Detection by multiplex PCR of Mycoplasma species associated with dairy cattle in Argentina

array:23 ["pii" => "S0325754121000791" "issn" => "03257541" "doi" => "10.1016/j.ram.2021.07.001" "estado" => "S300" "fechaPublicacion" => "2022-04-01" "aid" => "471" "copyright" => "Asociación Argentina de Microbiología" "copyrightAnyo" => "2021" "documento" => "article" "crossmark" => 1 "licencia" => "http://creativecommons.org/licenses/by-nc-nd/4.0/" "subdocumento" => "fla" "cita" => "Rev Argent Microbiol. 2022;54:158-61" "abierto" => array:3 [ "ES" => true "ES2" => true "LATM" => true ] "gratuito" => true "lecturas" => array:1 [ "total" => 0 ] "itemAnterior" => array:19 [ "pii" => "S0325754121000560" "issn" => "03257541" "doi" => "10.1016/j.ram.2021.05.002" "estado" => "S300" "fechaPublicacion" => "2022-04-01" "aid" => "466" "copyright" => "Asociación Argentina de Microbiología" "documento" => "article" "crossmark" => 1 "licencia" => "http://creativecommons.org/licenses/by-nc-nd/4.0/" "subdocumento" => "fla" "cita" => "Rev Argent Microbiol. 2022;54:152-7" "abierto" => array:3 [ "ES" => true "ES2" => true "LATM" => true ] "gratuito" => true "lecturas" => array:1 [ "total" => 0 ] "en" => array:14 [ "idiomaDefecto" => true "cabecera" => "Brief report" "titulo" => "The drop plate method as an alternative for Azospirillum spp. viable cell enumeration within the consensus protocol of the REDCAI network" "tienePdf" => "en" "tieneTextoCompleto" => "en" "tieneResumen" => array:3 [ 0 => "en" 1 => "en" 2 => "es" ] "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "152" "paginaFinal" => "157" ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "La técnica de la microgota como alternativa para el recuento de Azospirillum spp. dentro del protocolo de la Red de Control de Calidad de Inoculantes (REDCAI)" ] ] "contieneResumen" => array:2 [ "en" => true "es" => true ] "contieneTextoCompleto" => array:1 [ "en" => true ] "contienePdf" => array:1 [ "en" => true ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 378 "Ancho" => 1305 "Tamanyo" => 71558 ] ] "descripcion" => array:1 [ "en" => "Congo red (RC) Petri plates with 20μl drop volume of 10−4, 10−5, 10−6, 10−7 dilutions, in quadruplicate. a. Drops distribution on the RC medium surface. b. Typical colonies of Azospirillum spp. in numbers in accordance with the ten-fold dilutions. Photo credits: Dr. Mariana Puente (a) and Dr. Luciana Di Salvo (b)." ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Luciana P. Di Salvo, Julia E. García, Mariana L. Puente, Josefina Amigo, Analía Anríquez, Claudia Barlocco, Silvia Benintende, Tatiana Bochatay, Marta Bortolato, Fabricio Cassán, Carolina Castaño, Melina Catafesta, Anahí Coniglio, Marisa Díaz, Liliana R. Galián, Eugenia Gallace, Patricia García, Inés E. García de Salamone, Marianela Landa, Germán Liernur, María Laura Maneiro, Rosana Massa, Julieta Malinverni, Nicolás Marchessi, Emilia Monteleone, Silvina Oviedo, Lucrecia Pobliti, Gabriela Portela, Débora Radovancich, Silvia Righes, Rosina Rocha, Enrique Rodríguez Cáceres, Alejandro Rossi, Gisela Santella, María Laura Tortora, Nora Trejo, José A. Valenzuela, Daniela Vallejo" "autores" => array:38 [ 0 => array:2 [ "nombre" => "Luciana P." "apellidos" => "Di Salvo" ] 1 => array:2 [ "nombre" => "Julia E." "apellidos" => "García" ] 2 => array:2 [ "nombre" => "Mariana L." "apellidos" => "Puente" ] 3 => array:2 [ "nombre" => "Josefina" "apellidos" => "Amigo" ] 4 => array:2 [ "nombre" => "Analía" "apellidos" => "Anríquez" ] 5 => array:2 [ "nombre" => "Claudia" "apellidos" => "Barlocco" ] 6 => array:2 [ "nombre" => "Silvia" "apellidos" => "Benintende" ] 7 => array:2 [ "nombre" => "Tatiana" "apellidos" => "Bochatay" ] 8 => array:2 [ "nombre" => "Marta" "apellidos" => "Bortolato" ] 9 => array:2 [ "nombre" => "Fabricio" "apellidos" => "Cassán" ] 10 => array:2 [ "nombre" => "Carolina" "apellidos" => "Castaño" ] 11 => array:2 [ "nombre" => "Melina" "apellidos" => "Catafesta" ] 12 => array:2 [ "nombre" => "Anahí" "apellidos" => "Coniglio" ] 13 => array:2 [ "nombre" => "Marisa" "apellidos" => "Díaz" ] 14 => array:2 [ "nombre" => "Liliana R." "apellidos" => "Galián" ] 15 => array:2 [ "nombre" => "Eugenia" "apellidos" => "Gallace" ] 16 => array:2 [ "nombre" => "Patricia" "apellidos" => "García" ] 17 => array:2 [ "nombre" => "Inés E." "apellidos" => "García de Salamone" ] 18 => array:2 [ "nombre" => "Marianela" "apellidos" => "Landa" ] 19 => array:2 [ "nombre" => "Germán" "apellidos" => "Liernur" ] 20 => array:2 [ "nombre" => "María Laura" "apellidos" => "Maneiro" ] 21 => array:2 [ "nombre" => "Rosana" "apellidos" => "Massa" ] 22 => array:2 [ "nombre" => "Julieta" "apellidos" => "Malinverni" ] 23 => array:2 [ "nombre" => "Nicolás" "apellidos" => "Marchessi" ] 24 => array:2 [ "nombre" => "Emilia" "apellidos" => "Monteleone" ] 25 => array:2 [ "nombre" => "Silvina" "apellidos" => "Oviedo" ] 26 => array:2 [ "nombre" => "Lucrecia" "apellidos" => "Pobliti" ] 27 => array:2 [ "nombre" => "Gabriela" "apellidos" => "Portela" ] 28 => array:2 [ "nombre" => "Débora" "apellidos" => "Radovancich" ] 29 => array:2 [ "nombre" => "Silvia" "apellidos" => "Righes" ] 30 => array:2 [ "nombre" => "Rosina" "apellidos" => "Rocha" ] 31 => array:2 [ "nombre" => "Enrique" "apellidos" => "Rodríguez Cáceres" ] 32 => array:2 [ "nombre" => "Alejandro" "apellidos" => "Rossi" ] 33 => array:2 [ "nombre" => "Gisela" "apellidos" => "Santella" ] 34 => array:2 [ "nombre" => "María Laura" "apellidos" => "Tortora" ] 35 => array:2 [ "nombre" => "Nora" "apellidos" => "Trejo" ] 36 => array:2 [ "nombre" => "José A." "apellidos" => "Valenzuela" ] 37 => array:2 [ "nombre" => "Daniela" "apellidos" => "Vallejo" ] ] ] ] "resumen" => array:1 [ 0 => array:3 [ "titulo" => "Highlights" "clase" => "author-highlights" "resumen" => "<ul class="elsevierStyleList" id="lis0005"><li class="elsevierStyleListItem" id="lsti0005">•The drop plate method was validated by 55 participants in 3 independent trials.</li><li class="elsevierStyleListItem" id="lsti0010">•Reliability, minimal heterogeneity among results, and economic benefits were met.</li><li class="elsevierStyleListItem" id="lsti0015">•Both methods showed similar number of Azospirillum viable cells.</li><li class="elsevierStyleListItem" id="lsti0020">•Drop plate method will be included in the REDCAI consensus protocol for Azospirillum spp.</li></ul>" ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0325754121000560?idApp=UINPBA00004N" "url" => "/03257541/0000005400000002/v2_202206160255/S0325754121000560/v2_202206160255/en/main.assets" ] "en" => array:20 [ "idiomaDefecto" => true "cabecera" => "Brief report" "titulo" => "Detection by multiplex PCR of Mycoplasma species associated with dairy cattle in Argentina" "tieneTextoCompleto" => true "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "158" "paginaFinal" => "161" ] ] "autores" => array:1 [ 0 => array:4 [ "autoresLista" => "Verónica E. Neder, Ariel F. Amadio, Luis F. Calvinho" "autores" => array:3 [ 0 => array:3 [ "nombre" => "Verónica E." "apellidos" => "Neder" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] ] ] 1 => array:3 [ "nombre" => "Ariel F." "apellidos" => "Amadio" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] ] ] 2 => array:4 [ "nombre" => "Luis F." "apellidos" => "Calvinho" "email" => array:1 [ 0 => "calvinho.luis@inta.gob.ar" ] "referencia" => array:3 [ 0 => array:2 [ "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:2 [ "etiqueta" => "b" "identificador" => "aff0010" ] 2 => array:2 [ "etiqueta" => "*" "identificador" => "cor0005" ] ] ] ] "afiliaciones" => array:2 [ 0 => array:3 [ "entidad" => "Instituto de Investigación de la Cadena Láctea (INTA-CONCET), Estación Experimental Agropecuaria Rafaela, Instituto Nacional de Tecnología Agropecuaria, Rafaela, Santa Fe, Argentina" "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:3 [ "entidad" => "Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral, Esperanza, Santa