Characterization of Bovine Papillomavirus Types Detected in Cattle Rumen Tissues from Amazon Region, Brazil.

The Bos Taurus Papillomavirus, commonly known as bovine papillomavirus (BPV), can cause lesions in the mucosa of the gastrointestinal tract (GIT) in cattle and induce the formation of papillomas in organs such as the pharynx, esophagus, rumen and reticulum. GIT papillomas can lead to feeding and breathing distress. Moreover, the sample collection is challenging, which reduces the BPV diagnosis in these organs. BPV can cause exophytic nodular, cauliflower-like, flat, filiform or atypical-shape papillomas at the epidermis. Histologically, the papillomas demonstrate orthokeratotic/parakeratotic hyperkeratosis and koilocytosis and, currently, BPV comprises 45 described types. The aim of this study was to carry out the genetic characterization of BPV present in rumen neoplastic lesions of cattle raised extensively in the Western Amazon region, Brazil. A total of 100 papillomatous ruminal samples were collected from animals slaughtered in Ji-Paraná and Urupá municipalities from the Rondônia state, Brazil. The samples were submitted to PCR using the primer pair FAP59/FAP64 and sequenced by the Sanger method. Histopathological analysis was performed on 24 samples, which had enough material for this purpose. As a result, samples were histologically classified as fibropapilloma and squamous papilloma. Among the samples analyzed, it was possible to identify the BPVs 2, 13 (Delta PVs) and 44, with one sample classified as a putative new subtype of BPV44. The present study could identify BPV13 and 44 types in cattle rumen tissues from the Brazilian Amazon region for the first time.

Mamastrovirus 5 detected in a crab-eating fox (Cerdocyon thous): Expanding wildlife host range of astroviruses.

Astroviruses are a common cause of gastroenteritis in children worldwide and can also cause infection in a range of domestic and wild animal species. Canine astrovirus (formally named as Mamastrovirus 5, MAstV5) has been reported worldwide, and its role as an enteric pathogen is still controversial. Herein, we describe the genomic characterization of a MAstV5 (strain crab-eating fox/2016/BRA) identified in a wild canid (Cerdocyon thous) diagnosed with canine distemper virus (CDV) as causa mortis. The nearly complete genome comprised 6579 nt in length and displayed the archetypal organization of astroviruses. The present report is the first evidence of MAstV5 infection in an animal species other than the dog and highlights a possible natural astrovirus spillover between domestic and wild canids. Moreover, these results show the first evidence of extra-intestinal MAstV5, suggesting a virus systemic spread. This work is expected to contribute to a better understanding of the astroviruses biology and their interactions with the wildlife health.

Influence of vaccine strains on the evolution of canine distemper virus.

Canine distemper virus (CDV) is a major dog pathogen belonging to the genus Morbillivirus of the family Paramyxoviridae. CDV causes disease and high mortality in dogs and wild carnivores. Although homologous recombination has been demonstrated in many members of Paramyxoviridae, these events have rarely been reported for CDV. To detect potential recombination events, the complete CDV genomes available in GenBank up to June 2015 were screened using distinct algorithms to detect genetic conversions and incongruent phylogenies. Eight putative recombinant viruses derived from different CDV genotypes and different hosts were detected. The breakpoints of the recombinant strains were primarily located on fusion and hemagglutinin glycoproteins. These results suggest that homologous recombination is a frequent phenomenon in morbillivirus populations under natural replication, and CDV vaccine strains might play an important role in shaping the evolution of this virus.

Detecção do vírus da diarreia viral bovina em carrapatos Rhipicephalus (Boophilus) microplus alimentados e, bovino persistentemente infectado

Background:  :  :  : Bovine viral diarrhea virus (BVDV) is one of the main agents that cause economical losses in cattle worldwide. Congenitally infected calves that are born persistently infected (PI) to BVDV are the main sources of infection to susceptible cattle. Direct contact is the most important form of transmission, but indirect contact can also spread BVDV, not only inside herds, but also between them. Transmission of BVDV by haematophagous insects has been proven experimentally, but the role of ticks in the transmission of BVDV has never been investigated. Ticks can heavily infest cattle raised in tropical areas and Rhipicephalus (Boophilus) microplus is the most important among them. The present experiment was carried out to investigate the role of R. microplus ticks in the transmission of BVDV, experimentally infecting PI calf with ticks. Material, Methods and Results: Three calves were used in the experiment: one PI calf was identified from a natural outbreak; a second animal was infested with the progeny of a tick fed on the PI calf and the third was kept as a negative control, infested with negative ticks. Viral RNA investigation was performed by reverse transcription followed by polymerase chain reaction (RT-PCR) from the sera of the calves and from ticks (adult females, eggs and larvae that were the progeny of the experimentally contaminated adult females and f

