ABSTRACT
Human papillomavirus (HPV) consists in a great clinical importance worldwide, being involved in the most frequent sexually transmitted viral infection, genital warts, with high incidence in both men and women. Furthermore, HPV can also develop a broad spectrum of cancers, the most common is cervical cancer, being one of the main causes of morbidity and mortality in women worldwide, especially in low-and middle-income countries (LMICs). Here we discuss the importance of cervical cancer prevention and others related cancers caused by HPV, information about HPV vaccines, evaluation of single-dose use, low vaccination coverage, as well as other topics regarding viral transmission, biology, and epidemiology, aiming for the reduction of cervical carcinoma cases and others HPV associated diseases, reducing infections and death in the world as regarding to HPV, through the wide people immunization.
ABSTRACT
The correlation between human papillomavirus and induction of cervical cancer has been established since 1974, but the induction of transient idiopathic infertility, attributed to HPV, has also recently been addressed. Therefore, there is a clear need for a more effective combat against the virus, which has periodically been correlated with new diseases that indiscriminately affect men, women, and children of all social classes. Recent technological advances have contributed notably to the improvement of experimental work tools, a fact that provides scientists with new horizons to be explored in the development of new products, free from serious side effects, due to genetic manipulation in the construction of expression vectors directed to therapeutic targets of interest. Thus, this book aims to tell readers a little bit of the natural history about HPV and the advances in prophylaxis and minimally invasive therapy, aiming to eradicate the virus and treat infected patients, who have developed HPV-induced cancer. Periodic exams and vaccination are still the best options to prevent HPV infection and the development of cancers associated with the virus, as well as the possibility of transient infertility occurrence attributed to the HPV.
Desde 1974 foi estabelecida a correlação do papilomavírus humano com a indução de câncer cervical, mas recentemente tem sido abordada também a indução de infertilidade idiopática transiente, atribuída ao HPV. Portanto, fica evidente a necessidade de combate mais efetivo ao vírus, que periodicamente vem sendo correlacionado com novas doenças que afetam indiscriminadamente homens, mulheres e crianças de todas as classes sociais. Os avanços tecnológicos recentes têm contribuído de forma notável para o aprimoramento das ferramentas de trabalho experimental, fato que proporciona aos cientistas novos horizontes a serem explorados no desenvolvimento de novos produtos, isentos de efeitos colaterais graves, devido à manipulação genética na construção de vetores de expressão gênica direcionados a alvos terapêuticos de interesse. Assim, este livro pretende relatar aos leitores um pouco da história natural sobre HPV e os avanços em termos de profilaxia e de terapia minimamente invasiva, visando erradicar o vírus e tratar os pacientes infectados, que desenvolveram câncer induzido por HPV. Os exames periódicos e a vacinação ainda são as melhores opções para se evitar a infecção por HPV e o desenvolvimento de cânceres associados ao vírus, assim como a possibilidade de ocorrência da infertilidade transiente atribuída ao HPV.
ABSTRACT
Human papillomavirus (HPV) infection is a leading cause of morbidity and mortality in women worldwide. The virus is associated with benign warts and a broad spectrum of malignancies, including cervical cancer, considered a disease of high clinical relevance, especially in developing countries. In this study we developed the production of recombinant proteins HPV16 L1 and HPV16 L2 in human cells in suspension (293-F), which were transiently co-transfected with the pUF3L1h and pUF3L2h vectors. Expressions of recombinant HPV16 L1 and L2 capsid proteins was detected by laser scanning confocal microscopy and flow cytometry. Both proteins were identified intracellularly in the nucleus and cytoplasm of cells. The presence of these heterologous proteins and VLPs formation were detected by transmission electron microscopy (TEM) through colloidal gold immunolabeling and negative staining. Cell extracts containing recombinant proteins were purified by affinity chromatography and immunization of Balb/c mice with the formulation HPV16 L1/L2 VLPs containing adjuvant was able to induce higher titer of anti-HPV16 L1, when compared to HPV16 L2 antibodies by indirect ELISA assay. These data indicate that transient expression in 293-F cells was efficiently established. The results are promising for obtain recombinant proteins of the HPV capsid for future studies involving human papillomavirus, as well as to contribute for the development of other vaccine strategies for prevention against HPV.
