Molecular detection of hemotropic mycoplasmas (hemoplasmas) in domestic cats (Felis catus) in Romania.

BACKGROUND: The hemotropic mycoplasmas (hemoplasmas) of the genus Mycoplasma are recognized as important bacteria that parasitize red blood cells, causing hemolytic anemia in many mammalian species, including cats. No information is available concerning the presence of feline hemoplasma infections in cats in Romania. Thus, the objective of the present study was to provide data on the occurrence and molecular characterization of hemotropic mycoplasmas in client-owned cats in Romania. METHODS: Blood samples from 51 unhealthy cats, originating from Timisoara Municipality, Romania, were screened for the presence of hemoplasmas using conventional polymerase chain reaction (PCR) targeting the 16S rRNA gene and sequencing assays. PCR-positive samples were subsequently analyzed by phylogenetic and population genetic analysis. RESULTS: Molecular analysis revealed 11 (21.6%) positive samples, consisting of 8 (72.7%) Candidatus Mycoplasma haemominutum and 3 (27.3%) Mycoplasma haemofelis confirmed positives. Candidatus Mycoplasma turicensis was not detected, and no co-infections were registered. No significant associations (p > 0.05) were found between the hemoplasma infection status and age, gender, breed, presence of ectoparasites, feline leukemia virus/feline immunodeficiency virus positivity of cats, or the sampling season. However, outdoor access was positively associated (p = 0.049) with infection and could be considered a risk factor (OR = 4.1) in acquiring feline hemotropic mycoplasmas. Phylogenetic analysis revealed that our sequences clustered with those selected from the GenBank database in two distinct clades. The registered population genetic indices were strongly supportive of the great variance in sequences between the recorded Mycoplasma species. CONCLUSIONS: The findings support the occurrence of feline hemoplasma infections in previously uninvestigated territories of Europe, providing useful information for small animal practitioners. To our knowledge, the present survey is the first reported molecular evidence of feline hemoplasma infections in Romania.

A molecular survey of vector-borne pathogens and haemoplasmas in owned cats across Italy.

BACKGROUND: Feline vector-borne pathogens (FeVBPs) have been increasingly investigated for their impact on cat health and their zoonotic potential. The aim of the present study was to assess the prevalence of FeVBPs and haemoplasmas in cats across Italy and to identify potential risk factors linked to their occurrence. METHODS: Blood samples from 958 owned cats living in the North (n = 556), Centre (n = 173) and South (n = 229) of Italy were tested for Babesia spp., Hepatozoon spp., Ehrlichia spp., Anaplasma spp. and filarioids by conventional PCR (cPCR) and for haemoplasmas and Bartonella spp. by SYBR green real-time PCR. Cats included in the study represent a sub-sample from a larger number of animals enrolled in a previous study, which were selected based on the geographical origin. Data on cats' positivity for Leishmania infantum, feline leukaemia virus (FeLV) and for feline immunodeficiency virus (FIV), available from the previous study, were included and examined. Potential risk factors for pathogen infection were assessed in relationship to categorical variables including sex, geographical origin, breed, neutering status and age of cats. RESULTS: Out of the 958 cats, 194 (20.2%) were positive for at least one of the tested pathogens, 89 (16%) from the North, 32 (18.5%) from the Centre and 73 (31.9%) from the South of Italy. A high prevalence of FeVBPs was detected in male cats (n = 125, 27.8%), living in the southern part of the country (n = 73, 31.9%), younger than 18 months of age (n = 24, 22.4%) and not neutered (n = 39; 27.5%). In particular, 24 cats (2.5%) tested PCR-positive for Bartonella spp., of which 1.6% for B. henselae and 0.9% for B. clarridgeiae. A total of 111 cats scored PCR-positive for haemoplasmas (11.6%), specifically "Candidatus Mycoplasma haemominutum" (n = 95, 9.9%), M. haemofelis (n = 14, 1.5%) and "Candidatus Mycoplasma turicensis" (n = 2, 0.2%). Moreover, 39, 31 and 8 cats were positive for FeLV (4.1%), L. infantum (3.2%) and FIV (0.8%), respectively. Co-infections were registered for 19 (9.8%) cats. CONCLUSIONS: These results confirm the occurrence of haemoplasmas and FeVBPs throughout Italy. Preventive measures to protect both animal and human health should be carried out also for owned cats, even if no health status of animals has been assessed in this study.