Detecção do vírus da diarreia viral bovina em carrapatos Rhipicephalus (Boophilus) microplus alimentados e, bovino persistentemente infectado

Background:  :  :  : Bovine viral diarrhea virus (BVDV) is one of the main agents that cause economical losses in cattle worldwide. Congenitally infected calves that are born persistently infected (PI) to BVDV are the main sources of infection to susceptible cattle. Direct contact is the most important form of transmission, but indirect contact can also spread BVDV, not only inside herds, but also between them. Transmission of BVDV by haematophagous insects has been proven experimentally, but the role of ticks in the transmission of BVDV has never been investigated. Ticks can heavily infest cattle raised in tropical areas and Rhipicephalus (Boophilus) microplus is the most important among them. The present experiment was carried out to investigate the role of R. microplus ticks in the transmission of BVDV, experimentally infecting PI calf with ticks. Material, Methods and Results: Three calves were used in the experiment: one PI calf was identified from a natural outbreak; a second animal was infested with the progeny of a tick fed on the PI calf and the third was kept as a negative control, infested with negative ticks. Viral RNA investigation was performed by reverse transcription followed by polymerase chain reaction (RT-PCR) from the sera of the calves and from ticks (adult females, eggs and larvae that were the progeny of the experimentally contaminated adult females and f

First detection of canine parvovirus type 2c in Brazil

The presence of canine parvovirus type 2 (CPV-2), 2a and 2b has been described in Brazil, however, the type 2c had not been reported until now. In the current study, seven out of nine samples from dogs with diarrhea were characterized as CPV-2c, indicating that this virus is already circulating in the Brazilian canine population.

No Brasil, a presença do parvovírus canino do tipo 2 (CPV-2), 2a e 2b já havia sido descrita, contudo, ainda não havia sido verificada a presença do tipo 2c. No presente trabalho, sete de nove amostras de cães com diarréia foram caracterizadas como CPV-2c, indicando que este vírus já está circulando na população canina no Brasil.

Comparision of different cell cultures for replication of infectious laryngotracheitis virus from chickens

The propagation of infectious laryngotracheitis virus (ILTV) has been described using primary cell cultures derived from chicken embryo liver and kidney or embryonated eggs, but these cultures use Specific Pathogen Free (SPF) eggs that are time and cost expensive. Since cell line cultures are easier to maintain in laboratory conditions, the growth of ILTV was evaluated in five different cell cultures: chicken embryo related cells (CER), a cell hybrid derived from chicken embryo fibroblasts cells and BHK-21; Vero, from African green monkey kidney cells; HD11, a chicken macrophage cell line; CEC-32, an avian fibroblast cell line and a primary cell culture of chicken embryo fibroblasts (CEF). Cytophatic effect was observed until 96 hours following inoculation and the detection of the viral DNA was performed by PCR. The HD11 and CEC-32 cell lines did not support the virus growth but CEF and Vero, as already described were permissive cultures for propagation of ILTV.The results also showed that the CER cell line can be used for primary isolation and replication of ILTV.

Comparision of different cell cultures for replication of infectious laryngotracheitis virus from chickens

The propagation of infectious laryngotracheitis virus (ILTV) has been described using primary cell cultures derived from chicken embryo liver and kidney or embryonated eggs, but these cultures use Specific Pathogen Free (SPF) eggs that are time and cost expensive. Since cell line cultures are easier to maintain in laboratory conditions, the growth of ILTV was evaluated in five different cell cultures: chicken embryo related cells (CER), a cell hybrid derived from chicken embryo fibroblasts cells and BHK-21; Vero, from African green monkey kidney cells; HD11, a chicken macrophage cell line; CEC-32, an avian fibroblast cell line and a primary cell culture of chicken embryo fibroblasts (CEF). Cytophatic effect was observed until 96 hours following inoculation and the detection of the viral DNA was performed by PCR. The HD11 and CEC-32 cell lines did not support the virus growth but CEF and Vero, as already described were permissive cultures for propagation of ILTV.The results also showed that the CER cell line can be used for primary isolation and replication of ILTV.

