Hemoplasma Species Research Articles

Development of a novel triplex-PCR assay for the identification of feline hemoplasma species and survey of hemoplasma species in cats in Türkiye

Three hemoplasma species, Mycoplasma haemofelis, Candidatus Mycoplasma haemominutum, and Candidatus Mycoplasma turicensis, have been identified in domestic and wild felids. M. haemofelis may cause severe clinical manifestations in domestic cats, whereas others can lead to mild infections. Identification of these pathogens is done using molecular diagnostic tools like conventional-PCR or real-time PCR. However, these have disadvantages, such as the failure to differentiate species or high cost. This study aimed to develop a triplex-PCR method for the diagnosis and discrimination of feline hemoplasma species. Furthermore, it is aimed at providing molecular data on the epidemiology of feline hemoplasma species in Türkiye, where there is a paucity of information on these pathogens. Triplex-PCR primers amplifying the 16S rRNA gene regions of M. haemofelis (1022 bp), Ca. Mycoplasma haemominutum (607 bp), and Ca. Mycoplasma turicensis (456 bp) species were designed and optimized. Moreover, the detection limits of the method were also determined and it was found that the primers could detect 0.001 ng/μL amount of DNA for M. haemofelis, 0.0001 ng/μL for Ca. Mycoplasma haemominutum, and 0.0002 ng/μL for Ca. Mycoplasma turicensis in the sample. 286 cat blood samples obtained from Sivas province were researched for feline hemoplasma species. Feline hemoplasma species were detected in samples of 29 out of 286 cats (10.23 %). Five samples (1.74 %) were infected with only M. haemofelis, whereas 22 (7.69 %) were only infected with Ca. Mycoplasma haemominutum. Two samples (0.69 %) were found to be infected with both M. haemofelis and Ca. Mycoplasma haemominutum. Ca. Mycoplasma turicensis was not detected in this study. A triplex-PCR method that can be used for the identification and species differentiation of feline hemoplasma species in hosts was developed. Moreover, hemoplasma species were found to be circulating in cats in the study area, and it is recommended that veterinarians and animal owners take the necessary precautions to protect themselves and their cats from these pathogens.

Detection and characterization of hemotropic Mycoplasmas in Iberian wolves (Canis lupus signatus) of Cantabria, Spain

Hemoplasmas (hemotropic mycoplasmas) are uncultivable wall-less bacteria able to infect mammalian erythrocytes. Hemoplasmas can cause anemia, especially in immunocompromised hosts, predisposing to secondary infections and even leading to death. Between 2017 and 2023, spleen samples of 131 wild Iberian wolves (Canis lupus signatus) of Cantabria (Spain) were screened for Mycoplasma spp. using a real-time PCR able to amplify a 360 bp fragment of the 16S rRNA gene and confirmed by direct Sanger sequencing. Additional conventional PCRs were performed to screen for coinfections by different Mycoplasma species and to discriminate between Mycoplasma haemocanis/haemofelis (Mhc/Mhf). Overall, 24/131 (18.3%) animals were PCR-positive. Biological and environmental factors potentially promoting hemoplasma infection in this species were analyzed. Two different hemoplasma species were detected: Mhc/Mhf (18/131; 13.7%) and Candidatus Mycoplasma haematoparvum (CMhp) (3/131; 2.3%), each with one nucleotide sequence type (ntST); three other sequences were not classified. No Mhc/Mhf and CMhp coinfection were observed. The 12 Mhc/Mhf suitable for ribonuclease P RNA sequencing were confirmed as Mhc. Mhc ntST was 100% identical to a Mhc sequence previously obtained in domestic dogs (Canis lupus familiaris), and in wild Iberian wolves of northwestern Spain (Asturias and Galicia) at a similar prevalence to the one found herein, suggesting a high Mhc genetic homogeneity in this wild population. CMhp ntST was 100% identical to CMhp sequences from domestic dogs. To our knowledge, this is the first description of CMhp in the Iberian wolf. The high genetic similarity observed in Mhc and CMhp sequences, as well as their high similarity with domestic dog sequences, suggest its recent introduction, a high level of intraspecific transmission within the wild wolf population, and likely, interspecific transmission between wolves and domestic dogs.

Haemotrophic Mycoplasmas Infecting Pigs: A Review of the Current Knowledge.