Genetic diversity and lack of molecular evidence for hemoplasma cross-species transmission between wild and synanthropic mammals from Central-Western Brazil.

Globally, hemotropic mycoplasmas (hemoplasmas) comprise an emerging or remerging bacteria group that attaches to red blood cells of several mammal's species and in some cases, causing hemolytic anemia. Herein, we assessed the occurrence, genetic diversity, the factors coupled to mammals infection, and the phylogeographic distribution of hemoplasmas in sylvatic and synanthropic mammals and their associated ectoparasites from Brazil. We collected spleen and/or blood samples from synanthropic rodents (Rattus rattus [N = 39] and Mus musculus [N = 9]), sylvatic rodents (Hydrochoerus hydrochaeris [N = 14]) and opossums (Didelphis albiventris [N = 43]). In addition, ticks (Amblyomma spp. [N = 270] and lice (Polyplax spinulosa [N = 6]) specimens were also sampled. Using a PCR targeting the 16S rRNA region, out of 48 small rodents, 14 capybaras and 43 opossums DNA samples, hemoplasma DNA was found in 25%, 50%, and 32.5% animals, respectively. Besides, we reported hemoplasma DNA in Amblyomma sp. (22.2% [2/9]) and lice (100% [2/2]) pools samples from rats, and one female A. sculptum DNA sample (3% [1/33]) obtained from a capybara. Additionally, and in agreement with ML analysis, the network analyses showed a clear phylogenetic separation among the hemoplasmas genotypes found in the different host species sampled, thus, suggesting the absence of cross-species hemoplasmas transmission between the mammals trapped. Finally, using the NTC network analysis, we reported the same 16S rRNA Mycoplasma genotype circulating in Rattus sampled in Brazil, Hungary, and Japan.

Detection of Hemotropic Mycoplasma sp. in white-eared opossums (Didelphis albiventris) from Southern Brazil / Detecção de Mycoplasma sp. hemotrópico em gambás-de-orelha-branca (Didelphis albiventris) no Sul do Brasil

Abstract Opossums are marsupials from the New World of the genus Didelphis and known as synanthropic animals due to their proximity with human beings. To date, 'Candidatus Mycoplasma haemodidelphis' has been solely found infecting the North American opossum (Didelphis virginiana). Accordingly, the aim of this study was to screen eight white-eared opossums (Didelphis albiventris) from a public park in Maringa city, Paraná State, southern Brazil, for hemoplasma infection. Blood samples were taken from caudal venipuncture, and DNA was extracted and further screened by a pan-hemoplasma PCR assay. Seven out of eight (87.50%; CI 95%: 47.35-99.68%) white-eared opossums were positive for Mycoplasma spp. Sequencing of the 16S rRNA fragment showed 98,97% identity with 'Ca. M. haemodidelphis' detected in the USA. Three out of eight (37.50%; CI 95%: 8.52-75.51%) white-eared opossums were infested by Amblyomma dubitatum ticks. This is the first report on detection of a potentially novel hemotropic Mycoplasma sp. infecting opossums from South America.

Resumo Gambás são marsupiais do Novo Mundo, pertencentes ao gênero Didelphis, e considerados animais sinantrópicos devido à sua proximidade com seres humanos. Atualmente, a espécie 'Candidatus Mycoplasma haemodidelphis' só foi encontrada infectando gambá norte americano (Didelphis virginiana). O objetivo do presente estudo foi detectar a infecção por hemoplasmas em oito gambás-de-orelha-branca (Didelphis albiventris) capturados em um parque público da cidade de Maringá, no Estado do Paraná, sul do Brasil. Amostras de sangue foram coletadas por venopunção caudal para a extração do DNA e posterior análise pela PCR para espécies de hemoplasmas. Sete de oito animais (87,50%; CI 95%: 47,35-99,68%) foram considerados positivos para Mycoplasma spp. O sequenciamento do fragmento do gene 16S rRNA obtido apresentou 98.97% de similaridade com sequências de 'Ca. M. haemodidelphis' detectadas nos Estados Unidos. Três gambás (37,50%; CI 95%: 8,52-75,51%) estavam infestados por carrapatos da espécie Amblyomma dubitatum. Esse é o primeiro relato de detecção de uma potencial nova espécie de Mycoplasma hemotrópico infectando gambás na América do Sul.