Comparision of different cell cultures for replication of infectious laryngotracheitis virus from chickens

The propagation of infectious laryngotracheitis virus (ILTV) has been described using primary cell cultures derived from chicken embryo liver and kidney or embryonated eggs, but these cultures use Specific Pathogen Free (SPF) eggs that are time and cost expensive. Since cell line cultures are easier to maintain in laboratory conditions, the growth of ILTV was evaluated in five different cell cultures: chicken embryo related cells (CER), a cell hybrid derived from chicken embryo fibroblasts cells and BHK-21; Vero, from African green monkey kidney cells; HD11, a chicken macrophage cell line; CEC-32, an avian fibroblast cell line and a primary cell culture of chicken embryo fibroblasts (CEF). Cytophatic effect was observed until 96 hours following inoculation and the detection of the viral DNA was performed by PCR. The HD11 and CEC-32 cell lines did not support the virus growth but CEF and Vero, as already described were permissive cultures for propagation of ILTV.The results also showed that the CER cell line can be used for primary isolation and replication of ILTV.

Comparision of different cell cultures for replication of infectious laryngotracheitis virus from chickens

The propagation of infectious laryngotracheitis virus (ILTV) has been described using primary cell cultures derived from chicken embryo liver and kidney or embryonated eggs, but these cultures use Specific Pathogen Free (SPF) eggs that are time and cost expensive. Since cell line cultures are easier to maintain in laboratory conditions, the growth of ILTV was evaluated in five different cell cultures: chicken embryo related cells (CER), a cell hybrid derived from chicken embryo fibroblasts cells and BHK-21; Vero, from African green monkey kidney cells; HD11, a chicken macrophage cell line; CEC-32, an avian fibroblast cell line and a primary cell culture of chicken embryo fibroblasts (CEF). Cytophatic effect was observed until 96 hours following inoculation and the detection of the viral DNA was performed by PCR. The HD11 and CEC-32 cell lines did not support the virus growth but CEF and Vero, as already described were permissive cultures for propagation of ILTV.The results also showed that the CER cell line can be used for primary isolation and replication of ILTV.

Comparision of different cell cultures for replication of infectious laryngotracheitis virus from chickens

The propagation of infectious laryngotracheitis virus (ILTV) has been described using primary cell cultures derived from chicken embryo liver and kidney or embryonated eggs, but these cultures use Specific Pathogen Free (SPF) eggs that are time and cost expensive. Since cell line cultures are easier to maintain in laboratory conditions, the growth of ILTV was evaluated in five different cell cultures: chicken embryo related cells (CER), a cell hybrid derived from chicken embryo fibroblasts cells and BHK-21; Vero, from African green monkey kidney cells; HD11, a chicken macrophage cell line; CEC-32, an avian fibroblast cell line and a primary cell culture of chicken embryo fibroblasts (CEF). Cytophatic effect was observed until 96 hours following inoculation and the detection of the viral DNA was performed by PCR. The HD11 and CEC-32 cell lines did not support the virus growth but CEF and Vero, as already described were permissive cultures for propagation of ILTV.The results also showed that the CER cell line can be used for primary isolation and replication of ILTV.

Comparision of different cell cultures for replication of infectious laryngotracheitis virus from chickens

The propagation of infectious laryngotracheitis virus (ILTV) has been described using primary cell cultures derived from chicken embryo liver and kidney or embryonated eggs, but these cultures use Specific Pathogen Free (SPF) eggs that are time and cost expensive. Since cell line cultures are easier to maintain in laboratory conditions, the growth of ILTV was evaluated in five different cell cultures: chicken embryo related cells (CER), a cell hybrid derived from chicken embryo fibroblasts cells and BHK-21; Vero, from African green monkey kidney cells; HD11, a chicken macrophage cell line; CEC-32, an avian fibroblast cell line and a primary cell culture of chicken embryo fibroblasts (CEF). Cytophatic effect was observed until 96 hours following inoculation and the detection of the viral DNA was performed by PCR. The HD11 and CEC-32 cell lines did not support the virus growth but CEF and Vero, as already described were permissive cultures for propagation of ILTV.The results also showed that the CER cell line can be used for primary isolation and replication of ILTV.

