Quality control in veterinary blood banks: evaluation of canine platelet concentrates stored for five days.

BACKGROUND: Platelets undergo structural, biochemical and functional alterations when stored, and platelet storage lesions reduce platelet function and half-life after transfusion. The objective of this study was to evaluate stored canine platelet concentrates with platelet aggregation, flow cytometry and biochemistry assays. Twenty-two bags of canine platelet concentrates were obtained by the platelet-rich plasma method and were assessed on days 1, 3 and 5 after collection. Parameters such as platelet counts, residual leukocytes, platelet swirling, glucose, lactate, pH, CD62P expression (platelet activation), JC-1 (mitochondrial function) and annexin V (apoptosis and cell death) were assessed. RESULTS: Over the five days of storage there was a significant decrease in glucose, HCO3, pCO2, ATP, pH, swirling and mitochondrial function, associated with a significant increase in lactate levels and pO2. At the end of storage pH was 5.9 ± 0.6 and lactate levels were 2.8 ± 1.2 mmol/L. Results of the quality parameters evaluated were similar to those reported in human platelets studies. The deleterious effects of storage were more pronounced in bags with higher platelet counts (> 7.49 × 1010/unit), suggesting that canine platelet concentrates should not contain an excessive number of platelets. CONCLUSIONS: Quality parameters of canine platelets under standard storage conditions were similar to those observed in human platelets. Our results have potential to be used for the routine evaluation and quality control in veterinary blood banks.

Comparison of JC-1 and MitoTracker probes for mitochondrial viability assessment in stored canine platelet concentrates: A flow cytometry study.

Mitochondria perform crucial roles in many biochemical processes, and mitochondrial depolarization is an early sign of platelet apoptosis. The mitochondrial membrane potential is usually evaluated through JC-1 probe, but it can also be assessed with MitoTracker probes. Our aim was to evaluate mitochondrial viability in stored canine platelet concentrates (PCs) with the fluorescent probes JC-1 and MitoTracker. Platelets from 22 canine PCs were stained with JC-1 and MitoTracker probes on days 1, 3, and 5 of storage. Data on metabolic parameters were also collected for correlation studies. Results of JC-1 and MitoTracker revealed a decrease in mitochondrial membrane potential in day 5 of storage compared to days 1 and 3, providing evidence of mitochondrial depolarization, a finding that was confirmed by the data on metabolic parameters. MitoTracker probes also added information regarding platelet swelling. In conclusion, MitoTracker probes offered a more complete mitochondrial analysis in the evaluation of stored canine PCs. © 2018 International Society for Advancement of Cytometry.

Development of a quantitative PCR for the detection of Rangelia vitalii.

The aim of this study was to develop and validate a SYBR Green qPCR assay to detect and quantify a fragment of the 18S rRNA gene of Rangelia vitalii in canine blood. Repeatability of the qPCR was determined by the intra- and inter-assay variations. The qPCR showed efficiency of E=101.30 (r(2)=0.996), detecting as few as one copy of plasmid containing the target DNA. Specificity of the assay was performed using DNA samples of Babesia canis, B. gibsoni, Ehrlichia canis, E. ewingii and Leishmania sp. No cross-reactivity was observed. Field samples consisting of blood from 265 dogs from Porto Alegre, Brazil were also tested. A total of 24 (9.05%) samples were positive for R. vitalii. Amplicons of 50% of positive samples were confirmed to be R. vitalii by Sanger sequencing. The positive samples had an average of 3.5×10(5) organisms/mL of blood (range: 1.27×10(3)-1.88×10(6)) based on the plasmid-generated standard curve. In conclusion, the SYBR Green qPCR assay developed herein is sensitive and specific and can be used as a diagnostic tool for detection and quantification of R. vitalii in canine blood samples.

Hemoplasma prevalence and hematological abnormalities associated with infection in three different cat populations from Southern Brazil.

