From Haemobartonella to hemoplasma: molecular methods provide new insights

Hemotropic mycoplasmas (aka hemoplasmas) are the causative agents of infectious anemia in numerous mammalian species. Originally known as Haemobartonella and Eperythrozoon species, these organisms have been reclassified within the genus Mycoplasma. The development of new molecular assays has expanded our knowledge of this heterogeneous group of agents and allowed us to study their epidemiology and pathogenesis. The present review summarizes recently gained insights into feline hemotropic mycoplasmas, formerly known as Haemobartonella felis. Besides the two initially identified feline hemoplasma species, Mycoplasma haemofelis and Candidatus Mycoplasma haemominutum, we discovered a third novel hemoplasma in a Swiss pet cat; preliminary results suggest that the pathogenic potential of the latter agent depends on cofactors. In applying PCR-based assays, feline hemoplasma infections have been documented in domestic cats and wild felids worldwide. Differences between the three hemoplasmas in regard to response to antibiotic treatment and establishment of a carrier status have been reported. Additionally, besides an ostensible vector-borne transmission, direct transmission by aggressive interaction of cats or interspecies transmission might play a role in the epidemiology of these organisms. Based on a potential vector-borne and interspecies transmission, a zoonotic potential of hemoplasmas should be further investigated.     

Molecular identification of `Candidatus Mycoplasma haemovis' in sheep with hemolytic anemia

We examined the presence of hemoplasmas, hemotropic mycoplasmas, among 11 sheep (Ovis aries) with regenerative and hemolytic anemia and found six of them were positive by real-time PCR. The positive samples were then subjected to conventional PCR for direct sequencing of the 16S rRNA gene. Nucleotide sequences of all the positive samples were identified as the 16S rRNA gene of `Candidatus Mycoplasma haemovis' by phylogenetic analysis, demonstrating the infections with this particular hemoplasma species in Japan.     

Development and application of a real-time TaqMan(®) qPCR assay for detection and quantification of 'Candidatus Mycoplasma haemolamae' in South American camelids

Two alpacas from a herd in southwest Switzerland died for unknown reasons. Necropsy revealed chronic weight loss and pale mucous membranes. Infection with hemotropic mycoplasmas was suspected and subsequently confirmed by molecular methods. In order to investigate the epidemiological situation in this herd, a real-time TaqMan(?) qPCR assay for the specific detection and quantification of hemoplasma infection in South American camelids was developed. This assay was based on the 16S rRNA gene and amplified 'Candidatus Mycoplasma haemolamae' DNA, but not DNA from other hemoplasmas or non-hemotropic mycoplasma species. The lower detection limit was one copy/PCR, and the amplification efficiency was 97.4%. In 11 out of 24 clinically healthy herd mates of the two infected alpacas, 'Candidatus M. haemolamae' infection was confirmed. No correlation was found between bacterial load and clinical signs or anemia. The assay described herein enables to detect and quantify 'Candidatus M. haemolamae' and may be used in future studies to investigate the prevalence, pathogenesis and treatment follow-up of hemoplasma infections in South American camelids.     

Real-time PCR-based prevalence study, infection follow-up and molecular characterization of canine hemotropic mycoplasmas

Hemotropic mycoplasmas (hemoplasmas) have been reported in several mammalian species including dogs. Infections may lead to hemolytic anemia, but investigations in the dog had been hampered by the lack of adequate diagnostic methods. Only recently sensitive PCR-based assays were reported for the two canine hemoplasmas, Mycoplasma haemocanis and Candidatus Mycoplasma haematoparvum. By applying these assays, 15.4% of 460 dogs from the south of France tested hemoplasma positive. It was hypothesized that this high prevalence may be associated with the presence of Rhipicephalus sanguineus, a proposed vector for canine hemoplasmas. To address this hypothesis and expand the PCR-based knowledge on canine hemoplasmosis, we investigated dogs in a climatic zone that does not allow for the permanent establishment of R. sanguineus. Blood samples were collected throughout a year from 889 dogs in Switzerland: 1.2% of the dogs tested real-time PCR positive. The infection status was not significantly associated with anemia, age or gender. Phylogenetic analyses of Candidatus M. haematoparvum and M. haemocanis isolates revealed > or =99.8% identity to published sequences. All samples collected from three infected dogs throughout a follow-up period of < or =13 months tested PCR positive. Interestingly, the majority of the infected dogs either had been imported from or had visited regions where R. sanguineus is indigenous. Thus, canine hemoplasma prevalence was found to be low in a country with a climate incompatible with frequent occurrence of R. sanguineus. Nonetheless, veterinarians may expect hemoplasma infections in dogs with a travel history and/or after potential tick vector exposure.     

