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ESTUDOS
AVANÇADOS
INTERDISCIPLINARES
VOLUME 21
Organizadores:
Robson Antonio Tavares Costa
Estélio Silva Barbosa
ESTUDOS
AVANÇADOS
INTERDISCIPLINARES
VOLUME 21
Organizadores
Organizadores:
Organizador
Dr.Robson
ROBSONAntonio
ANTONIO
TAVARES
COSTA
Tavares
Costa
Robson Antonio Tavares Costa
Estélio Silva Barbosa
EDITORA ENTERPRISING
Direção Nadiane Coutinho
Gestão de Editoração Antonio Rangel Neto
Gestão de Sistemas João Rangel Costa
Conselho Editorial ·Antonio Augusto Teixeira Da Costa, Phd – Ulht – Pt
·Eraldo Pereira Madeiro, Dr – Unitins – Br
·Eugenia Maria Mariano da Rocha Barichello, Dra.
UFSM;
·Luama Socio, Dra. - Unitins - Br
·Ismael Fenner, Dr. - Fics – Py
·Francisco Horácio da Silva Frota, Dr. UECE;
·Tânia Regina Martins Machado, Dra. - Unitins – Br;
·Agnaldo de Sousa Barbosa, Dr. UNESP.
Copyright © 2023 da edição brasileira.
by Editora Enterprising.
Copyright © 2023 do texto.
by Autores.
Todos os direitos reservados.
Todo o conteúdo apresentado neste livro,
inclusive correção ortográfica e gramatical, é
de responsabilidade do(s) autor(es). Obra sob
o selo Creative Commons-Atribuição 4.0
Internacional. Esta licença permite que outros
remixem, adaptem e criem a partir do
trabalho, para fins não comerciais, desde que
lhe atribuam o devido crédito e que licenciem
as novas criações sob termos idênticos.
Diagramação
João Rangel Costa
Design da capa
Nadiane Coutinho
Revisão de texto
Os autores
EDITORA ENTERPRISING
www.editoraenterprising.net
E-mail: [email protected]
Tel. : +55 61 98229-0750
CNPJ: 40.035.746/0001-55
Robson Antonio Tavares Costa
Estélio Silva Barbosa
(Organizadores)
Estudos Avançados
Interdisciplinares
Volume 21
Brasília - DF
E82
Estudos Avançados Interdisciplinares Volume 21 / Robson Antonio
Tavares Costa (Organizador), Estélio Silva Barbosa (Organizador)Brasília: Editora Enterprising, 2023.
(Estudos Avançado Interdisciplinares Volume 21)
Livro em PDF
170p., il.
ISBN: 978-65-84546-43-1
DOI: 10.29327/5242655
1.Interdisciplinares. 2. Pesquisas. 3. Práticas. 4. Estudos.
I. Título.
CDD: 370
Acreditamos que o conhecimento é a grande estratégia de inclusão e integração, e a escrita é a grande
ferramenta do conhecimento, pois ela não apenas permanece, ela floresce e frutifica.
Equipe Editora Enterprising.
Sumário
08
APRESENTAÇÃO
CAPÍTULO 1:
A INCLUSÃO DOS ALUNOS COM DEFICIÊNCIA: DESAFIOS
SOCIOPOLÍTICOS E ORIENTAÇÕES NORMATIVO-PEDAGÓGICAS EM
ÂMBITO NACIONAL
09
Railene dos Santos Monteiro
Ricardo Figueredo Pinto
CAPÍTULO 2:
FATORES E MOTIVAÇÕES NA DÁDIVA DE SANGUE NO CONTEXTO
LUSOBRASILEIRO
26
Ivonete Ferreira Maciel
Danilele Ramos Guedes
Simone do Socorro Azevedo Lima
CAPÍTULO 3:
CORRIDA DE RUA E SISTEMA IMUNOLÓGICO RUNNING AND THE
IMMUNE SYSTEM
55
Gabriel Lemos da Costa
Moisés Simão Santa Rosa de Sousa
Virvalene Costa Melo
Ricardo Figueredo Pinto
CAPÍTULO 4:
PERFIL NUTRICIONAL DE PACIENTES RENAIS EM HEMODIÁLISE: UMA
REVISÃO INTEGRATIVA DA LITERATURA
65
Carliane de Jesus Louzeiro
Joseana Moreira Assis Ribeiro
Fábio Costa de Vasconcelos
CAPÍTULO 5:
CONTRIBUIÇÃO DAS ATIVIDADES FÍSICAS SOBRE OS IMPACTOS DO
TRABALHO PROLONGADO EM POSIÇÃO ORTOSTÁTICA EM
TRABALHADORES DO CENTRO COMERCIAL DE BELÉM-PA
Moisés Simão Santa Rosa de Sousa
Natália de Nazaré Martins Vasconcelos
Virvalene Costa Melo
Ricardo Figueredo Pinto
73
CAPÍTULO 6:
IMPORTÂNCIA DA NUTRIÇÃO PARA OS PRATICANTES DE
MUSCULAÇÃO
110
Lucas Levy Silva de Oliveira
Caroline Laila Segundo S. Nascimento
Joseana Moreira Assis Ribeiro
CAPÍTULO 7:
A IMPORTÂNCIA DA AJUDA DOS PAIS NO TRATAMENTO
ODONTOLÓGICO DOS FILHOS
122
Maria Betânia Furtado Araújo
Bruno de Souza Carvalho Tavares
CAPÍTULO 8:
LUTO E A PSICOLOGIA HOSPITALAR
132
Elizandra Vieira Nogueira
Bruno de Souza Carvalho Tavares
CAPÍTULO 9:
CRICOTIREOIDOSTOMIA:
EMERGÊNCIA
CIRÚRGICA
E
POR
PUNÇÃO
NA
144
Laisa Katrine Lemos do Carmo
Bruno de Souza Carvalho Tavares
CAPÍTULO 10:
A IMPORTÂNCIA DA AUDITORIA INTERNA PARA AS ORGANIZAÇÕES
Tayane Carmo da Silva
155
Apresentação
Prezados(as) leitores(as),
É com muita satisfação que apresentamos o vigésimo primeiro
volume
da
Coleção
intitulada
“ESTUDOS
AVANÇADOS
INTERDISCIPLINARES”, que reúne em seus capítulos pesquisadores
de diversas instituições com discussões e temáticas que circundam
uma gama de possibilidades de pesquisas e de relações dialógicas
que certamente podem ser relevantes para o desenvolvimento
social brasileiro a partir de uma ótica que contempla as mais
vastas questões da sociedade. Tal obra visa dar publicidade a
estudos e pesquisas frutos de árduos trabalhos acadêmicos que
decerto contribuem, cada um a seu modo, para o aprofundamento
de discussões em suas respectivas áreas pois são pesquisas
germinadas, frutificadas e colhidas de temas atuais que estão
sendo debatidos nas principais universidades nacionais e que
refletem o interesse de pesquisadores no desenvolvimento social e
científico que possam impactar positivamente a qualidade de vida
de homens e de mulheres.
Assim sendo, convidamos todos os leitores para exercitar diálogos
com os estudos aqui contemplados, esperamos que os textos
publicados contribuam para a formação intelectual e a reflexão
crítica dos alunos, professores e demais leitores. Desejamos
ressaltar, em nome de todos que compõem a Editora Enterprising,
a nossa gratidão para com os pesquisadores cujos trabalhos
aparecem aqui reunidos, que diante da dedicação, temos a
oportunidade de nos debruçar acerca de assuntos atuais e
pertinentes.
Sejam bem-vindos e tenham proveitosas leituras!
Equipe Editora Enterprising.
Capítulo 1
A INCLUSÃO DOS ALUNOS
COM DEFICIÊNCIA: DESAFIOS
SOCIOPOLÍTICOS E
ORIENTAÇÕES NORMATIVOPEDAGÓGICAS EM ÂMBITO
NACIONAL
DOI: 10.29327/5242655.1-1
Railene dos Santos Monteiro
Ricardo Figueredo Pinto
ESTUDOS AVANÇADOS INTERDISCIPLINARES Vol. 21
A INCLUSÃO DOS ALUNOS COM DEFICIÊNCIA: DESAFIOS
SOCIOPOLÍTICOS E ORIENTAÇÕES NORMATIVO-PEDAGÓGICAS EM
ÂMBITO NACIONAL
Railene dos Santos Monteiro
Ricardo Figueredo Pinto
RESUMO
Este artigo tem como objetivo analisar o desafio histórico-social da educação dos alunos com
deficiência na perspectiva da inclusão como direito humano/social, apresentar alguns elementos
fundamentais da política nacional de inclusão no ensino regular e verificar se são adequadas ao
desenvolvimento da capacidade pessoal e social, integral e plena do aluno, conforme reza o artigo
4º, III da LBD/96. Assim utilizou-se como metodologia uma pesquisa documental e bibliográfica a
fim de que houvesse resultados mais concisos e coerentes. De modo, que resultou nas discussões
sobre pressupostos sociopolíticos e orientações normativo-pedagógicas que a fundamentam,
estruturam e organizam a inclusão educacional no âmbito nacional, trazendo em um único estudo
apontamentos sobre a Constituição Federal de 1988; Declaração Mundial de Educação para Todos
de 1990; Declaração de Salamanca de 1994; Lei de Diretrizes e Bases da Educação Nacional de
1996; Resolução CNE/CEB nº 2, de 2001, paralelo ao que já foi produzido sobre a temática no
Brasil. Logo, este artigo trás discussões importantes ao campo da educação inclusiva e abre
horizontes para novos estudos.
Palavras-chaves: Alunos com deficiência. Desafios Sociopolíticos. Inclusão educacional.
ABSTRACT
This article aims to analyze the historical-social challenge of educating students with disabilities
from the perspective of inclusion as a human/social right, to present some fundamental elements of
the national policy of inclusion in regular education and to verify whether they are adequate for the
development of capacity personal and social, integral and full of the student, as stated in article 4,
III of LBD/96. Thus, a documentary and bibliographical research was used as a methodology in
order to have more concise and coherent results. Thus, it resulted in discussions on sociopolitical
assumptions and normative-pedagogical guidelines that underlie, structure and organize educational
inclusion at the national level, bringing in a single study notes on the Federal Constitution of 1988;
World Declaration on Education for All 1990; 1994 Salamanca Declaration; Law of Guidelines and
Bases of National Education of 1996; CNE/CEB Resolution nº 2, from 2001, parallel to what has
already been produced on the subject in Brazil. Therefore, this article brings important discussions
to the field of inclusive education and opens horizons for new studies.
Keywords: Students with disabilities. Sociopolitical Challenges. educational inclusion
10
ESTUDOS AVANÇADOS INTERDISCIPLINARES Vol. 21
RESUMEN
Este artículo tiene como objetivo analizar el desafío histórico-social de educar a los estudiantes con
discapacidad desde la perspectiva de la inclusión como derecho humano/social, presentar algunos
elementos fundamentales de la política nacional de inclusión en la educación regular y verificar si
son adecuados. para el desarrollo de la capacidad personal y social, integral y plena del alumno,
conforme lo dispuesto en el artículo 4, III de la LBD/96. Así, se utilizó como metodología una
investigación documental y bibliográfica para tener resultados más concisos y coherentes. Así,
resultó en discusiones sobre supuestos sociopolíticos y lineamientos normativo-pedagógicos que
sustentan, estructuran y organizan la inclusión educativa a nivel nacional, trayendo en un solo
apuntes de estudio sobre la Constitución Federal de 1988; Declaración Mundial sobre Educación
para Todos 1990; Declaración de Salamanca de 1994; Ley de Lineamientos y Bases de la
Educación Nacional de 1996; Resolución CNE/CEB nº 2, de 2001, paralela a lo ya producido sobre
el tema en Brasil. Por lo tanto, este artículo trae discusiones importantes para el campo de la
educación inclusiva y abre horizontes para nuevos estudios.
Palabras Clave: Estudiantes con discapacidad. Desafíos sociopolíticos. inclusión educative.
1. INTRODUÇÃO
A constituição de uma política nacional de educação inclusiva de pessoas com deficiência
possui um itinerário marcado por desafios sociopolíticos, no sentido de assegurá-la como direito
humano e social, assim como também desafiador é o estabelecimento de diretrizes-paradigmas de
orientações normativo-pedagógicas do processo de ensino-aprendizagem adequados e eficazes a
essa condição pessoal e social, dada a complexidade do fenômeno em questão.