ABSTRACT
Two allopatric populations of Brazilian diploid and tetraploid Odontophrynus americanus species complex, both from São Paulo state, had their blood hemoglobin biochemically analyzed. In addition, these specimens were cytogenetically characterized. Biochemical characterization of hemoglobin expression showed a distinct banding pattern between the allopatric specimens. Besides this, two distinct phenotypes, not linked to ploidy, sex, or age, were observed in adult animals of both populations. Phenotype A exhibits dark-colored body with small papillae, ogival-shaped jaw with reduced interpupillary distance and shorter hind limbs. Phenotype B shows yellowish-colored body with larger papillae, arch-shaped jaw with broader interpupillary distance and longer hind limbs. Intermediate phenotypes were also found. Considering the geographical isolation of both populations, differences in chromosomal secondary constrictions and distinct hemoglobins banding patterns, these data indicate that 2n and 4n populations represent cryptic species in the O. americanus species complex. The observed phenotypic diversity can be interpreted as population genetic variability. Eventually future data may indicate a probable beginning of speciation in these Brazilian frogs. Such inter- and intrapopulational differentiation/speciation process indicates that O. americanus species complex taxonomy deserves further evaluation by genomics and metabarcoding communities, also considering the pattern of hemoglobin expression, in South American frogs.
ABSTRACT
Two allopatric populations of Brazilian diploid and tetraploid Odontophrynus americanus species complex, both from São Paulo state, had their blood hemoglobin biochemically analyzed. In addition, these specimens were cytogenetically characterized. Biochemical characterization of hemoglobin expression showed a distinct banding pattern between the allopatric specimens. Besides this, two distinct phenotypes, not linked to ploidy, sex, or age, were observed in adult animals of both populations. Phenotype A exhibits dark-colored body with small papillae, ogival-shaped jaw with reduced interpupillary distance and shorter hind limbs. Phenotype B shows yellowish-colored body with larger papillae, arch-shaped jaw with broader interpupillary distance and longer hind limbs. Intermediate phenotypes were also found. Considering the geographical isolation of both populations, differences in chromosomal secondary constrictions and distinct hemoglobins banding patterns, these data indicate that 2n and 4n populations represent cryptic species in the O. americanus species complex. The observed phenotypic diversity can be interpreted as population genetic variability. Eventually future data may indicate a probable beginning of speciation in these Brazilian frogs. Such inter- and intrapopulational differentiation/speciation process indicates that O. americanus species complex taxonomy deserves further evaluation by genomics and metabarcoding communities, also considering the pattern of hemoglobin expression, in South American frogs.
ABSTRACT
Leptospirosis isazoonosis caused by pathogenic bacteria from the genus Leptospira.Thediseaserepresents a seriouspublic health problem inunderdeveloped tropical countries.Leptospire sinfecthosts throughsmallabrasions in the skin or mucousmembranesand they rapidly disseminateto target organs.Thecapacityof some pathogenic leptospira lstrains toacquire thenegative complementregulators factorH(FH)andC4bbindingpro teincorrelateswith their abilityto survivein humans e rum.Inthis study weasses sed the functional consequences oft heagemaculardegeneration-associatedpolymorphismFHHis402orFHTyr402onFH Leptospira interactions.Inbinding assaysusing sub-satura tingamounts ofFH, theFHTyr402 variant interacted with all the strainstested more stronglythanthe FHHis402variant.Athigher concentrations, differences tendedto disappear. We then compared co factor activities displayed by FH. His402and FH Tyr402 bound tothe surface ofL.interrogans. Bothvariantsex ibit similar activity as cofactors for FactorI mediated cleavage of C3b,thusindicating that they do not differin their capacity toregulate the complement cascade.