Occurrence of hemotropic mycoplasmas in non-human primates (Alouatta caraya,Sapajus nigritus and Callithrix jacchus) of southern Brazil

Hemoplasmas, the erythrocyte-associated mycoplasmas, have been detected in several primates, causing mostly subclinical infection. This study aimed to determine the prevalence of hemoplasma infection in captive and free-ranging monkeys from southern Brazil, as well as factors and hematological abnormalities associated with infection. Blood samples from 40 non-human primates (NHP) were tested for hemoplasmas and coinfections. An overall of 10/40 (25.0%) NHP tested positive for hemoplasmas using PCR-based assays, including 9/14 (64.3%) black howler monkeys (Alouatta caraya) and 1/24 (4.2%) black-horned capuchin (Sapajus nigritus). Infection was not statistically associated with anemia, but wild-born monkeys and male black howler monkeys were more likely to be positive when compared with captive-born animals and female black howler monkeys, respectively. The sequences from the black howler monkey hemoplasma were similar (94% identity) to the squirrel monkey hemoplasma (“Candidatus Mycoplasma kahanei”) and were phylogenetically located in a different cluster when compared to the human hemoplasma (“Candidatus Mycoplasma haemohominis”).     

Feline hemotropic mycoplasmosis (feline hemobartonellosis).

Hemotropic mycoplasmas represent an important cause of anemia in cats worldwide. Previously known as Haemobartonella species, sequencing of the 16S rRNA genes of these organisms has led to their reclassification as mycoplasmas. Two species have been identified in cats, M haemofelis and "Candidatus M haemominutum." The latter organism alone has not been associated with disease in naturally infected cats but may cause anemia in FeLV-infected cats and accelerate development of FeLV-induced myeloproliferative disease. The mode of transmission of these organisms remains enigmatic. Nevertheless, development of sensitive DNA-based tests for these unculturable organisms has improved the understanding of the epidemiology and pathogenesis of FHM. Cats with clinical signs and laboratory abnormalities consistent with FHM should be treated with doxycycline; enrofloxacin may represent an effective alternative. Transfusion with packed red blood cells after cross-matching may be required for severely anemia cats, and addition of prednisone may be required if the diagnosis of FHM is uncertain, or response to antimicrobials alone is insufficient. Affected cats should be tested for FeLV, the most common concurrent infection in cats with FHM.     

PCR检测新疆南疆部分地方品种绵羊的嗜血支原体和泰勒虫感染情况

为了解新疆南疆部分地方品种绵羊嗜血支原体和泰勒虫的感染情况,采用PCR法检测新疆南疆5个地方品种绵羊共100份血液DNA样本。结果表明:新疆南疆部分地方品种绵羊泰勒虫和嗜血支原体感染率分别为35.0%(35/100)和3.0%(3/100)。仅和田羊和多浪羊上检测到嗜血支原体感染,感染率分别为8.0%(2/25)和5.0%(1/20),不同品种羊的嗜血支原体感染率无统计学差异(P>0.05);泰勒虫感染率多浪羊最高,为60.0%(12/20),卡拉库尔羊感染率最低,为15.0%(3/20),不同品种羊泰勒虫感染率差异显著(P<0.05)。散养绵羊嗜血支原体感染率6.0%(3/50),圈养绵羊未发现感染,两者间无统计学差异(P>0.05);散养和圈养绵羊的泰勒虫感染率分别为26.0%(13/50)和44.0%(22/50),两者之间无统计学差异(P>0.05)。将PCR产物进行双向测序,对序列进行比对分析,发现3个嗜血支原体阳性样本均被鉴定为绵羊支原体,35个泰勒虫阳性样本均被签定为绵羊泰勒虫。说明新疆南疆部分地方品种绵羊的泰勒虫感染均较为普遍,而嗜血支原体的感染情况有待于进一步调查。     

Mycoplasms of the swine--A review]