Na concepção tradicional de atendimento pedagógico sustentava-se a integração escolar do
aluno às estruturas físicas, administrativa, curricular, pedagógica e política da escola, onde teria que
ser capaz de aprender no nível pré-estabelecido pelo sistema de ensino.
“Numa perspectiva de integração, o indivíduo com deficiência não é recusado no ambiente
escolar. Ele pode participar, desde que se adapte, desde que reúna condições individuais
necessárias para estar em um dado ambiente.” (CAMARGO, 2008, p. 76-77).
Da integração dessas pessoas na escola, partiu-se para uma concepção de inclusão escolar,
cujo processo de ensino-aprendizagem envolve uma inserção total e incondicional do aluno e exige
a transformação da escola, pois defende a inclusão de alunos com quaisquer deficiências e
necessidades, preferencialmente no sistema de ensino regular, cabendo às escolas adaptarem-se às
suas especificidades.
As escolas inclusivas propõem um modo de organização do sistema educacional que
considera as necessidades de todos os alunos e que é estruturado em função dessas
necessidades. (MANTOAN, 2003, p. 24).
11
ESTUDOS AVANÇADOS INTERDISCIPLINARES Vol. 21
O Brasil fez opção pela construção de um sistema educacional inclusivo ao concordar com a
Declaração Mundial de Educação para Todos, firmada em Jomtien, na Tailândia, em 1990, e ao
mostrar consonância com os postulados produzidos em Salamanca (Espanha, 1994) na Conferência
Mundial sobre Necessidades Educacionais Especiais: Acesso e Qualidade. Desses documentos,
ressaltam-se alguns princípios inspiradores que orientam a política nacional de atendimento de
alunos com deficiência, presentes na Constituição Federal de 1988 (CF/88), na Lei de Diretrizes e
Bases da Educação (1996) e na Resolução CNE/CEB nº 2, de 11 de setembro de 2001, que
estabelece as Diretrizes Nacionais para a Educação Especial na Educação Básica. Deste modo, a
necessidade e o amplo debate sobre a constituição de uma política de educação inclusiva levou o
Brasil a adotar a concepção e proposta de inclusão de alunos com deficiências preferencialmente na
rede regular, conforme preceitua os artigos 208, III da CF/88 e 4º, III e 58 da LDB/96.
O amplo contexto da reflexão histórico-social e teórica, os grandes embates na elaboração e
implantação de paradigmas para as orientações normativo-pedagógicas e a complexidade do
processo de ensino-aprendizagem adequados à essa condição pessoal e social, faz entender que a
inclusão escolar desses alunos é um processo desafiador que envolve todos os atores que atuam e
influem o ambiente institucional escolar (família, pais, amigos, direção, coordenação pedagógica,
serviço especializado, alunos, professores), como também, a interação e inserção nos grupos
sociais (BRASIL/MEC, 2001, p. 6).
O objetivo desse sucinto trabalho é analisar esse desafio da inclusão escolar da pessoa com
deficiência no sentido de assegurar a educação como direito humano e social, como também estudar
de onde emanam e como se dá princípios e as orientações normativo-pedagógicas do processo de
ensino-aprendizagem, tendo como finalidade verificar se são adequadas ao desenvolvimento da
capacidade pessoal e social, integral e plena do aluno, conforme reza o artigo 4º, III e V da LBD/96.
2. O DESAFIO DA EDUCAÇÃO DA PESSOA COM DEFICIÊNCIA COMO DIREITO
HUMANO E SOCIAL
Segundo Sassaki (1997), o processo de inclusão e em particular, o de inclusão educacional,
desencadeou-se a partir da reflexão histórico-social e cultural de conceitos, mentalidades e
comportamentos considerados deficitários/diferentes/minoritários no padrão social. Quatro períodos
norteariam a história das pessoas com deficiência:
12
ESTUDOS AVANÇADOS INTERDISCIPLINARES Vol. 21
a) Período da exclusão: é aquele identificado antes do século XX, onde as pessoas com deficiências
não participavam de qualquer tipo de educação em escolas, sendo pessoas rejeitadas, perseguidas e
ignoradas pela sociedade;
b) Período da segregação: ocorre já no século XX e se estende até mais ou menos à década de
cinquenta. O atendimento às crianças com deficiências é dado exclusivamente nas grandes
instituições especializadas. Na década de 60, surgem as escolas especiais e mais tarde as classes
especiais dentro das escolas comuns. O que faz surgir no sistema educacional brasileiro dois
subsistemas: a educação comum e a educação especial;
c) Período da integração: localizado na década de 70, onde as escolas comuns passaram a aceitar os
alunos com deficiências em salas comuns. Os alunos tinham que se adaptar às escolas, seus
currículos, sua estrutura, enfim, tudo o que ela proporcionava. E aqueles que não se adaptavam
eram então excluídos. Surgem as classes especiais dentro das escolas regulares;
d) Período da Inclusão: surge na forma em que se encontra hoje; tem seu início a partir da segunda
metade da década de 80, implementou-se na década de 90 e permeia as ideias deste século (XXI) e
abre possibilidades acerca da visão futura. Neste último período, a proposta é de adaptar a escola
para receber os alunos com deficiência, onde as diferenças devem ser aceitas e, assim, a escola
comum se tornar inclusiva.
Tratando-se de Brasil, vê-se que sua história da educação está fortemente marcada pela
exclusão escolar. Desde a Colonização, os alunos eram diferenciados e classificados de acordo com
a sua classe social, cor, gênero, raça, tipo de deficiência entre outras classificações excludentes. As
oportunidades eram para poucos e somente a elite tinha acesso à escola, sendo este o quadro
educacional do país até a primeira metade do século XX (MAZZOTA, 2005, p. 16).
“Falar sobre a educação especial no Brasil implica, necessariamente, a consideração de dois
aspectos constitutivos de nossa historia: a desigualdade e a diversidade. O país é construído
a partir da diversidade de populações e de suas histórias, mas de forma extremamente
desigual. A formação econômica do Brasil e as características de sua organização social
fizeram com que o país passasse a conviver com vários problemas que impactaram
diretamente a vida de crianças e jovens brasileiros, muitos dos quais presentes ate hoje:
crianças abandonadas nas ruas das grandes cidades, desde o século XVII (Lima &
Venâncio, 1991), restrita cobertura escolar e, consequentemente, um numero grande de
analfabetos (Gomes, 2001; Lima, 2011), entre outros. Findado o regime escravocrata,
muitas famílias não foram incorporadas diretamente ao setor produtivo, passando a
sobreviver nas grandes cidades, sem acesso a condições de vida minimamente satisfatórias.
Na República, a massa de brasileiros desempregada e considerada iletrada foi identificada
como marginal e seus hábitos vistos como indecentes e de transgressão aos bons costumes,
13
ESTUDOS AVANÇADOS INTERDISCIPLINARES Vol. 21
aos olhos de uma elite que tomava seu país como atrasado em relação a Europa.”
Somente a partir da segunda metade do século XX, com a expansão de instituições privadas
de educação especial (de cunho assistencialista) (ROSADO, 2009, p. 3), as mudanças sóciopolíticas da década de 1950/60 (período pós-guerra “era de ouro”, desenvolvimentismo moderno de
JK) e a grande demanda de “pessoas especiais” que não conseguiam ser incluídas nas instituições
públicas, começou-se a refletir no meio político e a elaborar, de forma organizada e
institucionalizada, o direito à educação escolar das “pessoas com deficiência”, mas com o objetivo
de adequar o sistema educacional nacional ao discurso da formação de recursos humanos para o
desenvolvimento do país, numa referência à teoria do capital humano (PUZIOL e SILVA, 2013):
“Em 1967, a primeira Constituição após o Golpe de Estado de 1964 previu o
estabelecimento dos planos nacionais de educação. A Emenda Constitucional de 1969
estabeleceu a execução dos planos nacionais e regionais de desenvolvimento. A legislação
sobre as diretrizes e bases da educação nacional foi revista e, em 1971, a Lei Educacional
n° 5.692 passou a obrigatoriedade da escolarização brasileira para oito anos. Esse período
foi decisivo para o início da formatação da Educação Especial como uma política de
Estado, com a criação de um órgão vinculado ao Ministério da Educação e Cultura, o
Centro Nacional de Educação Especial (CENESP), que passou a ter a responsabilidade de
formular e impulsionar as ações de Educação Especial no Brasil. A criação deste órgão e a
implantação de suas ações encontraram subsídio na perspectiva desenvolvimentista adotada
pelo regime militar à época” (KASSAR, 2001, p. 44).
Nesse contexto, por meio de Campanhas Nacionais (ROSADO, 2009, p. 5) e de amplo debate
para promover um atendimento educacional mais adequado aos deficientes em todo o território
nacional, a educação especial começa a se constituir enquanto direito subjetivo e social. A primeira
Lei de Diretrizes e Bases da Educação (Lei nº. 4.024/61), estabelece a diretriz normativa e
pedagógica de incluir preferencialmente o aluno “excepcional” no sistema educacional comum, mas
de ainda contar com as parcerias de instituições privadas, demostrando ainda a omissão do Estado,
como se podem ver no Título X, artigos 88 e 89 da referida lei:
“Art. 88. A educação de excepcionais deve no que for possível, enquadrar-se no sistema
geral de educação, a fim de integrá-los na comunidade.
Art. 89. Toda iniciativa privada considerada eficiente pelos conselhos estaduais de
educação, e relativa à educação de excepcionais, receberá dos poderes públicos tratamento
14
ESTUDOS AVANÇADOS INTERDISCIPLINARES Vol. 21
especial mediante bolsas de estudo, empréstimos e subvenções.”
Em 1971, com a reforma do ensino de 1º e 2º graus, a LDB de 1961 foi alterada, passando a
vigorar a Lei 5.692/71. Nesta Lei, a educação direcionada aos deficientes está presente no artigo 9º,
referente ao capítulo do ensino de 1º e 2º graus, significando que a educação dos “excepcionais”
está incluída na educação básica.
Com o advento da CF/88, é definida a educação como um direito de todos, assegurando o
pleno desenvolvimento da pessoa, o exercício da cidadania e a qualificação para o trabalho (artigo
205). É estabelecida a igualdade de condições de acesso e permanência na escola como um dos
princípios para o ensino (artigo 206, inciso I). A garantia do direito à educação das pessoas com
deficiência é dever do Estado e sua oferta de atendimento educacional especializado deve acontecer
preferencialmente na rede regular de ensino (artigo 208, inciso III). Fica definida, portanto, uma
concepção de educação inclusiva numa perspectiva ampla com atendimento a todas as pessoas em
idade escolar, tendo o Estado (União, Estados, Municípios e Distrito Federal) como responsável por
garantir o direito à educação e prestar o serviço às pessoas com deficiências preferencialmente no
ensino regular.
Convém lembrar que a proposta de uma política nacional de educação de pessoas com
deficiências como direito humano e social, conforme os referidos diplomas e dispositivos legais,
segundo Laplane (2004, p. 18), ainda está distante de ser alcançado efetivamente, devido a um
conjunto de fatores que delineiam um quadro complexo de exclusão social e econômico no país,
que levam, muitas vezes, o atendimento aos moldes tradicionais de não-segregação (integração ao
sistema educacional pré-estabelecido) ou a verdadeira ausência de atendimento educacional:
O discurso contradiz a realidade educacional brasileira, caracterizada por salas
superlotadas, instalações físicas insuficientes, quadros docentes cuja formação deixa a
desejar. Essas condições de existência do nosso sistema educacional levam a questionar a
própria ideia de inclusão como política que, simplesmente, insira alunos nos contextos
escolares existentes.
Sobre esse quadro complexo de exclusão social e econômico no país, Kassar (2012, p. 11) se
expressa:
“Na última década, diferentes trabalhos (Goncalves, 2008; Kassar, 2006; Pletsch, 2010)
apontam para situações de fracasso de alunos com deficiências nas escolas comuns,
inclusive quando todos os quesitos previstos pela legislação educacional estão presentes
(professores formados, salas adaptadas, número de alunos reduzidos por sala, frequência
15
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em salas de recursos multifuncionais no contraturno escolar, entre outros). Os resultados
encontrados em pesquisas brasileiras parecem discrepar dos resultados de pesquisas em
outros países de economia central, como as relatadas por Downing e MacFarland (2010).
