Clonality test by PCR - PARR in real time of canine lymphomas

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. 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 and IgH Minor for B lymphocytes (LB), and TCRy for lymphocytes T (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 reaction control. After amplification, the melting curve gradually increased the temperature by 1o C/5 s to 95o C 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. 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 analysis may 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 ± 3o C when compared to that in the control sample for LB and LT, 83.5o C and 80o C, respectively: in the literature, there is a relative difference in this temperature, which may vary up to 4o C. 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.(AU)

Cross-sectional survey for Toxoplasma gondii infection in humans in Fernando de Noronha island, Brazil / Estudo transversal da infecção por Toxoplasma gondii em humanos da Ilha de Fernando de Noronha, Brasil

Abstract Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is zoonotic disease and is one of the most important foodborne parasitic diseases globally. The prevalence in humans is highly variable, being influenced by cultural habits, socioeconomic, and environmental conditions. The objective of this study was to estimate the prevalence of T. gondii infection in humans on the archipelago of Fernando de Noronha, Pernambuco State, Brazil, and to identify the risk factors associated with this infection. The seroprevalence of immunoglobulin G anti-T. gondii antibodies was 50.4% (172/341, 95% CI: 45.2%-55.7%). Factors associated with the infection were consumption of well water or rainwater (odds ratio [OR]: 2.43, p=0.020) and consumption of game meat (OR: 1.80, p=0.026). This is the first study to provide epidemiological information of T. gondii infection among the residents of the Island of Fernando de Noronha, revealing a considerable antibody seroprevalence in this population. This study provides information for the adoption of prevention and control measures in island environments.

Resumo A toxoplasmose, causada pelo protozoário Toxoplasma gondii, é uma zoonose e uma das doenças parasitárias transmitidas por alimentos mais importantes em todo o mundo. A prevalência em humanos é altamente variável, sendo influenciada por hábitos culturais, condições socioeconômicas e ambientais. O objetivo deste estudo foi estimar a prevalência de infecção por T. gondii em humanos, no arquipélago de Fernando de Noronha, Pernambuco, Brasil, e identificar os fatores de risco associados a essa infecção nesse contexto insular. A soroprevalência de anticorpos IgG anti-T. gondii nos ilhéus foi de 50,4% (172/341, 95% CI: 45,2%-55,7%). Os fatores associados à infecção encontrados foram o consumo de água do poço ou de água da chuva (Odds ratio [OR]: 2,43, p=0,020) e consumo de carne de caça (OR: 1,80, p=0,026). Este é o primeiro estudo a fornecer informações epidemiológicas da infecção por T. gondii entre os moradores da Ilha de Fernando de Noronha, revelando uma considerável soroprevalência de anticorpos nessa população. Este estudo fornece informações para subsidiar a adoção de medidas de prevenção e controle em ambientes insulares.

Methicillin-resistant Staphylococcus epidermidis in handler of cheese made with goat's milk in Brazil / Staphylococcus epidermidis resistente à meticilina em manipulador de queijo elaborado com leite de cabra no Brasil

ABSTRACT: In the Northeastern Brazil, artisanal cheese production is an important local economic activity for small producers. Methicillin-resistant Staphylococcus is responsible for causing infection in animals and humans. This study described the first detection of methicillin-resistant S. epidermidis isolated in the nasal cavity of a handler of coalho cheese made with goat's milk in Northeastern Brazil. This brief communication highlighted the importance of adopting biosafety measures by cheese handlers, in order to reduce possible contamination and the spread of pathogens in the production chain of this type of artisanal cheese in Brazil.

RESUMO: Na região Nordeste do Brasil, a cadeia de produção de queijo artesanal é uma atividade local importante para pequenos produtores. Staphylococcus resistentes à meticilina são responsáveis por causar infecções em animais e seres humanos. Neste estudo descreve-se a primeira detecção de S. epidermidis resistente à meticilina isolado da cavidade nasal de um manipulador de queijo coalho elaborado com leite de cabra no Nordeste do Brasil. Este relato destaca a importância da adoção de medidas de biossegurança por manipuladores de queijo, a fim de reduzir possíveis contaminações e a disseminação de patógenos na cadeia produtiva deste tipo de queijo artesanal no Brasil.

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.