Subject(s)
Complement Factor H/analysis , Complement Factor H/immunology , Leptospira interrogans serovar pomona/genetics , Leptospira interrogans serovar pomona/immunology , Leptospira/immunology , Complement Factor H/isolation & purification , Polymorphism, Genetic/immunologyABSTRACT
We investigated the ability of the sPLA(2), known as MT-III, isolated from the viperid snake Bothrops asper, to induce LB formation in macrophages and the major cellular signaling pathways involved in this process. The effects of MT-III on ADRP localization and expression and macrophage ultrastructure were assessed. Our results showed that this sPLA(2) induced a marked increase in LB numbers in macrophages, induced the recruitment of ADRP in macrophages, and up-regulated ADRP expression. Ultrastructural analysis showed the presence of weakly and strongly osmiophilic LBs in sPLA(2)-stimulated cells. Enlargement of the ER and Golgi cisterns was also observed. Pretreatment of cells with H7 or staurosporine (PKC inhibitors), LY294002 or wortmannin (PI3K inhibitors), SB202190 or PD98059 (p38(MAPK) and ERK1/2 inhibitors, respectively), or Pyr-2 or Bel (cPLA(2) and iPLA(2) inhibitors, respectively) significantly reduced sPLA(2)-induced LB formation. Herbimycin (a PTK inhibitor) and indomethacin or etoricoxib (COX inhibitors) failed to alter sPLA(2)-induced effects. In conclusion, our results show for the first time the ability of a venom sPLA(2) to induce the formation of LBs and the expression of ADRP in macrophages. Venom PLA(2)-induced LB formation is dependent on PKC, PI3K, p38(MAPK), ERK1/2, cPLA(2), and iPLA(2) signaling pathways but not on PTK, COX-1, or COX-2 pathways. Activation of the ER and Golgi complex may play an important role in the formation of LBs induced by this sPLA(2) in macrophages.
Subject(s)
Group II Phospholipases A2/metabolism , Inclusion Bodies/metabolism , Macrophages/metabolism , Signal Transduction/physiology , Animals , Blotting, Western , Bothrops , Crotalid Venoms/metabolism , Crotalid Venoms/pharmacology , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Immunohistochemistry , Inclusion Bodies/ultrastructure , Lipids , Macrophages/drug effects , Macrophages/ultrastructure , Male , Mice , Microscopy, Electron, TransmissionABSTRACT
Leptospirosis is a zoonosis caused by pathogenic bacteria from the genus Leptospira. The disease represents a serious public health problem in underdeveloped tropical countries. Leptospires infect hosts through small abrasions in the skin or mucous membranes and they rapidly disseminate to target organs. The capacity of some pathogenic leptospiral strains to acquire the negative complement regulators factor H (FH) and C4b binding protein correlates with their ability to survive in human serum. In this study we assessed the functional consequences of the age macular degeneration-associated polymorphism FH His4°² or FH Tyr4°² on FH-Leptospira interactions. In binding assays using sub-saturating amounts of FH, the FH Tyr4°² variant interacted with all the strains tested more strongly than the FH His4°² variant. At higher concentrations, differences tended to disappear. We then compared cofactor activities displayed by FH His4°² and FH Tyr4°² bound to the surface of L. interrogans. Both variants exhibit similar activity as cofactors for Factor I-mediated cleavage of C3b, thus indicating that they do not differ in their capacity to regulate the complement cascade.
ABSTRACT
Human papillomaviruses (HPVs) are responsible for the most common human sexually transmitted viral infections. Infection with high-risk HPVs, particularly HPV16, is associated with the development of cervical cancer. The papillomavirus L1 major capsid protein, the basis of the currently marketed vaccines, self-assembles into virus-like particles (VLPs). Here, we describe the expression, purification and characterization of recombinant HPV16 L1 produced by a methylotrophic yeast. A codon-optimized HPV16 L1 gene was cloned into a non-integrative expression vector under the regulation of a methanol-inducible promoter and used to transform competent Pichia pastoris cells. Purification of L1 protein from yeast extracts was performed using heparin-sepharose chromatography, followed by a disassembly/reassembly step. VLPs could be assembled from the purified L1 protein, as demonstrated by electron microscopy. The display of conformational epitopes on the VLPs surface was confirmed by hemagglutination and hemagglutination inhibition assays and by immuno-electron microscopy. This study has implications for the development of an alternative platform for the production of a papillomavirus vaccine that could be provided by public health programs, especially in resource-poor areas, where there is a great demand for low-cost vaccines.