Fe, Argentina" "etiqueta" => "b" "identificador" => "aff0010" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "⁎" "correspondencia" => "Corresponding author." ] ] ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "Detección por PCR multiplex de especies de Mycoplasma asociadas a ganado lechero en Argentina" ] ] "textoCompleto" => "Mycoplasmas are common inhabitants of the respiratory mucosa, the urogenital tract, conjunctival surfaces, the gastrointestinal tract and the mammary gland; they can be commensals, opportunistic pathogens or primary pathogens associated with respiratory disease, otitis, mastitis, arthritis, reproductive disease, meningitis and conjunctivitis in cattle <a class="elsevierStyleCrossRefs" href="#bib0110">5,11,13</a>. Some Mycoplasma species are highly contagious, spread rapidly within a herd, and respond poorly to antibiotic therapy, which often determines the need of segregating or culling affected animals<a class="elsevierStyleCrossRef" href="#bib0150">13</a>. Hence, a rapid and accurate diagnosis is of outmost importance both for the control and for prevention of disease outbreaks. Early studies using biochemical methods detected the presence of M.bovis associated with mastitis in dairy cattle in Argentina<a class="elsevierStyleCrossRef" href="#bib0100">3</a>. In the last few years, Mycoplasma infections in cattle have received increasing attention, being the organisms identified either by biochemical and molecular methods<a class="elsevierStyleCrossRefs" href="#bib0145">12,14,15</a> or by immunohistochemistry<a class="elsevierStyleCrossRef" href="#bib0135">10</a>.Several conventional PCR methods have been developed in different countries to identify Mycoplasma species either from isolates grown through classical culture or directly from milk samples<a class="elsevierStyleCrossRef" href="#bib0150">13</a>. However, so far only few Mycoplasma species have been isolated from dairy cattle in Argentina and there is scarce information about their frequency and the epidemiological and clinical features associated with their presence in local dairy herds. Hence, there is a need to generate methods for rapid identification at the species level to be used as a diagnostic tool both during outbreaks and epidemiological surveys. We have recently reported the isolation of M.leachii associated with arthritis in dairy calves and the development of a multiplex PCR for detecting both this organism and M.californicum<a class="elsevierStyleCrossRef" href="#bib0145">12</a>. Since other species have been reported to be associated with diseases in dairy cattle in Argentina<a class="elsevierStyleCrossRefs" href="#bib0135">10,14,15</a>, the objectives of this study were to develop a multiplex PCR for identifying other Mycoplasma species (M. bovis and M.canadense) and to describe the frequency of Mycoplasma species isolated from clinical samples submitted to a diagnostic laboratory.A total of 1548 samples were submitted to the Microbiology Laboratory of the Rafaela Experiment Station of INTA from January 2009 to July 2016 by veterinarians who suspected the presence of Mycoplasma as etiologic agent of diseases in dairy cattle. One hundred and seven of these samples were from bulk tank milk, 1412 from composite milk from individual cows and 29 from various organs and biological fluids of dairy calves. The samples came from dairy farms located in the central dairy area of Argentina (Santa Fe and Córdoba provinces) and in the Mar y Sierras dairy area (Buenos Aires province). In a first stage, biological specimens were processed by the classical methodology using modified Hayflick medium at 37°C in a 10% CO2 atmosphere incubated for 7–10 days and Mycoplasmas were presumptively identified on the basis of their colony morphology under a microscope at 15–25× magnification as previously described<a class="elsevierStyleCrossRef" href="#bib0145">12</a>. DNA from bacterial growth in culture medium from colonies suspected to be Mycoplasma spp. was extracted using the procedure previously described<a class="elsevierStyleCrossRef" href="#bib0145">12</a> and subjected to a PCR test to confirm that isolates belonged to this genus<a class="elsevierStyleCrossRef" href="#bib0095">2</a>. For further identification of M. leachii and M. californicum a multiplex PCR to amplify specific DNA fragments from the conserved spacer region between the 16S and 23S ribosomal RNA gene was carried out as previously described<a class="elsevierStyleCrossRef" href="#bib0145">12</a>.For designing a multiplex PCR for amplifying specific DNA fragments of M. bovis and M. canadense two steps were followed. First, the conserved spacer regions between the 16S and 23S ribosomal RNA gene<a class="elsevierStyleCrossRef" href="#bib0090">1</a> of M. bovis ATCC 25025 and of six unidentified isolates previously characterized as belonging to genus Mycoplasma by a genus-specific PCR<a class="elsevierStyleCrossRef" href="#bib0095">2</a> were amplified and sequenced (ABI3130xl, Applied Biosystems). The sequences obtained were aligned against the NCBI database using BLASTN and then compared with the reported hits in GenBank using the ClustalX multiple sequence alignment program. The 16S-23S rRNA gene sequence obtained from the unidentified strain showed 99% identity with M. canadense strain 275C (DQ847417) while the other was confirmed as M. bovis. The analysis of the 16S-23S rRNA region allowed the design of species-specific primers, but with very similar amplicon size. Then, specific primers only for M. canadense were designed using FastPCR (<a href="https://primerdigital.com/tools/pcr.html">https://primerdigital.com/tools/pcr.html</a>) for the 16S-23S rRNA region. A multiplex-reaction was set up using those specific primers for M. canadense while primers for M. bovis were those previously described to amplify a conserved segment of the uvrC gene suitable for specific diagnosis<a class="elsevierStyleCrossRef" href="#bib0170">17</a>. Primers for M. bovis were UVRF (5′-TTACGCAAGAGAATGCTTCA-3′) and UVRR (5′-TAGGAAAGCACCCTATTGAT-3′) and for M. canadense were CANDF (5′-GCGGAACATTAGTTAGTTGGTA-3′) and CANDR (5′-CGTTAGCTGCGTCAGTGAATT-3′). Expected DNA amplification fragments were 1626bp for M. bovis and 623bp for M. canadense, respectively. The amplification program was: 94°C 2min, 30 cycles of 94°C 30s, 54°C 30s, 72°C 60s, 72°C 5min. Primers designed for amplifying specific fragments of the 16S-23S rRNA region for differentiation of M. canadense as well as those previously described<a class="elsevierStyleCrossRef" href="#bib0145">12</a> for M. californicum and M. leachii were tested for specificity using DNA from the other Mycoplasma species included in this study. The individual 25μl reaction mixture consisted of: 2.5μl of 10× enzyme buffer, 2mM MgCl2, 0.2mM of each dNTP, 0.03μM of each primer, 1.25 U of Taq DNA polymerase (Productos Bio-Lógicos®) and 50ng of genomic DNA. Cycling consisted of an initial denaturation step at 94°C for 2min, and then 30 cycles of the following sequence: 94°C for 30s, 54°C for 30s and 72°C for 1min, with a final extension at 72°C for 5min. In all reactions a negative control using the same previously described mixture, but replacing the DNA by sterile distilled water, was used. The DNA products obtained were visualized in 1.5% agarose gels. The gels were prepared in 1× TAE buffer, adding GelRed TM 10000× to a final concentration of 1×, the electrophoretic runs were performed at 90V. A 100bp ladder step marker was used. DNA fragments resolved in the gel were visualized in a UV light transilluminator (255nm).Of the total samples processed, 38 isolates were phenotypically identified as Mycoplasma spp. This preliminary identification was confirmed by the genus-specific PCR technique. Identification of the isolates at species level was achieved using both multiplex PCR described. <a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a> summarizes the frequency of isolated Mycoplasma species and their origin.