The mycoplasmas constitute a group of microorganisms placed between bacteria and virus. The name, Mycoplasma, is derived from the mycelial morphology of the organisms. The minimal reproductive unit, the elementary body, measures 0.2-0.5 mum. Unlike bacteria, mycoplasmas are not confined by a rigid cell wall, but just by a thin membrane. For their cultivation, though common bacteriological technique is adequate, especially enriched media are required. Antibiotics, as a rule penicillins, are added to the medium for inhibition of bacteria. Up to the present, 5 porcine species of mycoplasma are known: Mycoplasma suipneumoniae, Mycoplasma hyorhinis, Mycoplasma hyosynoviae, Mycoplasma flocculare, and Acholeplasma granularum. The 4 species first mentioned are very common among swine in Denmark. A. granularum has not been demonstrated so far. Occasionally, other species of mycoplasma are found in swine. M. suipneumoniae is by far the most important porcine mycoplasma, being to-day regarded as the primary etiologic agent in porcine enzootic pneumonia. A pure mycoplasma infection usually results in only weak clinical signs of pneumonia, but the disease may be aggravated by secondary factors as bacteria, parasites, and bad housing conditions. Enzootic pneumonia is usually prevalent only in fattening units, where it tends to persist indefinitely. The mycoplasma infection is practically incurable. Control of the disease is attempted by the SPF-program launched by the Danish Meat Research Institute, Roskilde. In this connexion the high sensitivity of mycoplasmas to physico-chemical influence is of advantage, because it results in a low rate of survival of the organisms outside the host. A further advantage is afforded by the fast that M. suipneumoniae is a definitely swine-specific organism. The rest of the porcine mycoplasmas are of far lesser importance. Yet, M. hyorhinis may produce a sero-fibrinous inflammation of serous cavities and joints in pigs less than 10 weeks old, and M. hyosynoviae may produce arthritis in fattening pigs.     

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.     

Genetic diversity and hematological and biochemical alterations in Alouatta primates naturally infected with hemoplasmas in Brazil

Mycoplasma spp. and Bartonella spp. are Gram-negative bacteria transmitted by arthropod vectors that infect red blood cells of several mammal species. This study investigated the occurrence and genetic diversity of hemoplasmas and Bartonella spp. in 68 howler monkeys kept in captivity in São Paulo, a southeastern state in Brazil. In addition, possible hematological, biochemical and electrophoretic changes of serum proteins associated with the occurrence of hemoplasmas and Bartonella spp. in captive primates were also investigated. The cPCR results showed that all sampled howler monkeys were negative for Bartonella spp. based on the gltA gene. The cPCR results indicated that 18 (26.47%) non-human primates (NHP) were positive for hemoplasmas based on the 16S rRNA gene. Monocyte and lymphocyte counts were higher in hemoplasma-positive howlers (P < 0.05). Platelet counts decreased in nonhuman primates (NHP) positive for hemoplasmas (P < 0.05). The results from the blood serum proteinogram and biochemistry analyses were not significantly different between NHPs positive and negative for hemotrophic mycoplasmas. Phylogenetic analysis using Bayesian Inference (BI) based on the 16S rRNA gene positioned the obtained sequences close to ‘Candidatus Mycoplasma kahanei’. The analysis of sequence diversity of the 16S rRNA gene showed that 5 different genotypes are circulating in NHP in Brazil and in the world; besides, a clear separation between the sequences of hemoplasmas that infect NHP of the Sapajus and Alouatta genus in Brazil was found, probably corresponding to two different species. The pathogenic potential of this hemoplasma species in NHP should be further investigated.     

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.     

Flow cytometry: clinical applications in equine medicine

The use of flow cytometry in veterinary diagnostics is becoming a valuable clinical tool with a broad range of applications. Physical characteristics of cells can be determined by the flow cytometer laser and electronics through the measurement of changes in light scatter properties. Other components and functions of cells can be defined through the application of fluorochrome dyes that have an affinity for cellular components. Traditionally, common clinical applications are immunophenotyping of cells of the hematopoietic system with fluorescent-labeled antibodies raised against specific cell surface proteins. Other approaches have been used to elucidate changes in cell function and DNA content. This review is intended to provide the reader with the fundamental uses of flow cytometry. Examples of clinical applications in equine patients include immune-mediated hemolytic anemia, immune-mediated thrombocytopenia (IMT), chronic inflammatory disease, and neoplasia.     