Haemotrophic mycoplasmas (haemoplasmas) are a group of highly specific and adapted bacteria. Three different haemoplasma species in pigs are known to date: Mycoplasma (M.) suis, M. parvum and 'Candidatus (Ca.) M. haemosuis'. Even though these bacteria have been known in pig farming for a long time, it is difficult to draw general conclusions about the relevance of their infections in pigs. This review summarizes the current knowledge on the three porcine haemoplasma species with regards to clinical and pathological descriptions, pathobiology, epidemiology and diagnostics as well as prevention and therapy. Overall, it is clear that considerably more data are available for M. suis than for the other two species, but generally, porcine haemoplasmas were found to be highly prevalent all over the world. Mycoplasma suis is the most virulent species, causing acute infectious anaemia in pigs (IAP), whereas M. parvum usually results in chronic and subclinical infections associated with performance losses. Little is known about the clinical significance of the recently discovered third porcine species 'Ca. M. haemosuis'. So far, the described pathogenic mechanisms mainly include direct destruction of erythrocytes via adhesion, invasion, eryptosis and nutrient scavenging, indirect erythrocyte lysis due to immune-mediated events and immune dysregulation processes. A review of published diagnostic data confirms PCR assays as the current standard method, with various cross-species and species-specific protocols. Overall, there is a need for further examination to obtain valuable insights for practical application, specifically regarding the importance of subclinical infections in naturally infected animals. An essential requirement for this will be to gain a more comprehensive understanding of the mechanisms operating between the host and the pathogen.

Multi-locus sequence analysis of ‘Candidatus Mycoplasma haematomacacae’ in free-ranging macaques from Thailand suggestive of a closer relationship to hemotropic mycoplasmas in capuchins and potential origin from bats

Although ‘Candidatus Mycoplasma haematomacacae’ (formerly known as ‘Candidatus Mycoplasma haemomacaque’) has been reported on extensively in macaques from Thailand, the USA, Japan, and Brazil, its genetic characterization has primarily been restricted to the 16S rRNA sequences with no exploration on multi-locus sequence analysis. The primary goal of this study was to characterize ‘Ca. M. haematomacacae’ among Thai macaques based on multiple genetic markers. Between April 2018 and November 2021, blood samples were taken from 580 free-ranging macaques (560 Macaca fascicularis and 20 Macaca nemestrina) in 15 locations encompassing 10 provinces throughout Thailand. Using the conventional PCR assay targeting the 16S ribosomal RNA (16S rRNA) gene, 338 out of 580 macaques (58.27 %) tested hemoplasma-positive. Of these, 40 positive samples were further subjected to DNA sequencing, and all were identified as ‘Ca. M. haematomacacae’. Subsequently, the partial nucleotide sequences of 23S ribosomal RNA (23S rRNA) and RNase P RNA (rnpB) genes of this particular hemoplasma species were amplified through nested PCR assay. The analysis of multi-locus genetic markers revealed that the 23S rRNA and rnpB sequences exhibited higher levels of genetic diversity than the 16S rRNA sequences. Furthermore, the 16S rRNA analyses demonstrated that ‘Ca. M. haematomacacae’ infecting Old World monkeys (Macaca spp.) was most closely related to hemotropic Mycoplasma spp. in black-capped capuchins (Sapajus apella) and Marcgrave's capuchins (Sapajus flavius) from Brazil, as well as establishing a common ancestor clade with hemotropic Mycoplasma spp. from the Neotropical bats in Belize and Peru and an Old World bat in Spain. The 23S rRNA analyses likewise evidenced that ‘Ca. M. haematomacacae’ formed a sister clade with hemotropic Mycoplasma spp. in Neotropical bats from Belize and Panama. Thus, the present findings, based on multi-locus sequence analysis, suggest a potential origin of ‘Ca. M. haematomacacae’ from Neotropical and Old World bats. To the best of the authors’ knowledge, this study provided the largest dataset so far of multi-locus genetic sequences of ‘Ca. M. haematomacacae’ isolated from Thai macaques and enhanced the accuracy of phylogenetic analyses, providing insights into their origins among hemotropic Mycoplasma spp. discovered worldwide.

Molecular Detection and Characterization of Mycoplasma spp. in Marine Mammals, Brazil.