First molecular detection of Mycoplasma ovis (Hemotropic mycoplasmas) from Sheep in Brazil / Primeira detecção molecular de Mycoplasma ovis (Micoplasmas Hemotrópicos) em Ovinos no Brasil

Abstract Mycoplasma ovis is an emerging zoonotic pathogen with a worldwide distribution and can cause mild to severe hemolytic anemia, icterus, and poor weight gain in animals. Although M. ovis has been described in small ruminants worldwide, data on M. ovis in sheep in Brazil is unknown. The objective of the present study was to present the first report of hemotropic mycoplasma (HM) in sheep from Brazil. We evaluated factors associated with this infection, such age group, tick presence, and anemia. Blood samples were collected from 33 sheep from a farm in southern Brazil and screened for hemoplasmas using PCR. Out of 33 samples, 26 (78.8%) tested positive for M. ovis. The sequencing of positive samples showed 100% identity with multiple M. ovis 16S rDNA sequences. No association was observed between the presence of M. ovis and the FAMACHA© score (p = 0.620). Rhipicephalus (Boophilus) microplus (15/33, 45.4%) was the tick species found on the animals. No significant association between M. ovis infection and presence of ticks (p = 0.4134) and age group (p = 0.4221) was observed. This is the first report of M. ovis infection in sheep from Brazil and only the second report of this pathogen in sheep in Latin America.

Resumo Mycoplasma ovis é um patógeno zoonótico emergente com distribuição mundial e pode causar anemia hemolítica de leve a grave, icterícia e baixo ganho de peso em animais. Embora M. ovis tenha sido descrito em pequenos ruminantes em todo o mundo, os dados sobre M. ovis em ovinos no Brasil são desconhecidos. O objetivo deste estudo foi apresentar o primeiro relato de micoplasmas hemotrópicos em ovinos no Brasil. Avaliamos os fatores associados a essa infecção, como faixa etária, presença de carrapatos e anemia. Amostras de sangue foram coletadas de 33 ovelhas de uma fazenda no sul do Brasil e testadas para hemoplasmas usando a PCR. Das 33 amostras, 26 (78,8%) apresentaram resultado positivo. O sequenciamento das amostras positivas mostrou 100% de identidade com múltiplas sequências de M. ovis 16S rDNA. Não foi observada associação entre a presença de M. ovis e o escore FAMACHA© (p = 0,620). Rhipicephalus (Boophilus) microplus (15/33, 45,4%) foi a espécie de carrapato encontrada nos animais. Não houve associação significativa entre infecção por M. ovis e presença de carrapatos (p = 0,4134) e faixa etária (p = 0,4221). Este é o primeiro relato de infecção por M. ovis em ovinos no Brasil e o segundo relato deste patógeno em ovinos na América Latina.

First molecular detection of Mycoplasma suis in the pig louse Haematopinus suis (Phthiraptera: Anoplura) from Argentina.

Porcine haemoplasmosis caused by Mycoplasma suis affects the global pig industry with significant economic losses. The main transmission route of M. suis is through the blood and some haematophagous arthropods, like flies and mosquitoes, could be the vectors to this pathogen. However, the presence of M. suis in pig haematophagous ectoparasites in natural conditions has not yet been studied. The most frequent ectoparasite in pigs is the blood-sucking louse Haematopinus suis, an obligate and permanent parasite. Therefore, this work aims to study the occurrence of M. suis in H. suis samples from both domestic and wild pig populations from Argentina; using the 16S rRNA gene. A total of 98 sucking lice, collected from domestic and wild pigs from Buenos Aires Province in central Argentina, were examined. We found M. suis DNA in 15 H. suis samples (15.30%). Positive lice were detected from all studied populations. This is the first report of M. suis presence in H. suis, being also the first detection in a pig ectoparasite species. We conclude that H. suis could serve as a mechanical vector for M. suis. This information not only extends the knowledge about the pathogen spectrum potentially transmitted by H. suis, but may be also useful in epidemiological studies about Mycoplasma.

Molecular detection of Mycoplasma suis in extensive pig production systems in the State of Maranhão, northeast Brazil / Detecção molecular de Mycoplasma suis em suínos de criações extensivas no estado do Maranhão, nordeste do Brasil

Abstract Mycoplasma suis is a bacterium that causes hemoplasmosis in pigs. This agent is capable of adhering to the surface of porcine erythrocytes, inducing structural changes on these cells. In Brazil, there are few reports about the disease, its causal agent, and the economic impact of this pathogen on pig production systems and farm sanitation. The present study aimed to investigate the occurrence of M. suis in extensive swine farms located in the counties of Itapecuru Mirim, Santa Rita and Rosario, State of Maranhão, northeast Brazil. For such purpose, 64 blood samples of pigs from these facilities were tested for M. suis using a 16S rRNA gene-based quantitative real-time PCR (qPCR); 82.3%, 65.2% and 25% of blood samples of swine from farms in the cities of Itapecuru Mirim, Santa Rita and Rosario were positive for M. suis by qPCR, respectively. This study shows, for the first time, that M. suis circulates in pig populations from the state of Maranhão, Northeast Brazil.