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.     

Hemotropic mycoplasmas in bats captured near human settlements in Nigeria

The presence of DNA of hemotropic mycoplasmas (hemoplasmas) was investigated for the first time in bats in Africa. Blood samples from 90 bats captured within or near human settlements in nine study areas from five states in Nigeria belonging to six genera of the families Pteropodidae, Rhinolophidae, and Molossidae were analyzed using conventional PCR protocol targeting a 391 bp part of the 16S rRNA gene. Of these, 32 samples (35 %) resulted positive. Eight nucleotide sequence types were identified, which were assigned to five genotypes showing between 93–99 % similarity with hemoplasmas from bats and/or rodents from other parts of the world, and/or Candidatus Mycoplasma haemohominis from a human patient. Network analysis showed genetic structure of hemoplasma sequences among bat species, but some sequences were shared among bats of different taxonomic groups and distant study areas. Further characterization of the samples using a protocol targeting ∼1200 bp of the 16S rRNA gene resulted in seven sequences that confirmed the results of the screening protocol. Hemoplasmas in Nigerian bats are prevalent, widely distributed and genetically diverse. The zoonotic risk to local inhabitants should not be neglected, due to the high similarity of some of the retrieved sequences with the human pathogen C. M. haemohominis.     

Microbiological and serological studies on caprine pneumonias in Oman

Eight of 10 typical cases of contagious caprine pleuro-pneumonia in Oman yielded strain F38-like mycoplasmas from the lungs in high titre, but no other mycoplasmas: both negative animals had been treated with tylosin shortly before death. Among 21 other lungs examined three of six cases of acute pneumonia yielded Mycoplasma ovipneumoniae; one also yielded M capricolum. M ovipneumoniae was also isolated from all eight cases of chronic pneumonia sampled from an abattoir, and from the lungs of three animals which died without overt signs of pneumonia. A single isolate of M arginini and three of unidentified mycoplasmas were also obtained from goats with and without pneumonia. Various bacterial species were isolated, none of which predominated. Antibodies to M mycoides subspecies capri (M m capri) and strain F38 were detected in sera from eight different sources. Assuming titres of 1 in 40 or more as positive in the indirect haemagglutination test used, 29 per cent of 422 serum samples had antibodies to M m capri alone, 2.6 per cent to strain F38 alone and 3.6 per cent to both organisms. These results confirm the presence of F38-like mycoplasmas in Oman, and indicate also widespread infection with M m capri. The role of the latter in caprine pneumonias in Oman requires elucidation.     

Immune-mediated Hemolytic Anemia and Thrombocytopenia in the Dog: A retrospective study of 55 cases diagnosed from 1979 through 1983 at the Western College of Veterinary Medicine

All recognized cases (n = 55) of immune-mediated hemolytic anemia and immune-mediated thrombocytopenia in dogs presented to the Western College of Veterinary Medicine from 1969 through 1983 were reviewed. Specific areas of concern were: association with other conditions, therapeutic response, prognosis, relapse rate and final outcome. Of these 55 cases, 19 were immune-mediated hemolytic anemia, 26 were immune-mediated thrombocytopenia and 10 were both immune-mediated hemolytic anemia and thrombocytopenia. Females were slightly over-represented and the mean age was 6.4 years. Therapy consisted of various combinations of immuno-suppressive drugs and in some cases, whole blood transfusion and splenectomy. No firm conclusions could be made regarding therapeutic efficacy, as a result of variation in treatment protocol and the occasional unavailability of follow-up data. Well over half of all cases were diagnosed as idiopathic. Precipitating factors or diseases most frequently implicated in secondary immune-mediated thrombocytopenia or hemolytic anemia were: recent vaccination, drug therapy, obstetrical complications, stress, recent viral infection and neoplasia. Twice as many cases of immune-mediated hemolytic anemia were seen in the cooler months (October to March), although this could not be related to antibody class or thermal reactivity. Immune-mediated thrombocytopenia both as a single disease and combined with immune-mediated hemolytic anemia had no seasonal incidence. History, clinical findings and hematological and clinical chemistry findings were consistent with data previously reported, with the exception of icterus, which appeared to be of higher incidence than most reports, being present in almost 50% of immune-mediated hemolytic anemia cases. Just over half of all dogs survived, although the survival rate was highest for immune-mediated hemolytic anemia, followed closely by immune-mediated thrombocytopenia and lowest for the combined disease. Immune-mediated thrombocytopenia most frequently ran a relapsing course requiring long-term or intermittent therapy.     

