amplification of the collagen-binding protein encoding gene cbpA. All these molecular targets might help to improve the future identification and further characterization of A. pyogenes which, as demonstrated in the present study, could also be isolated from reptile specimens.     

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.     

Analysis of 16S rDNA sequences from pathogenic Leptospira serovars and use of single nucleotide polymorphisms for rapid speciation by D-HPLC

Leptospira have a worldwide distribution and include important zoonotic pathogens yet diagnosis and differentiation still tend to rely on traditional bacteriological and serological approaches. In this study a 1.3 kb fragment of the rrs gene (16S rDNA) was sequenced from a panel of 22 control strains, representing serovars within the pathogenic species Leptospira interrogans, Leptospiraborgpetersenii, and Leptospirakirschneri, to identify single nucleotide polymorphisms (SNPs). These were identified in the 5′ variable region of the 16S sequence and a 181 bp PCR fragment encompassing this region was used for speciation by Denaturing High Performance Liquid Chromatography (D-HPLC). This method was applied to eleven additional species, representing pathogenic, non-pathogenic and intermediate species and was demonstrated to rapidly differentiate all but 2 of the non-pathogenic Leptospira species. The method was applied successfully to infected tissues from field samples proving its value for diagnosing leptospiral infections found in animals in the UK.     

The effect of various Mycoplasma bovis isolates on bovine leukocyte responses

Mycoplasma bovis (M. bovis) contributes to a number of clinical syndromes in cattle; in particular, chronic pneumonia that is poorly responsive to therapy has been increasingly recognized as an important cause of morbidity, mortality, and financial loss. M. bovis impairs host immune function, but little is known about whether field isolates vary significantly in their effect on immune function. This research tested the hypothesis that different field isolates vary in their ability to suppress cellular metabolism and cellular production of radical oxygen species (ROS) by bovine leukocytes. Total blood leukocytes from 6 cattle were exposed to six field isolates, two diagnostic lab isolates, and two high passage laboratory isolates of M. bovis, and ROS production was measured by oxidation of dihydrorhodamine 123 (DHR-123). Cellular metabolism was measured by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Significant differences in the response to some field isolates were identified. Three field isolates and both diagnostic lab isolates significantly decreased ROS production by leukocytes from multiple cattle, while the high pass laboratory isolates did not. In contrast, MTT reduction was not significantly impaired by any of the M. bovis strains tested. M. bovis impairs ROS production by bovine leukocytes; the magnitude of the effect appears to be isolate-dependent, and is not related to a general impairment of cellular metabolism. Chronic M. bovis infection in some cattle may be related to impaired ability of leukocytes to produce ROS when exposed to M. bovis.     

Occurrence of canine hemotropic mycoplasmas in domestic dogs from urban and rural areas of the Valdivia Province,southern Chile

This is the first study to investigate the occurrence, risk factors and hematological findings of hemoplasmas in dogs from Chile. Complete blood count and 16S rRNA conventional PCR for Mycoplasma spp. were performed in 278 blood samples from rural (n = 139) and urban (n = 139) dogs in Valdivia. Real time 16S rRNA PCR (qPCR) allowed species identification. Mycoplasma spp. occurrence was 24.8%. ‘Candidatus M. haematoparvum’ (CMhp) was identified in 12.2% and Mycoplasma haemocanis (Mhc) in 11.9% dogs. It was not possible to identify species in two Mycoplasma spp. samples by qPCR. Sequencing allowed identifying one of them as ‘Candidatus M. turicensis’ (CMt). Frequency in rural localities was higher (41.7%) than in urban (7.9%). Rural locality, maleness and older age were risk factors for hemoplasmosis. Hemoplasma-positive dogs had a higher total protein. This is the first report of Mhc, CMhp and CMt in dogs from Chile, with a high occurrence in rural localities.     

新疆地区3株驴源马链球菌马亚种新SeM基因型的鉴定与进化分析

从新疆地区某驴养殖场获得了3株驴腺疫链球菌分离株HTP133、HTP123和HTP232.为了解这3株驴腺疫链球菌的生物学特性和确定其分子分型,本研究对其进行了生化特性、药敏特性的检测,对16S rRNA进行序列对比分析,并利用PCR扩增SeM等位基因和测序鉴定其基因型.研究结果表明3株分离菌均为马链球菌马亚种.药敏试...     

牛支原体新疆株的分离鉴定

为调查新疆规模化奶牛场病牛死亡原因并确定病原,本研究无菌采集7份肺炎病死牛病变肺组织样,通过牛支原体液体培养基和固体培养基分离到1株支原体,采用形态学观察和生化试验鉴定该分离株,采用支原体特异性引物和牛支原体16S rRNA通用引物扩增基因序列并测序,使用DNAStar软件将分离菌株测序结果与GenBank中的标准株序列进行同源性比对,采用Mega 6.0软件中的邻接法(Neighbor-Joining,NJ)依据16S rRNA序列构建分离株系统进化树。结果显示,分离株菌落呈典型的"煎蛋样",菌落中心凹陷深入培养基,周边菲薄而透明,经Dienes染液染色后,菌落中心呈深蓝色。该分离株不分解葡萄糖、尿素、不水解精氨酸,血细胞吸附试验和溶血试验均呈阴性,氯化三苯基四氮唑还原反应呈阳性,产生膜和斑。PCR反应扩增出大小为1 911 bp的牛支原体特异性目的片段;分离株16S rRNA基因序列与牛支原体标准株PG45的序列同源性为99.8%,与牛支原体地方株(Mb NM2012、Mb HB0801、Mb Hubei-1、Mb Ningxia-1、Mb CQ-W70和Mb 08M)的同源性为99.3%～99.7%。系统进化树显示,分离株16S rRNA基因与Mb Ningxia-1株和Mb 08M株亲缘关系较近,处于同一分支。本研究结果证实了引起病牛死亡的病原为牛支原体,为新疆牛支原体病的防治提供了科学依据。