Resumo Mycoplasma suis é uma bactéria que causa a hemoplasmose em suínos. Este agente é capaz de se aderir à superfície dos eritrócitos de suínos, ocasionando deformações estruturais nestas células. No Brasil, poucos são os relatos acerca do parasita, da infecção e de seus impactos econômicos nas esferas produtiva e sanitária. O objetivo deste estudo foi investigar, por meio da PCR em tempo real quantitativa (qPCR) baseada no gene 16S rRNA, a ocorrência de M. suis em 64 amostras de sangue de suínos de criações extensivas dos municípios de Itapecuru Mirim, Santa Rita e Rosário, localizados no estado do Maranhão. Foram obtidos um percentual de 82,3%, 65,2% e 25% de amostras positivas na qPCR para M. suis nos municípios de Itapecuru Mirim, Santa Rita e Rosário, respectivamente. Este estudo mostra que M. suis circula entre os suínos de criações extensivas no estado do Maranhão.

HIGH PREVALENCE OF MYCOPLASMA AND EIMERIA SPECIES IN FREE-RANGING EASTERN WILD TURKEYS ( MELEAGRIS GALLOPAVO SILVESTRIS) IN ONTARIO, CANADA.

Following extirpation from Ontario, Canada in the early 1900s, Eastern Wild Turkeys (EWTs; Meleagris gallopavo silvestris) were successfully reintroduced to the province in 1984. Despite the subsequent establishment of robust populations and biannual hunting seasons, data on the circulation of potential pathogens in these birds are lacking. Similarly, the interface between EWTs and poultry is poorly understood and includes possible bidirectional pathogen transmission via direct or indirect contact. Mycoplasma and Eimeria spp. are potential pathogens in Galliformes, and our objective was to determine their prevalence and distribution in Ontario EWTs. During the 2015 spring hunting season (April and May), oropharyngeal swabs from 147 hunter-harvested and five opportunistically collected EWTs from southern Ontario were cultured for Mycoplasma spp. The intestinal or cloacal contents of 107 of these birds and an additional 24 opportunistically and biologist-collected EWTs were analyzed for Eimeria spp. using PCR or fecal flotation. At least one Mycoplasma spp. was isolated from 98.7% (150/152) of EWTs, with six species identified. Mycoplasma gallopavonis was identified most commonly in 96.7% (147/152), followed by Mycoplasma gallinaceum in 23.7% (36/152). Potential poultry pathogens ( Mycoplasma meleagridis, Mycoplasma iowae, and Mycoplasma synoviae) were isolated from swabs of five (3.3%) EWTs. Coinfections with up to three Mycoplasma spp. were detected in 36.8% (56/152) of EWTs. Most EWTs tested positive for Eimeria spp. oocysts (75.6%; 99/131). A subset of positive samples ( n=16) were characterized by PCR, which detected the following species: Eimeria meleagrimitis (93.8%), Eimeria adenoeides (93.8%), Eimeria gallopavonis (56.3%), and Eimeria meleagridis (12.5%). The majority (93.8%) of these samples were positive for more than one Eimeria spp. We showed that numerous, mostly nonpathogenic Mycoplasma and Eimeria spp. circulate in EWTs across southern Ontario, and this helped to establish baseline information for comparison with future surveillance and monitoring.

Phylogenetic framework for the phylum Tenericutes based on genome sequence data: proposal for the creation of a new order Mycoplasmoidales ord. nov., containing two new families Mycoplasmoidaceae fam. nov. and Metamycoplasmataceae fam. nov. harbouring Eperythrozoon, Ureaplasma and five novel genera.