Epidemiology of Mycoplasma agalactiae and Mycoplasma mycoides cluster in flocks of northeastern Brazil / Epidemiologia de Mycoplasma agalactiae e Mycoplasma mycoides cluster em rebanhos do nordeste do Brasil

ABSTRACT: The present study aimed to investigate contagious agalactia (CA) in flocks from Pernambuco State. The study involved 225 goats and 63 ewes; 288 milk samples and 100 vaginal swabs were collected in total. The PCR assays were carried out using specific primers to Mycoplasma agalactiae and the Mycoplasma mycoides cluster. Among the goat's milk samples,12.0% (27/225) were positive for Mycoplasma agalactiae DNA, while 5.3% (12/225) contained the Mycoplasma mycoides cluster. Of the vaginal swabs taken from goats, 15.4% (12/78) were positive for Mycoplasma agalactiae DNA and 3.8% (3/78) contained the Mycoplasma mycoides cluster. In the case of ewes, 4.3% (1/23) of the milk samples contained Mycoplasma agalactiae DNA, and 7.5% (3/40) were positive for the Mycoplasma mycoides cluster. Vaginal swabs taken from sheep´s were negative. Analysis of risk factors for mycoplasmosis, showed that goats and sheep flocks on the extensive breeding system are more likely to have mycoplasmosis than those on the intensive breeding system (odds ratio (OR) 6.2; p=0.004); meat goat and sheep flocks are more likely to have infection compared to dairy flocks (OR 4.8; p=0.011); unclean animal housing increases the chances of infection (OR 5.0; p=0.031) and not performing quarantine increases the chances of mycoplasmosis (OR 4.6; p=0.042). Based on these findings we conclude that CA syndrome in the semiarid region of Pernambuco state can be associated with Mycoplasma agalactiae and Mycoplasma mycoides cluster.

RESUMO: O objetivo deste estudo foi investigar a Agalaxia contagiosa em rebanhos do estado de Pernambuco. Foram examinadas 225 cabras e 63 ovelhas, das quais foram colhidas 288 amostras de leite e 100 suabes vaginais. Foram realizadas reações da PCR com iniciadores específicos para Mycoplasma agalactiae e Mycoplasma mycoides cluster. A frequência total de Mycoplasma agalactiae em amostras de leite caprino foi de 12,0% (27/225) e de 5,3% (12/225) para Mycoplasma mycoides cluster. Dos suabes vaginais de cabras as frequências detectadas na PCR foram de 15,4% (12/78) para Mycoplasma agalactiaee 3,8% (3/78) para Mycoplasma mycoides cluster. Em leite de ovelhas a frequência de Ma foi de 4.3% (1/23) e de 7,5% (3/40) para Mycoplasma mycoides cluster. Na análise dos fatores de risco para micoplasmoses verificou-se que rebanhos de caprinos e ovinos mantidos no sistema extensivo são mais prováveis de adquirir micoplasmose quando comparados com o sistema intensivo (odds ratio (OR) 6,2; p=0,004); rebanhos de caprinos e ovinos de corte são mais prováveis de adquirir micoplamsose do que rebanhos de leite (OR 4,8; p=0,011); não realizar limpeza das instalações aumenta as chances de infecção (OR 5,0; p=0,031); não realizar quarentena aumenta as chances das micoplasmoses estudadas (OR 4,6; p=0,042). Conclui-se que M. agalactiae e Mycoplasma mycoides cluster estão envolvidos na síndrome de CA em rebanhos de caprinos e ovinos do semiárido pernambucano.

Detecção de Toxoplasma gondii em órgãos do sistema reprodutivo de carneiros naturalmente infectados no Brasil / Detection of Toxoplasma gondii in the reproductive organs of rams in Brazil

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.

The aim of the study was to detect genomic DNA of Toxoplasma gondii in testicle and epididymis samples from rams sold in abattoirs in the state of Pernambuco, Northeast Brazil. Fifty (50) blood serum samples were collected, as well as 50 testicle and epididymis samples. Indirect Immunofluorescence (IIF) was used during screening of the rams. The Polymerase Chain Reaction (PCR) was used with animals that were positive in serology. Our results confirmed that 24% (12/50) of the rams were positive in IIF. Genomic DNA was detected in the epididymis at 8.3% (1/12) of the animals. The molecular identity of the amplified products was confirmed through sequencing. This paper reports the first occurrence of T. gondii DNA in the reproductive organs of naturally infected rams in Brazil.