Clonality Test by PCR - PARR in Real Time of Canine Lymphomas

Background: Lymphoma is a neoplasm of hematopoietic origin that affects canines. The proper establishment of prognosis and rapid institution of treatment are essential for a better quality of life, and immunophenotyping is one of the tools used for this purpose. The objective of this study was to perform a clonality test for immunophenotypic characterization of canine lymphomas using the polymerase chain reaction (PCR) for antigen receptor rearrangements (PARR) technique in real-time from samples fixed in formalin and embedded in paraffin.Materials, Methods & Results: The 23 analyzed samples were fixed in formalin and embedded in paraffin canine lymphoma from the Collection Laboratory of Histopathology of the Animal Pathology Area of the Departament of Veterinary Medicine - Federal Rural University of Pernambuco UFRPE. Samples were processed, their DNA was extracted, quantified, diluted, and standardized at a concentration of 50 ng/µL. After extraction, all samples were subjected to conventional PCR for endogenous control (detection of the IgM target region), in which the extracted DNA was amplified in a final volume of 25 µL. The 128 bp amplified product was detected by 1.5% agarose gel electrophoresis. Of the 23 samples analyzed for the detection of the conserved region referring to the endogenous gene, 91.30% (21/23) amplified the conserved region Cµ by conventional PCR, and two samples 8.70% (2/23) were negative. Endogenous control positive samples were subjected to real-time PCR-PARR for detection of IgH Major (LB), IgH Minor (LB), and TCRγ (LT) target regions. All reactions were performed in duplicate to reduce the risk of false-positive or false-negative results due to technical errors. Samples previously confirmed by immunohistochemistry were used as positive controls for T cell and B cell lymphoma, and MilliQ water was used as a negative control. After amplification, the melting curve gradually increased the temperature by 1C/5 s to 95C during continuous fluorescence monitoring. Of the 21 samples analyzed, 100.00% (21/21) demonstrated clonal amplification. Of these, 57.15% (12/21) were positive for phenotype B, and 42.85% (9/21) were positive for phenotype T.Discussion: Due to the importance of researching and confirming samples from files fixed and embedded in paraffin samples in laboratories, PCR-PARR is a good tool for this purpose. In the present study, real-time PCR analysis demonstrated greater sensitivity in the characterization of the immunophenotype of lymphomas from old samples fixed in formalin and embedded in paraffin. The temperature of melting curve analysismay vary depending on the amount of DNA and its quality. In the present study, it was found that the average melting temperature in the samples varied between ± 3C when compared to that in the control sample for LB and LT, 83.5C and 80C, respectively: in the literature, there is a relative difference in this temperature, which may vary up to 4C. Real-time PCR-PARR was satisfactory in the characterization of the immunophenotype of canine lymphomas from formalin-fixed and paraffin-embedded samples; therefore, its use is recommended for both retrospective studies. The use of PCR-PARR associated with histopathological and/or cytopathological examination in cases of canine lymphomas strongly helps pathologists, provide a safe establishment of the immunophenotype, minimize errors, and optimize the diagnosis, thus directly contributing to the establishment of the prognosis.Keywords: immunophenotyping, lymphoproliferative disease, real-time PCR, TCRγ, IgH