Mycoplasma spp. are wall-less bacteria able to infect mammals and are classified as hemotropic (hemoplasma) and nonhemotropic. In aquatic mammals, hemoplasma have been reported in California sea lions (Zalophus californianus) and river dolphins (Inia spp.). We investigated Mycoplasma spp. in blood samples of West Indian manatees (Trichechus manatus), pinnipeds (5 species), and marine cetaceans (18 species) that stranded or were undergoing rehabilitation in Brazil during 2002-2022. We detected Mycoplasma in blood of 18/130 (14.8%) cetaceans and 3/18 (16.6%) pinnipeds. All tested manatees were PCR-negative for Mycoplasma. Our findings indicate that >2 different hemoplasma species are circulating in cetaceans. The sequences from pinnipeds were similar to previously described sequences. We also detected a nonhemotropic Mycoplasma in 2 Franciscana dolphins (Pontoporia blainvillei) that might be associated with microscopic lesions. Because certain hemoplasmas can cause disease and death in immunosuppressed mammals, the bacteria could have conservation implications for already endangered aquatic mammals.

Expanded diversity of novel hemoplasmas in rare and undersampled Neotropical bats

Hemotropic mycoplasmas are emerging as a model system for studying bacterial pathogens in bats, but taxonomic coverage of sampled host species remains biased. We leveraged a long-term field study in Belize to uncover novel hemoplasma diversity in bats by analyzing 80 samples from 19 species, most of which are infrequently encountered. PCR targeting the partial 16S rRNA gene found 41% of bats positive for hemoplasmas. Phylogenetic analyses found two novel host shifts of hemoplasmas, four entirely new hemoplasma genotypes, and the first hemoplasma detections in four bat species. One of these novel hemoplasmas (from Neoeptesicus furinalis) shared 97.6% identity in the partial 16S rRNA gene to a human hemoplasma (Candidatus Mycoplasma haemohominis). Additional analysis of the partial 23S rRNA gene allowed us to also designate two novel hemoplasma species, in Myotis elegans and Phyllostomus discolor, with the proposed names Candidatus Mycoplasma haematomyotis sp. nov. and Candidatus Mycoplasma haematophyllostomi sp. nov., respectively. Our analyses show that additional hemoplasma diversity in bats can be uncovered by targeting rare or undersampled host species.

Hemotropic Mycoplasma spp. in Aquatic Mammals, Amazon Basin, Brazil.

Hemotropic Mycoplasma spp. (hemoplasmas) are uncultivable bacteria that infect mammals, including humans. We detected a potentially novel hemoplasma species in blood samples from wild river dolphins in the Amazon River Basin, Brazil. Further investigation could determine pathogenicity and zoonotic potential of the detected hemoplasma.

Evaluating the association between blood genotype or phenotype and haemoplasma infection in UK and Italian cats.

In humans, blood groups are associated with varying prevalence of infections. The aim of this study was to determine if associations exist between the feline AB blood group system and haemoplasma infection. Data from two studies were combined. In the first study, DNA samples from 131 haemoplasma-infected and 132 haemoplasma-uninfected UK cats underwent pyrosequencing to determine their blood genotype as AA, Ab or bb. In the second study, blood samples from 160 Italian cats of known blood phenotype A, B or AB underwent PCR testing for feline haemoplasma species DNA. Haemoplasma infection was demonstrated in cats of all phenotypes and genotypes. A significantly higher number of Ab genotype cats tested positive for overall haemoplasma infection status (p = 0.04) and for Mycoplasma haemofelis infection (p = 0.03). Haemoplasma-infected Italian cats were few, possibly increasing the chance of type II error, and the presence of purebred cats in the sample population may have had a confounding effect. Feline haemoplasmas do not appear to preferentially use either blood type A or B antigens as attachment sites for erythrocyte colonisation. Further investigations in a larger number of haemoplasma-infected cats of known blood phenotype are warranted to explain the association between genotype Ab and haemoplasma infection.

Retrospective prevalence and associated risk factors of Mycoplasma haemofelis infection in owned cats.

Data on the prevalence and associated risk factors of naturally occurring haemoplasmosis in owned cats in Malaysia is limited. Being the most pathogenic of the three known feline haemoplasma species, Mycoplasma haemofelis (Mhf) infection was analysed from 2016 to 2019 to determine the periodical prevalence and associated risk factors in Northeastern Malaysia - Kelantan. Archived patient data of 77 clinically ill cats suspected of having M. haemofelis infection were reviewed in this study. Out of the 77 suspected cases, 53 (68.8%) were clinically diagnosed with haemoplasmosis amongst which 46 (59.7%) of the subpopulation were further confirmed with polymerase chain reaction (PCR). Risk factors for M. haemofelis infection (age, breed, ectoparasitism, household condition, roaming status, and sex) were analysed. There was no significant association of breed, ectoparasitism, household condition (number of cats) and occurrence of clinical signs with feline mycoplasmosis. Young, male and roamer cats were more likely to be diagnosed of mycoplasmosis than other categories of cats in this study. There was also a significant association between cats infected with 'Candidatus Mycoplasma haemominutum' with M. haemofelis. Thus, the coinfection of these two haemoplasma species is not uncommon. This study indicates that infection by M. haemofelis in anaemic cats is a common find in client-owned cats from Northeastern Malaysia. As the natural mode of transmission of haemoplasma infection remain unestablished, information in this study may highlight the importance of this disease and contribute to effective prevention and control strategies to minimize feline infectious anaemia (FIA) caused by M. haemofelis.