<elsevierMultimedia ident="tbl0005"></elsevierMultimedia>Since convenience samples were used, the results of this study are not indicative of the prevalence of the different isolated species. The low number of isolates in relation to the total of processed samples indicates that the presence of this genus detected by classical culture is not a frequent finding. No systematic studies have been performed in Argentina to determine prevalence of the different Mycoplasma species associated with defined syndromes in dairy cattle; rather, disease outbreaks and clinical cases have been reported so far. Three isolates (7.9%) were identified as M. bovis. Within the Mycoplasma genus, M. bovis is currently considered the most frequently isolated species associated with disease in cattle worldwide<a class="elsevierStyleCrossRef" href="#bib0140">11</a>. Since the first report causing mastitis in a dairy herd, M. bovis has been isolated from cases of respiratory disease, mastitis and arthritis in cattle, being considered an emergent pathogen in several countries<a class="elsevierStyleCrossRefs" href="#bib0110">5,11</a>. In Argentina its presence was first reported in a mastitis outbreak in a dairy farm located in Buenos Aires province where 11.8% of cows presented clinical mastitis that in various cases affected the four mammary quarters, caused a marked decrease in milk production and did not respond to antibiotic therapy. The organism was isolated from composite milk samples from cows with clinical and subclinical mastitis, bulk tank milk and a preputial swab from a bull by classical culture and characterized as M. bovis by biochemical tests and indirect immunofluorescence of the isolated colonies<a class="elsevierStyleCrossRef" href="#bib0100">3</a>. More recently, M. bovis was detected in milk samples both from cows with clinical and subclinical mastitis and bulk tank milk from dairy herds from Buenos Aires and Córdoba provinces and characterized by amplifying a fragment of the uvrC gene<a class="elsevierStyleCrossRef" href="#bib0160">15</a>; however, no further information was provided about the cases from which the processed samples came. In addition, M. bovis was identified by immunohistochemistry in cases of bronchopneumonia unresponsive to antibiotic treatment both in beef and dairy calves from Córdoba and Santa Fe provinces<a class="elsevierStyleCrossRef" href="#bib0135">10</a>. These results, together with those obtained in the present study confirm the importance of M. bovis as a causative agent of mastitis, bronchopneumonia and arthritis as has been pointed out in different countries<a class="elsevierStyleCrossRefs" href="#bib0110">5,11</a> and indicate the need to include diagnostic methodology in routine laboratories to identify this pathogen.M. canadense was isolated from a single case of intramammary (IMI) infection from a dairy herd in Buenos Aires province. Diseases and clinical cases associated with this organism are not as well characterized as those associated with M. bovis. This species has been reported to cause not only IMI<a class="elsevierStyleCrossRef" href="#bib0110">5</a>, but also to be associated with granulopapular vulvovaginitis in dairy cattle in single or mixed infections with Mycoplasmabovigenitalium<a class="elsevierStyleCrossRef" href="#bib0125">8</a> and with arthritis in a calf<a class="elsevierStyleCrossRef" href="#bib0130">9</a>. In Argentina, this species was isolated from two bulk tank milk samples from a dairy farm located in Buenos Aires province<a class="elsevierStyleCrossRef" href="#bib0160">15</a>.M. californicum is considered to be the second most frequently isolated Mycoplasma species from bovine mastitis cases in USA after M.bovis<a class="elsevierStyleCrossRef" href="#bib0110">5</a>, sharing with the latter similar characteristics regarding both the clinical mastitis features and the importance of the presence of asymptomatic carriers in the dissemination of the organism among cows and dairy herds<a class="elsevierStyleCrossRef" href="#bib0115">6</a>. In the present study, this organism was isolated from 4 cows during an outbreak of mastitis refractory to antibiotic therapy, as well as from two bulk tank milk samples from the same dairy farm located in Córdoba province. In Argentina, the isolation of M. californicum was reported from bulk tank milk samples<a class="elsevierStyleCrossRef" href="#bib0160">15</a> and clinical mastitis samples from the same dairy herd<a class="elsevierStyleCrossRef" href="#bib0155">14</a> located in Buenos Aires province.Twenty eight isolates (73.7%) were characterized as M. leachii. These isolates belonged to a single dairy farm where cases of polyarthritis in calves were previously detected<a class="elsevierStyleCrossRef" href="#bib0145">12</a>. In this farm, milk samples were taken from clinical mastitis cases and also from individual cows (composite milk samples) as part of a control program based on the detection of asymptomatic carriers. In addition, bulk tank milk samples were taken for monitoring the presence of the organism in the lactating dairy herd as the control program progressed. M. leachii, previously referred to as Mycoplasma sp. bovine Group 7<a class="elsevierStyleCrossRef" href="#bib0165">16</a>, was early recognized as a cause of polyarthritis, mastitis and abortion in dairy cattle in Australia<a class="elsevierStyleCrossRef" href="#bib0120">7</a> and recent studies characterized clinical signs and histopathological lesions induced by experimental IMI with this pathogen<a class="elsevierStyleCrossRef" href="#bib0105">4</a>. The present study, as well as our previous report<a class="elsevierStyleCrossRef" href="#bib0145">12</a> confirms the ability of this organism to cause diverse clinical syndromes.In conclusion, the multiplex PCR technique for detecting M. bovis and M. canadense described in this study, along with the one previously described for detecting M. californicum and M. leachii, provide a useful diagnostic tool for characterizing some of the Mycoplasma species that cause disease in cattle in Argentina. The results of this study confirm the presence of several species of Mycoplasma associated with different disease syndromes in dairy cattle. The techniques used in the present study can be useful to broaden knowledge about Mycoplasma infections in cattle, since the search for these organisms is not usually included in routine diagnoses.Conflict of interestThe authors declare that they have no conflicts of interest." "textoCompletoSecciones" => array:1 [ "secciones" => array:8 [ 0 => array:3 [ "identificador" => "xres1733194" "titulo" => "Highlights" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abst0005" ] ] ] 1 => array:3 [ "identificador" => "xres1733192" "titulo" => "Abstract" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abst0010" ] ] ] 2 => array:2 [ "identificador" => "xpalclavsec1529460" "titulo" => "Keywords" ] 3 => array:3 [ "identificador" => "xres1733193" "titulo" => "Resumen" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abst0015" ] ] ] 4 => array:2 [ "identificador" => "xpalclavsec1529459" "titulo" => "Palabras clave" ] 5 => array:2 [ "identificador" => "sec0005" "titulo" => "Conflict of interest" ] 6 => array:2 [ "identificador" => "xack611775" "titulo" => "Acknowledgments" ] 7 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2020-10-20" "fechaAceptado" => "2021-07-04" "PalabrasClave" => array:2 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec1529460" "palabras" => array:3 [ 0 => "Dairy cattle" 1 => "Mycoplasma spp." 2 => "Multiplex PCR" ] ] ] "es" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Palabras clave" "identificador" => "xpalclavsec1529459" "palabras" => array:3 [ 0 => "Ganado lechero" 1 => "Mycoplasma spp." 2 => "PCR multiplex" ] ] ] ] "tieneResumen" => true "highlights" => array:2 [ "titulo" => "Highlights" "resumen" => "<ul class="elsevierStyleList" id="lis0005"><li class="elsevierStyleListItem" id="lsti0005">•Mycoplasma species are not routinely diagnosed in dairy cattle in Argentina.