The genus Mycoplasma, including species earlier classified in the genera Eperythrozoon and Haemobartonella, contains ~ 120 species and constitutes an extensively polyphyletic assemblage of bacteria within the phylum Tenericutes. Due to their small genome sizes and lack of unique characteristics, the relationships among the mycoplasmas/Tenericutes are not reliably discerned. Using genome sequences for 140 Tenericutes, their evolutionary relationships were examined using multiple independent approaches. Phylogenomic trees were constructed for 63 conserved proteins, 45 ribosomal proteins, three main subunits of RNA polymerase and 16S rRNA gene sequences. In all of these trees, Tenericutes species reliably grouped into four main clades designated as the "Acholeplasma", "Spiroplasma", "Pneumoniae" and "Hominis" clusters. These clades are also distinguished based on a similarity matrix constructed based on 16S rRNA gene sequences. Mycoplasma species were dispersed across 3 of these 4 clades highlighting their extensive polyphyly. In parallel, our comparative genomic analyses have identified > 100 conserved signature indels (CSIs) and 14 conserved signature proteins (CSPs), which are uniquely shared by the members of four identified clades, strongly supporting their monophyly and identifying them in molecular terms. Mycoplasma mycoides, the type species of the genus Mycoplasma, and a small number of other Mycoplasma species, formed a strongly supported clade within the "Spiroplasma" cluster. Nine CSIs and 14 CSPs reliably distinguish this clade from all other Mycoplasmatales species. The remainder of the Mycoplasmatales species are part of the "Pneumoniae" and "Hominis" clusters, which group together in phylogenetic trees. Here we are proposing that the order Mycoplasmatales should be emended to encompass only the Mycoplasma species within the "Spiroplasma" cluster and that a new order, Mycoplasmoidales ord. nov., should be created to encompass the other Mycoplasma species. The "Pneumoniae" and the "Hominis" clusters are proposed as two new families, Mycoplasmoidaceae fam. nov., which includes the genera Eperythrozoon, Ureaplasma, and the newly proposed genera Malacoplasma and Mycoplasmoides, and Metamycoplasmataceae fam. nov. to contain the newly proposed genera Metamycoplasma, Mycoplasmopsis, and Mesomycoplasma. The results presented here allow reliable discernment, both in phylogenetic and molecular terms, of the members of the two proposed families as well as different described genera within these families including members of the genus Eperythrozoon, which is comprised of uncultivable organisms. The taxonomic reclassifications proposed here, which more accurately portray the genetic diversity among the Tenericutes/Mycoplasma species, provide a new framework for understanding the biological and clinical aspects of these important microbes.

Risk factors of different hemoplasma species infections in cats.

BACKGROUND: Hemoplasma species (spp.) commonly cause infections in cats worldwide. However, data on risk factors for infections are limited. The aim of this study was to determine the prevalence of hemoplasma spp. infections in cats in Southern Germany and to assess risk factors associated with infection. RESULTS: DNA was extracted from blood samples of 479 cats presented to different veterinary hospitals for various reasons. DNA of feline hemoplasmas was amplified by use of a previously reported PCR assay. Direct sequencing was used to confirm all purified amplicons and compared to hemoplasma sequences reported in GenBank. Results were evaluated in relation to the age, sex, housing conditions, feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) status of the cats. The overall hemoplasma prevalence rate was 9.4% (45/479; 95% CI: 7.08-12.36). 'Candidatus Mycoplasma (M.) haemominutum' (Mhm) DNA was amplified from 42 samples, M. haemofelis from 2, and M. haemocanis from 1 sample. There was a significantly higher risk of hemoplasma infection in cats from multi-cat households, in outdoor cats, as well as in cats with FIVinfection and in cats with abortive FeLV infection, but not in cats with progressive or regressive FeLV infection. CONCLUSIONS: Mhm infection is common in cats in Southern Germany. Higher prevalence in multi-cat households and associations with FeLV infection likely reflect the potential for direct transmission amongst cats. Outdoor access, male gender, and FIV infection are additional risk factors that might relate to aggressive interactions and exposure to vectors.

Assessment of the ability of Aedes species mosquitoes to transmit feline Mycoplasma haemofelis and ' Candidatus Mycoplasma haemominutum'.