No entanto, uma análise que enfoque a escolaridade das crianças com deficiências em
contraste com a educação geral brasileira nos permite dizer que aqueles alunos não são os
únicos a não demonstrar bom desempenho escolar. Expandindo nosso foco para o ensino
fundamental brasileiro, e possível identificar vários problemas. Inicialmente, pode-se
apontar sua ineficiência em relação a matricular – de fato – toda a população. Lima (2011)
explica que, desde a década de 1990, apesar do ensino fundamental brasileiro apresentar
condições físicas (capacidade instalada) para atender a todos os indivíduos na faixa etária
adequada (numero de escolas sufi cientes), dado o considerável numero de indivíduos fora
da faixa etária na escola (distorção idade x série) e de indivíduos em idade própria fora
dela, ainda não cumpriu esta obrigação. As análises de Pinto e Alves (2010) também
contribuem para entender a dinâmica presente em nossas escolas. Além dos problemas
apontados por Lima (2011), temos baixo investimento de recursos em educação pública,
quando comparamos recursos do Fundo de Manutenção e Desenvolvimento da Educação
Básica e de Valorização dos Profissionais da Educação (Fundeb) de diferentes estados e o
investimento em educação pública de países de economia central e mesmo de escolas de
elite do estado de São Paulo.”
Apesar das políticas públicas preconizarem a garantia de acesso escolar e a aceitação da
pessoa com deficiência, com um esforço coletivo na equiparação de oportunidades, a literatura
aponta muitas dificuldades para sua implementação na realidade brasileira. Esse descompasso entre
o que está na legislação e o que se constata na prática leva a pensar o desafio da inclusão escolar
como parte da inclusão social e econômica, sendo um “processo pelo qual a sociedade se adapta
para poder incluir, em seus sistemas sociais gerais, pessoas com necessidades especiais e,
simultaneamente, estas se preparam para assumir seus papéis na sociedade” (SASSAKI, 1997, p.
41).
Há uma clara necessidade de que os sujeitos da escola e da comunidade participem de sua
construção, reconhecendo as diferenças entre os alunos de modo a desenvolver um projeto político
pedagógico a partir de suas necessidades e das experiências vivenciadas na sala de aula, tendo como
garantia a qualidade da educação básica para uma formação integral e plena de acordo com a
capacidade de cada um (LDB/96, artigo 4º, V).
Portanto, para construir uma educação que abranja todos os segmentos da sociedade e cada
um dos seus cidadãos é necessária uma estratégia de ação que tenha princípios e diretrizes
normativo-pedagógicas acerca do processo de ensino-aprendizagem de alunos com deficiência
baseado na inclusão de todos, quaisquer que sejam suas limitações e possibilidades individuais e
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sociais.
3. PRINCÍPIOS
E
DIRETRIZES
NORMATIVO-PEDAGÓGICAS
ACERCA
DO
PROCESSO DE ENSINO-APRENDIZAGEM DE ALUNOS COM DEFICIÊNCIA
Para uma ampla e profunda análise dos princípios e diretrizes normativo-pedagógicas que
orientam o processo de ensino-aprendizagem em âmbito nacional de pessoas com deficiência, além
do estudo dos dois grandes marcos referenciais nacionais - a Lei de Diretrizes e Bases da Educação
Nacional (LDB/96) e a Resolução CNE/CEB n.º 02, de 11 de setembro de 2001, que institui as
Diretrizes Nacionais para a Educação Especial na Educação Básica -, faz-se necessário estudar
alguns marcos referenciais internacionais, considerados pressupostos sociopolíticos e princípios
inspiradores dessas diretrizes normativas e pedagógicas nacionais.
No Brasil, a apropriação do discurso favorável à inclusão foi fortemente influenciada por
movimentos e declarações internacionais, desde o final da década de 40, com a Declaração
Universal dos Direitos Humanos, tomando maior impulso a partir dos anos 90 em favor da
implantação das reformas neoliberais, com a já citada teoria do capital humano a favor das teses
desenvolvimentistas do país (PUZIOL e SILVA, 2013).
A Assembleia Geral da Organização das Nações Unidas produziu vários documentos
internacionais, norteadores para o desenvolvimento de políticas públicas de seus países membros,
entre esses, o Brasil, que reconhece seus conteúdos e os respeita na elaboração das políticas
públicas nacionais. Dentre os documentos produzidos, cita-se: Declaração Universal dos Direitos
Humanos (1948); Declaração Mundial Sobre Educação para Todos (1990); Declaração de
Salamanca (1994); Convenção Interamericana para a Eliminação de Todas as Formas de
Discriminação Contra as Pessoas Portadoras de Deficiência (Convenção da Guatemala/1999) e a
Declaração de Montreal sobre a Inclusão (Canadá/2001). Destes, apresentar-se-á alguns pontos
relevantes da Declaração Mundial Sobre Educação para Todos e da Declaração de Salamanca.
Em 1990, organismos internacionais (Banco Mundial, Programa das Nações Unidas para o
Desenvolvimento - PNUD, Fundo das Nações Unidas para a Infância - UNICEF e Organização das
Nações Unidas para a Educação e Cultura - UNESCO) organizaram a Conferência de Educação
Mundial Para Todos (Conferência de Jomtien/Tailândia), em que foram discutidos problemas
relativos à escolaridade da população nos países em desenvolvimento. Ao seu término, foi aprovada
a Declaração de Jomtien (com 10 artigos e o Plano de Ação com 50 itens) onde foram propostas
metas a serem cumpridas pelos países participantes e signatários. Essa Conferência destacou, em
especial, o princípio da universalização e da equidade de acesso à educação, bem expresso no seu
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artigo 3º – Universalizar o acesso à educação e promover a equidade (ONU/UNICEF, 1990):
“1. A educação básica deve ser proporcionada a todas as crianças, jovens e adultos. Para
tanto, é necessário universalizá-la e melhorar sua qualidade, bem como tomar medidas
efetivas para reduzir as desigualdades. 2. Para que a educação básica se torne equitativa, é
mister oferecer a todas as crianças, jovens e adultos, a oportunidade de alcançar e manter
um padrão mínimo de qualidade da aprendizagem. 3. A prioridade mais urgente é melhorar
a qualidade e garantir o acesso à educação para meninas e mulheres, e superar todos os
obstáculos que impedem sua participação ativa no processo educativo. Os preconceitos e
estereótipos de qualquer natureza devem ser eliminados da educação. 4. Um compromisso
efetivo para superar as disparidades educacionais deve ser assumido. Os grupos excluídos –
os pobres; os meninos e meninas de rua ou trabalhadores; as populações das periferias
urbanas e zonas rurais; os nômades e os trabalhadores migrantes; os povos indígenas; as
minorias étnicas, raciais e linguísticas; os refugiados; os deslocados pela guerra; e os povos
submetidos a um regime de ocupação – não devem sofrer qualquer tipo de discriminação no
acesso às oportunidades educacionais. 5. As necessidades básicas de aprendizagem das
pessoas portadoras de deficiências requerem atenção especial. Posteriormente, em 1994, a Conferência Mundial Sobre Necessidades Educativas Especiais:
Acesso e Qualidade, ocorrida em Salamanca/Espanha, sistematizada em 85 artigos em três grandes
partes, teve como resultado a Declaração de Salamanca Sobre Princípios, Política e Práticas na Área
das Necessidades Educativas Especiais. O documento aprovado amplia o conceito de necessidades
especiais na perspectiva da inclusão, inserindo crianças excluídas da escola por trabalho infantil e
abuso sexual e as que têm necessidades especiais graves, afirmando que todas devem ser atendidas
no mesmo ambiente de ensino. Sua Estrutura de Ação em Educação Especial é orientada por vários
princípios, entre esse o de que as escolas devem “acolher todas as crianças, independente de suas
condições físicas, intelectuais, sociais, emocionais, linguísticas ou outras” (artigo 3º).
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Foi durante esta Conferência que o conceito de escola inclusiva passou a ser discutido de
forma mais sistemática, de uma educação acolhedora, adaptada ao aluno com deficiência, bem
articulada politicamente e preparada para receber e ensinar todos em suas singularidades e
particularidades, sendo a escola que deve se adaptar ao aluno e não o contrário, como
historicamente tem acontecido, estabelecendo como princípio fundamental da escola inclusiva
(artigo 7º):
“todas as crianças devem aprender juntas, sempre que possível, independentemente de
quaisquer dificuldades ou diferenças que elas possam ter. Escolas inclusivas devem
reconhecer e responder às necessidades diversas de seus alunos, acomodando ambos os
estilos e ritmos de aprendizagem e assegurando uma educação de qualidade a todos através
de um currículo apropriado, arranjos organizacionais, estratégias de ensino, uso de recurso
e parceria com as comunidades. Na verdade, deveria existir uma continuidade de serviços e
apoio proporcional ao contínuo de necessidades especiais encontradas dentro da escola”
(UNESCO, 1994).
Nos artigos 8º e 9º a Declaração de Salamanca traça orientações referentes à escola especial
e a escola inclusiva:
“8. O encaminhamento de crianças a escolas especiais ou a classes especiais ou a sessões
especiais dentro da escola em caráter permanente deveriam constituir exceções, a ser
recomendado somente naqueles casos infrequentes onde fique claramente demonstrado que
a educação na classe regular seja incapaz de atender às necessidades educacionais ou
sociais da criança ou quando sejam requisitados em nome do bem-estar da criança ou de
outras crianças. 9. Finalmente, escolas especiais ou unidades dentro das escolas inclusivas
podem continuar a prover a educação mais adequada a um número relativamente pequeno
de crianças portadoras de deficiências que não possam ser adequadamente atendidas em
classes ou escolas regulares” (UNESCO, 1994).
Com base nos artigos 3º, 7º, 8º e 9º, percebe-se a concepção de inclusão desta Declaração que
reconhece o princípio de igualdade de oportunidade para todas as pessoas com necessidas especiais
ou não, sempre que possível em escolas regulares e que, exceções a essa regra, devem ser
consideradas individualmente, caso a caso, quando a educação em instituição específica seja
requerida. E ainda que, nos casos excepcionais das escolas especiais, a educação não precisa ser
inteiramente segregada, estabelecendo um fluxo de movimento da escola especial para a regular e a
organização de um trabalho integrado.
Nesse contexto sociopolítico macro, as Declarações de Jomtien e de Salamanca influenciaram
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fortemente a elaboração e a promulgação da LDB/96, em seus treze títulos e cento e vinte artigos,
considerada uma lei baseada no princípio do direito universal à educação. A LDB/96, no Título II,
Dos Princípios e Fins da Educação Nacional, art. 2º e 4º; Titulo III, Do Direito à Educação e do
Dever de Educar, artigos 4º e 5º; e no Título IV, Da Organização da Educação Nacional, artigo 8º,
10º e 11º, fundamenta-se em princípios da CF/88, das Declarações de Jomtien e de Salamanca. O
Estado (União, os Estados, o Distrito Federal e os Municípios) é garantidor de direitos e prestador
de serviços a todos às pessoas e deve se organizar em regime de colaboração entre os seus entes
federados para efetivo atendimento aos mesmos.
A nova LDB/96 reservou um capítulo específico para a educação especial, o Capítulo V Da
Educação Especial, artigos 58, 59 e 60, fato relevante para uma área tão pouco contemplada
historicamente no conjunto das políticas públicas educacionais no Brasil.
Contudo, ao se analisar o Capítulo V desta Lei, percebe-se que o artigo 58 caracteriza a
educação especial como “modalidade especial”, definição que apresenta um “caráter circular, vago
e genérico”, pois prevê, nos parágrafos 1º e 2º, a existência de apoio especializado no ensino regular
e de serviços especiais separados quando não for possível a inclusão.
O artigo 59 aponta as providências ou apoio, de ordem escolar ou de assistência, que os
sistemas de ensino deverão assegurar aos alunos considerados público alvo da educação especial.
Preconiza que os sistemas de ensino devem assegurar aos alunos currículo, métodos, recursos e
organização específica para atender às suas necessidades; assegura a terminalidade específica
àqueles que não atingiram o nível exigido para a conclusão do ensino fundamental, em virtude de
suas deficiências; e garante a aceleração de estudos aos superdotados para conclusão do programa
escolar.
O artigo 60 prevê o estabelecimento de critérios de caracterização das instituições privadas de
educação especial, através dos órgãos normativos dos sistemas de ensino, para o recebimento de
apoio técnico e financeiro público; ao mesmo tempo em que reafirma, em seu parágrafo único, a
preferência pela ampliação do atendimento no ensino regular público.