Bovine Mastitis Caused by Multidrug-Resistant Nocardia farcinica

Background: Mastitis caused by Nocardia is characterized by pyogranulomatous inflammation related to inadequate hygiene conditions and is difficult to treat. Prompted by the absence of documentation of Nocardia farcinica associated to bovine mastitis in the Northeast region of Brazil, this is the first report to describe bovine mastitis caused by multidrug-resistant N. farcinica. Case: Four milk samples (one from each teat) obtained from a 3-year-old Jersey cow raised on a property located in the metropolitan region of Recife, Pernambuco state, Brazil, were submitted to the Laboratory of Infectious-Contagious Diseases of the Veterinary Hospital at Universidade Federal Rural de Pernambuco. At the laboratory, samples were cultured in base agar enriched with 7% sheep blood (blood agar) in a microbiological incubator at 37°C under aerobic conditions for 72 h. After only 48 h, however, pure bacterial colony growth was observed in all samples. Macroscopic analysis revealed small colonies, with an irregular shape, dry aspect, and greyish in color. Gram-positive rods forming filaments and/or ramifications were observed using a Gram staining method. Nocardia spp. were identified according to morphotinctorial characteristics. Susceptibility testing using the disc-diffusion method in agar (antibiogram) was performed using the following antibiotics: penicillin (10 IU), tetracycline (30 µg), amoxicillin (10 µg), gentamicin (10 µg), cephalexin (30 µg), erythromycin (15 µg), cephalothin (30 µg) and ampicillin (30 µg). However, the organism exhibited resistance to all drugs; as such, a new milk sample was obtained at the same location the initial samples were collected. Samples (approximately 5 mL) were collected aseptically and separately from all four teats in sterile bottles, during which the presence of granular material was noted. Bacterial culture was performed as previously described and, after 48 h, colony growth with the same characteristics as the first isolation were observed, and with same morphotinctorial characteristics in the Gram stain. A resistance profile was observed for 14 of the antimicrobial drugs tested; sensitivity was verified only for ciprofloxacin and amoxicillin with clavulanic acid. One bacterial colony was selected and sent to the Center of Strategic Technology of Northeast (CETENE-PE) for species identification using a matrix-associated laser desorption ionization-time of flight (MALDI-TOF/MS) technique, which confirmed the species to be N. farcinica. Molecular characterization of 16s ribosomal DNA was performed using polymerase chain reaction with universal prokaryotic primers 516F-13R. Subsequently, the amplified product was subjected to sequencing, and the result was analyzed for quality using Phred base calling software; bases with a Phred value > 20 were kept. The sequence was evaluated using GenBank, in which the isolate exhibited 99% similarity to N. farcinica. Discussion: Clinical findings and animal history associated with microbiological culture and bacterial identification using the MALDI-TOF technique, as well as DNA sequencing, confirmed the case of clinical mastitis to be caused by N. farcinica. These bacteria are considered saprophytes, and their occurrence is associated with deficiencies in hygienic-sanitary management, such as not using pre- and post-dipping, which may favor mammary gland infection. Treatment of N. farcinica mastitis is effective only when properly performed, with agent identification and antibiotic sensitivity tests in vitro associated with the adoption of hygienic-sanitary measures. This is the first description of bovine mastitis caused by N. farcinica in the northeast of Brazil. Multidrug resistance should raise awareness of producers searching for laboratory aids in agent identification as well as antibiotic sensitivity tests, and to develop a proper therapeutic protocol based on results obtained in laboratory examinations

Occurrence of Apicomplexa protozoa in wild birds in the Northeast region of Brazil

Abstract Protozoa of the Apicomplexa phylum are worldwide distributed with capacity to infect endothermic animals. The study of these protozoa in wild birds in Brazil is scarce. This study aimed to evaluate the occurrence of apicomplexan protozoa in wild birds in the Northeast of Brazil. From October to December 2019, brain tissue samples were collected from 71 captive birds from the Wild Animal Screening Center of the Pernambuco State (CETRAS-Tangara) and 25 free-living birds from the Caatinga biome in Rio Grande do Norte, totaling 96 animals (41 species). Brain fragments were subjected to molecular diagnosis by nested PCR for the 18s rDNA gene of Apicomplexa parasites, followed by DNA sequencing. This gene was detected in 25% (24/96) of the samples, and it was possible to perform DNA sequencing of 14 samples, confirming three genera: Isospora, Sarcocystis and Toxoplasma from eight bird species (Amazona aestiva, Coereba flaveola, Egretta thula, Paroaria dominicana, Sporophila nigricollis, Cariama cristata, Columbina talpacoti, Crypturellus parvirostris). The occurrence these coccidia in wild birds provides important epidemiological information for the adoption of preventive measures for its conservation. Future studies are needed to better understand the consequence of Apicomplexa infection in birds in Caatinga and Atlantic Forest biomes

Detecção de Brucella sp. em leite informal comercializado no estado de Alagoas, Brasil: um breve alerta à saúde pública