Análise do poder discriminatório da SE-AFLP para Salmonella Enteritidis frente a outras técnicas fenotípicas e genotípicas

Salmonella  (S.) Enteritidis tem sido uma das bactérias mais implicadas em casos de infecções alimentares, sendo a investigação de sua epidemiologia realizada por diferentes métodos fenotípicos e genotípicos. Dentre as técnicas de tipificação de bactérias, a técnica de single-enzyme amplified fragment length polymorphism (SE-AFLP) é um dos métodos mais recentemente descritos, apresentando boa confiabilidade e fácil aplicação. No presente trabalho, a SE-AFLP foi comparada com as técnicas fenotípicas de fagotipificação (PT) e determinação da sensibilidade a antimicrobianos (DSA) e genotípicas de rep-PCR (seqüências repetitivas REP, ERIC e BOX) e detecção de genes de virulência (genes spvR e spvC). Foram analisadas 20 amostras de S. Enteritidis, sendo onze isoladas de suínos da Região Sul do Brasil e nove amostras oriundas de outros países. A caracterização destas amostras pelas técnicas de PT, DSA, presença de genes de virulência e rep-PCR foi descrita em um trabalho anterior. O poder discriminatório obtido pelas técnicas foi calculado pelo índice de diversidade de Simpson (D). A SE-AFLP encontrou um número maior de perfis e obteve uma maior capacidade discriminatória do que as outras técnicas genotípicas, embora seu D tenha sido menor do que o das técnicas fenotípicas. Desta forma, ficou demonstrada a importância da utilização conjunta de técnicas fenotípicas e genotípicas na ca

Use of a modified AFLP protocol to discriminate Salmonella enterica subsp.enterica serovar Enteritidis isolates

Salmonella enterica  subsp. enterica (S.) serovar Enteritidis is one of the main pathogens involved in food-borne diseases worldwide. In epidemiological investigations of food-related salmonellosis, subtyping is necessary to improve preventive and control measures. Single-enzyme amplified fragment length polymorphism (SE-AFLP) analysis is a modified AFLP that uses only one restriction enzyme to produce DNA fragments that are selectively amplified by PCR. In order to assess the applicability of SE-AFLP in S. Enteritidis typing, one hundred and eight strains isolated from poultry, swine and also from human salmonellosis outbreaks in Southern Brazil were analyzed. Strains from other countries and six different S. enterica serovars were also included as controls. SE-AFLP was able to distinguish S. Enteritidis from the other S. enterica serovars analyzed. However, most of S. Enteritidis strains isolated from poultry, salmonellosis outbreaks and most of the strains from other countries shared the same predominant pattern. The low genetic diversity identified in S. Enteritidis suggests that the strains analyzed are clonally related and one predominant SE-AFLP genotype is widely spread in Southern Brazil.

Detecção de Salmonella sp. em emas (Rhea americana) através de suabes cloacais

A ema ( Rhea americana) é uma ratita nativa da América do Sul que está sendo criada comercialmente em fazendas no sul do Brasil pela sua carne e penas. Devido ao potencial que as aves têm de transmitir sorovares de Salmonella sp capazes de causar toxinfecção alimentar, torna-se fundamental conhecer o estado sanitário das emas em relação a este patógeno. Com este objetivo, procurou-se verificar a viabilidade da utilização do suabe cloacal para o isolamento de Salmonella sp. em emas. Vísceras e suabes cloacais foram coletadas de 26 aves abatidas em um frigorífico no Rio Grande do Sul. Utilizando-se o isolamento em fígado e/ou ceco como método diagnóstico padrão, determinou-se alta especificidade (80%) e valor preditivo positivo (90,91%); porém, verificou-se baixa sensibilidade (47,62%) e valor preditivo negativo (26,7%). Das 11 cepas isoladas a partir de suabes, duas (18,2%) foram identificadas como S. enterica enterica rugosa, três (27,3%) como S. Newport e seis (54,5%) como S. Typhimurium. Através desse estudo, verificou-se que a utilização de suabe cloacal para detecção de Salmonella sp. em emas deve ser criteriosa, uma vez que o número de falsos negativos é elevado.