</li><li class="elsevierStyleListItem" id="lsti0010">•Two multiplex PCR allowed identification of Mycoplasma species of interest.</li><li class="elsevierStyleListItem" id="lsti0015">•These methods can contribute to increase knowledge of Mycoplasma infections in cattle.</li></ul>" ] "resumen" => array:2 [ "en" => array:2 [ "titulo" => "Abstract" "resumen" => "There is scarce information about the frequency and epidemiological and clinical features associated with the presence of Mycoplasma spp. in Argentine dairy herds. The objectives of this study were to develop a multiplex PCR for identifying M.bovis and M.canadense and to describe the frequency of Mycoplasma spp. isolated from clinical samples submitted to a diagnostic laboratory. Of a total of 1548 samples from intramammary infections, bulk tank milk and biological fluids, 38 Mycoplasma isolates were obtained. M. bovis, M. canadense, M.californicum and M.leachii were detected by using two multiplex PCRs, confirming their presence in clinical conditions in dairy cattle. The techniques used in the present study can be useful to broaden the knowledge about Mycoplasma infections in cattle, since the search for these organisms is not usually included in routine diagnoses." ] "es" => array:2 [ "titulo" => "Resumen" "resumen" => "Existe poca información sobre la frecuencia, así como las características epidemiológicas y clínicas asociadas con la presencia de Mycoplasma en los rodeos lecheros argentinos. Los objetivos de este estudio fueron desarrollar una PCR multiplex para identificar M.bovis y M.canadense y describir la frecuencia de especies de Mycoplasma aisladas de muestras clínicas enviadas a un laboratorio de diagnóstico. De un total de 1.548 muestras de infecciones intramamarias, leche de tanque de frío y fluidos biológicos, se obtuvieron 38 aislamientos de Mycoplasma. Mediante 2 PCR multiplex se detectaron M. bovis, M. canadense, M.californicum y M.leachii, confirmando su presencia en síndromes clínicos en ganado lechero. Las técnicas utilizadas en el presente estudio pueden ser útiles para ampliar el conocimiento sobre las infecciones por Mycoplasma en bovinos, ya que la búsqueda de estos organismos no suele incluirse en los diagnósticos de rutina." ] ] "multimedia" => array:1 [ 0 => array:8 [ "identificador" => "tbl0005" "etiqueta" => "Table 1" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "detalles" => array:1 [ 0 => array:3 [ "identificador" => "at1" "detalle" => "Table " "rol" => "short" ] ] "tabla" => array:1 [ "tablatextoimagen" => array:1 [ 0 => array:1 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Mycoplasma species \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Number and percentage of total isolates \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Origin (number of isolates) \t\t\t\t\t\t\n \t\t\t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td-with-role" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">M. bovis \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">3 (7.9%) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">Bulk tank milk (2), arthritis (1) \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">M. californicum \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">6 (15.8%) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">Intramammary infection (4), bulk tank milk (2) \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">M. canadense \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">1 (2.6%) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">Intramammary infection (1) \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">M. leachii \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">28 (73.7%) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">Intramammary infection (21), bulk tank milk (6), mandibular abscess (1) \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">Total \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">38 (100%) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] ] ] ] "descripcion" => array:1 [ "en" => "Frequency and origin of Mycoplasma isolated from biological samples obtained from dairy cattle." ] ] ] "bibliografia" => array:2 [ "titulo" => "References" "seccion" => array:1 [ 0 => array:2 [ "identificador" => "bibs0015" "bibliografiaReferencia" => array:17 [ 0 => array:3 [ "identificador" => "bib0090" "etiqueta" => "1" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Sequence analysis of the 16S-23S spacer region in rRNA operons of the Mollicutes" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:6 [ 0 => "K. 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"vista" => "all" ] ] ] "idiomaDefecto" => "en" "url" => "/03257541/0000005400000002/v2_202206160255/S0325754121000791/v2_202206160255/en/main.assets" "Apartado" => array:4 [ "identificador" => "37862" "tipo" => "SECCION" "en" => array:2 [ "titulo" => "Microbiología agrícola, ambiental e industrial" "idiomaDefecto" => true ] "idiomaDefecto" => "en" ] "PDF" => "https://static.elsevier.es/multimedia/03257541/0000005400000002/v2_202206160255/S0325754121000791/v2_202206160255/en/main.pdf?idApp=UINPBA00004N&text.app=https://www.elsevier.es/" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0325754121000791?idApp=UINPBA00004N"]

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Detección por PCR multiplex de especies de Mycoplasma asociadas a ganado lechero en Argentina

Verónica E. Nedera, Ariel F. Amadioa, Luis F. Calvinhoa,b,

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calvinho.luis@inta.gob.ar

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a Instituto de Investigación de la Cadena Láctea (INTA-CONCET), Estación Experimental Agropecuaria Rafaela, Instituto Nacional de Tecnología Agropecuaria, Rafaela, Santa Fe, Argentina

b Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral, Esperanza, Santa Fe, Argentina

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  "cita" => "Rev Argent Microbiol. 2022;54:158-61"
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    "cita" => "Rev Argent Microbiol. 2022;54:152-7"
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      "cabecera" => "<span class="elsevierStyleTextfn">Brief report</span>"
      "titulo" => "The drop plate method as an alternative for <span class="elsevierStyleItalic">Azospirillum</span> spp&#46; viable cell enumeration within the consensus protocol of the REDCAI network"
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          "titulo" => "La t&#233;cnica de la microgota como alternativa para el recuento de <span class="elsevierStyleItalic">Azospirillum</span> spp&#46; dentro del protocolo de la Red de Control de Calidad de Inoculantes &#40;REDCAI&#41;"
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            "en" => "<p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">Congo red &#40;RC&#41; Petri plates with 20<span class="elsevierStyleHsp" style=""></span>&#956;l drop volume of 10<span class="elsevierStyleSup">&#8722;4</span>&#44; 10<span class="elsevierStyleSup">&#8722;5</span>&#44; 10<span class="elsevierStyleSup">&#8722;6</span>&#44; 10<span class="elsevierStyleSup">&#8722;7</span> dilutions&#44; in quadruplicate&#46; a&#46; Drops distribution on the RC medium surface&#46; b&#46; Typical colonies of <span class="elsevierStyleItalic">Azospirillum</span> spp&#46; in numbers in accordance with the ten-fold dilutions&#46; Photo credits&#58; Dr&#46; Mariana Puente &#40;a&#41; and Dr&#46; Luciana Di Salvo &#40;b&#41;&#46;</p>"
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          "autoresLista" => "Luciana P&#46; Di Salvo, Julia E&#46; Garc&#237;a, Mariana L&#46; Puente, Josefina Amigo, Anal&#237;a Anr&#237;quez, Claudia Barlocco, Silvia Benintende, Tatiana Bochatay, Marta Bortolato, Fabricio Cass&#225;n, Carolina Casta&#241;o, Melina Catafesta, Anah&#237; Coniglio, Marisa D&#237;az, Liliana R&#46; Gali&#225;n, Eugenia Gallace, Patricia Garc&#237;a, In&#233;s E&#46; Garc&#237;a de Salamone, Marianela Landa, Germ&#225;n Liernur, Mar&#237;a Laura Maneiro, Rosana Massa, Julieta Malinverni, Nicol&#225;s Marchessi, Emilia Monteleone, Silvina Oviedo, Lucrecia Pobliti, Gabriela Portela, D&#233;bora Radovancich, Silvia Righes, Rosina Rocha, Enrique Rodr&#237;guez C&#225;ceres, Alejandro Rossi, Gisela Santella, Mar&#237;a Laura Tortora, Nora Trejo, Jos&#233; A&#46; Valenzuela, Daniela Vallejo"