A produção de leite constitui relevante atividade agropecuária no Brasil. O leite informal é o produto vendido diretamente do produtor ao consumidor, sem garantia de ter sido submetido a tratamentos térmicos ou obedecidas as condições mínimas de higiene exigidas para captação, transporte e comercialização. Devido aos riscos iminentes à saúde pública quanto à transmissão de patógenos, a exemplo de Brucella sp., a comercialização do leite in natura (informal) é proibida pela legislação brasileira. Objetivou-se realizar a identificação molecular Brucella sp. em amostras de leite informal comercializado no estado de Alagoas, Brasil. Foram coletadas amostras de leite bovino in natura provenientes de 100 pontos de venda informal, distribuídos em 80 municípios do estado de Alagoas. As amostras foram submetidas à centrifugação, extração de material genômico e reação em cadeia da polimerase. Dos 102 municípios que compõem o Estado de Alagoas, foi identificada a existência do comércio informal de leite em 80 destes (78,4%). Das 100 amostras analisadas de leite informal, 1% apresentou resultado positivo ao gênero Brucella. Conclui-se que, embora a baixa frequência verificada, o comércio informal do leite in natura comercializado em Alagoas pode conferir riscos para transmissão da brucelose à população consumidora do estado

Isolamento e caracterização genética de Toxoplasma gondii de gatos ferais no Arquipélago de Fernando de Noronha, Pernambuco, Brasil

Toxoplasma gondii é um coccídeo intracelular obrigatório formador de cistos teciduais, responsável pela toxoplasmose, zoonose de grande impacto na saúde pública. É capaz de infectar a maioria dos animais homeotérmicos, incluindo humanos. Os felídeos são os únicos hospedeiros definitivos, apresentando grande importância na epidemiologia da toxoplasmose pela capacidade de eliminar oocistos nas fezes, contaminando o ambiente. Este estudo teve como objetivo isolar e genotipar T. gondii em gatos ferais (Felis catus) do Arquipélago de Fernando de Noronha, Estado de Pernambuco, Brasil. Durante o período de um ano, gatos ferais fracos foram capturados pelo Centro de Vigilância Animal do Arquipélago. Após contenção química (quetamina 10% e xilazina 1%) foram coletadas amostras de sangue de 31 felinos ferais de diferentes localidades da Ilha. Esses animais doentes, posteriormente, vieram a óbito e fragmentos de encéfalo, coração, pulmão, diafragma e fígado foram coletados. As amostras de sangue foram destinadas à sorologia por meio da Reação de Imunofluorescência Indireta (RIFI) para pesquisa de anticorpos (IgG) anti-T.gondii e os fragmentos de tecido deos felinos positivos na RIFI foram submetidos ao bioensaio em camundongos para isolamento do protozoário. Anticorpos anti-Toxoplasma gondii foram detectados em 18/31 (58%) felinos. Sete animais tiveram seus tecidos submetidos ao bioensaio, obtendo-se dois isolados de T. gondii não patogênicos para camundongos. A genotipagem foi realizada por meio da PCR-RFLP multilocus, utilizando 10 marcadores genéticos (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 e Apico). Uma cepa atípica de T. gondii (ToxoDB #146) foi identificada, sendo este o primeiro relato deste genótipo em gatos ferais no mundo. Os resultados obtidos neste trabalho contribuem para o estudo da epidemiologia molecular deste agente e permitem concluir que a infecção por T. gondii ocorre na população de felinos do Arquipélago. Medidas de controle baseadas na educação sanitária devem ser reforçadas para prevenir a infecção dos felinos e reduzir as fontes de infecção para outros hospedeiros intermediários, sobretudo para a população humana desta Ilha.Toxoplasma gondii is an obligate intracellular coccidian, tissue cyst forming, which causes toxoplasmosis, zoonotic disease of great impact in public health. T. gondii is able to infect most of warm-blooded animals, including humans. Felids are recognized as the only definitive hosts and are important in toxoplasmosis epidemiology, shedding oocysts in feces, contaminating environment. The purpose of this study was to isolate and genotyping T. gondii in feral cats (Felis catus) from the Fernando de Noronha Archipelago, Pernambuco state, Brazil. During one year sick feral cats were caught by Archipelago Animal Vigilance Center. After chemical restraint (ketamine 10% and xylazine 1%) blood samples of 31 feral cats from different locations on the island were collected. These weak animals posteriorly died and fragments of brain, heart, lung, diaphragm, and liver were collected. Blood samples were intended to serology by Indirect Immunofluorescence Assay (IFA) for IgG antibody detection and tissue fragments were submitted to mouse bioassay for T. gondii isolation. Anti-T.gondii antibodies were detected in 18/31 (58%) felines. Tissues from seven animals were submitted to bioassay, obtaining two T. gondii isolates non-pathogenic for mouse. Genotyping was performed by PCR-RFLP using 10 molecular markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 e Apico), identifying an atypical strain of T. gondii (ToxoDB #146). This is the first report of this genotype in feral cats worldwide. The results obtained contribute to molecular epidemiology studies of this pathogen and show/demonstrate T. gondii infection in feline population of the Archipelago. Control measures based on health education should be reinforced to prevent the cat infection and to reduce infection sources for definitive and intermediate hosts, especially to human population of this island