Phenotypic and genotypic characterization of Salmonella Enteritidis isolates

In order to study the epidemiology of Salmonella Enteritidis outbreaks and determine the source of contamination so that a recurrence can be avoided, detailed characterization is necessary. Thus, the purpose of this study was to verify whether rep-PCR was able to discriminate among Salmonella Enteritidis isolates. Phage typing, detection of virulence genes and antimicrobial resistance testing were also associated to rep-PCR results. One hundred and two S. Enteritidis isolates from broiler carcasses, food, human, pigs, poultry-related samples, and nine isolates from other countries were genotypically typed by REP-PCR, ERIC-PCR and BOX-PCR, collectively called rep-PCR. Phage typing, detection of virulence genes and antimicrobial resistance testing were also performed. Only three fingerprinting profiles were obtained with each rep-PCR method, with the majority of isolates belonging to the same profile. No relationship was observed between genotypic profile and year, place of isolation or source of infection. However, the less frequent rep-PCR profiles showed single antimicrobial resistance patterns. Although few strains isolated from swine were analyzed, different antimicrobial resistance patterns were observed. Furthermore, phage type 4 was not found in swine isolates. rep-PCR showed a lower discriminatory power as compared with antimicrobial resistance and phage typing, but the combination of genotypic and phenotypic methods was more discriminatory than any method alone, resulting in 48 different types.

Uma caracterização detalhada de Salmonella Enteritidis é necessária para que possa ser desenvolvido o estudo da epidemiologia dos surtos causados por este organismo, bem como a determinação da fonte de contaminação, evitando que ocorram novos surtos. Assim, o objetivo deste estudo foi verificar se a rep-PCR era capaz de diferenciar isolados de S. Enteritidis. A fagotipagem, a detecção de genes de virulência e a determinação de resistência antimicrobiana foram associadas aos resultados da rep-PCR. Cento e duas S. Enteritidis isoladas de carcaças de frango, alimentos prontos para consumo, humanos suínos, amostras relacionadas a aves, e nove isolados de outros países foram genotipicamente tipados por REP-PCR, ERIC-PCR e BOX-PCR, juntamente chamados de rep-PCR. A fagotipagem, a detecção de genes de virulência e a determinação de resistência antimicrobiana também foram realizadas. Somente três padrões de fingerprinting foram obtidos com cada método de rep-PCR, sendo que a maioria dos isolados pertenceu ao mesmo perfil. Nenhuma relação foi observada entre o perfil genotípico e o ano, o local de isolamento e a fonte de infecção. Entretanto, os perfis menos freqüentes de rep-PCR apresentaram padrões de resistência antimicrobiana únicos. Embora poucas amostras de suínos tenham sido analisadas, diferentes padrões de resistência antimicrobiana foram observados. Além disso, o fagotipo 4 não foi encontrado em isolados de suínos. A rep-PCR apresentou um menor poder discriminatório quando comparada com a resistência antimicrobiana e com a fagotipagem, mas a combinação dos métodos genotípicos e fenotípicos foi mais discriminatória do que qualquer método isolado, resultando em 48 tipos diferentes.

Use of a modified AFLP protocol to discriminate Salmonella enterica subsp.enterica serovar Enteritidis isolates

Salmonella enterica  subsp. enterica (S.) serovar Enteritidis is one of the main pathogens involved in food-borne diseases worldwide. In epidemiological investigations of food-related salmonellosis, subtyping is necessary to improve preventive and control measures. Single-enzyme amplified fragment length polymorphism (SE-AFLP) analysis is a modified AFLP that uses only one restriction enzyme to produce DNA fragments that are selectively amplified by PCR. In order to assess the applicability of SE-AFLP in S. Enteritidis typing, one hundred and eight strains isolated from poultry, swine and also from human salmonellosis outbreaks in Southern Brazil were analyzed. Strains from other countries and six different S. enterica serovars were also included as controls. SE-AFLP was able to distinguish S. Enteritidis from the other S. enterica serovars analyzed. However, most of S. Enteritidis strains isolated from poultry, salmonellosis outbreaks and most of the strains from other countries shared the same predominant pattern. The low genetic diversity identified in S. Enteritidis suggests that the strains analyzed are clonally related and one predominant SE-AFLP genotype is widely spread in Southern Brazil.