First Report of 'Candidatus Mycoplasma haematomacacae' in Laboratory-Kept Rhesus Monkeys (Macaca mulatta) Maintained in Rio de Janeiro, Brazil.

Simple SummaryHealth assessment in animals used in research studies are essential, since only animals that present no diseases are considered suitable for these purposes. In laboratory kept animals, a bacterium that infects red blood cells, named hemotropic Mycoplasma (also called hemoplasmas), has been described as problem for research studies. Different hemoplasma species have been detected infecting monkeys from Brazil. However, the occurrence of these bacteria in monkeys maintained in laboratory in Brazil have never been described. Accordingly, this study aimed: (1) to screen laboratory-kept rhesus monkeys for hemoplasmas; (2) to verify if any of the hemoplasma-positive animals demonstrate a decrease in their red blood cells counts; and (3) to investigate the genetic diversity of hemoplasma species in monkeys from Brazil. Five out of eight (62.5%) rhesus monkeys tested positive for hemoplasmas using a technique that detects DNA from these bacteria in monkey’s blood. Further analysis demonstrated that rhesus monkeys were infected by a species named ‘Candidatus Mycoplasma haematomacacae’ that had already been described occurring in monkeys from Japan and USA. Although no decreases on red blood cells count were perceived in rhesus monkeys evaluated herein, future studies are needed to elucidate if ‘Ca. M. haematomacacae’ is a problem for research studies that use rhesus monkeys.Health monitoring programs in animals used as experimental models are essential, since only disease-free subjects are considered suitable for research purposes. In laboratory-kept animals, hemoplasmas have been described as an important confounding variable. Different hemoplasma species have been detected infecting non-human primates (NHP) from Brazil. However, the occurrence of hemoplasma species in laboratory-kept NHP in Brazil has not-yet been assessed. Accordingly, this study aimed (i) to screen laboratory-kept rhesus monkeys for hemoplasmas, (ii) to verify if any of the hemoplasma-positive animals demonstrate hematological abnormalities, and (iii) to assess the genotype diversity of hemoplasma species in NHP from Brazil. Five out of eight (62.5%; 95% CI: 3.05–8.63) rhesus monkeys tested positive for hemotropic Mycoplasma spp. by PCR. Sequencing, phylogenetic, distance, and genotype diversity analyses of partial 16S rRNA gene demonstrate that rhesus monkeys were infected by ‘Candidatus Mycoplasma haematomacacae’ (formerly ‘Candidatus Mycoplasma haemomacaque’). Assessments of partial 16S rRNA diversity of hemoplasma species in NHP suggest that at least four genetically diverse groups may occur in Brazil. Although no hematological abnormalities were demonstrated in rhesus monkeys evaluated herein, future studies are needed to elucidate the influence of ‘Ca. M. haematomacacae’ as a confounding variable on research studies.

Molecular detection of Mycoplasma wenyonii and its closely related hemotropic Mycoplasma sp. in blood-sucking flies from a buffalo farm in Chachoengsao province, Thailand