Além desses artigos que tratam especificamente da educação especial, aponta-se, também,
como fundamentais para o estabelecimento de princípios da inclusão escolar de pessoas com
deficiências, o artigo 3º, inciso I, que garante igualdade de condições para o acesso e permanência
na escola e inciso III, que afirma o direito ao pluralismo de ideias e de concepções pedagógicas; e o
artigo 4º, inciso III, que garante o atendimento educacional especializado gratuito aos educandos
com deficiência, transtornos globais do desenvolvimento e altas habilidades ou superdotação,
transversal a todos os níveis, etapas e modalidades, preferencialmente na rede regular de ensino, e
inciso V, que assegura acesso aos níveis mais elevados do ensino, da pesquisa e da criação artística,
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segundo a capacidade de cada um.
Para implementar e viabilizar esse processo de mudanças sociopolíticas e contribuir para a
normatização da proposta de inclusão educacional prevista na LDB/96, é expedida pela Câmara
Nacional de Educação (CNE), através de sua Câmara de Educação Básica (CEB), a Resolução
CNE/CEB nº 2 de 11 de setembro de 2001, que estabelece as Diretrizes Nacionais para a Educação
Especial na Educação Básica, com base no Parecer CNE/CEB nº 17/2001. A finalidade é atender
alunos que apresentem deficiência em todas as suas etapas e modalidades, tendo início na educação
infantil, nas creches e pré-escolas, assegurando-lhes os serviços de educação especial sempre que se
evidencie, mediante avaliação e interação com a família e a comunidade, a necessidade de
atendimento educacional especializado.
Através desta Resolução, as Diretrizes Nacionais para Educação Especial na Educação Básica
assumem a educação especial como modalidade da educação escolar, entendendo-a como:
“um processo educacional definido por uma proposta pedagógica que assegure recursos e
serviços educacionais especiais, organizados institucionalmente para apoiar, complementar,
suplementar e, em alguns casos, substituir os serviços educacionais comuns, de modo a
garantir a educação escolar e promover o desenvolvimento das potencialidades dos
educandos que apresentam necessidades educacionais especiais, em todas as etapas e
modalidades da educação básica” (BRASIL, 2001, artigo 3º).
Segundo o artigo 4º, a educação especial deverá considerar as situações singulares, os perfis
dos estudantes, as características biopsicossociais dos alunos e suas faixas etárias e se pautar em
princípios éticos, políticos e estéticos de modo a assegurar:
“I - a dignidade humana e a observância do direito de cada aluno de realizar seus projetos
de estudo, de trabalho e de inserção na vida social; II - a busca da identidade própria de
cada educando, o reconhecimento e a valorização das suas diferenças e potencialidades,
bem como de suas necessidades educacionais especiais no processo de ensino e
aprendizagem, como base para a constituição e ampliação de valores, atitudes,
conhecimentos, habilidades e competências; III - o desenvolvimento para o exercício da
cidadania, da capacidade de participação social, política e econômica e sua ampliação,
mediante o cumprimento de seus deveres e o usufruto de seus direitos”.
As especificidades do aluno com público alvo da educação especial seriam aquelas vistas
durante o processo educacional, tais como: I - dificuldades acentuadas de aprendizagem ou
limitações no processo de desenvolvimento que dificultem o acompanhamento das atividades
curriculares, compreendidas em dois grupos: a) aquelas não vinculadas a uma causa orgânica
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específica; b) aquelas relacionadas a condições, disfunções, limitações ou deficiências; II –
dificuldades de comunicação e sinalização diferenciadas dos demais alunos, demandando a
utilização de linguagens e códigos aplicáveis; III - altas habilidades/superdotação, grande facilidade
de aprendizagem que os leve a dominar rapidamente conceitos, procedimentos e atitudes (artigo 5º).
Segundo o referido documento, os fundamentos da proposta para uma política “inclusiva”
devem valorizar a igualdade de oportunidade e a diversidade do processo educativo, determinando
que as escolas da rede regular de ensino devem prever e prover na organização de suas classes
comuns, entre outros pontos, os seguintes (artigo 8º):
“I - professores das classes comuns e da educação especial capacitados e especializados,
respectivamente, para o atendimento às necessidades educacionais dos alunos;
II - distribuição dos alunos com necessidades educacionais especiais pelas várias classes do
ano escolar em que forem classificados, de modo que essas classes comuns se beneficiem
das diferenças e ampliem positivamente as experiências de todos os alunos, dentro do
princípio de educar para a diversidade;
III – flexibilizações e adaptações curriculares que considerem o significado prático e
instrumental dos conteúdos básicos, metodologias de ensino e recursos didáticos
diferenciados e processos de avaliação adequados ao desenvolvimento dos alunos que
apresentam necessidades educacionais especiais, em consonância com o projeto
pedagógico da escola, respeitada a freqüência obrigatória;
VII – sustentabilidade do processo inclusivo, mediante aprendizagem cooperativa em sala
de aula, trabalho de equipe na escola e constituição de redes de apoio, com a participação
da família no processo educativo, bem como de outros agentes e recursos da comunidade.”
Dessa forma, com essas diretrizes nacionais, o que se pretende é garantir que todos os alunos
tenham acesso escolar e aceitação; que as diferenças sejam acolhidas e que ocorra um esforço
coletivo na equiparação de oportunidades de desenvolvimento com qualidade. Não basta que uma
criança público alvo da educação especial seja incluída em uma escola regular para que haja uma
inclusão progressiva e se promova o sucesso escolar do aluno. Para tanto se faz premente que as
condições necessárias para uma educação de qualidade para todos sejam efetivadas, como consta na
Legislação Educacional e nas Diretrizes Nacionais para a Educação Especial na Educação Básica.
4. CONSIDERAÇÕES FINAIS
Conforme analisado neste trabalho, vivencia-se um momento em que mundialmente se debate
e articula a inclusão dos alunos com deficiência preferencialmente na rede regular de ensino.
Constitui-se, na realidade, um desafio histórico-social a educação na perspectiva da inclusão como
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direito humano e social.
A legislação educacional nacional, com seus princípios e diretrizes normativas, são explícitas
quanto à obrigatoriedade em acolher e matricular todos os alunos, independente de suas
necessidades ou diferenças. Entretanto, não é suficiente apenas esse acolhimento, mas que o aluno
tenha condições efetivas de aprendizagem e de desenvolvimento de suas potencialidades. É
necessário e urgente, assim, que os sistemas de ensino se organizem para além de assegurar essas
matrículas, assegurem também a permanência de todos os alunos, sem perder de vista a
intencionalidade pedagógica e a qualidade do ensino, desenvolvendo mecanismos didáticopedagógicos (operacionais) adequados a essa condição pessoal e social.
O princípio da inclusão representa um grande desafio e demanda mudanças em diversos
níveis, desde o diagnóstico dos alunos, a composição e formação de recursos humanos, passando
pelos currículos, pela metodologia de ensino, até a participação da família e da comunidade escolar
na elaboração do projeto político pedagógico.
Como foi discutido quando se tratou da educação como direito humano e social, a inclusão é
um processo histórico-social-político e, como tal, vem para desconstituir paradigmas passados de
segregação e exclusão. A parceria entre ensino comum e ensino especializado é indicada como uma
possibilidade para a melhoria da educação em geral. A inclusão que se pretende efetivar nas escolas
só é possível onde houver respeito às diferenças e, consequentemente, práticas pedagógicas que
permitam a todas as crianças aprenderem de acordo com seu ritmo e suas potencialidades.
Este trabalho trás contribuições significativas e concisas sobre a temática de inclusão
educacional. Para além deste estudo, sugere-se uma análise detalhada de outros documentos:
nacionais como, tais como, resolução nº 4, de 2 de outubro de 2009 (institui diretrizes operacionais
para o atendimento educacional especializado na educação básica, modalidade
de educação
especial ); decreto nº 7.611, de 17 de novembro de 2011( dispõe sobre a educação especial, o
atendimento educacional especializado; lei nº 13.146/2015(institui a lei brasileira de inclusão da
pessoa com deficiência (Estatuto da Pessoa com Deficiência).
REFERÊNCIAS
BEYER, Hugo Otto. A Educação Inclusiva: incompletudes escolares e perspectivas de ação.
Revista de Educação Especial do Centro de Educação, 2012. Disponível em:
<https://periodicos.ufsm.br/educacaoespecial/article/view/5003>. Acesso em: 22/05/2023.
BRASIL. Lei nº. 4.024, de 20 de dezembro de 1961. Fixa as diretrizes e bases da educação
nacional.
Brasília,
DF,
1961.
Disponível
em:
<http://www.
planalto.gov.br/ccivil_03/Leis/L4024.htm>. Acesso em: 22/05/2023.
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BRASIL. Resolução CNE/CEB nº 2, de 11 de setembro de 2001. Institui Diretrizes Nacionais
para a Educação Especial na Educação Básica. Brasília, DF, 2001. Disponível em:
<http://portal.mec.gov.br/cne/arquivos/pdf/CEB0201.pdf>. Acesso em: 22/05/2023.
BRASIL. Parecer CNE/CEB nº 17/2001. Diretrizes Nacionais para a Educação Especial na
Educação
Básica.
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Disponível
em:
<http://portal.mec.gov.br/seesp/arquivos/pdf/parecer17.pdf>. Acesso em: 22/05/2023.
BRASIL. Secretaria de Educação Fundamental. Parâmetros Curriculares Nacionais: Adaptações
Curriculares. Estratégias para a Educação de Alunos com Necessidades Educacionais
Especiais. Brasília-DF: MEC/SEF/SEESP, 1998.
BRASIL/MEC. Diretrizes Nacionais para a Educação Especial na Educação Básica. BrasíliaDF: SEESP, 2001.
BRASIL. Política Nacional de Educação Especial na Perspectiva da Educação Inclusiva. Portal
MEC,
Brasília,
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em:
<http://portal.mec.gov.br/arquivos/pdf/politicaeducespecial.pdf>. Acesso em: 22/05/2023.
CAMARGO, Éder Pires. Ensino de Física e Deficiência Visual: Dez anos de investigação no
Brasil. São Paulo: Plêiade/FAPESP, 2008.
GIL, A. C. Métodos e técnicas da pesquisa social. 6. ed. São Paulo: Atlas, 2008.