Caracterização genética e biológica de isolados de Toxoplasma gondii (Nicolle e Manceaux, 1909) de animais e humano na região Nordeste do Brasil

Toxoplasma gondii é um coccídio intracelular obrigatório, formador de cistos teciduais, causador da toxoplasmose, doença zoonótica de grande impacto à Saúde Pública. Foram realizados três estudos, divididos em capítulos que abordam a infecção por T. gondii em humano, animais de produção e silvestre. O primeiro estudo teve como objetivo isolar e caracterizar cepas de T. gondii obtidas de tecidos de animais de produção abatidos para consumo humano no Arquipélago de Fernando de Noronha, Pernambuco. Foram obtidos dois isolados de T. gondii, sendo um de ovino e um de suíno e ambos foram caracterizados como genótipo ToxoDB #146, descrevendo-se o primeiro relato deste genótipo nestas espécies no mundo. Os isolados apresentaram perfil diferente para virulência fenotípica e molecular. O segundo estudo teve como objteivo relatar um caso de toxoplasmose congênita severa envolvendo o genótipo atípico ToxoDB #162, isolado a partir de sangue do cordão umbilical de recém-nascido em Alagoas. O resultado da genotipagem dos genes ROP5 e ROP18 foi compatível com o perfil de elevada virulência do isolado em camundongo. O terceiro estudo teve como objetivo identificar protozoários do subfilo Apicomplexa em tecido cardíaco de 39 saguis (Callithrix jacchus) de Pernambuco por meio do sequenciamento de fragmento do gene 18S rDNA onde foi detectado o DNA de T. gondii em sete animais (7/39 - 17,9%). Os resultados obtidos neste estudo contribuem com importantes informações biológicas e genéticas sobre T. gondii em animais e humano nos estados de Alagoas e Pernambuco.Toxoplasma gondii is an obligate intracellular coccidian, tissue cyst forming, which causes toxoplasmosis, zoonotic disease of great impact in public health. Three studies were conducted and divided into chapters, which address T. gondii infection in human, farm and wild animals. The first study aimed to isolate and characterize T. gondii strains from tissues of farm animals slaughtered for human consumption on Fernando de Noronha Archipelago, Pernambuco. Two T. gondii isolates were obtained, onde from sheep and one from pig, both isolates were characterized as genotype ToxoDB #146. This is the first report of this genotype in pig and sheep worldwide. The isolates showed different phenotypic and molecular profiles. The second study reported a case of severe congenital toxoplasmosis in newborn baby from Alagoas, that involved the atypical genotype ToxoDB #162, isolated from umbilical cord blood. Genotype result of ROP5 and ROP18 genes could predict the high virulence of the isolate in mice. The third study investigated the presence of apicomplexan protozoans in heart tissue from 39 common marmoset (Callithrix jacchus) from Pernambuco by sequencing a fragment of 18S rDNA gene, by which T. gondii DNA was detected in seven animals (7/39 - 17,9%). Results obtained in the present study contribute to biological and genetic information of T. gondii in animals and human from Alagoas and Pernambuco state.Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNP

Clonality Test by PCR - PARR in Real Time of Canine Lymphomas

Background: Lymphoma is a neoplasm of hematopoietic origin that affects canines. The proper establishment of prognosis and rapid institution of treatment are essential for a better quality of life, and immunophenotyping is one of the tools used for this purpose. The objective of this study was to perform a clonality test for immunophenotypic characterization of canine lymphomas using the polymerase chain reaction (PCR) for antigen receptor rearrangements (PARR) technique in real-time from samples fixed in formalin and embedded in paraffin.Materials, Methods & Results: The 23 analyzed samples were fixed in formalin and embedded in paraffin canine lymphoma from the Collection Laboratory of Histopathology of the Animal Pathology Area of the Departament of Veterinary Medicine - Federal Rural University of Pernambuco UFRPE. Samples were processed, their DNA was extracted, quantified, diluted, and standardized at a concentration of 50 ng/µL. After extraction, all samples were subjected to conventional PCR for endogenous control (detection of the IgM target region), in which the extracted DNA was amplified in a final volume of 25 µL. The 128 bp amplified product was detected by 1.5% agarose gel electrophoresis. Of the 23 samples analyzed for the detection of the conserved region referring to the endogenous gene, 91.30% (21/23) amplified the conserved region Cµ by conventional PCR, and two samples 8.70% (2/23) were negative. Endogenous control positive samples were subjected to real-time PCR-PARR for detection of IgH Major (LB), IgH Minor (LB), and TCRγ (LT) target regions. All reactions were performed in duplicate to reduce the risk of false-positive or false-negative results due to technical errors. Samples previously confirmed by immunohistochemistry were used as positive controls for T cell and B cell lymphoma, and MilliQ water was used as a negative control. After amplification, the melting curve gradually increased the temperature by 1C/5 s to 95C during continuous fluorescence monitoring. Of the 21 samples analyzed, 100.00% (21/21) demonstrated clonal amplification. Of these, 57.15% (12/21) were positive for phenotype B, and 42.85% (9/21) were positive for phenotype T.Discussion: Due to the importance of researching and confirming samples from files fixed and embedded in paraffin samples in laboratories, PCR-PARR is a good tool for this purpose. In the present study, real-time PCR analysis demonstrated greater sensitivity in the characterization of the immunophenotype of lymphomas from old samples fixed in formalin and embedded in paraffin. The temperature of melting curve analysismay vary depending on the amount of DNA and its quality. In the present study, it was found that the average melting temperature in the samples varied between ± 3C when compared to that in the control sample for LB and LT, 83.5C and 80C, respectively: in the literature, there is a relative difference in this temperature, which may vary up to 4C. Real-time PCR-PARR was satisfactory in the characterization of the immunophenotype of canine lymphomas from formalin-fixed and paraffin-embedded samples; therefore, its use is recommended for both retrospective studies. The use of PCR-PARR associated with histopathological and/or cytopathological examination in cases of canine lymphomas strongly helps pathologists, provide a safe establishment of the immunophenotype, minimize errors, and optimize the diagnosis, thus directly contributing to the establishment of the prognosis.Keywords: immunophenotyping, lymphoproliferative disease, real-time PCR, TCRγ, IgH

Co-Infection by Sarcoptes scabiei and Microsporum gypseum in Free-Ranging Crab-Eating Fox, Cerdocyon thous (Linnaeus, 1766).

ABSTRACT The aim of the present study was to describe the clinical manifestation, treatment and outcome of a case of co- infection by Sarcoptes scabiei and Microsporum gypseum in Cerdocyon thous (crab-eating fox) from Northeastern Brazil

Detecção de Toxoplasma gondii em órgãos do sistema reprodutivo de carneiros naturalmente infectados no Brasil

Objetivou-se com esse estudo detectar o DNA genômico de T. gondii em amostras de testículo e epidídimo de ovinos comercializados em abatedouros do Estado de Pernambuco Região Nordeste do Brasil. Foram coletadas 50 amostras de soro sanguíneo, 50 amostras de testículos e 50 de epidídimos. Para a triagem dos animais foi utilizada a técnica de Imunofluorescência Indireta (RIFI) e posteriormente empregou-se a Reação em Cadeia da Polimerase (PCR) nos animais positivos na sorologia. Observou-se 24% (12/50) dos animais positivos na RIFI e o DNA genômico foi detectado no epidídimo em 8,3% (1/12) das amostras. A identidade molecular dos produtos amplificados foi confirmada por sequenciamento. Relata-se a primeira ocorrência da presença do DNA de T. gondii em órgãos do sistema reprodutivo de carneiros naturalmente infectados no Brasil