Three hemoplasma species are recognized in domestic cats: Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum' and 'Candidatus Mycoplasma turicensis'. We report the prevalence and hematological abnormalities of hemoplasma infection in 369 domestic cats from three different populations (blood donors, hospitalized cats and shelter cats) from Southern Brazil. Complete blood counts were performed at the time of blood collection, and DNA was extracted and tested by conventional PCR for each hemoplasma species. A total of 79 samples (21.40%) were positive for at least one species. The most prevalent hemoplasma was 'Candidatus Mycoplasma haemominutum', with 50/369 (13.55%) positive cats, followed by 'Candidatus Mycoplasma turicensis', 10/369 (2.71%), and Mycoplasma haemofelis, 8/369 (2.16%). Mycoplasma haemofelis and 'Candidatus Mycoplasma haemominutum' coinfection was observed in 4/369 (1.08%), whereas 'Candidatus Mycoplasma haemominutum' and 'Candidatus Mycoplasma turicensis' in 5/369 (1.35%). Three cats (0.81%) were infected with all three hemoplasmas. There was no association between infection and the different populations. Anemia was associated with Mycoplasma haemofelis and 'Candidatus Mycoplasma haemominutum', but not with 'Candidatus Mycoplasma turicensis'. Male cats and cats with outdoor access were more likely to be infected. Although 'Candidatus Mycoplasma haemominutum' is believed to cause minimal or no hematological alterations, the infected cats studied herein were more likely to be anemic.

Hemoplasma prevalence and hematological abnormalities associated with infection in three different cat populations from Southern Brazil / Prevalência da infecção por hemoplasmas e alterações hematológicas associadas à infecção em três diferentes populações de gatos do sul do Brasil

Three hemoplasma species are recognized in domestic cats: Mycoplasma haemofelis, ‘Candidatus Mycoplasma haemominutum’ and ‘Candidatus Mycoplasma turicensis’. We report the prevalence and hematological abnormalities of hemoplasma infection in 369 domestic cats from three different populations (blood donors, hospitalized cats and shelter cats) from Southern Brazil. Complete blood counts were performed at the time of blood collection, and DNA was extracted and tested by conventional PCR for each hemoplasma species. A total of 79 samples (21.40%) were positive for at least one species. The most prevalent hemoplasma was ‘Candidatus Mycoplasma haemominutum’, with 50/369 (13.55%) positive cats, followed by ‘Candidatus Mycoplasma turicensis’, 10/369 (2.71%), and Mycoplasma haemofelis, 8/369 (2.16%). Mycoplasma haemofelis and ‘Candidatus Mycoplasma haemominutum’ coinfection was observed in 4/369 (1.08%), whereas ‘Candidatus Mycoplasma haemominutum’ and ‘Candidatus Mycoplasma turicensis’ in 5/369 (1.35%). Three cats (0.81%) were infected with all three hemoplasmas. There was no association between infection and the different populations. Anemia was associated with Mycoplasma haemofelis and ‘Candidatus Mycoplasma haemominutum’, but not with ‘Candidatus Mycoplasma turicensis’. Male cats and cats with outdoor access were more likely to be infected. Although ‘Candidatus Mycoplasma haemominutum’ is believed to cause minimal or no hematological alterations, the infected cats studied herein were more likely to be anemic.

Três espécies de hemoplasmas são reconhecidas em gatos domésticos: Mycoplasma haemofelis, ‘Candidatus Mycoplasma haemominutum’ e ‘Candidatus Mycoplasma turicensis’. A prevalência e alterações hematológicas associadas à infecção por hemoplasmas foi estudada, em 369 gatos domésticos de três populações distintas (doadores de sangue, hospitais e gatos de abrigo) do Sul do Brasil. Foram realizados hemogramas completos no momento da coleta de sangue e as amostras tiveram seu DNA extraído e testado por PCR convencional para cada espécie de hemoplasmas. Setenta e nove amostras (21,40%) foram positivas para pelo menos uma espécie. O mais prevalente foi ‘Candidatus Mycoplasma haemominutum’ com 50/369 (13,55%) gatos positivos, seguidos por ‘Candidatus Mycoplasma turicensis’ com 10/369 (2,71%) e Mycoplasma haemofelis com 8/369 (2,16%). Coinfecção por Mycoplasma haemofelis e ‘Candidatus Mycoplasma haemominutum’ foi observada em 4/369 (1,08%), enquanto ‘Candidatus Mycoplasma haemominutum’ e ‘Candidatus Mycoplasma turicensis’ coinfectaram 5/369 (1,35%) gatos. Três (0,81%) gatos apresentaram infecção pelos três hemoplasmas. Não houve associação entre a infecção e as diferentes populações. Anemia foi associada com a infecção por Mycoplasma haemofelis e ‘Candidatus Mycoplasma haemominutum’, mas não com ‘Candidatus Mycoplasma turicensis’. Gatos machos e com acesso à rua apresentaram maior probabilidade de serem infectados. Embora se acredite que ‘Candidatus Mycoplasma haemominutum’ possa causar alterações hematológicas mínimas ou ausentes, gatos infectados encontrados neste estudo foram mais propensos à anemia.

Molecular detection of Ehrlichia canis and Anaplasma platys in dogs in Southern Brazil.