Bovine hemoplasmosis is a disease in buffaloes and cattle caused by hemotropic mycoplasmas or hemoplasmas. Only two bovine hemoplasma species, Mycoplasma wenyonii and Candidatus Mycoplasma haemobos, have been described in several countries. Hemoplasmas induce acute hemolytic anemia or chronic infection, leading to production loss. Bovine hemoplasma DNA was also detected in blood-sucking arthropods, suggesting vector transmission in farms. To date, no studies of the molecular detection of bovine hemoplasmas in Thai buffaloes and arthropod vectors have been reported. This study aimed to study the 1-year diversity of hematophagous flies in a buffalo farm located in Chachoengsao province, Thailand, and to investigate the molecular occurrence of bovine hemoplasmas in those flies using a polymerase chain reaction (PCR) assay and sequence analyses. A total of 1,488 mosquitoes, 867 stable flies, and 312 tabanid flies were collected during this study. The most abundant mosquitoes, stable flies, and tabanid flies were Culex tritaeniorhynchus, Stomoxys calcitrans, and Tabanus megalops, respectively. A total of 249 genomic DNA samples of flies were tested using a PCR assay based on the 16S rRNA gene; 23.69% (59/249) of the insect samples were positive in this assay. Positive samples (n = 8) were subjected to bidirectional sequencing. The BLAST results showed that only three samples from Stomoxys calcitrans and two samples from Tabanus megalops showed 99.90% and 99.17% similarities to the M. wenyonii isolate B003 (MG948626/Water buffalo/Cuba) and the M. wenyonii isolate C124 (MG948625/Cattle/Cuba), respectively. This molecular occurrence of bovine hemoplasmas in blood-sucking flies suggested that those flies are the mechanical vectors for bovine hemoplasmas in Thailand. Based on the phylogenetic analysis of the 16S rRNA gene, the sequences of M. wenyonii were likely classified into two subgroups (A and B), suggesting closely related bovine hemoplasma species. Finally, the genetic analysis of the 23S rRNA gene from these two subgroups revealed that subgroup A could be M. wenynoii and subgroup B may be a subspecies of M. wenyonii or another putative novel species. However, further investigation should be conducted in buffaloes, cattle, and blood-sucking flies to gain more 16S rRNA and 23 rRNA gene sequences of bovine hemoplasmas.

Prevalence and Risk Factors for Bartonella spp. and Haemoplasma Infections in Cats from Greece.

Simple SummaryBartonellosis and haemoplasmosis are diseases with global impact on the health of domestic cats and of zoonotic importance. This is the first study investigating the risk factors for Bartonella spp. and haemoplasma species infections in cats from Greece. In addition, this study determined the serologic and molecular prevalence of Bartonella spp. and haemoplasma species infections in different populations of cats living in different regions of Greece. A total of 452 cats were enrolled into the study. Blood was collected from each cat for the serological detection of Bartonella henselae antibodies and the molecular detection of Bartonella spp. and haemoplasma species infections. Overall, the seroprevalence of B. henselae was 35.4%, while the molecular prevalence of Bartonella spp. and haemoplasma species was 2.9% and 19%, respectively. The results of this study indicate that cats with partial or exclusive outdoor access and cats with flea infestation are at the greatest risk for B. henselae seropositivity. Furthermore, cats living in warmer regions such as Attica and Crete are at the greatest risk. Lack of ectoparasiticide use was identified as a risk factor for haemoplasma species infection. This suggests that the use of ectoparasiticides in cats may be an effective means of preventing haemoplasma species infection in cats.Bartonellosis and haemoplasmosis are vector-borne diseases with global impact on the health of domestic cats and of zoonotic importance. The aim of this study was to describe the epidemiological aspects of various populations of cats infected with Bartonella spp. or haemoplasma species. The populations evaluated included client-owned cats, stray cats and cats that live in breeding catteries in Greece. A total of 452 cats were prospectively enrolled into the study. A commercially available indirect immunofluorescence antibody testkit was used for the detection of Bartonella henselae IgG antibodies in serum. PCRs for the detection of Bartonella spp. and haemoplasma species DNA in the blood were also performed in a subgroup of 242 of the 452 cats. Risk factors for B. henselae seropositivity and infection with the haemoplasma species were determined using multivariable analysis. Overall, 160 (35.4%) of the 452 cats were seropositive for B. henselae. Seven (2.9%) and 46 (19%) of the 242 cats were PCR-positive for Bartonella spp. and haemoplasma species, respectively. The factors associated with B. henselae seropositivity, based on multivariate analysis, included older age, outdoor access, living region and flea infestation. Non-administration of ectoparasiticides was associated with haemoplasma species infection. This study shows a high prevalence of seropositivity for B. henselae and a relatively high prevalence of infection with haemoplasma species. Therefore, it is necessary to establish optimal strategies for the prevention of Bartonella spp. and haemoplasma species infections, considering the high-risk groups of cats identified in this study.