Subject(s)
Capsid Proteins/metabolism , Human papillomavirus 16/metabolism , Oncogene Proteins, Viral/metabolism , Pichia/virology , Blotting, Western , Capsid Proteins/isolation & purification , Cell Transformation, Viral/physiology , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation, Viral , Hemagglutination Inhibition Tests , Human papillomavirus 16/ultrastructure , Microscopy, Electron, Transmission , Oncogene Proteins, Viral/isolation & purification , Papillomavirus Infections/metabolism , Pichia/metabolismABSTRACT
Human papillomaviruses (HPVs) are responsible for the most common human sexually transmitted viral infections. Infection with high-risk HPVs, particularly HPV16, is associated with the development of cervical cancer. The papillomavirus L1 major capsid protein, the basis of the currently marketed vaccines, self-assembles into virus-like particles (VLPs). Here, we describe the expression, purification and characterization of recombinant HPV16 L1 produced by a methylotrophic yeast. A codon-optimized HPV16 L1 gene was cloned into a non-integrative expression vector under the regulation of a methanol-inducible promoter and used to transform competent Pichia pastoris cells. Purification of L1 protein from yeast extracts was performed using heparin-sepharose chromatography, followed by a disassembly/reassembly step. VLPs could be assembled from the purified L1 protein, as demonstrated by electron microscopy. The display of conformational epitopes on the VLPs surface was confirmed by hemagglutination and hemagglutination inhibition assays and by immuno-electron microscopy. This study has implications for the development of an alternative platform for the production of a papillomavirus vaccine that could be provided by public health programs, especially in resource-poor areas, where there is a great demand for low-cost vaccines.
Subject(s)
Humans , /isolation & purification , Papillomavirus Infections/prevention & control , Papillomavirus VaccinesABSTRACT
Infections with human papillomavirus type 16 (HPV-16) are closely associated with the development of human cervical carcinoma, which is one of the most common causes of cancer death in women worldwide. At present, the most promising vaccine against HPV-16 infection is based on the L1 major capsid protein, which self-assembles in virus-like particles (VLPs). In this work, we used a lactose-inducible system based on the Lactobacillus casei lactose operon promoter (plac) for expression of the HPV-16 L1 protein in L. casei. Expression was confirmed by Western blotting, and an electron microscopy analysis of L. casei expressing L1 showed that the protein was able to self-assemble into VLPs intracellularly. The presence of conformational epitopes on the L. casei-produced VLPs was confirmed by immunofluorescence using the anti-HPV-16 VLP conformational antibody H16.V5. Moreover, sera from mice that were subcutaneously immunized with L. casei expressing L1 reacted with Spodoptera frugiperda-produced HPV-16 L1 VLPs, as determined by an enzyme-linked immunosorbent assay. The production of L1 VLPs by Lactobacillus opens the possibility for development of new live mucosal prophylactic vaccines.
Subject(s)
Capsid Proteins/metabolism , Lacticaseibacillus casei/metabolism , Oncogene Proteins, Viral/metabolism , Recombination, Genetic , Viral Vaccines , Virion/metabolism , Animals , Antibodies, Viral/blood , Antibodies, Viral/immunology , Capsid Proteins/administration & dosage , Capsid Proteins/genetics , Capsid Proteins/immunology , Cells, Cultured , Human papillomavirus 16/metabolism , Humans , Immunization , Lacticaseibacillus casei/genetics , Lactose/pharmacology , Mice , Mice, Inbred BALB C , Oncogene Proteins, Viral/administration & dosage , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/immunology , Operon , Promoter Regions, Genetic , Protein Conformation , Spodoptera , Viral Vaccines/administration & dosage , Viral Vaccines/genetics , Viral Vaccines/immunologyABSTRACT
Infections with human papillomavirus type 16 (HPV-16) are closely associated with the development of human cervical carcinoma, which is one of the most common causes of cancer death in women worldwide. At present, the most promising vaccine against HPV-16 infection is based on the L1 major capsid protein, which self-assembles in virus-like particles (VLPs). In this work, we used a lactose-inducible system based on the Lactobacillus casei lactose operon promoter (plac) for expression of the HPV-16 L1 protein in L. casei. Expression was confirmed by Western blotting, and an electron microscopy analysis of L. casei expressing L1 showed that the protein was able to self-assemble into VLPs intracellularly. The presence of conformational epitopes on the L. casei-produced VLPs was confirmed by immunofluorescence using the anti-HPV-16 VLP conformational antibody H16.V5. Moreover, sera from mice that were subcutaneously immunized with L. casei expressing L1 reacted with Spodoptera frugiperda-produced HPV-16 L1 VLPs, as determined by an enzyme-linked immunosorbent assay. The production of L1 VLPs by Lactobacillus opens the possibility for development of new live mucosal prophylactic vaccines.