KASSAR, Mônica de Carvalho Magalhães. Educação especial no Brasil: desigualdades e
desafios no reconhecimento da diversidade. Portal SCIELO, São Paulo, 2012. Disponível em:
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_________. Educação especial na perspectiva da educação inclusiva: desafios da implantação
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Development of an Automated Imaging Pipeline for the Analysis of the Zebrafish Larval Kidney
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Jens H. Westhoff
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English
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Spoken
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Jens H. Westhoff1*, Stefan Giselbrecht2, Miriam Schmidts3, Sebastian Schindler1, Philip L. Beales3,
Burkhard Tönshoff1, Urban Liebel4,6*, Jochen Gehrig5,6* Jens H. Westhoff1*, Stefan Giselbrecht2, Miriam Schmidts3, Sebastian Schindler1, Phil
Burkhard Tönshoff1, Urban Liebel4,6*, Jochen Gehrig5,6* 1 Department of Pediatrics I, University Children’s Hospital, University of Heidelberg, Heidelberg, Germany;, 2 Institute for Biological Interfaces , Karlsruhe
Institute of Technology, Eggenstein-Leopoldshafen, Germany, 3 Molecular Medicine Unit, Birth Defects Research Centre, University College London (UCL),
Institute of Child Health, London, United Kingdom, 4 Institute for Applied Computer Science, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen,
Germany, 5 Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany, 6 Accelerator Laboratory, Innovation
Department, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany; Abstract 241955) to PLB, the Stiftung Landesbank Baden-Württemberg to JHW, the Dutch Kidney foundation (CP11.18) to PLB and MS and institutional
funding from Innovation Department, Karlsruhe Institute of Technology (KIT), Germany. MS was funded by an Action Medical Research UK clinical training
fellowship (RTF-1411) and PLB is a Wellcome Trust Senior Research Fellow. We acknowledge support by Deutsche Forschungsgemeinschaft and Open
Access Publishing Fund of Karlsruhe Institute of Technology. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript. Competing interests: The authors have the following conflicts. UL is co-founder and executive director, and JG is application strategist at Acquifer GmbH. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies
on sharing data and materials. * E-mail: [email protected] (JHW); [email protected] (UL); [email protected] (JG) Received July 25, 2013; Accepted October 21, 2013; Published December 4, 2013 Copyright: © 2013 Westhoff et al. This is an open-access article distributed under the terms of the Creative Commons Attributi
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. thoff et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
tion, and reproduction in any medium, provided the original author and source are credited. Funding: This project was supported by the European Commission Seventh Framework Programme funded projects DOPAMINET (http://
www.dopaminet.eu/; grant no. 223744) and Eurenomics (http://www.eurenomics.eu/; grant no. 305608) to UL, and SYSCILIA (http://www.syscilia.org/;
grant no. 241955) to PLB, the Stiftung Landesbank Baden-Württemberg to JHW, the Dutch Kidney foundation (CP11.18) to PLB and MS and institutional
funding from Innovation Department, Karlsruhe Institute of Technology (KIT), Germany. MS was funded by an Action Medical Research UK clinical training
fellowship (RTF-1411) and PLB is a Wellcome Trust Senior Research Fellow. We acknowledge support by Deutsche Forschungsgemeinschaft and Open
Access Publishing Fund of Karlsruhe Institute of Technology. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript. Competing interests: The authors have the following conflicts. UL is co-founder and executive director, and JG is application strategist at Acquifer GmbH. There are no patents, products in development or marketed products to declare. Abstract This does not alter the authors' adherence to all the PLOS ONE policies
on sharing data and materials. * E-mail: [email protected] (JHW); [email protected] (UL); [email protected] (JG) Citation: Westhoff JH, Giselbrecht S, Schmidts M, Schindler S, Beales PL, et al. (2013) Development of an Automated Imaging Pipeline for the Analysis of
the Zebrafish Larval Kidney. PLoS ONE 8(12): e82137. doi:10.1371/journal.pone.0082137 Abstract The analysis of kidney malformation caused by environmental influences during nephrogenesis or by hereditary
nephropathies requires animal models allowing the in vivo observation of developmental processes. The zebrafish
has emerged as a useful model system for the analysis of vertebrate organ development and function, and it is
suitable for the identification of organotoxic or disease-modulating compounds on a larger scale. However, to fully
exploit its potential in high content screening applications, dedicated protocols are required allowing the consistent
visualization of inner organs such as the embryonic kidney. To this end, we developed a high content screening
compatible pipeline for the automated imaging of standardized views of the developing pronephros in zebrafish
larvae. Using a custom designed tool, cavities were generated in agarose coated microtiter plates allowing for
accurate positioning and orientation of zebrafish larvae. This enabled the subsequent automated acquisition of stable
and consistent dorsal views of pronephric kidneys. The established pipeline was applied in a pilot screen for the
analysis of the impact of potentially nephrotoxic drugs on zebrafish pronephros development in the Tg(wt1b:EGFP)
transgenic line in which the developing pronephros is highlighted by GFP expression. The consistent image data that
was acquired allowed for quantification of gross morphological pronephric phenotypes, revealing concentration
dependent effects of several compounds on nephrogenesis. In addition, applicability of the imaging pipeline was
further confirmed in a morpholino based model for cilia-associated human genetic disorders associated with different
intraflagellar transport genes. The developed tools and pipeline can be used to study various aspects in zebrafish
kidney research, and can be readily adapted for the analysis of other organ systems. Citation: Westhoff JH, Giselbrecht S, Schmidts M, Schindler S, Beales PL, et al. (2013) Development of an Automated Imaging Pipeline for the Analysis of
the Zebrafish Larval Kidney. PLoS ONE 8(12): e82137. doi:10.1371/journal.pone.0082137
Editor: Sheng-Ping Lucinda Hwang, Institute of Cellular and Organismic Biology, Taiwan
Received July 25, 2013; Accepted October 21, 2013; Published December 4, 2013
Copyright: © 2013 Westhoff et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This project was supported by the European Commission Seventh Framework Programme funded projects DOPAMINET (http://
www.dopaminet.eu/; grant no. 223744) and Eurenomics (http://www.eurenomics.eu/; grant no. 305608) to UL, and SYSCILIA (http://www.syscilia.org/;
grant no. Automated Screening Platform for Zebrafish Kidneys Despite rodent studies and observational reports in humans,
detailed
data
on
potential
harmful
side
effects
on
nephrogenesis is still missing for many drugs and chemical
compounds [4,5]. Additionally, there is a lack of large-scale
chemical screens for modifiers of hereditary nephropathies. This is mainly due to the very laborious and time-consuming
testing of substance specific effects on normal and abnormal
organogenesis using standard experimental setups, thus
hampering investigations on a larger scale. Additionally, in vivo
imaging of kidney development in a spatiotemporal context is
not feasible in rodents, necessitating animal models that are
experimentally
more
accessible. Furthermore,
dedicated
imaging techniques are required which enable the in vivo
visualization of developing organs and tissues on a larger
scale. function of the zebrafish pronephros can aid in the
understanding of the role of genes mutated in kidney disease,
or the impact of compounds on renal development and function
in humans [17]. Thus, the combination of this in vivo model
system with automated imaging technologies could serve as a
tool for the large scale analysis of kidney phenotypes. However, to our current knowledge, a screening platform
compatible with in vivo imaging of zebrafish larval kidneys has
not been described yet. Here, we delineate the development of an automated HCS
compatible imaging pipeline designed for live imaging of
zebrafish kidneys in chemical screening scenarios. Using a
custom designed orientation tool, embryos could be accurately
positioned in wells of microtiter plates allowing consistent
imaging of dorsal views of the pronephros. Subsequent
automated imaging was performed on a standard widefield
screening microscope and a data handling and visualization
pipeline was developed. A pilotscreen for morphological kidney
abnormalities was performed using a subset of potentially
nephrotoxic drugs applied to larvae of the Tg(wt1b:EGFP)
transgenic line in which the developing pronephros is
highlighted by GFP expression [19]. The obtained in vivo data
was cross-validated by histological analysis. In addition, we
demonstrate that the established microscopy platform can also
be utilized for genetic disease models. The
development
of
high
content
screening
(HCS)
technologies has had a major impact on biomedical and
pharmaceutical research, as these platforms are suitable for a
wide range of large-scale investigations using in vitro and in
vivo model systems [6]. Due to its small size and various other
experimental advantages, the zebrafish can be readily
employed in large scale in vivo assays. Automated Screening Platform for Zebrafish Kidneys Moreover, the high
degree of anatomical and physiological homology to higher
vertebrates renders it a relevant model for biomedical research
[7]. Thus, the zebrafish has emerged as the main vertebrate
model system for whole organism screening experiments. Consequently, the zebrafish has been successfully used in
chemical, toxicological, behavioral and genetic screening
experiments [4,78910–11]. Moreover, its transparency in
combination with the wealth of mutant and transgenic zebrafish
strains available facilitates the large scale analysis of tissue-
specific phenotypes. This includes, for example, the search for
anti-inflammatory, anti-angiogenic or neuroactive compounds
[1213–14]. Fish keeping and embryo handling Adult zebrafish of the Tg(wt1b:EGFP) transgenic line [19]
were maintained according to reference [20]. Eggs were
collected
from
pairwise
and
batch
crossings. The
developmental stage of embryos was determined as previously
described [21]. Embryos were raised in fish water at 28°C. At
24 hpf embryos were enzymatically dechorionated using 10
mg/ml Pronase. Embryos were transferred to a beaker, washed
twice with 400 ml of fish water and transferred into clean petri
dishes [22]. Prior to transferring into agarose coated microtiter
plates, 48 or 72 hpf old larvae were anesthetized using 0.03%
tricaine. Ethics statement All zebrafish husbandry and experimental procedures were
performed in accordance with the German animal protection
standards and were approved by the Government of Baden-
Württemberg,
Regierungspräsidium
Karlsruhe,
Germany
(Aktenzeichen 35-9185.64). Despite its widespread usage in large scale experiments, it
remains challenging to fully exploit the advantages of this
model system in HCS experiments. Zebrafish embryos are
largely incompatible with standard HCS protocols developed
for other model systems, one of the reasons being that their
relatively large size and complex three-dimensional shape
often lead to random positioning and orientation of embryos
within wells of microtiter plates. This especially hampers
reproducibility and detailed visualization of cell or tissue
morphology, as well as developmental processes. Whilst
several studies and recent technological advances have tried
to address these challenges, they often require sophisticated
technical setups [15] or are incompatible with chemical
screening [16]. Thus, there remains a demand of easy-to-use
screening protocols and dedicated tools which can facilitate
performing complex zebrafish HCS assays. Introduction preterm newborns with ongoing nephrogenesis can interfere
with nephron generation and thus can cause short- and
potentially
long-term
kidney
impairment
[2]. Besides
environmental factors, hereditary nephropathies due to
mutations in genes encoding ciliary proteins are the most
common cause of end-stage renal disease in children due to
polycystic, cystic-dysplastic and/or nephronophthisis like renal
phenotypes [3]. Adverse environmental conditions and genetic influences
can have a major impact on organogenesis. According to
statistical surveys, 22-50% of all pregnant women take drugs
within the first trimenon of gestation, often whilst they are
unaware of their pregnancy [1]. However, nephrotoxic
medication taken by pregnant women or administered to December 2013 | Volume 8 | Issue 12 | e82137 1 PLOS ONE | www.plosone.org Automated Screening Platform for Zebrafish Kidneys Morpholino injections Antisense splice blocking morpholino oligonucleotides (Gene
Tools, LLC, Philomath, USA) were designed against the exon1-
intron1 and exon2 –intron2 boundary of zebrafish ift172 gene,
and ift80 morpholino has been previously published [23]. Morpholinos were injected into 1-cell stage embryos at 500 nM
concentration. Both ift172 morpholinos resulted in similar
phenotypes comparable to the phenotype previously published
[2425–26]. A standard control oligonucleotide was used as
control. Morpholino sequences: ift80 splice blocking MO: 5’-
AGGTGTATGTGGAACCTGTGATAAG-3’, ift172 Exon2 splice
donor
blocking
MO:
5’- Data handling, deconvolution and visualization Data handling, generation of multilayer tiffs and generation of
maximum projections were carried out using custom written
Perl scripts and Fiji [28] macros available on request. Fluorescence z-stacks were deconvolved with Huygens