Molecular detection of haemophilic pathogens reveals evidence of Candidatus Mycoplasma haemobos in dogs and parasitic ticks in central China

BackgroundIn addition to Mycoplasma haemocanis and Candidatus Mycoplasma haematoparvum, a few hemoplasma species that mainly infect other livestock have been detected in dogs. ‘Candidatus Mycoplasma haemobos’ (Ca. M. haemobos) has been found in a variety of animals in China. The present study was aimed to investigate the occurrence of ‘Ca. M. haemobos’ infections in dogs and ticks collected from the Henan province, China.ResultsOverall, 55 dog blood samples and 378 ticks on skins were collected from anemic and healthy dogs, and these samples were subjected to PCR, sequence analysis, and identification. The results showed that Haemaphysalis longicornis (266) and Rhipicephalus (Boophilus) microplus (112) were the only two parasitic ticks on dogs. Molecular detection revealed that 163 M. haemocanis, 88 ‘Ca. M. haemobos’ and 32 Anaplasma platys positive amplicons could be amplified from dogs, H. longicornis and R. (B.) microplus. In addition, co-infections (M. haemocanis + A. platys and ‘Ca. M. haemobos’+ A. platys) could be also detected.ConclusionsTo the best of our knowledge, this is the first molecular evidence of ‘Ca. M. haemobos’ natural infection in dogs and tick species identified as H. longicornis and R. (B.) microplus from China.

Molecular prevalence of bovine hemoplasmosis in Turkey with first detection of Mycoplasma wenyonii and Candidatus Mycoplasma haemobos in cattle and water buffalo.

Hemoplasma species can cause infection varying from mild to severe in a wide range of hosts, including cattle and water buffalo. Two hemoplasma species, Mycoplasma wenyonii and Candidatus Mycoplasma haemobos, have been reported in cattle and water buffalo from different parts of the world to date. There was a lack of information on the presence and distribution of these pathogens in Turkey despite the negative economic impact on livestock production. This study aimed to develop a duplex PCR assay amplifying the 16S rRNA gene, in order to analyze DNA samples obtained from 297 cattle and 360 water buffaloes, and to determine the molecular prevalence of bovine hemoplasma species in Sivas province. Bovine hemoplasma species were found in 94 of 297 (31.64%) cattle and in 17 of 360 (4.72%) water buffaloes in this study. Randomly selected six positives PCR products (three samples each species) obtained from cattle and water buffaloes were sequenced, and the consensus sequences were uploaded to GenBank. Nucleotide similarity of 96.97-100% was determined between M. wenyonii isolates obtained in this study and those of M. wenyonii isolates present in the GenBank database, whereas C. Mycoplasma haemobos isolates from this study shared 99.04-100% homology with the C. Mycoplasma haemobos isolates uploaded to the GenBank. With the current study, the molecular presence of M. wenyonii and C. Mycoplasma haemobos were documented for the first time in cattle and water buffaloes in Turkey. Considering the rate of prevalence, veterinarians should take precautions against bovine hemoplasma species to protect animal health.

First study on microscopic and molecular evidences of two bovine hemoplasma species in cattle herds in Al-Qadisiyah Province, Iraq.

Background and Aim:Hemotropic Mycoplasmas are small epierythrocytic bacteria that cause infectious anemia in several livestock species and in humans. Several reports have been made on hemoplasma infections in the south and north of Iraq, but there have been no studies in the middle Euphrates of Iraq. This study aimed to evaluate the presence of hemoplasma species in cattle in Al-Qadisiyah Province, Iraq.Materials and Methods:Two hundred blood samples were collected from cattle with pale mucous membrane from regions with heavy tick endemicity. The samples were analyzed for the presence of Rickettsia pathogens using thin blood smears and the Diff-Quik stains. All the samples were also examined using polymerase chain reaction (PCR) to amplify the 16S ribosomal ribonucleic acid (rRNA) gene to confirm the presence of the smear-identified microorganisms. Ten PCR positive samples were subjected to 16S rRNA partial gene sequencing to identify the species.Results:The findings uncovered positivity in 68 (34%) blood smears. PCR revealed positive confirmation in 18 (9%) of the 200 blood samples. Mycoplasma wenyonii and Candidatus mycoplasma hemobos were identified from 10 PCR positive samples. The nucleotide sequences of the isolates were closely related to isolates from cattle, buffalo, and dogs in Vietnam, Cuba, India, and Germany.Conclusion:Bovine hemoplasma infections are prevalent in cattle in the Al-Qadisiyah Province in Iraq. Our results may have significance for the development of control programs.

Molecular detection and genetic analysis of porcine haemoplasmas in commercial pig farms from Thailand reveal a putative novel species.