Objectives The objective of this study was to evaluate wild-caught mosquitoes for evidence of hemotropic Mycoplasma species DNA and to determine whether the feline hemoplasmas, Mycoplasma haemofelis (Mhf) and ' Candidatus Mycoplasma haemominutum' (Mhm), can be transmitted by Aedes aegypti mosquitoes in a laboratory setting. Methods Wild-caught mosquito pools (50 mosquitoes per pool, 84 pools) utilized in routine public health department disease surveillance programs were tested for hemotropic Mycoplasma species DNA using PCR with primers designed to amplify all known hemoplasmas. Additionally, mosquitoes were trapped in the vicinity of known feral cat colonies, pooled (50 mosquitoes per pool) and tested (84 pools). Purpose-bred cats housed in a research facility were infected with Mhf or Mhm and then colonized laboratory A aegypti were fed upon the bacteremic cats. After a 7 day incubation period, mosquitoes previously fed on infected cats were allowed to feed again on naive cats, which were monitored for bacteremia for 10 weeks. Results Mycoplasma wenyonii DNA was confirmed in one wild-caught mosquito pool by DNA sequencing. While 7% of cats tested in feral colonies were hemoplasma positive, none of the mosquitoes trapped near colonies were positive. Hemoplasma DNA was amplified from A aegypti by PCR immediately after the infectious blood meal, but DNA was not detected at 7 and 14 days after feeding. Although evidence for uptake of organisms existed, hemoplasma DNA was not amplified from the experimentally infested cats in the 10 week observation period. Conclusions and relevance While wild-caught mosquitoes contained hemoplasma DNA and laboratory reared A aegypti mosquitoes take up hemoplasmas during the blood meal, there was no evidence of biologic transmission in this model.

Occurrence of Mycoplasma haemocanis in dogs infested by ticks in Campo Grande, Mato Grosso do Sul, Brazil / Ocorrência de Mycoplasma haemocanis em cães infestados por carrapatos em Campo Grande, Mato Grosso do Sul, Brasil

Abstract Hemotropic mycoplasmas in dogs, such as Mycoplasma haemocanis, have been described worldwide. Recently, these pathogens have been reported to be causative agent of zoonosis. It is known that its transmission may occur through the action of blood-sucking arthropods (e.g. ticks or fleas), through blood transfusion, contaminated fomites and/or transplacentally. In Brazil, M. haemocanis is present in practically all regions and the tick Rhipicephalus sanguineus sensu lato is suspected the main vector. In the municipality of Campo Grande, state of Mato Grosso do Sul, there is little information about infection of dogs by M. haemocanis, or on the main epidemiological features associated with it. Thus, the aim of the present study was to determine the occurrence of M. haemocanis among dogs infested by ticks and to assess possible associations with some epidemiological factors. The polymerase chain reaction (PCR) and DNA sequencing were used to analyze dog blood samples (n = 94). DNA from M. haemocanis was detected in four samples. No significant associations were observed with any epidemiological parameter analyzed here. However, the results from this study confirm that this pathogen is circulating in this region and should be considered in the differential diagnosis of diseases among anemic dogs.

Resumo Micoplasmas hemotrópicos de cães, como Mycoplasma haemocanis, já foram descritos em todo o mundo. Recentemente, esses patógenos têm sido apontados como causadores de zoonoses. É sabido que a transmissão pode ocorrer devido à ação de artrópodes sugadores de sangue (carrapatos, pulgas), transfusão sanguínea e/ou fômites contaminados e por via transplacentária. No Brasil, Mycoplasma haemocanis está presente em praticamente todas as regiões, e o carrapato Rhipicephalus sanguineus sensu lato é suspeito como principal vetor. No município de Campo Grande, Estado de Mato Grosso do Sul, Brasil não existem muitas informações acerca de infecções de cães por M. haemocanis, assim como quais são os principais aspectos epidemiológicos associados a este patógeno. Assim, o objetivo, no presente estudo, foi determinar a ocorrência de M. haemocanis em cães infestados por carrapatos e analisar possíveis associações com alguns fatores epidemiológicos. A Reação em Cadeia da Polimerase (PCR) e o sequenciamento de DNA foram utilizados para analisar amostras de sangue de cães (n = 94). DNA de M. haemocanis foi identificado em quatro amostras. Não foram observadas associações significativas com qualquer parâmetro epidemiológico analisado. No entanto, os resultados deste estudo confirmam que esse patógeno está circulando na região e deve ser considerado no diagnóstico diferencial de causas de anemia em cães.

A Novel Mycoplasma sp. Associated with Phallus Disease in Goose Breeders: Pathological and Bacteriological Findings.