The aims of this study were to determine the occurrence of Anaplasma platys and Ehrlichia canis infection in dogs in Porto Alegre, Southern Brazil; and to investigate their association with hematological abnormalities. Serum samples from 196 dogs were first tested using dot-ELISA for antibodies against Anaplasma spp. and Ehrlichia canis. Peripheral blood samples from 199 dogs were subjected to 16S rRNA nested PCR (nPCR) for A. platys and E. canis, followed by DNA sequencing to ensure pathogen identity. A total of 19/196 samples (9.69%) were positive for Anaplasma spp. using ELISA and 28/199 (14.07%) samples were positive for A. platys by nested PCR. All the dog samples were negative for E. canis, both in anti-E. canis antibody tests and in nested PCR. There were no significant differences in hematological parameters between A. platys-PCR positive and negative dogs and Anaplasma spp. serologically positive dogs, except for basophil counts, which were higher in nPCR-positive dogs. This is the first report showing A. platys presence in dogs in Southern Brazil. In conclusion, hematological parameters may not be sufficient to diagnose A. platys infection in dogs in Southern Brazil, probably due either to low pathogenicity or to chronic infection. On the other hand, E. canis may either have very low occurrence or be absent in dogs in Porto Alegre.

Molecular detection of Ehrlichia canis and Anaplasma platys in dogs in southern Brazil / Detecção molecular de Ehrlichia canis e Anaplasma platys em cães do sul do Brasil

The aims of this study were to determine the occurrence of Anaplasma platys and Ehrlichia canis infection in dogs in Porto Alegre, Southern Brazil; and to investigate their association with hematological abnormalities. Serum samples from 196 dogs were first tested using dot-ELISA for antibodies against Anaplasma spp. and Ehrlichia canis. Peripheral blood samples from 199 dogs were subjected to 16S rRNA nested PCR (nPCR) for A. platys and E. canis, followed by DNA sequencing to ensure pathogen identity. A total of 19/196 samples (9.69%) were positive for Anaplasma spp. using ELISA and 28/199 (14.07%) samples were positive for A. platys by nested PCR. All the dog samples were negative for E. canis, both in anti-E. canis antibody tests and in nested PCR. There were no significant differences in hematological parameters between A. platys-PCR positive and negative dogs and Anaplasma spp. serologically positive dogs, except for basophil counts, which were higher in nPCR-positive dogs. This is the first report showing A. platys presence in dogs in Southern Brazil. In conclusion, hematological parameters may not be sufficient to diagnose A. platys infection in dogs in Southern Brazil, probably due either to low pathogenicity or to chronic infection. On the other hand, E. canis may either have very low occurrence or be absent in dogs in Porto Alegre.

O objetivo deste estudo foi determinar a ocorrência de Anaplasma platys e Ehrlichia canis em cães de Porto Alegre, sul do Brasil, sua detecção molecular e associação com anormalidades hematológicas. Amostras séricas de 196 cães foram inicialmente triadas por dot-ELISA para a presença de anticorpos contra Anaplasma spp. e Ehrlichia canis. Amostras de sangue periférico de 199 cães foram submetidas à nested PCR (16S rRNA) para A. platys e E. canis, seguido de sequenciamento do DNA para confirmar a identidade do agente. Do total, 19/196 (9,69%) amostras foram positivas para Anaplasma spp. por dot-ELISA e 28/199 (14,07%) por nPCR. Todas as amostras dos cães foram negativas para E. canis no teste sorológico anti-E. canis e também na nPCR. Não houve diferença significativa nos parâmetros hematológicos, exceto a contagem de basófilos, que apresentou valores mais altos em cães positivos na nPCR para A. platys. Este é o primeiro relato da presença de A. platys no Rio Grande do Sul, e a primeira detecção molecular do agente no sul do Brasil. Em conclusão, parâmetros hematológicos não são suficientes para diagnosticar a infecção por A. platys em cães, provavelmente devido sua baixa patogenicidade ou infecção crônica. Por outro lado, E. canis parece ter ocorrência baixa ou mesmo nula em cães de Porto Alegre.