Haemoplasma is a trivial name for haemotropic Mycoplasma spp., which can attach to the surface of red blood cells leading to deformity and anaemia in a wide range of mammalian animals, including pigs. In Thailand, there is only one study that reported the occurrence of Mycoplasma suis without other haemoplasma species. In this study, we examined the molecular occurrence and genetic diversity of porcine haemoplasmas in Thai domestic pigs (Sus scrofa domesticus) from commercial farms using a PCR assay targeting the 16S rRNA gene, DNA sequencing, nucleotide sequence type (ntST) analysis and phylogenetic analysis. A total of 665 blood samples were collected from pigs at thirteen farms located in eight provinces of Thailand during 2019-2020. Genomic DNA was extracted from blood samples and tested by PCR. The frequency of haemoplasma infection was 37.1% (247/665, 95% CI: 33.5%-40.9%) in all pigs. Among 247 PCR positive samples, 194 were sequenced and analysed by nucleotide BLAST, ntST diversity, phylogenetic trees and ntST networks. The results of this genetic analysis indicated that at least four species with 27 nucleotide sequence types (Mycoplasma suis, Mycoplasma parvum, Candidatus Mycoplasma haemosuis and a putative novel species) of porcine haemoplasmas were identified. Thus, it appears that haemoplasmas show a high genetic diversity in the Thai pig population. In addition, a putative novel species was genetically characterized by other markers, namely, the 23S rRNA and RNase P RNA (rnpB) genes. For phylogenetic analysis, Candidatus Mycoplasma haemosuis was placed into the Mycoplasma haemofelis group, and the three remaining species were placed into the Mycoplasma suis group in all trees containing the 16S rRNA, 23S rRNA and rnpB genes. Further studies, such as pathobiology and epidemiology, should be conducted to better characterize this putative novel species.

Molecular investigation of haemotropic mycoplasmas and Coxiella burnetii in free-living Xenarthra mammals from Brazil, with evidence of new haemoplasma species.

Although mammals of the superorder Xenarthra are considered hosts of a wide range of zoonotic agents, works aiming at investigating the role of these animals as hosts for bacteria with zoonotic potential are rare. The present study aimed to investigate the occurrence and molecularly characterize Coxiella burnetii and haemoplasma (haemotropic mycoplasmas) DNA in blood and spleen samples from 397 free-living Xenarthra mammals (233 sloths, 107 anteaters and 57 armadillos) in five Brazilian states (Mato Grosso do Sul, São Paulo, Pará, Rondônia and Rio Grande do Sul). All biological samples from Xenarthra were negative in the qPCR for Coxiella burnetii based on the IS1111 gene. The absence of C. burnetii DNA in blood and spleen samples from Xenarthra suggests that these mammals may not act as possible hosts for this agent in the locations studied. When performed conventional PCR assays for the endogenous (gapdh) mammalian gene, 386 samples were positive. When screened by molecular assays based on the 16S rRNA gene of haemoplasmas, 81 samples were positive, of which 15.54% (60/386) were positive by conventional PCR and 5.44% (21/386) were positive by real-time PCR; three samples were positive in both assays. Of these, 39.74% (31/78) were also positive for the 23S rRNA gene and 7.69% (6/78) for the haemoplasma RNAse P gene. Among the samples positive for haemoplasmas, 25.64% (20/78) were obtained from anteaters (Tamandua tetradactyla and Myrmecophaga tridactyla), 39.74% (31/78) from sloths (Bradypus tridactylus, Bradypus sp. and Choloepus sp.) 34.61% (27/78) from armadillos (Priodontes maximus, Euphractus sexcinctus and Dasypus novemcinctus). A haemoplasma 16S rRNA sequence closely related and showing high identity (99.7%) to Mycoplasma wenyonii was detected, for the first time, in B. tridactylus. Based on the low identity and phylogenetic positioning of 16S rRNA and 23S rRNA sequences of haemoplasmas detected in anteaters and armadillos, the present study showed, for the first time, the occurrence of putative new Candidatus haemotropic Mycoplasma spp. ("Candidatus Mycoplasma haematotetradactyla" and "Candidatus Mycoplasma haematomaximus") in Xenarthra mammals from Brazil.