Análise do poder discriminatório da SE-AFLP para Salmonella Enteritidis frente a outras técnicas fenotípicas e genotípicas

Salmonella  (S.) Enteritidis tem sido uma das bactérias mais implicadas em casos de infecções alimentares, sendo a investigação de sua epidemiologia realizada por diferentes métodos fenotípicos e genotípicos. Dentre as técnicas de tipificação de bactérias, a técnica de single-enzyme amplified fragment length polymorphism (SE-AFLP) é um dos métodos mais recentemente descritos, apresentando boa confiabilidade e fácil aplicação. No presente trabalho, a SE-AFLP foi comparada com as técnicas fenotípicas de fagotipificação (PT) e determinação da sensibilidade a antimicrobianos (DSA) e genotípicas de rep-PCR (seqüências repetitivas REP, ERIC e BOX) e detecção de genes de virulência (genes spvR e spvC). Foram analisadas 20 amostras de S. Enteritidis, sendo onze isoladas de suínos da Região Sul do Brasil e nove amostras oriundas de outros países. A caracterização destas amostras pelas técnicas de PT, DSA, presença de genes de virulência e rep-PCR foi descrita em um trabalho anterior. O poder discriminatório obtido pelas técnicas foi calculado pelo índice de diversidade de Simpson (D). A SE-AFLP encontrou um número maior de perfis e obteve uma maior capacidade discriminatória do que as outras técnicas genotípicas, embora seu D tenha sido menor do que o das técnicas fenotípicas. Desta forma, ficou demonstrada a importância da utilização conjunta de técnicas fenotípicas e genotípicas na ca

Detecção de Salmonella sp. em emas (Rhea americana) através de suabes cloacais

A ema ( Rhea americana) é uma ratita nativa da América do Sul que está sendo criada comercialmente em fazendas no sul do Brasil pela sua carne e penas. Devido ao potencial que as aves têm de transmitir sorovares de Salmonella sp capazes de causar toxinfecção alimentar, torna-se fundamental conhecer o estado sanitário das emas em relação a este patógeno. Com este objetivo, procurou-se verificar a viabilidade da utilização do suabe cloacal para o isolamento de Salmonella sp. em emas. Vísceras e suabes cloacais foram coletadas de 26 aves abatidas em um frigorífico no Rio Grande do Sul. Utilizando-se o isolamento em fígado e/ou ceco como método diagnóstico padrão, determinou-se alta especificidade (80%) e valor preditivo positivo (90,91%); porém, verificou-se baixa sensibilidade (47,62%) e valor preditivo negativo (26,7%). Das 11 cepas isoladas a partir de suabes, duas (18,2%) foram identificadas como S. enterica enterica rugosa, três (27,3%) como S. Newport e seis (54,5%) como S. Typhimurium. Através desse estudo, verificou-se que a utilização de suabe cloacal para detecção de Salmonella sp. em emas deve ser criteriosa, uma vez que o número de falsos negativos é elevado.

Análise do poder discriminatório da SE-AFLP para Salmonella Enteritidis frente a outras técnicas fenotípicas e genotípicas

Salmonella  (S.) Enteritidis tem sido uma das bactérias mais implicadas em casos de infecções alimentares, sendo a investigação de sua epidemiologia realizada por diferentes métodos fenotípicos e genotípicos. Dentre as técnicas de tipificação de bactérias, a técnica de single-enzyme amplified fragment length polymorphism (SE-AFLP) é um dos métodos mais recentemente descritos, apresentando boa confiabilidade e fácil aplicação. No presente trabalho, a SE-AFLP foi comparada com as técnicas fenotípicas de fagotipificação (PT) e determinação da sensibilidade a antimicrobianos (DSA) e genotípicas de rep-PCR (seqüências repetitivas REP, ERIC e BOX) e detecção de genes de virulência (genes spvR e spvC). Foram analisadas 20 amostras de S. Enteritidis, sendo onze isoladas de suínos da Região Sul do Brasil e nove amostras oriundas de outros países. A caracterização destas amostras pelas técnicas de PT, DSA, presença de genes de virulência e rep-PCR foi descrita em um trabalho anterior. O poder discriminatório obtido pelas técnicas foi calculado pelo índice de diversidade de Simpson (D). A SE-AFLP encontrou um número maior de perfis e obteve uma maior capacidade discriminatória do que as outras técnicas genotípicas, embora seu D tenha sido menor do que o das técnicas fenotípicas. Desta forma, ficou demonstrada a importância da utilização conjunta de técnicas fenotípicas e genotípicas na ca