Frequencies of DEA blood types in a purebred canine blood donor population in Porto Alegre, RS, Brazil / Frequências dos tipos sanguíneos do sistema DEA em uma população de cães de raça doadores de sangue em Porto Alegre, RS

The study of canine immunohematology is very important for veterinary transfusion medicine. The objective of this study was to determine the DEA blood type frequencies in a purebred canine blood donor population from Porto Alegre, RS, Brazil. One hundred clinically healthy purebred dogs were chosen, 20 dogs from each breed (Great Dane, Rottweiler, Golden Retriever, German Shepherd and Argentine Dogo). Blood samples were taken in ACD-A tubes and the MSU hemagglutination tube test (MI, USA) was used to determine the blood types. The studied population presented general frequencies of 61 percent for DEA 1.1, 22 percent for DEA 1.2, 7 percent for DEA 3, 100 percent for DEA 4, 9 percent for DEA 5 and 16 percent for DEA 7. A significant association was found between breeds and certain combinations of blood types in this population. The results are in agreement with the literature since most part of the canine population studied was positive for DEA 1.1, the most antigenic blood type in dogs. Differences were found among the studied breeds and those should be considered when selecting a blood donor. The knowledge of blood types frequencies and their combinations in different canine populations, including different breeds, is important because it shows the particularities of each group, helps to keep a data bank of local frequencies and minimizes the risks of transfusion reactions.

O estudo da imunohematologia canina é muito importante para a medicina veterinária transfusional. O objetivo deste estudo foi determinar as frequências dos tipos sanguíneos do sistema DEA em uma população de cães de raça que fazem parte de um programa de cães doadores de sangue em Porto Alegre, RS, Brasil. Cem cães saudáveis de raça pura foram selecionados, vinte de cada raça (Dogue Alemão, Rottweiler, Golden Retriever, Pastor Alemão e Dogo Argentino). Amostras de sangue foram coletadas em tubos contendo ACD-A e o teste de hemaglutinação em tubo de ensaio da MSU (MI, EUA) foi utilizado para determinar os tipos sanguíneos. A população estudada apresentou frequências gerais de 61 por cento para DEA 1.1, 22 por cento para DEA 1.2, 7 por cento para DEA 3, 100 por cento para DEA 4, 9 por cento para DEA 5 and 16 por cento para DEA 7. Uma associação significativa foi encontrada entre as raças e certas combinações de tipos sanguíneos nesta população. Os resultados estão de acordo com a literatura, visto que a maioria da população canina estudada foi positiva para DEA 1.1, o tipo sanguineo mais antigênico em cães. Foram encontradas diferenças entre as raças estudadas e estas devem ser consideradas na seleção de um doador de sangue. O conhecimento das frequências dos tipos sanguíneos e de suas combinações em diferentes populacões caninas, incluindo diferentes raças, é importante, pois demonstra as particularidades de cada grupo, auxilia na manutenção de um banco de dados sobre as frequências locais e minimiza os riscos de reações transfusionais.

Infecção por Hepatozoon canis em canino doméstico na região Sul do Brasil confirmada por técnicas moleculares / Hepatozoon canis infection in a domestic dog in Southern Brazil confirmed by molecular techniques

Hepatozoonose canina é uma doença transmitida por carrapatos e causada pelo protozoário Hepatozoon spp. No Brasil, existem poucos relatos da infecção e dados sobre sua epidemiologia, patogenicidade, seus vetores e sua caracterização genética. No presente estudo, são utilizadas técnicas moleculares para o diagnóstico e a caracterização do parasita. Foi estudado um canino que apresentava emagrecimento progressivo, anemia regenerativa, neutropenia e hiperglobulinemia. Gamontes de Hepatozoon spp. foram visualizados no esfregaço sanguíneo. DNA foi extraído do sangue, e o diagnóstico foi confirmado pela técnica da reação em cadeia da polimerase (PCR). O produto da PCR foi purificado a partir do gel e clonado, e o fragmento de 625pb foi sequenciado. A sequência foi submetida ao GenBank como isolado de Porto Alegre, e a análise molecular revelou homologia de 98-100 por cento com Hepatozoon canis e máxima de 92 por cento com Hepatozoon americanum. Este estudo representa a primeira confirmação molecular da presença de H. canis no sul do Brasil.

Canine Hepatozoonosis is a vector-borne disease caused by the protozoa Hepatozoon spp. There are few case reports in Brazil and the epidemiology, pathogenicity, vectors and molecular characterization is poorly understood. The present study used a canine presenting weight loss, regenerative anemia, neutropenia and hyperglobulinemia. Hepatozoon spp. gamonts were observed in the blood smear by microscopy. DNA was extracted from blood, and the diagnosis was confirmed by PCR assay. The PCR product was purified from gel and cloned. A fragment of 625bp was sequenced and submitted to the GenBank database as Isolate Porto Alegre. Molecular analysis showed a homology of 98-100 percent with Hepatozoon canis and less than 92 percent for H. americanum. This study represents the first molecular confirmation that H. canis is present in Southern Brazil.