Hemotropic mycoplasmas (hemoplasmas) in free-ranging bats from Southern Brazil

Hemotropic mycoplasmas (hemoplasmas) are bacteria distributed worldwide and affect domestic and wildlife animals and human beings. Hemoplasmas have been described infecting hematophagous and non-hematophagous bats; however, transmission risk and zoonotic potential in vampire bats remain to be fully established. This study aimed to evaluate the presence of hemotropic mycoplasma species in free-ranging bats from this area using a universal PCR protocol for hemoplasmas. Accordingly, ten blood samples were collected from six male common vampire bats (Desmodus rotundus), two male hairy-legged vampire bats (Diphylla ecaudata), and two female non-hematophagous Pallas's mastiff bats (Molossus sp.) from the Curitiba’s region, Paraná State, Southern Brazil. A total of eight (8/10) blood samples were positive byconventional PCR; five (5/6) Desmodus rotundus, two (2/2) Diphylla ecaudata, and one (1/2) Molossus sp. bats. The analyses of the partial sequence of the 16S rDNA gene suggest that the hemoplasma detected in Desmodus rotundus in South Brazil has a high identity compared to the hemoplasma circulating in vampire bats from Central and South America.     

The central role of lipoproteins in the pathogenesis of mycoplasmoses

Mycoplasmas are a diverse group of pathogens responsible for disease in a wide range of animal species. In recent years there have been considerable advances in knowledge of the proteins and structures involved in adherence in some mycoplasmas, but understanding of the biochemical functions and roles in virulence of another central feature of mycoplasmas, their lipoproteins, continues to develop. The aim of this review is to examine current knowledge of the roles of lipoproteins in the pathogenicity and the evolution of virulence in those mycoplasmas causing disease in domestic animals. Those lipoproteins that have been characterised have roles in adherence, in transport of nutrients into the mycoplasma cell, and in enzymatic interactions with the host. Furthermore they appear to play a prominent role in both inducing the host immune response to infection and in facilitating evasion of this response, particularly through the generation of dramatic levels of antigenic variation on the cell surface. Recent genomic comparisons of several pathogenic mycoplasmas have identified a further level of interaction between lipoproteins and pathogenicity. In several pathogens large scale horizontal gene transfer between distantly related mycoplasma species has resulted in the acquisition of a large number of genes, including those encoding lipoproteins thought to play a role in virulence, by one mycoplasma from another inhabiting the same host species. The interactions between these horizontally transferred genes, their new mycoplasma host and the animal that it infects may be an important contributing factor in the pathogenesis of some mycoplasmoses.     

Mycoplasma ovis in captive cervids: prevalence, molecular characterization and phylogeny

Hemotrophic mycoplasmas (hemoplasmas) are bacteria that attach to red blood cells of mammals, leading to acute and/or subclinical disease in infected animals. It has been suggested that Mycoplasma ovis, a hemoplasma that infects sheep and goats worldwide, may also infect deer. The aim of this study was to evaluate whether South American deer are infected with M. ovis. EDTA-anticoagulated blood samples from a herd of 32 captive South American deer were collected. DNA extraction of blood samples was performed followed by PCR amplification of the 16S and 23S rRNA genes, and sequencing of products. Using M. ovis PCR, 27/31 (87%) were positive, including 21/22 Mazama nana; 2/3 Mazama americana and 4/6 Blastocerus dichotomus. Sequencing of the nearly entire 16S rRNA gene of 26/27 positive samples showed 98.2-98.8% identity to M. ovis of sheep (GenBank, AF338268) and 98.6-99.4% identity to M. ovis-like of a fawn (FJ824847); the 23S rRNA gene from one of these isolates and the fawn's had 97.6% identity. The remaining isolate had just 94.9% identity to the 16S rRNA gene of M. ovis and only 89.4% identity to the 23S rRNA gene of the fawn's M. ovis. This is the first report of M. ovis in captive South American deer, revealing a high prevalence of hemoplasma infection in these animals.