In April 2014, poor fertility in a major commercial goose breeder operation in California triggered the submission of six live affected Toulouse ganders ( Anser anser ) to the California Animal Health and Food Safety Laboratory, Turlock branch (University of California-Davis). Toulouse were principally affected among all breeds, and their egg fertility dropped from 65.7% to less than 33.9% in the first 40 days of the 2014 breeding season. The flock consisted of 410 adult birds, 90 males and 320 females, between 2 and 5 yr of age. Inspection of the flock revealed that 44.4% of the Toulouse ganders had severe phallic deformities that prevented them from mating. At postmortem examination, severe yellowish fibrocaseous exudate disrupted the architecture of the phallus and occasionally produced fistulating tracts through the wall of the organ. Microscopically, multifocal lymphoid nodules were noted in the mucosa and submucosa of the phallus and were associated with extensive granulomatous reaction, intralesional bacteria, and spermatozoa. Mycoplasma spp. were isolated from the phallus of affected and nonaffected birds, and PCR protocols targeting the 16S-23S ribosomal RNA intergenic spacer regions and the RNA polymerase beta subunit gene were performed to identify the isolates. Three distinct species were identified on sequencing and analysis using the National Center for Biotechnology Information basic local alignment search tool: Mycoplasma cloacale , Mycoplasma anseris , and an unknown novel Mycoplasma sp. Additionally, Pasteurella multocida , in combination with other bacteria, was also isolated from the phallic lesions and identified as serotype 3 with a DNA profile of 1511 (National Veterinary Service Laboratory). This is the first report of these Mycoplasma spp. and other bacteria associated with reproductive disease in ganders in the United States.

Arginine deiminase: recent advances in discovery, crystal structure, and protein engineering for improved properties as an anti-tumor drug.

Arginine deiminase (ADI) is an important arginine-degrading enzyme with wide applications, in particular as an anti-cancer agent for the therapy of arginine-auxotrophic tumors. In recent years, novel ADIs with excellent properties have been identified from various organisms, and crystal structures of ADI were investigated. To satisfy the requirements of potential therapeutic applications, protein engineering has been performed to improve the activity and properties of ADIs. In this mini-review, we systematically summarized the latest progress on identification and crystal structure of ADIs, and protein engineering strategies for improved enzymatic properties, such as pH optimum, K m and k cat values, and thermostability. We also outlined the PEGylation of ADI for improved circulating half-life and immunogenicity, as well as their performance in clinical trials. Finally, perspectives on extracellular secretion and property improvement of ADI were discussed.

Molecular investigation of hemotropic mycoplasms in human beings, dogs and horses in a rural settlement in Southern Brazil / Investigação molecular de espécies de micoplasmas hemotrópicos em cães, equinos e humanos de um assentamento rural do Sul do Brasil

SUMMARYThe aims of this study were to determine the prevalence of hemoplasmas in a rural Brazilian settlement's population of human beings, their dogs and horses, highly exposed to tick bites; to identify the tick species parasitizing dogs and horses, and analyze factors associated with their infection. Blood samples from 132 dogs, 16 horses and 100 humans were screened using a pan-hemoplasma SYBR green real-time PCR assay followed by a species-specific TaqMan real-time PCR. A total of 59/132 (44.7%) dog samples were positive for hemoplasmas (21 Mycoplasma haemocanisalone, 12 ' CandidatusMycoplasma haematoparvum' alone and 21 both). Only 1/100 (1.0%) human sample was positive by qPCR SYBR green, with no successful amplification of 16S rRNA or 23 rRNA genes despite multiple attempts. All horse samples were negative. Dogs >1 year of age were more likely to be positive for hemoplasmas ( p= 0.0014). In conclusion, although canine hemoplasma infection was highly prevalent, cross-species hemoplasma transmission was not observed, and therefore may not frequently occur despite overexposure of agents and vectors.

RESUMOOs objetivos deste estudo foram determinar a prevalência de hemoplasmas numa população restrita de cães, equinos e humanos altamente expostos a picadas de carrapatos em assentamento rural brasileiro; identificar as espécies de carrapatos parasitando cães e equinos, e analisar os fatores associados à infecção. Amostras de sangue de 132 cães, 16 cavalos e 100 humanos foram avaliadas utilizando um protocolo pan-hemoplasma em PCR quantitativas em tempo real (qPCR) com SYBR green, seguido de qPCR TaqMan espécie-específicos. Cinquenta e nove/132 (44,7%) cães foram positivos para hemoplasmas (21 Mycoplasma haemocanis, 12 ' Candidatus Mycoplasmahaematoparvum' e 21 para ambos). Uma amostra humana do total de 100 (1%) foi positiva pelo qPCR SYBR green, mas os genes 16S rRNA ou 23S rRNA não foram amplificados com sucesso, apesar de inúmeras tentativas. Todas as amostras de cavalos foram negativas. Cães > 1 ano apresentaram mais chance de serem positivos para hemoplasmas ( p= 0,0014). Concluindo, embora infecções por hemoplasmas caninos sejam altamente prevalentes, a transmissão de hemoplasmas entre espécies não foi observada, e desta forma podem não ocorrer de forma frequente apesar da alta exposição aos agentes e vetores.