Professional deconvolution software (SVI, Hilversum, The
Netherlands) using a theoretical point spread function based on
microscope parameters. Batch deconvolution was carried out
on a workstation with 24 CPU cores and 64 Gigabyte of
memory. Cropping of images and generation of overview
images was carried out using a Fiji macro (Macro S1). In brief,
maximum projection images were duplicated, automatically
thresholded and the resulting binary images were eroded. The
position of kidneys was detected by measuring the center of
mass. The corresponding coordinates were restored on original
images, a bounding box was defined and images were cropped
accordingly. Cropped images were loaded into a stack and
overview images were generated using the ‘Make Montage’
function of Fiji. ACGAGATGAGAGCTTACTTTTGGGT-3’, ift172 Exon1 splice
blocking
MO:
5’-ACGCTGTCAATATTTTACCTGAGGC-3’,
Standard
control
(ctr)
oligonucleotide:
5’-
CCTCTTACCTCAGTTACAATTTATA-3’. Drug treatment of embryos A subset of certain drug classes was chosen for which an
adverse effect on the developing kidney had been described in
animal and/or human studies [2]. To evaluate concentration-
dependent toxicity, 5 different concentrations of each drug (2.5
mM, 5 mM, 10 mM, 20 mM, 40 mM) were tested. 24 hpf
dechorionated embryos were transferred to 6-well-plates and
treated with the following drugs dissolved in E3 solution with
0.003% 1-pheny-2-thiourea (PTU, Alfa Aesar, Karlsruhe, In the zebrafish larva, the functional pronephros comprises
only 2 nephrons with fused glomeruli located ventrally to the
dorsal aorta. Interestingly, despite tremendous differences in
nephron number, the composition of a single nephron shows
great homology at the cellular and molecular level between
human and zebrafish [17,18]. Additionally, kidney development
and function largely depend on the same orthologous genes for
all vertebrate kidneys. Therefore, studying formation and PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e82137 2 Automated Screening Platform for Zebrafish Kidneys Germany): penicillin G potassium salt (AppliChem, Darmstadt,
Germany), ampicillin sodium salt (AppliChem, Darmstadt,
Germany), gentamicin sulfate (Sigma-Aldrich, St. Louis, USA),
kanamycin sulfate (AppliChem, Darmstadt, Germany), captopril
(CalBiochem, Darmstadt, Germany), losartan potassium salt
(Molekula,
Gillingham,
Dorset,
United
Kingdom),
acetaminophen (Caesar und Loretz, Hilden, Germany),
indomethacin sodium salt (AppliChem, Darmstadt, Germany). Treatment period was 24 hours. For indomethacin, lower
concentrations (0.01 mM, 0.025 mM, 0.05 mM, 0.075 mM and
0.1 mM) had to be applied due to 100% lethality rates at higher
concentrations. 2-4 repeats of each experiment were
performed. Total number of embryos that underwent drug
treatment is shown in Table S1. The pH was adjusted for each
experiment. Following drug treatment, the number of dead
larvae was assessed and the living larvae were transferred to
0.003% PTU containing E3 solution. For imaging studies,
0.03% tricaine was added to the medium. 4°C. Embryos were transferred in 100 µl fish water containing
PTU and tricaine (see above) and manually arrayed and
oriented under a stereomicroscope. To aid in positioning of
regions of interest within the grid-based and fixed field views of
the Scan^R system, embryos were positioned in such a way
that all yolk sacs were approximately at the same position
within cavities using features of the well plate as guidelines. Image acquisition Embryos were imaged on a standard Scan^R high-content
screening microscope (Olympus, Hamburg, Germany) [27] as
previously described [16,22]. Data was acquired using 33 z-
slices (Δz = 15μm) per embryo and channel using a 4x (N.A. =
0.13) objective. Integration times were fixed (80 ms for GFP). Imaging times were approximately 1 hour for a full 96 well
plate. For each experimental plate the A1 positions of the
imaging grid was re-centered to compensate for minor
differences in positioning of embryos. Generation of a 96 well template tool The tool is made from brass and consists of a base plate
with 96 perpendicular pins arranged in a certain way to match
the positions of the wells of a microtiter plate. The tool was
produced by CNC milling (Datron, M7). In a first step, the base
plate with the dimensions of 140 mm x 100 mm x 21 mm was
milled by a four flute end mill (20 mm). Secondly, the array of
96 pins with 11 mm height and rectangular footprint (5.9 mm x
1 mm) was created by partly thinning the base plate down to
9 mm between the pins (Four Flute End Mill, 6 mm and 3 mm,
respectively). Finally, the ends of the 96 pins were tapered to a
conical tip with 60° by utilizing a standard engraving tool. Analysis of morphological and pronephric phenotypes Analysis of morphological and pronephric phenotypes
Overall morphology was scored on a Leica MZ10 F
stereomicroscope (Leica Microsystems, Wetzlar, Germany). Lethality and pericardial/yolk sac edema were rated for each
experiment. Blinded analysis of tubular and glomerular
phenotypes was performed by SS and JHW on maximum
projections of deconvolved z-stacks. Using ImageJ, 2
parameters were manually measured for tubular structures: i)
maximum distance between the tubules and ii) angle between
neck segment and proximal convoluted tubule [18] being visible
in Tg(wt1b:EGFP) zebrafish. For description of glomerular
changes, the distance between the glomeruli was determined
and glomeruli were classified as either normal or showing
glomerular malformation judged by the glomerular area and
structure. Heatmaps were generated using matrix2png [29]. Standard positioning of embryos for chemical
screening The visualization of bilateral symmetric organs of zebrafish
embryos usually demands dorsal or ventral views. However,
consistent large scale imaging of zebrafish embryos remains
challenging in automated screening experiments, as stable and
reproducible positioning is complicated by the size and
complex three-dimensional shape of embryos. The screening system used in this study stores data as
single tiff files in one folder per experimental plate. To reduce
file number and thus facilitate subsequent data handling and
analysis tasks, multilayer tiff files were generated for each
imaging position and channel. Spatial widefield data usually
suffers from out-of-focus blur thus reducing overall image
quality [30]. To restore images, fluorescent datasets were
batch deconvolved with Huygens Professional software using a
theoretical point spread function [16]. Subsequently, maximum
projection images were generated from deconvolved z-stacks
(Figure 2E). The positions of larval kidneys were automatically
detected within maximum projections of deconvolved data
using the center of mass of the corresponding binary image
after automatic thresholding. To restrict image data to the
pronephric region and remove unnecessary areas, a bounding
box was defined around the center of mass and images were
cropped accordingly (Figure 2F). Subsequently, overview
images
were
generated
from
cropped
kidney
images
representing all kidneys in one 96-well plate, allowing for rapid
manual assessment of morphological phenotypes (Figure 2G). In summary, this pipeline allows consistent imaging and rapid
evaluation of gross morphological abnormalities of the
developing zebrafish kidney after compound treatment or in
genetic screens. It can also be easily adapted for the analysis
of other tissues and organs that require consistent imaging. To enable a simplified handling and precise positioning of
zebrafish larvae and to achieve consistent visualization of
tissues in high content screening scenarios, we have
developed a tool to create agarose molds in a standard
microtiter plate. The tool allows the preparation of agarose
coated 96 well plates in a single replication step. The tool
consists of a base plate with 96 rectangular pins, whose
positions exactly match the centers of wells of standard and
commercially available 96 well microtiter plates (Figure 1A, B). The pins end with a keel shaped geometry, which was
previously shown to be suitable for accurate ventral positioning
of zebrafish larvae [16]. Statistical analysis Data were evaluated using IBM® SPSS® Statistics Version
21. For lethality and edema rates and glomerular fusion and
malformation, statistical analysis was performed by Chi-square
test. Datasets of low sample sizes were additionally tested
using Fisher’s exact test. For tubular angle and distance,
means among treatment groups were compared using ANOVA
with Bonferroni correction for multiple comparisons as a post-
hoc test. Significance was defined as p<0.05. Pipeline for automated pronephros imaging To visualize and score renal phenotypes, we developed a
protocol for automated imaging of dorsal views of zebrafish
larval kidneys (Figure 2A). Prior to imaging, compound treated
or microinjected embryos of the Tg(wt1b:EGFP) stable
transgenic line were raised to the desired developmental stage
(48 or 72 hpf) and then transferred into microtiter plates
containing agarose cavities as described above (Figure 2B, C). To automatically image zebrafish kidneys, larvae were
manually positioned and oriented in the agarose cavities and
subsequently imaged on a standard widefield HCS microscope. To ensure capture of entire organs and compensate for minor
variations in z-positioning, each larva was acquired using z-
stacks with 33 slices in the bright field and GFP channel
(Figure 2D). The cavities in the plate allow larvae to be
positioned with high enough accuracy, so that regions of
interest (e.g. pronephros) are located within the limited field of
view for all plated embryos. Thus, the tool permits the organ
and
tissue
specific
screening
on
standard
screening
microscopes using a fixed field of view for all wells, without the
necessity of additional software modules for automatic
detection and centering the region of interests [16]. Histological analysis Histological analysis device allows generation of molds in each well independently,
thus avoiding cross-contamination, which was a major
limitation of the previous design. Furthermore, the restriction to
single wells and the depths of the cavities drastically minimizes
movement of surrounding medium leading to a massively
improved stability of orientation of embryos in comparison to
the silicone template. Thus, plates with oriented embryos and
larvae could be used in combination with automated plate
handling and stacking systems. Larvae were fixed in 4% paraformaldehyde overnight at 4°C. Samples were dehydrated through an ethanol series and
processed for embedding in Paraffin (Surgipath® Paraplast®,
Leica Biosystems, Wetzlar, Germany). 3 μm sections were cut
using a Leica RM 2165 microtome (Leica Microsystems,
Nussloch, Germany). Sections were deparaffinized in xylene
and rehydrated through graded washes of ethanol in water
before staining with hematoxylin and eosin. The stained
sections were imaged with a Leica DMI4000 B microscope
equipped with a Leica DFC320 digital camera. Automated Screening Platform for Zebrafish Kidneys Automated Screening Platform for Zebrafish Kidneys Preparation of agarose molds in microtiter plates Preparation of agarose molds in microtiter plates
70 µl of 1% agarose in fish water was added to each well of
a 96 well microtiter plate (Cat.-No. 655180, Greiner,
Frickenhausen, Germany) using a multi-channel pipette. The
agarose coated well plates were pre-cooled at room
temperature for one minute. To generate grooves the brass
tool was inserted, while adjusting the penetration depth of pins
using spacers. After solidification the tool was carefully
removed and plates were optionally stored in plastic bags at December 2013 | Volume 8 | Issue 12 | e82137 PLOS ONE | www.plosone.org 3 Standard positioning of embryos for chemical
screening The tool was fabricated out of a solid
block of brass using CNC milling (Figure 1A, B), giving rise to a
precise work piece with identical xyz-dimensions of each pin
allowing the generation of deep keel-shaped cavities in wells of
a 96-well plate filled with agarose (Figure 1C). Such prepared
plates can be readily used to manually position specimen
enabling the subsequent automated acquisition of dorsal views
using inverted screening microscope systems (Figure 1D). using inverted screening microscope systems (Figure 1D). We recently demonstrated an alternative protocol for
automated dorsal imaging of oriented larvae using a silicone
tool to generate an array of 96 agarose molds [16]. However,
while plates generated with this tool can be employed for the
consistent automated imaging of tissues, the design prevents
utilization in chemical or drug screening applications. The novel December 2013 | Volume 8 | Issue 12 | e82137 PLOS ONE | www.plosone.org 4 4 Automated Screening Platform for Zebrafish Kidneys Figure 1. Standardized orientation of zebrafish embryos. (A,B) Photographs of the brass tool for the simultaneous generation
of agarose grooves within 96 well microtiter plates: (A) top view and (B) tilted view. For dimensions of the plate see Materials and
Methods section. (C) Schematic depiction of a single well with a ventrally oriented embryo within an agarose cavity. Drawing is not
to scale. (D) Illustrative example of aligned and oriented embryos. Shown are dorsal views of 48 hpf embryos acquired using a 2.5x
objective on an inverted wide field screening microscope. doi: 10.1371/journal.pone.0082137.g001 Figure 1. Standardized orientation of zebrafish embryos. (A,B) Photographs of the brass tool for the simultaneous generation
of agarose grooves within 96 well microtiter plates: (A) top view and (B) tilted view. For dimensions of the plate see Materials and
Methods section. (C) Schematic depiction of a single well with a ventrally oriented embryo within an agarose cavity. Drawing is not
to scale. (D) Illustrative example of aligned and oriented embryos. Shown are dorsal views of 48 hpf embryos acquired using a 2.5x
objective on an inverted wide field screening microscope. doi: 10.1371/journal.pone.0082137.g001 A pilot screen for drug-related effects on kidney