Hemotropic mycoplasmas in bats from forest fragments, state of Paraná, southern Brazil

The order Chiroptera is the second largest group of mammals with bats being identified as reservoir of several viral zoonoses, although, little is known about their role in other groups of pathogens, including hemotropic Mycoplasma spp. To date, hemoplasma species have been found infecting several species of bats with high genetic diversity between 16S rRNA gene sequences. On this study, we aimed to identify the occurrence and characterize 16S and 23S rRNA genes of hemoplasma species in four bats species (Artibeus lituratus, Carollia perspicillata, Sturnira lilium and Sturnira tildae) from forest fragments in Paraná State, southern Brazil, using PCR-based assays. Spleen tissue samples were collected, DNA extracted and further screened by a pan‑hemoplasma PCR assay. All samples consistently amplified the mammal endogenous gapdh gene. One out of 15 (6.66%; 95% CI: 0.2-31%) bats tested positive for hemotropic Mycoplasma sp. by the PCR assay targeting the 16S rRNA gene. Sequencing of the 16S rRNA gene fragment from the hemoplasma-positive bat showed 99.14% identity with hemotropic Mycoplasma sp. detected in Sturnira parvidens from Belize. Sequencing of the 23S rRNA gene fragment from the hemoplasma-positive bat showed 86.17% identity with ‘Candidatus Mycoplasma haemosphiggurus’ detected in orange-spined hairy dwarf porcupines (Sphiggurus villosus) from Southern Brazil.

Implications of Mycoplasma hemofelis Bacterial Load on the Hematological Parameters of Naturally Infected Cats

Mycoplasma hemofelis is the most pathogenic hemoplasma species that affect cats. M. hemofelis may cause an acute infection that leads to hemolytic anaemia. The objective of this study was to detect and to quantify the load of M. hemofelis in cats by conventional polymerase chain reaction (PCR) and by quantitative PCR (qPCR) and to describe the possible hematological changes. M. hemofelis DNA was detected in 28.6% of the randomly selected cats (42 of 147) attended at the Veterinary Hospital of the Federal University of Mato Grosso, Brazil. The agreement between conventional PCR and qPCR was substantive (k 0.6). Females were twice as likely to acquire infection as males (odds ratio, 2.31). There was no statistically significant association (P > .05) and little/no correlation between the hematological parameters and the average of bacterial load. The results indicate that M. hemofelis infection is not related to clinical signs and bacterial blood load in cats. The agreement between conventional and quantitative PCR made it possible to detect infection by M. hemofelis in a larger number of cats.

'Candidatus Mycoplasma haematohydrochoerus', a novel hemoplasma species in capybaras (Hydrochoerus hydrochaeris) from Brazil.

Three different species of hemoplasmas have been described in rodents, Mycoplasma coccoides, 'Candidatus Mycoplasma haemomuris' and 'Candidatus Mycoplasma haemosphiggurus'. Additionally, potentially novel hemoplasma species have been detected in wild rodents from Brazil, including capybaras (Hydrochoerus hydrochaeris). Capybaras are the largest rodent in the world and are well adapted to live within close proximity to humans, which increases the risk to spread of zoonotic pathogens. Herein, we investigate the occurrence and genetic diversity of hemoplasmas infecting free-ranging capybaras from southern Brazil. Blood samples and ticks from 17 capybaras were collected. Packed cell volume and total plasma protein were measured, DNA was extracted, and further screened by species-specific and pan-hemoplasma PCR assays targeting the 16S rRNA gene of hemoplasmas. Sixteen out of 17 (94.12%; 95% CI: 73.02-98.95%) were anemic. Only one young female was hypoproteinemic. All capybaras were infested by adults and nymphs of Amblyomma dubitatum ticks. Using the PCR assay targeting the 16S rRNA gene of M. coccoides, 13/17 (76.47%; 95% CI: 52.74-90.44%) capybaras were positive for hemoplasmas. When DNA samples were tested by the pan-hemoplasma PCR, 16/17 (94.12%; 95% CI: 73.02-98.95%) animals were positive. One out of 11 (9.09%) adult ticks salivary glands tested positive for hemoplasma by the pan-hemoplasma PCR assay. Sequencing and phylogenetic analysis of the 16S and 23S rRNA gene fragments confirmed that animals were infected by a novel hemotropic Mycoplasma sp. previously reported in capybaras from Brazil. Additionally, sequencing and phylogenetic analysis of the 23S rRNA gene from three hemoplasma-positive capybaras samples from a previous study performed in midwestern Brazil also confirm our findings. Based on phylogenetic and Neighbor-Net network analysis of the 16S rRNA and 23S rRNA genes, the name 'Candidatus Mycoplasma haematohydrochoerus' is proposed for this novel organism.