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          "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall"><ul class="elsevierStyleList" id="lis0005"><li class="elsevierStyleListItem" id="lsti0005"><span class="elsevierStyleLabel">&#8226;</span><p id="par0005" class="elsevierStylePara elsevierViewall">The drop plate method was validated by 55 participants in 3 independent trials&#46;</p></li><li class="elsevierStyleListItem" id="lsti0010"><span class="elsevierStyleLabel">&#8226;</span><p id="par0010" class="elsevierStylePara elsevierViewall">Reliability&#44; minimal heterogeneity among results&#44; and economic benefits were met&#46;</p></li><li class="elsevierStyleListItem" id="lsti0015"><span class="elsevierStyleLabel">&#8226;</span><p id="par0015" class="elsevierStylePara elsevierViewall">Both methods showed similar number of <span class="elsevierStyleItalic">Azospirillum</span> viable cells&#46;</p></li><li class="elsevierStyleListItem" id="lsti0020"><span class="elsevierStyleLabel">&#8226;</span><p id="par0020" class="elsevierStylePara elsevierViewall">Drop plate method will be included in the REDCAI consensus protocol for <span class="elsevierStyleItalic">Azospirillum</span> spp&#46;</p></li></ul></p></span>"
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    "titulo" => "Detection by multiplex PCR of <span class="elsevierStyleItalic">Mycoplasma</span> species associated with dairy cattle in Argentina"
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        "titulo" => "Detecci&#243;n por PCR multiplex de especies de <span class="elsevierStyleItalic">Mycoplasma</span> asociadas a ganado lechero en Argentina"
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    "textoCompleto" => "<span class="elsevierStyleSections"><p id="par0020" class="elsevierStylePara elsevierViewall">Mycoplasmas are common inhabitants of the respiratory mucosa&#44; the urogenital tract&#44; conjunctival surfaces&#44; the gastrointestinal tract and the mammary gland&#59; they can be commensals&#44; opportunistic pathogens or primary pathogens associated with respiratory disease&#44; otitis&#44; mastitis&#44; arthritis&#44; reproductive disease&#44; meningitis and conjunctivitis in cattle <a class="elsevierStyleCrossRefs" href="#bib0110"><span class="elsevierStyleSup">5&#44;11&#44;13</span></a>&#46; Some <span class="elsevierStyleItalic">Mycoplasma</span> species are highly contagious&#44; spread rapidly within a herd&#44; and respond poorly to antibiotic therapy&#44; which often determines the need of segregating or culling affected animals<a class="elsevierStyleCrossRef" href="#bib0150"><span class="elsevierStyleSup">13</span></a>&#46; Hence&#44; a rapid and accurate diagnosis is of outmost importance both for the control and for prevention of disease outbreaks&#46; Early studies using biochemical methods detected the presence of <span class="elsevierStyleItalic">M&#46;</span><span class="elsevierStyleItalic">bovis</span> associated with mastitis in dairy cattle in Argentina<a class="elsevierStyleCrossRef" href="#bib0100"><span class="elsevierStyleSup">3</span></a>&#46; In the last few years&#44; <span class="elsevierStyleItalic">Mycoplasma</span> infections in cattle have received increasing attention&#44; being the organisms identified either by biochemical and molecular methods<a class="elsevierStyleCrossRefs" href="#bib0145"><span class="elsevierStyleSup">12&#44;14&#44;15</span></a> or by immunohistochemistry<a class="elsevierStyleCrossRef" href="#bib0135"><span class="elsevierStyleSup">10</span></a>&#46;</p><p id="par0025" class="elsevierStylePara elsevierViewall">Several conventional PCR methods have been developed in different countries to identify <span class="elsevierStyleItalic">Mycoplasma</span> species either from isolates grown through classical culture or directly from milk samples<a class="elsevierStyleCrossRef" href="#bib0150"><span class="elsevierStyleSup">13</span></a>&#46; However&#44; so far only few <span class="elsevierStyleItalic">Mycoplasma</span> species have been isolated from dairy cattle in Argentina and there is scarce information about their frequency and the epidemiological and clinical features associated with their presence in local dairy herds&#46; Hence&#44; there is a need to generate methods for rapid identification at the species level to be used as a diagnostic tool both during outbreaks and epidemiological surveys&#46; We have recently reported the isolation of <span class="elsevierStyleItalic">M&#46;</span><span class="elsevierStyleItalic">leachii</span> associated with arthritis in dairy calves and the development of a multiplex PCR for detecting both this organism and <span class="elsevierStyleItalic">M&#46;</span><span class="elsevierStyleItalic">californicum</span><a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">12</span></a>&#46; Since other species have been reported to be associated with diseases in dairy cattle in Argentina<a class="elsevierStyleCrossRefs" href="#bib0135"><span class="elsevierStyleSup">10&#44;14&#44;15</span></a>&#44; the objectives of this study were to develop a multiplex PCR for identifying other <span class="elsevierStyleItalic">Mycoplasma</span> species &#40;<span class="elsevierStyleItalic">M&#46; bovis</span> and <span class="elsevierStyleItalic">M&#46;</span><span class="elsevierStyleItalic">canadense</span>&#41; and to describe the frequency of <span class="elsevierStyleItalic">Mycoplasma</span> species isolated from clinical samples submitted to a diagnostic laboratory&#46;</p><p id="par0030" class="elsevierStylePara elsevierViewall">A total of 1548 samples were submitted to the Microbiology Laboratory of the Rafaela Experiment Station of INTA from January 2009 to July 2016 by veterinarians who suspected the presence of <span class="elsevierStyleItalic">Mycoplasma</span> as etiologic agent of diseases in dairy cattle&#46; One hundred and seven of these samples were from bulk tank milk&#44; 1412 from composite milk from individual cows and 29 from various organs and biological fluids of dairy calves&#46; The samples came from dairy farms located in the central dairy area of Argentina &#40;Santa Fe and C&#243;rdoba provinces&#41; and in the Mar y Sierras dairy area &#40;Buenos Aires province&#41;&#46; In a first stage&#44; biological specimens were processed by the classical methodology using modified Hayflick medium at 37<span class="elsevierStyleHsp" style=""></span>&#176;C in a 10&#37; CO<span class="elsevierStyleInf">2</span> atmosphere incubated for 7&#8211;10 days and Mycoplasmas were presumptively identified on the basis of their colony morphology under a microscope at 15&#8211;25&#215; magnification as previously described<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">12</span></a>&#46; DNA from bacterial growth in culture medium from colonies suspected to be <span class="elsevierStyleItalic">Mycoplasma</span> spp&#46; was extracted using the procedure previously described<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">12</span></a> and subjected to a PCR test to confirm that isolates belonged to this genus<a class="elsevierStyleCrossRef" href="#bib0095"><span class="elsevierStyleSup">2</span></a>&#46; For further identification of <span class="elsevierStyleItalic">M&#46; leachii</span> and <span class="elsevierStyleItalic">M&#46; californicum</span> a multiplex PCR to amplify specific DNA fragments from the conserved spacer region between the 16S and 23S ribosomal RNA gene was carried out as previously described<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">12</span></a>&#46;</p><p