Molecular detection of haemotropic Mycoplasma species in urban and rural cats from Portugal.

OBJECTIVES: The aim of the present study was to evaluate the prevalence of haemoplasma infection in cats in Portugal and to assess risk factors for infection. METHODS: Real-time polymerase chain reaction techniques were used to assess 236 urban and rural cats from central and southern Portugal. RESULTS: The overall prevalence of haemoplasma in the target population was 27.1% (64/236), with individual species' prevalences as follows: 17.8% (42/236) 'Candidatus Mycoplasma haemominutum' (CMhm), 14.4% (34/236) Mycoplasma haemofelis (Mhf) and only 5.9% (14/236) 'Candidatus Mycoplasma turicensis' (CMt). Multiple infections were detected in 8.1% (19/236) of the samples, with triple and double infections with Mhf and CMhm being most commonly detected (5.9% [14/236] of cats). Haemoplasma infection was significantly higher in shelter cats (P = 0.015) than in cats with other lifestyles (eg, free-roaming/house pet/blood donors). Haemoplasma prevalence was also higher in cats with feline immunodeficiency virus infection (FIV; P = 0.011). Although sex was not significantly associated with haemoplasma infection (P = 0.050), CMt was predominantly found in males (P = 0.032). Also, the presence of haemoplasma multiple infections was statistically associated with being in a shelter (P = 0.021), male (P = 0.057) and with FIV co-infection (P = 0.004). No evidence of an association between haemoplasma infection and geographical location, age or feline leukaemia virus co-infection was found. CONCLUSIONS AND RELEVANCE: The results obtained in our study are consistent with the documented worldwide prevalence of feline haemoplasma infections, suggesting that the three main feline haemoplasma species are common in Portugal.

American kestrel (Falco spaverius) fledgling with severe bilateral periorbital swelling and infection with Mycoplasma buteonis, Avibacterium (Pasteurella) gallinarum, and Staphylococcus pasteuri.

Abstract: A female American kestrel (Falco spaverius) fledgling was found on the ground with a suspected trauma to the right eye and open-mouth breathing. During the first 2 days of hospitalization, the bird developed severe bilateral periorbital cellulitis, blepharoedema, and sinusitis. The periocular tissues of the right globe were devitalized and communicated with a fistula at the commissure of the right side of the beak. The blepharoedema of the left eye was aspirated and yielded a dark colored malodorous fluid, which was submitted for aerobic bacterial and Mycoplasma cultures. Results showed a mixed infection with Mycoplasma buteonis, Avibacterium gallinarum, and Staphylococcus pasteuri, all of which are not commonly isolated from birds of prey. With antimicrobial therapy, supportive care, and surgical debridement of the right periocular necrotic tissues and adhesed phthisical globe, the kestrel recovered from this severe mixed upper respiratory infection.

Identification, occurrence and clinical findings of canine hemoplasmas in southern Brazil.

Hemoplasmas are ubiquitous pleomorphic and epicellular bacteria detected in erythrocytes in several species. In Brazil, studies on hemoplasmas have not included information on occurrence, clinical signs, and risk factors in dogs. This paper investigates the occurrence of hemoplasmas in dogs, focusing on risk factors and clinical status. Conventional PCR for the four types of canine hemoplasmas was performed in 331 blood samples collected from dogs clinically treated at a teaching veterinary hospital. Of all samples, 17/331 (5.1%) were positive for Mycoplasma haemocanis and 6/331 (1.8%) were positive for a 'Candidatus Mycoplasma haemominutum-like' organism. Risk factors included the presence of vectors, old age, dog bite wounds, and neoplastic diseases. In the multivariate analysis, a 4.40 odds ratio in dogs with vector-borne diseases indicated risk for hemoplasmosis. There was correlation between hemoplasma infection and neoplastic disease, suggesting that neoplastic conditions are a risk factor for hemoplasma infection in dogs.