Subject(s)
Female , Humans , /classification , VaccinesABSTRACT
Os mais importantes reservatórios do vírus influenza são os pássaros. A manutenção do vírus influenza em hospedeiros naturais, inclusive o homem, permite que esse vírus realize rearranjos entre as suas cepas. O recente relato de uma cepa influenza aviária A(H5N1), em humanos, se deu em uma criança com doença respiratória fatal, na China em 1977. O presente estudo foi conduzido para elucidar o transporte da influenza por pássaros que migram, anualmente, através de ambos hemisférios o do Norte e do Sul, com especial atenção voltada à espécies Vireo olivaceo [Juruviara(BR) e Red-eyed vireo(USA)] que viaja do USA para o Brasil, e vice-versa, e a espécie Elaenia mesoleuca [Tuque(BR) e (USA)] que voa por todo o Hemisfério Sul. Essas espécies de pássaros, que residem e migram em São Paulo, e que demonstram transportar o vírus influenza, foram selecionadas. As partículas virais isoladas foram observadas por microscópio eletrônico. O vírus influenza foi detectado pelos testes: House Duplex/PCR e Gloria. Os resultados revelam que os pássaros das espécies: Elaenia mesoleuca e Vireo olivaceus são transportes do vírus influenza enquanto cruzam ambos Hemisférios. Para o conhecimento da função que os pássaros migratórios podem desempenhar na epidemia de influenza, no Brasil, caracterização dos subtipos deste vírus estão sendo realizados.
Subject(s)
Influenza A virus , Viruses , Birds , In Vitro Techniques , Animal Migration , Culture Media , MethodsABSTRACT
The objective was to study Orthomyxovirus in heterothermic animals. Blood samples from snakes (genus Bothrops and Crotalus) and from toads and frogs (genus Bufo and Rana) were collected to evaluate the red cell receptors and antibodies specific to influenza virus by the hemagglutination and hemagglutination inhibition tests, respectively. Both snakes and toads kept in captivity presented receptors in their red cells and antibodies specific to either influenza virus type A (human and equine origin) or influenza type B. The same was observed with recently captured snakes. Concerning the influenza hemagglutination inhibition antibodies protective levels were observed in the reptiles' serum, against influenza type A and type B. Unlike the toads, 83.3% of the frogs presented mean levels of Ab 40HIU for some influenza strains. It was concluded that heterothermic animals could offer host conditions to the influenza virus and also susceptibility to the infection.
Subject(s)
Alphainfluenzavirus/isolation & purification , Antibodies, Viral/blood , Anura/virology , Betainfluenzavirus/isolation & purification , Viperidae/virology , Animals , Hemagglutination Inhibition Tests , Alphainfluenzavirus/immunology , Betainfluenzavirus/immunologyABSTRACT
O objetivo foi pesquisar Ortomyxovirus em animais heterotérmicos. Coletou-se sangue de serpentes dos gêneros Bothrops e Crotalus e de sapo e rãs dos gêneros Bufo e Rana, para a detecção dos receptores de hemácias e anticorpos específicos, ao vírus influenza, pelos testes de hemaglutinação e inibição da hemaglutinação, respectivamente. Pelo teste de hemaglutinação, verificou-se que serpentes e sapos em cativeiro apresentaram receptores em suas hemácias para o vírus influenza, humano e eqüino do tipo A e tipo B. O mesmo ocorreu com serpentes recém chegadas. Quanto ao teste de inibição da hemaglutinação dos soros dos répteis observou-se títulos protetores de anticorpos aos vírus influenza tipo A (origens humana e eqüina) e tipo B. Com soro de sapo não se observou reação de inibição da hemaglutinação porém, 83,3 por cento das rãs obtiveram médias de 40UIH para algumas cepas. Conclui-se que animais heterotérmicos podem oferecer condições de hospedeiros aos vírus influenza, assim como susceptibilidade à infecção.