id="par0035" class="elsevierStylePara elsevierViewall">For designing a multiplex PCR for amplifying specific DNA fragments of <span class="elsevierStyleItalic">M&#46; bovis</span> and <span class="elsevierStyleItalic">M&#46; canadense</span> two steps were followed&#46; First&#44; the conserved spacer regions between the 16S and 23S ribosomal RNA gene<a class="elsevierStyleCrossRef" href="#bib0090"><span class="elsevierStyleSup">1</span></a> of <span class="elsevierStyleItalic">M&#46; bovis</span> ATCC 25025 and of six unidentified isolates previously characterized as belonging to genus <span class="elsevierStyleItalic">Mycoplasma</span> by a genus-specific PCR<a class="elsevierStyleCrossRef" href="#bib0095"><span class="elsevierStyleSup">2</span></a> were amplified and sequenced &#40;ABI3130xl&#44; Applied Biosystems&#41;&#46; The sequences obtained were aligned against the NCBI database using BLASTN and then compared with the reported hits in GenBank using the ClustalX multiple sequence alignment program&#46; The 16S-23S rRNA gene sequence obtained from the unidentified strain showed 99&#37; identity with <span class="elsevierStyleItalic">M&#46; canadense</span> strain 275C &#40;DQ847417&#41; while the other was confirmed as <span class="elsevierStyleItalic">M&#46; bovis</span>&#46; The analysis of the 16S-23S rRNA region allowed the design of species-specific primers&#44; but with very similar amplicon size&#46; Then&#44; specific primers only for <span class="elsevierStyleItalic">M&#46; canadense</span> were designed using FastPCR &#40;<a href="https://primerdigital.com/tools/pcr.html">https&#58;&#47;&#47;primerdigital&#46;com&#47;tools&#47;pcr&#46;html</a>&#41; for the 16S-23S rRNA region&#46; A multiplex-reaction was set up using those specific primers for <span class="elsevierStyleItalic">M&#46; canadense</span> while primers for <span class="elsevierStyleItalic">M&#46; bovis</span> were those previously described to amplify a conserved segment of the <span class="elsevierStyleItalic">uvrC</span> gene suitable for specific diagnosis<a class="elsevierStyleCrossRef" href="#bib0170"><span class="elsevierStyleSup">17</span></a>&#46; Primers for <span class="elsevierStyleItalic">M&#46; bovis</span> were UVRF &#40;5&#8242;-TTACGCAAGAGAATGCTTCA-3&#8242;&#41; and UVRR &#40;5&#8242;-TAGGAAAGCACCCTATTGAT-3&#8242;&#41; and for <span class="elsevierStyleItalic">M&#46; canadense</span> were CANDF &#40;5&#8242;-GCGGAACATTAGTTAGTTGGTA-3&#8242;&#41; and CANDR &#40;5&#8242;-CGTTAGCTGCGTCAGTGAATT-3&#8242;&#41;&#46; Expected DNA amplification fragments were 1626<span class="elsevierStyleHsp" style=""></span>bp for <span class="elsevierStyleItalic">M&#46; bovis</span> and 623<span class="elsevierStyleHsp" style=""></span>bp for <span class="elsevierStyleItalic">M&#46; canadense</span>&#44; respectively&#46; The amplification program was&#58; 94<span class="elsevierStyleHsp" style=""></span>&#176;C 2<span class="elsevierStyleHsp" style=""></span>min&#44; 30 cycles of 94<span class="elsevierStyleHsp" style=""></span>&#176;C 30<span class="elsevierStyleHsp" style=""></span>s&#44; 54<span class="elsevierStyleHsp" style=""></span>&#176;C 30<span class="elsevierStyleHsp" style=""></span>s&#44; 72<span class="elsevierStyleHsp" style=""></span>&#176;C 60<span class="elsevierStyleHsp" style=""></span>s&#44; 72<span class="elsevierStyleHsp" style=""></span>&#176;C 5<span class="elsevierStyleHsp" style=""></span>min&#46; Primers designed for amplifying specific fragments of the 16S-23S rRNA region for differentiation of <span class="elsevierStyleItalic">M&#46; canadense</span> as well as those previously described<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">12</span></a> for <span class="elsevierStyleItalic">M&#46; californicum</span> and <span class="elsevierStyleItalic">M&#46; leachii</span> were tested for specificity using DNA from the other <span class="elsevierStyleItalic">Mycoplasma</span> species included in this study&#46; The individual 25<span class="elsevierStyleHsp" style=""></span>&#956;l reaction mixture consisted of&#58; 2&#46;5<span class="elsevierStyleHsp" style=""></span>&#956;l of 10&#215; enzyme buffer&#44; 2<span class="elsevierStyleHsp" style=""></span>mM MgCl<span class="elsevierStyleInf">2</span>&#44; 0&#46;2<span class="elsevierStyleHsp" style=""></span>mM of each dNTP&#44; 0&#46;03<span class="elsevierStyleHsp" style=""></span>&#956;M of each primer&#44; 1&#46;25 U of <span class="elsevierStyleItalic">Taq</span> DNA polymerase &#40;Productos Bio-L&#243;gicos&#174;&#41; and 50<span class="elsevierStyleHsp" style=""></span>ng of genomic DNA&#46; Cycling consisted of an initial denaturation step at 94<span class="elsevierStyleHsp" style=""></span>&#176;C for 2<span class="elsevierStyleHsp" style=""></span>min&#44; and then 30 cycles of the following sequence&#58; 94<span class="elsevierStyleHsp" style=""></span>&#176;C for 30<span class="elsevierStyleHsp" style=""></span>s&#44; 54<span class="elsevierStyleHsp" style=""></span>&#176;C for 30<span class="elsevierStyleHsp" style=""></span>s and 72<span class="elsevierStyleHsp" style=""></span>&#176;C for 1<span class="elsevierStyleHsp" style=""></span>min&#44; with a final extension at 72<span class="elsevierStyleHsp" style=""></span>&#176;C for 5<span class="elsevierStyleHsp" style=""></span>min&#46; In all reactions a negative control using the same previously described mixture&#44; but replacing the DNA by sterile distilled water&#44; was used&#46; The DNA products obtained were visualized in 1&#46;5&#37; agarose gels&#46; The gels were prepared in 1&#215; TAE buffer&#44; adding GelRed TM 10<span class="elsevierStyleHsp" style=""></span>000&#215; to a final concentration of 1&#215;&#44; the electrophoretic runs were performed at 90<span class="elsevierStyleHsp" style=""></span>V&#46; A 100<span class="elsevierStyleHsp" style=""></span>bp ladder step marker was used&#46; DNA fragments resolved in the gel were visualized in a UV light transilluminator &#40;255<span class="elsevierStyleHsp" style=""></span>nm&#41;&#46;</p><p id="par0040" class="elsevierStylePara elsevierViewall">Of the total samples processed&#44; 38 isolates were phenotypically identified as <span class="elsevierStyleItalic">Mycoplasma</span> spp&#46; This preliminary identification was confirmed by the genus-specific PCR technique&#46; Identification of the isolates at species level was achieved using both multiplex PCR described&#46; <a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a> summarizes the frequency of isolated <span class="elsevierStyleItalic">Mycoplasma</span> species and their origin&#46;</p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia><p id="par0045" class="elsevierStylePara elsevierViewall">Since convenience samples were used&#44; the results of this study are not indicative of the prevalence of the different isolated species&#46; The low number of isolates in relation to the total of processed samples indicates that the presence of this genus detected by classical culture is not a frequent finding&#46; No systematic studies have been performed in Argentina to determine prevalence of the different <span class="elsevierStyleItalic">Mycoplasma</span> species associated with defined syndromes in dairy cattle&#59; rather&#44; disease outbreaks and clinical cases have been reported so far&#46; Three isolates &#40;7&#46;9&#37;&#41; were identified as <span class="elsevierStyleItalic">M&#46; bovis&#46;</span> Within the <span class="elsevierStyleItalic">Mycoplasma</span> genus&#44; <span class="elsevierStyleItalic">M&#46; bovis</span> is currently considered the most frequently isolated species associated with disease in cattle worldwide<a class="elsevierStyleCrossRef" href="#bib0140"><span class="elsevierStyleSup">11</span></a>&#46; Since the first report causing mastitis in a dairy herd&#44; <span class="elsevierStyleItalic">M&#46; bovis</span> has been isolated from cases of respiratory disease&#44; mastitis and