development dependent effects on overall survival rates, edema formation
and pronephric phenotypes. Detailed results are listed in Table
S1-S3. Color coded overview maps were also generated
(Figure 3I-M). To validate phenotypic alterations observed in
the transgenic model and thus confirm the utility of the
proposed screening pipeline, histological analysis of glomerular
and tubular cross-sections of 48 hpf larvae treated at the
highest non-lethal concentration was carried out (Figure S1). The pipeline was subsequently evaluated in a pilot screen to
investigate the impact of potentially nephrotoxic drugs on the
development of the zebrafish pronephros. To this end, a subset
of drugs from different classes was chosen for which an
adverse effect on the developing kidney was previously
identified in animal studies and/or human observations [2]. Dechorionated 24 hpf old embryos were treated with 8 different
drugs in increasing concentrations for 24 hours. Following drug
treatment, live larvae were imaged and data was visualized as
described above. Impact of tested compounds on kidney development Impact of tested compounds on kidney development
In humans, due to the putative absence of fetal toxicity at
therapeutic doses, penicillin antibiotics are widely prescribed to
pregnant women and frequently administered to preterm
newborns [31]. In our study, penicillin G potassium salt
administration increased lethality rates dose-dependently
(Figure 3I). Concomitantly, minor pronephric alterations were
observed including incompletely fused glomeruli (Figure 3B). On the other hand, ampicillin sodium salt did not cause higher
lethality, increased edema rates or major phenotypic renal
alterations (Figure 3C, I-M). Cross-sections of larvae treated
with penicillin G or ampicillin did not show major glomerular or
tubular alterations when compared to untreated control larvae
(Figure
S1A-C),
thus
confirming
results
obtained
by
fluorescence microscopy. Taken together, this indicates only To score lethality rates and development of pericardial and
yolk sac edema, treated larvae were examined on a stereo
microscope. The detailed results are listed in Table S1. To objectively quantify morphological abnormalities of the
pronephros following drug treatment, glomerular and tubular
alterations were discriminated in the Tg(wt1b:EGFP) transgenic
line. Glomerular alterations were subdivided into (i.) glomerular
malformation indicated by abnormal or reduced glomerular
shape and area, and (ii.) incomplete glomerular fusion
representing
aberrant
pronephros
development. Tubular
parameters were classified into (i.) the angle between the
tubular neck segment and the proximal convoluted tubule and
(ii.) variations in the maximum distance between the 2 tubular
systems (Figure 3A). Several drugs showed concentration PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e82137 5 Automated Screening Platform for Zebrafish Kidneys Figure 2. Overview of workflow for the automated imaging of the developing zebrafish pronephros. (A) Overview of the
workflow for screening larval kidneys. The flowchart illustrates the different steps carried out to obtain overview images of kidneys. (B) Initial compound treatment or microinjection of embryos prior to sample preparation and imaging. (C) Schematic illustrating the
transfer of embryos into agarose coated microtiter plates, and alignment and orientation of embryos. (D-G) Acquisition and
processing of image Data. D to F show data of the same embryo. (D) Automated acquisition of z-stacks (33 z-slices, dZ=15µm) on
an inverted widefield screening microscope. (E) Deblurring of images using deconvolution. Shown are maximum projections of z-
stacks of raw data (left panel) and deconvolved data (right panel). (F) Automated detection and cropping of the kidney region. The
red square indicates the position and dimensions of the cropped region. Impact of tested compounds on kidney development (G) Automated generation of overview images for quick
assessment of overall morphological changes. Indomethacin skeletal formula in (A) taken from (http://en.wikipedia.org/wiki/
File:Indometacin_skeletal.svg). d i 10 1371/j
l
0082137
002 Figure 2. Overview of workflow for the automated imaging of the developing zebrafish pronephros. (A) Overview of the
workflow for screening larval kidneys. The flowchart illustrates the different steps carried out to obtain overview images of kidneys. (B) Initial compound treatment or microinjection of embryos prior to sample preparation and imaging. (C) Schematic illustrating the
transfer of embryos into agarose coated microtiter plates, and alignment and orientation of embryos. (D-G) Acquisition and
processing of image Data. D to F show data of the same embryo. (D) Automated acquisition of z-stacks (33 z-slices, dZ=15µm) on
an inverted widefield screening microscope. (E) Deblurring of images using deconvolution. Shown are maximum projections of z-
stacks of raw data (left panel) and deconvolved data (right panel). (F) Automated detection and cropping of the kidney region. The
red square indicates the position and dimensions of the cropped region. (G) Automated generation of overview images for quick
assessment of overall morphological changes. Indomethacin skeletal formula in (A) taken from (http://en.wikipedia.org/wiki/
File:Indometacin_skeletal.svg). doi: 10 1371/journal pone 0082137 g002 minor effects of the 2 β-lactam antibiotics on pronephros
development. only a minor increase in lethality rates without significant
effects on edema formation (Figure 3I, J). Nevertheless,
glomerular malformation and incomplete glomerular fusion
were found for higher drug concentrations. Tubular angle was
slightly widened at higher doses (Figure 3D, M). Histological
analysis of gentamicin-treated larvae revealed no gross
morphological abnormalities, although a mild rarefication of Aminoglycosides,
although
not
recommended
during
pregnancy [31], are often used for treatment of neonatal
sepsis,
even
in
premature
newborns
with
on-going
nephrogenesis. However, serum drug concentrations can be
monitored to minimize the risk of renal and auditory toxicity
[32]. In our study, gentamicin sulfate administration caused PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e82137 6 Automated Screening Platform for Zebrafish Kidneys Figure 3. Overview of compound concentration-dependent pronephric phenotypes. Illustrative examples of pronephroi of a
(A) non-treated embryo, and after treatment with (B) 20 mM penicillin, (C) 40 mM ampicillin, (D) 40 mM gentamicin, (E) 40 mM
kanamycin, (F) 40 mM acetaminophen, (G) 40 mM captopril, (H) 10 mM losartan. For examples of phenotypes after Indomethacin
treatment see Figure 4A-E. Impact of tested compounds on kidney development Arrow and arrowheads in (A) indicate the different morphological parameters of the pronephros scored
to evaluate compound effect on the developing kidney. Arrow: fused glomeruli; Arrowhead: angle between the neck segment and
the proximal convoluted tubule. (I-M) Heatmaps showing (I) lethality rates, (J) edema rates and (K-M) changes in morphological
parameters of the pronephros. In detail, (K) incomplete glomerular fusion, (L) glomerular malformation and (M) tubular angle. For
further details see Materials and Methods and Tables S1-S3. Colour codes indicate the percentage of embryos (I-L) with particular
phenotype, or the angle between neck segment and proximal convoluted tubule (M) as indicated by the colour coded legend. Grey
squares indicate missing data points. Concentration ranges used are indicated above the heatmaps, or below for Indomethacin,
respectively. Abbreviations: penicillin (Pen), ampicillin (Amp), gentamicin (Gen), kanamycin (Kan), acetaminophen (Ace), captopril
(Cap), losartan (Los) and indomethacin (Ind). *p<0.05, **p<0.001. doi: 10.1371/journal.pone.0082137.g003 Figure 3. Overview of compound concentration-dependent pronephric phenotypes. Illustrative examples of pronephroi of a
(A) non-treated embryo, and after treatment with (B) 20 mM penicillin, (C) 40 mM ampicillin, (D) 40 mM gentamicin, (E) 40 mM
kanamycin, (F) 40 mM acetaminophen, (G) 40 mM captopril, (H) 10 mM losartan. For examples of phenotypes after Indomethacin
treatment see Figure 4A-E. Arrow and arrowheads in (A) indicate the different morphological parameters of the pronephros scored
to evaluate compound effect on the developing kidney. Arrow: fused glomeruli; Arrowhead: angle between the neck segment and
the proximal convoluted tubule. (I-M) Heatmaps showing (I) lethality rates, (J) edema rates and (K-M) changes in morphological
parameters of the pronephros. In detail, (K) incomplete glomerular fusion, (L) glomerular malformation and (M) tubular angle. For
further details see Materials and Methods and Tables S1-S3. Colour codes indicate the percentage of embryos (I-L) with particular
phenotype, or the angle between neck segment and proximal convoluted tubule (M) as indicated by the colour coded legend. Grey
squares indicate missing data points. Concentration ranges used are indicated above the heatmaps, or below for Indomethacin,
respectively. Abbreviations: penicillin (Pen), ampicillin (Amp), gentamicin (Gen), kanamycin (Kan), acetaminophen (Ace), captopril
(Cap), losartan (Los) and indomethacin (Ind). *p<0.05, **p<0.001. doi: 10.1371/journal.pone.0082137.g003 Figure 3. Overview of compound concentration-dependent pronephric phenotypes. Automated microscopy screening of genetic kidney
disease models Additionally, captopril and losartan treated larvae displayed
slightly altered glomerular structure in histological sections that
appeared less dense compared to controls (Figure S1G,
H).This data is partially consistent with animal studies showing
renal abnormalities after treatment with ACE inhibitors or
angiotensin receptors [4748–49]. g
y
y
[
]
Splice blocking morpholinos for ift80 and ift172 were
designed as described in the Methods section. By using the
standard positioning tool as described above, automated
imaging
of
dorsally
positioned
morpholino-injected
Tg(wt1b:EGFP) zebrafish (3 dpf) was performed in 96 well
plates (Figure 5A). Microscopy revealed a ventral curvature of
the tail (Figure 5B-D) affecting approximately 90% of all
morpholino
injected
embryos
and
consistent
with
the
phenotype previously described for ift80 morphants [23]. Morphological
alterations
in
fluorescence
microscopy
predominantly consisted of large cystic glomeruli (Figure 5E-G)
that were consistently reproducible [23], while embryos treated
with
standard
morpholino
showed
normal
glomerular
morphology. Cross-sections
of
both
ift80-
and
ift172-
morpholino injected Tg(wt1b:EGFP) zebrafish confirmed the
formation of large pronephric cysts (Figure S2A-C). Tubular
dilatation and epithelial flattening was observed both in
fluorescence images (Figure 5F, G) and histological sections
(Figure S2B, C). This further demonstrates that our pipeline is
suitable for large scale therapeutic screening investigations in
genetic models of renal disease such as ciliopathies. However,
the applicability largely depends on the morphological
phenotypes as severe malformations impair position accuracy
within cavities. NSAIDs are widely used for closure of patent ductus
arteriosus in preterms and are administered during pregnancy
for prevention and treatment of toxemia, polyhydramnions and
premature birth. However, exposure to NSAIDs during
pregnancy can cause hypoperfusion of the fetal kidneys and
acute renal failure in newborns with cystic changes of
developing nephrons and long-term renal dysfunction [2,50]. In
our study, concentrations of the non-selective COX1/COX2
inhibitor indomethacin had to be lowered due to 100% lethality
at higher doses. Strikingly, even at drastically lower
concentrations there was a severe phenotype (Figure 4A-E). This included concentration dependent increases in edema
formation
and
lethality
(Figure
4D,
F). Fluorescence
microscopy (Figure 4A, E) revealed significant concentration
dependent
increases
in
glomerular
malformation
and
incomplete glomerular fusion (Figure 4G). Furthermore, tubular
angles widened and tubular distances slightly shortened
(Figure 4H). Severe renal phenotypes were also confirmed in
histological sections of indomethacin treated larvae. Here, no
regular glomerulus was detectable ventrally to the dorsal aorta
and laterally seen glomerular structures appeared strongly
malformed or were not identifiable. Automated microscopy screening of genetic kidney
disease models been considered safe [38]. Its hepatotoxicity at high doses is
well described [39] and has recently been investigated in
zebrafish [40]. In addition, animal data further revealed fetal
kidney damage following acetaminophen administration to
pregnant rats [41]. In our study, acetaminophen caused
concentration dependent significant alterations of pronephros
morphology and an increase in edema formation, whereas
lethality
rates
remained
unchanged
(Figure
3F,
I-M). Histological sections confirmed severe renal phenotypes
following acetaminophen administration. No fused glomerulus
was detectable ventrally to the dorsal aorta and glomerular
structures appeared strongly malformed. In addition, tubular
epithelium was flattened (Figure S1F). These results match
previously published data showing dose-, duration- and onset-
dependent changes in pronephros morphology following
acetaminophen administration in zebrafish larvae [42]. Beyond performing toxicological screens for kidney damage,
the presented automated microscopy pipeline can be utilized in
the analysis of genetic disease models. Gene-knockdown or
knock-out
models
can
potentially
be
used
for
HCS
investigations for the search of therapeutic strategies for
hereditary kidney diseases. To test the utility of the developed
pipeline, we focused on cilia-associated human genetic
disorders. Intraflagellar transport (IFT) constitutes the bidirectional
transport of protein complexes along axonemal microtubules. IFT plays an essential role in the assembly and function of cilia