Subject(s)
Animals , Anura , Orthomyxoviridae , Influenza B virus , Viperidae , Influenza, Human , Antibodies, Viral , Hemagglutination Inhibition TestsABSTRACT
EPEC e EHEC constituem um risco significativo para a saúde pública em diferentes partes do mundo. Ambas colonizam a mucosa intestinal e subvertem as funções celulares do epitélio intestinal ao produzirem uma lesão histopatológica característica, conhecida por lesão A/E (attaching-and-effacing), na qual a intimina é uma das proteínas envolvidas. A família das intiminas apresenta também uma região conservada, que compreende os aminoácidos de 388 a 667 (Int 388-667). O objetivo do presente trabalho foi a obtenção de um anticorpo policlonal contra a região conservada de intimina. A caracterização fenotípica das amostras de EPEC e EHEC utilizando este anticorpo permitiu observar-se a maneira variável que ele reconhece os diversos subtipos de intimina e sugere que ele seja uma ferramenta para detecção destes patógenos, sendo o ensaio de immuno-dot o método de captura de escolha.
ABSTRACT
Intimins are outer membrane proteins expressed by enteric bacterial pathogens capable of inducing intestinal attachment-and-effacement lesion (A/E). Through immunoblotting, immunofluorescence, flow citometry and immunogold we observed that the obtained polyclonal antibody against conserved intimin region recognizes the different intimin subtypes and suggests that it can be used as a tool for EPEC and EHEC detection. Besides, immuno-dot assay seems to be a possible alternative as a capture method.
EPEC e EHEC constituem um risco significativo para a saúde pública em diferentes partes do mundo. Ambas colonizam a mucosa intestinal e subvertem as funções celulares do epitélio intestinal ao produzirem uma lesão histopatológica característica, conhecida por lesão A/E (attaching-and-effacing), na qual a intimina é uma das proteínas envolvidas. A família das intiminas apresenta também uma região conservada, que compreende os aminoácidos de 388 a 667 (Int 388-667). O objetivo do presente trabalho foi a obtenção de um anticorpo policlonal contra a região conservada de intimina. A caracterização fenotípica das amostras de EPEC e EHEC utilizando este anticorpo permitiu observar-se a maneira variável que ele reconhece os diversos subtipos de intimina e sugere que ele seja uma ferramenta para detecção destes patógenos, sendo o ensaio de immuno-dot o método de captura de escolha.
ABSTRACT
Intimins are outer membrane proteins expressed by enteric bacterial pathogens capable of inducing intestinalattachment-and-effacement lesion (A/E). Through immunoblotting, immunofluorescence, flow citometry andimmunogold we observed that the obtained polyclonal antibody against conserved intimin region recognizesthe different intimin subtypes and suggests that it can be used as a tool for EPEC and EHEC detection.Besides, immuno-dot assay seems to be a possible alternative as a capture method.
Subject(s)
Antibodies , Antibodies/immunologyABSTRACT
Realizou-se um acompanhamento da resposta destes anfíbios à indução de anemia hemolítica pela fenilhidrazina, dentro de uma cinética de 7 pontos, abrangendo o estágio pré-anêmico, considerado normal e no 5º, 10º, 15º, 20º, 30º e 50º dias após a anemização, durante a maturação eritrocitária, por diferentes métodos morfológicos, através das microscopias de luz e eletrônica, e por citometria de fluxo. Conjuntamente, os resultados experimentais sugerem que a atividade gênica nas células eritróides de Odontophrynus americanus 4n é mais intensa quando comparada com as células eritróides de O. americanus 2n, em resposta ao estresse fisiológico. Avaliou-se a expressão da hemoglobina em O. americanus 2n e 4n, através da determinação dos valores hematológicos, eletroforese de hemoglobinas em acetato de celulose em pH alcalino, agar em pH ácido, focalização isoelétrica e eletroforese de cadeias globínicas, a fim de obter informações quanto à regulação da expressão gênica no sistema diplóide-tetraplóide. [...] Tentou-se correlacionar o papel das mitocôndrias com a biossíntese final das moléculas de hemoglobina, dentro do contexto da hemossomogênese, em células eritróides íntegras e fracionadas, através do mapeamento ultra-estrutural imunocitoquímico da hemoglobina. [...] Os resultados experimentais aliados aos dados obtidos na literatura sugerem a proposição de nova nomenclatura científica para os O. americanus 2n e 4n, classificando-os como espécies distintas.