arthritis in cattle&#44; being considered an emergent pathogen in several countries<a class="elsevierStyleCrossRefs" href="#bib0110"><span class="elsevierStyleSup">5&#44;11</span></a>&#46; In Argentina its presence was first reported in a mastitis outbreak in a dairy farm located in Buenos Aires province where 11&#46;8&#37; of cows presented clinical mastitis that in various cases affected the four mammary quarters&#44; caused a marked decrease in milk production and did not respond to antibiotic therapy&#46; The organism was isolated from composite milk samples from cows with clinical and subclinical mastitis&#44; bulk tank milk and a preputial swab from a bull by classical culture and characterized as <span class="elsevierStyleItalic">M&#46; bovis</span> by biochemical tests and indirect immunofluorescence of the isolated colonies<a class="elsevierStyleCrossRef" href="#bib0100"><span class="elsevierStyleSup">3</span></a>&#46; More recently&#44; <span class="elsevierStyleItalic">M&#46; bovis</span> was detected in milk samples both from cows with clinical and subclinical mastitis and bulk tank milk from dairy herds from Buenos Aires and C&#243;rdoba provinces and characterized by amplifying a fragment of the <span class="elsevierStyleItalic">uvrC</span> gene<a class="elsevierStyleCrossRef" href="#bib0160"><span class="elsevierStyleSup">15</span></a>&#59; however&#44; no further information was provided about the cases from which the processed samples came&#46; In addition&#44; <span class="elsevierStyleItalic">M&#46; bovis</span> was identified by immunohistochemistry in cases of bronchopneumonia unresponsive to antibiotic treatment both in beef and dairy calves from C&#243;rdoba and Santa Fe provinces<a class="elsevierStyleCrossRef" href="#bib0135"><span class="elsevierStyleSup">10</span></a>&#46; These results&#44; together with those obtained in the present study confirm the importance of <span class="elsevierStyleItalic">M&#46; bovis</span> as a causative agent of mastitis&#44; bronchopneumonia and arthritis as has been pointed out in different countries<a class="elsevierStyleCrossRefs" href="#bib0110"><span class="elsevierStyleSup">5&#44;11</span></a> and indicate the need to include diagnostic methodology in routine laboratories to identify this pathogen&#46;</p><p id="par0050" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">M&#46; canadense</span> was isolated from a single case of intramammary &#40;IMI&#41; infection from a dairy herd in Buenos Aires province&#46; Diseases and clinical cases associated with this organism are not as well characterized as those associated with <span class="elsevierStyleItalic">M&#46; bovis</span>&#46; This species has been reported to cause not only IMI<a class="elsevierStyleCrossRef" href="#bib0110"><span class="elsevierStyleSup">5</span></a>&#44; but also to be associated with granulopapular vulvovaginitis in dairy cattle in single or mixed infections with <span class="elsevierStyleItalic">Mycoplasma</span><span class="elsevierStyleItalic">bovigenitalium</span><a class="elsevierStyleCrossRef" href="#bib0125"><span class="elsevierStyleSup">8</span></a> and with arthritis in a calf<a class="elsevierStyleCrossRef" href="#bib0130"><span class="elsevierStyleSup">9</span></a>&#46; In Argentina&#44; this species was isolated from two bulk tank milk samples from a dairy farm located in Buenos Aires province<a class="elsevierStyleCrossRef" href="#bib0160"><span class="elsevierStyleSup">15</span></a>&#46;</p><p id="par0055" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">M&#46; californicum</span> is considered to be the second most frequently isolated <span class="elsevierStyleItalic">Mycoplasma</span> species from bovine mastitis cases in USA after <span class="elsevierStyleItalic">M&#46;</span><span class="elsevierStyleItalic">bovis</span><a class="elsevierStyleCrossRef" href="#bib0110"><span class="elsevierStyleSup">5</span></a>&#44; sharing with the latter similar characteristics regarding both the clinical mastitis features and the importance of the presence of asymptomatic carriers in the dissemination of the organism among cows and dairy herds<a class="elsevierStyleCrossRef" href="#bib0115"><span class="elsevierStyleSup">6</span></a>&#46; In the present study&#44; this organism was isolated from 4 cows during an outbreak of mastitis refractory to antibiotic therapy&#44; as well as from two bulk tank milk samples from the same dairy farm located in C&#243;rdoba province&#46; In Argentina&#44; the isolation of <span class="elsevierStyleItalic">M&#46; californicum</span> was reported from bulk tank milk samples<a class="elsevierStyleCrossRef" href="#bib0160"><span class="elsevierStyleSup">15</span></a> and clinical mastitis samples from the same dairy herd<a class="elsevierStyleCrossRef" href="#bib0155"><span class="elsevierStyleSup">14</span></a> located in Buenos Aires province&#46;</p><p id="par0060" class="elsevierStylePara elsevierViewall">Twenty eight isolates &#40;73&#46;7&#37;&#41; were characterized as <span class="elsevierStyleItalic">M&#46; leachii&#46;</span> These isolates belonged to a single dairy farm where cases of polyarthritis in calves were previously detected<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">12</span></a>&#46; In this farm&#44; milk samples were taken from clinical mastitis cases and also from individual cows &#40;composite milk samples&#41; as part of a control program based on the detection of asymptomatic carriers&#46; In addition&#44; bulk tank milk samples were taken for monitoring the presence of the organism in the lactating dairy herd as the control program progressed&#46; <span class="elsevierStyleItalic">M&#46; leachii</span>&#44; previously referred to as <span class="elsevierStyleItalic">Mycoplasma</span> sp&#46; bovine Group 7<a class="elsevierStyleCrossRef" href="#bib0165"><span class="elsevierStyleSup">16</span></a>&#44; was early recognized as a cause of polyarthritis&#44; mastitis and abortion in dairy cattle in Australia<a class="elsevierStyleCrossRef" href="#bib0120"><span class="elsevierStyleSup">7</span></a> and recent studies characterized clinical signs and histopathological lesions induced by experimental IMI with this pathogen<a class="elsevierStyleCrossRef" href="#bib0105"><span class="elsevierStyleSup">4</span></a>&#46; The present study&#44; as well as our previous report<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">12</span></a> confirms the ability of this organism to cause diverse clinical syndromes&#46;</p><p id="par0065" class="elsevierStylePara elsevierViewall">In conclusion&#44; the multiplex PCR technique for detecting <span class="elsevierStyleItalic">M&#46; bovis</span> and <span class="elsevierStyleItalic">M&#46; canadense</span> described in this study&#44; along with the one previously described for detecting <span class="elsevierStyleItalic">M&#46; californicum</span> and <span class="elsevierStyleItalic">M&#46; leachii</span>&#44; provide a useful diagnostic tool for characterizing some of the <span class="elsevierStyleItalic">Mycoplasma</span> species that cause disease in cattle in Argentina&#46; The results of this study confirm the presence of several species of <span class="elsevierStyleItalic">Mycoplasma</span> associated with different disease syndromes in dairy cattle&#46; The techniques used in the present study can be useful to broaden knowledge about <span class="elsevierStyleItalic">Mycoplasma</span> infections in cattle&#44; since the search for these organisms is not usually included in routine diagnoses&#46;</p><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0030">Conflict of interest</span><p id="par0070" class="elsevierStylePara elsevierViewall">The authors declare that they have no conflicts of interest&#46;</p></span></span>"
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          "palabras" => array:3 [
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            1 => "<span class="elsevierStyleItalic">Mycoplasma</span> spp&#46;"
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    "highlights" => array:2 [
      "titulo" => "Highlights"
      "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall"><ul class="elsevierStyleList" id="lis0005"><li class="elsevierStyleListItem" id="lsti0005"><span class="elsevierStyleLabel">&#8226;</span><p id="par0005" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Mycoplasma</span> species are not routinely diagnosed in dairy cattle in Argentina&#46;</p></li><li class="elsevierStyleListItem" id="lsti0010"><span class="elsevierStyleLabel">&#8226;</span><p id="par0010" class="elsevierStylePara elsevierViewall">Two multiplex PCR allowed identification of <span class="elsevierStyleItalic">Mycoplasma</span> species of interest&#46;</p></li><li class="elsevierStyleListItem" id="lsti0015"><span class="elsevierStyleLabel">&#8226;</span><p id="par0015" class="elsevierStylePara elsevierViewall">These methods can contribute to increase knowledge of <span class="elsevierStyleItalic">Mycoplasma</span> infections in cattle&#46;</p></li></ul></p></span>"
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        "resumen" => "<span id="abst0010" class="elsevierStyleSection elsevierViewall"><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">There is scarce information about the frequency and epidemiological and clinical features associated with the presence of <span class="elsevierStyleItalic">Mycoplasma</span> spp&#46; in Argentine dairy herds&#46; The objectives of this study were to develop a multiplex PCR for identifying <span class="elsevierStyleItalic">M&#46;</span><span class="elsevierStyleItalic">bovis</span> and <span class="elsevierStyleItalic">M&#46;</span><span class="elsevierStyleItalic">canadense</span> and to describe the frequency of <span class="elsevierStyleItalic">Mycoplasma</span> spp&#46; isolated from clinical samples submitted to a diagnostic laboratory&#46; Of a total of 1548 samples from intramammary infections&#44; bulk tank milk and biological fluids&#44; 38 <span class="elsevierStyleItalic">Mycoplasma</span> isolates were obtained&#46; <span class="elsevierStyleItalic">M&#46; bovis</span>&#44; <span class="elsevierStyleItalic">M&#46; canadense</span>&#44; <span class="elsevierStyleItalic">M&#46;</span><span class="elsevierStyleItalic">californicum</span> and <span class="elsevierStyleItalic">M&#46;</span><span class="elsevierStyleItalic">leachii</span> were detected by using two multiplex PCRs&#44; confirming their presence in clinical conditions in dairy cattle&#46; The techniques used in the present study can be useful to broaden the knowledge about <span class="elsevierStyleItalic">Mycoplasma</span> infections in cattle&#44; since the search for these organisms is not usually included in routine diagnoses&#46;</p></span>"
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        "resumen" => "<span id="abst0015" class="elsevierStyleSection elsevierViewall"><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Existe poca informaci&#243;n sobre la frecuencia&#44; as&#237; como las caracter&#237;sticas epidemiol&#243;gicas y cl&#237;nicas asociadas con la presencia de <span class="elsevierStyleItalic">Mycoplasma</span> en los rodeos lecheros argentinos&#46; Los objetivos de este estudio fueron desarrollar una PCR multiplex para identificar <span class="elsevierStyleItalic">M&#46;</span><span class="elsevierStyleItalic">bovis</span> y <span class="elsevierStyleItalic">M&#46;</span><span class="elsevierStyleItalic">canadense</span> y describir la frecuencia de especies de <span class="elsevierStyleItalic">Mycoplasma</span> aisladas de muestras cl&#237;nicas enviadas a un laboratorio de diagn&#243;stico&#46; De un total de 1&#46;548 muestras de infecciones intramamarias&#44; leche de tanque de fr&#237;o y fluidos biol&#243;gicos&#44; se obtuvieron 38 aislamientos de <span class="elsevierStyleItalic">Mycoplasma</span>&#46; Mediante 2 PCR multiplex se detectaron <span class="elsevierStyleItalic">M&#46; bovis</span>&#44; <span class="elsevierStyleItalic">M&#46; canadense</span>&#44; <span class="elsevierStyleItalic">M&#46;</span><span class="elsevierStyleItalic">californicum</span> y <span class="elsevierStyleItalic">M&#46;</span><span class="elsevierStyleItalic">leachii&#44;</span> confirmando su presencia en s&#237;ndromes cl&#237;nicos en ganado lechero&#46; Las t&#233;cnicas utilizadas en el presente estudio pueden ser &#250;tiles para ampliar el conocimiento sobre las infecciones por <span class="elsevierStyleItalic">Mycoplasma</span> en bovinos&#44; ya que la b&#250;squeda de estos organismos no suele incluirse en los diagn&#243;sticos de rutina&#46;</p></span>"
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                  <table border="0" frame="\n
                  \t\t\t\t\tvoid\n
                  \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
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                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black"><span class="elsevierStyleItalic">Mycoplasma</span> species&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
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                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Number and percentage of total isolates&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Origin &#40;number of isolates&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
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                  \t\t\t\t"><span class="elsevierStyleItalic">M&#46; bovis</span>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">3 &#40;7&#46;9&#37;&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Bulk tank milk &#40;2&#41;&#44; arthritis &#40;1&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t"><span class="elsevierStyleItalic">M&#46; californicum</span>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">6 &#40;15&#46;8&#37;&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Intramammary infection &#40;4&#41;&#44; bulk tank milk &#40;2&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t"><span class="elsevierStyleItalic">M&#46; canadense</span>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t">1 &#40;2&#46;6&#37;&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Intramammary infection &#40;1&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t"><span class="elsevierStyleItalic">M&#46; leachii</span>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">28 &#40;73&#46;7&#37;&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Intramammary infection &#40;21&#41;&#44; bulk tank milk &#40;6&#41;&#44; mandibular abscess &#40;1&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t"><span class="elsevierStyleItalic">Total</span>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t">38 &#40;100&#37;&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t  " align="" valign="\n
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Article information

ISSN: 03257541
Original language: English

DOI:10.1016/j.ram.2021.07.001

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Year/Month	Html	Pdf	Total

2024 October	14	2	16
2024 September	47	3	50
2024 August	38	1	39
2024 July	33	6	39
2024 June	26	0	26
2024 May	20	5	25
2024 April	28	3	31
2024 March	38	7	45
2024 February	39	3	42
2024 January	42	5	47
2023 December	44	12	56
2023 November	57	14	71
2023 October	67	11	78
2023 September	37	5	42
2023 August	36	9	45
2023 July	38	7	45
2023 June	57	6	63
2023 May	93	8	101
2023 April	64	8	72
2023 March	29	4	33
2023 February	26	5	31
2023 January	21	3	24
2022 December	17	11	28
2022 November	18	10	28
2022 October	17	9	26
2022 September	23	18	41
2022 August	29	17	46
2022 July	26	16	42
2022 June	46	17	63
2022 May	37	11	48
2022 April	24	11	35
2022 March	19	10	29
2022 February	24	9	33
2022 January	30	4	34
2021 December	54	13	67
2021 November	14	9	23
2021 October	13	16	29
2021 September	12	5	17

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