and flagella by contributing to cell motility, sensory perception
and cilium-based signaling [51,52]. IFT80 and IFT172 both are
members of the IFT-B subcomplex [53] and while IFT80
mutations in humans have been identified to cause Jeune
asphyxiating thoracic dystrophy [23], a congenital ciliary
chondrodysplasia condition associated with renal disease in
approximately 20% of cases [54], no human mutations in
IFT172 have been identified to date. However, abrogation of
Ift172 function in mice leads to a VACTERL-like phenotype
including renal malformations [55], indicating that IFT172 plays
an important role for kidney development in mammals. In
zebrafish, the insertional mutant line ift172hi2211Tg exhibits
glomerular cysts and a ventral body curvature [56]. Intake of ACE inhibitors and angiotensin receptor blockers
during pregnancy has been associated with fetopathies
including renal pathologies in humans [4344–45] and other
mammals [46]. In our study, captopril at 40 mM significantly
increased edema formation and induced concentration
dependent alterations in glomerular and tubular parameters
(Figure 3G, I-M). Losartan increased lethality rates and
glomerular and tubular parameters at higher concentrations,
while edema rates were unchanged (Figure 3H, I-M). Impact of tested compounds on kidney development Illustrative examples of pronephroi of a
(A) non-treated embryo, and after treatment with (B) 20 mM penicillin, (C) 40 mM ampicillin, (D) 40 mM gentamicin, (E) 40 mM
kanamycin, (F) 40 mM acetaminophen, (G) 40 mM captopril, (H) 10 mM losartan. For examples of phenotypes after Indomethacin
treatment see Figure 4A-E. Arrow and arrowheads in (A) indicate the different morphological parameters of the pronephros scored
to evaluate compound effect on the developing kidney. Arrow: fused glomeruli; Arrowhead: angle between the neck segment and
the proximal convoluted tubule. (I-M) Heatmaps showing (I) lethality rates, (J) edema rates and (K-M) changes in morphological
parameters of the pronephros. In detail, (K) incomplete glomerular fusion, (L) glomerular malformation and (M) tubular angle. For
further details see Materials and Methods and Tables S1-S3. Colour codes indicate the percentage of embryos (I-L) with particular
phenotype, or the angle between neck segment and proximal convoluted tubule (M) as indicated by the colour coded legend. Grey
squares indicate missing data points. Concentration ranges used are indicated above the heatmaps, or below for Indomethacin,
respectively. Abbreviations: penicillin (Pen), ampicillin (Amp), gentamicin (Gen), kanamycin (Kan), acetaminophen (Ace), captopril
(Cap), losartan (Los) and indomethacin (Ind). *p<0.05, **p<0.001. doi: 10.1371/journal.pone.0082137.g003 capillary loops could be seen (Figure S1D). Kanamycin caused
a concentration-dependent increase in lethality and edema
formation (Figure 3I, J). However, glomerular and tubular
parameters remained unaltered (Figure 3E). Concordantly, no
major glomerular or tubular alterations were observed in
histological
sections
of
larvae
following
kanamycin
administration (Figure S1E). In other studies, microinjection of
gentamicin into the cardiac venous sinus led to acute renal
failure [33]. As only minor effects of gentamicin were observed in our study, it suggests that this may have been due to poorer
penetration into inner organs. Several human and animal
studies report on aminoglycoside-induced glomerular and
tubular damage in pre- and at-term newborns [3435–36]. Substance-specific differences in the degree of ototoxic and
nephrotoxic side effects among various aminoglycosides are
well known [37]. In humans, the intake of acetaminophen at therapeutic doses
during gestation and administration to preterm newborns has PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 7 December 2013 | Volume 8 | Issue 12 | e82137 December 2013 | Volume 8 | Issue 12 | e82137 Automated Screening Platform for Zebrafish Kidneys Automated microscopy screening of genetic kidney
disease models Additionally, pronephric
tubular epithelium appeared flattened (Figure S1I). Thus, our
data in larval zebrafish matches previously published data from
other animal models confirming severe renal side effects of
indomethacin on kidney development during nephrogenesis
[50]. Conclusions Here, we demonstrate the development of an automated
screening pipeline for imaging developing kidneys in the
zebrafish larvae. This novel methodology allows for the
consistent acquisition of dorsal views of pronephric kidneys on
a standard inverted screening microscope. The platform can PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e82137 8 Automated Screening Platform for Zebrafish Kidneys Figure 4. Impairment of pronephros development upon indomethacin treatment. (A) Overview of pronephric alterations in
zebrafish larvae (50 hpf) following indomethacin administration for 24 hours. Row 1 shows control embryos, rows 2-6 zebrafish
embryos following indomethacin administration in increasing concentrations (row 2, 0.01 mM; row 3, 0.025 mM; row 4, 0.05 mM;
row 5, 0.075 mM and row 6, 0.1 mM). (B-E) Comparison of (B-C) 50 hpf control larva and (D-E) indomethacin (0.1 mM) treated
larva. (D) Brightfield image shows edema formation following indomethacin administration. (E) Fluorescence image showing
nephron (glomerular and tubular) malformation. (F) Quantification of lethality rates and edema formation following indomethacin
administration. (G) Concentration-dependent increases in glomerular malformation and decreases in glomerular fusion rates
following indomethacin administration. (H) Widened tubular angles between neck segment and proximal convoluted tubule following
indomethacin administration. Data are shown as mean ± SD. *p<0.05, **p<0.001. doi: 10.1371/journal.pone.0082137.g004 Figure 4. Impairment of pronephros development upon indomethacin treatment. (A) Overview of pronephric alterations in
zebrafish larvae (50 hpf) following indomethacin administration for 24 hours. Row 1 shows control embryos, rows 2-6 zebrafish
embryos following indomethacin administration in increasing concentrations (row 2, 0.01 mM; row 3, 0.025 mM; row 4, 0.05 mM;
row 5, 0.075 mM and row 6, 0.1 mM). (B-E) Comparison of (B-C) 50 hpf control larva and (D-E) indomethacin (0.1 mM) treated
larva. (D) Brightfield image shows edema formation following indomethacin administration. (E) Fluorescence image showing
nephron (glomerular and tubular) malformation. (F) Quantification of lethality rates and edema formation following indomethacin
administration. (G) Concentration-dependent increases in glomerular malformation and decreases in glomerular fusion rates
following indomethacin administration. (H) Widened tubular angles between neck segment and proximal convoluted tubule following
indomethacin administration. Data are shown as mean ± SD. *p<0.05, **p<0.001. doi: 10.1371/journal.pone.0082137.g004 the imaging protocol is easy-to-use and can be readily modified
for studying other organ systems or tissues such as the brain
region. Furthermore, in combination with HCS software tools
which enable automated feature of interest detection, it can be serve as a convenient tool in kidney research, e.g. Supporting Information Figure S1. Cross-sections of pronephric regions after
compound exposure. Shown are glomerular (upper panels)
and tubular (lower panels) sections at 48 hours post
fertilization. (A) control, (B) penicillin (20 mM), (C) ampicillin (40
mM), (D) gentamicin (40 mM), (E) kanamycin (40 mM), (F)
acetaminophen (40 mM), (G) captopril (40 mM), (H) losartan
(10 mM) and (I) indomethacin (0.75 mM) treatment. (TIF) Figure S1. Cross-sections of pronephric regions after
compound exposure. Shown are glomerular (upper panels)
and tubular (lower panels) sections at 48 hours post
fertilization. (A) control, (B) penicillin (20 mM), (C) ampicillin (40
mM), (D) gentamicin (40 mM), (E) kanamycin (40 mM), (F)
acetaminophen (40 mM), (G) captopril (40 mM), (H) losartan
(10 mM) and (I) indomethacin (0.75 mM) treatment. (TIF) The current pipeline only allows for scoring of gross
morphological abnormalities of the pronephros. Therefore, we
validated the impact of treatments on pronephros formation by
histological analysis, which largely confirmed the phenotypes
observed in the transgenic model. This further demonstrates
the utility of the established screening pipeline to score
pronephric phenotypes. Moreover, the histological analysis
revealed additional alterations, such as epithelial flattening,
which are more difficult to score in the fluorescence data, thus
complementing the in vivo approach. Although protocols for
large scale histology experiments exist [57], the fast filtering of
compound libraries by in vivo screening is considerably more
time- and cost effective. Therefore, we propose that in a
genuine large scale experiment, histological analysis is only
carried out as a follow-up experiment in combination with
molecular methods to characterize hits identified in a screen. Figure S2. Cross-sections of pronephric regions after
morpholino injections. Shown are glomerular (upper panels)
and tubular (lower panels) sections at 72 hours post
fertilization. (A) control-MO, (B) ift80-MO and (C) ift172-MO
injection. (TIF) Figure S2. Cross-sections of pronephric regions after
morpholino injections. Shown are glomerular (upper panels)
and tubular (lower panels) sections at 72 hours post
fertilization. (A) control-MO, (B) ift80-MO and (C) ift172-MO
injection. (TIF) Table S1. Lethality and edema formation in zebrafish
larvae following drug treatment. (DOCX) The mode of drug administration imposes another limitation
of the established pipeline, as penetration to inner organs can
be hampered by the chemical and physical properties of the
noxa and the biological barrier of the larval zebrafish skin. This
can be overcome by microinjection of drugs into the blood
stream as described by Hentschel et al. [33]. Conclusions in chemical
studies as a primary screening tool to identify organotoxic
substances or to search for potential therapeutic compounds
that attenuate renal pathology in disease models. Importantly, PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e82137 9 Automated Screening Platform for Zebrafish Kidneys 5. Detection of cystic kidneys after Ift80 and Ift172 knockdown. (A) Automatically generated overview thumbnail i
kidneys of morpholino injected larvae: standard ctr-MO (row 1-2), ift80-MO (row 3-5) and ift172-MO (row 6-8). ypic alterations of zebrafish larvae at 4 dpf after morpholino microinjection; (C) ift80-MO and (D) ift172-MO injected em
glomerular cyst (arrow) and a ventrally curved tail compared to (B) standard ctr-MO injected embryos. (E-G) In-
ation of glomerular cysts at 72 hpf in (F) ift80-MO and (G) ift172-MO injected embryos compared to standard ctr-MO inj
s (E) using the Tg(wt1b:EGFP) transgenic line. Figure 5. Detection of cystic kidneys after Ift80 and Ift172 knockdown. (A) Automatically generated overview thumbnail image
of 96 kidneys of morpholino injected larvae: standard ctr-MO (row 1-2), ift80-MO (row 3-5) and ift172-MO (row 6-8). (B-D)
Phenotypic alterations of zebrafish larvae at 4 dpf after morpholino microinjection; (C) ift80-MO and (D) ift172-MO injected embryos
exhibit glomerular cyst (arrow) and a ventrally curved tail compared to (B) standard ctr-MO injected embryos. (E-G) In-detail
visualisation of glomerular cysts at 72 hpf in (F) ift80-MO and (G) ift172-MO injected embryos compared to standard ctr-MO injected
embryos (E) using the Tg(wt1b:EGFP) transgenic line. doi: 10.1371/journal.pone.0082137.g005 Detection of cystic kidneys after Ift80 and Ift172 knockdown. (A) Autom
dneys of morpholino injected larvae: standard ctr-MO (row 1-2)
ift80-MO Figure 5. Detection of cystic kidneys after Ift80 and Ift172 knockdown. (A) Automatically generated overview thumbnail image
of 96 kidneys of morpholino injected larvae: standard ctr-MO (row 1-2), ift80-MO (row 3-5) and ift172-MO (row 6-8). (B-D)
Phenotypic alterations of zebrafish larvae at 4 dpf after morpholino microinjection; (C) ift80-MO and (D) ift172-MO injected embryos
exhibit glomerular cyst (arrow) and a ventrally curved tail compared to (B) standard ctr-MO injected embryos. (E-G) In-detail
visualisation of glomerular cysts at 72 hpf in (F) ift80-MO and (G) ift172-MO injected embryos compared to standard ctr-MO injected
embryos (E) using the Tg(wt1b:EGFP) transgenic line. Macro S1. Fiji macro to crop images and generate
overview images.
(ZIP) Macro S1. Fiji macro to crop images and generate
overview images. (ZIP) Conclusions doi: 10.1371/journal.pone.0082137.g005 December 2013 | Volume 8 | Issue 12 | e82137 10 PLOS ONE | www.plosone.org Automated Screening Platform for Zebrafish Kidneys be developed or modified to be compatible with automated
imaging assays, respectively [61]. used
for
the
automated
acquisition
of
standardized
multidimensional high resolution datasets. The pilot screen for nephrotoxic drugs during nephrogenesis
revealed
a
concentration
dependent
effect
of
several
compounds on nephrogenesis. Thus, considering the lingering
lack of data for many substances, subsequent large-scale
investigations performed with this screening pipeline might
contribute
to
our
understanding
of
substance-specific
nephrotoxic side effects. Acknowledgements Genetic research over the last years has demonstrated that
the zebrafish pronephros is a valuable model system for the
study of hereditary human nephropathies as abnormalities in
podocyte gene function, renal epithelial primary cilia genes and
renal ion channels and transporters lead to defective
pronephric kidney function in the zebrafish mimicking human
disease [59]. However, screening for disease modulating
compounds in a zebrafish model requires convenient and
accessible protocols. Here, we demonstrate that the developed
imaging pipeline can also be utilized to detect abnormal
phenotypes in genetic disease models. Thus, it could serve as
a platform for prospective high-content drug screening
experiments. We thank Sebastian Hötzel for fish care, J. Bohn for CNC
milling, B. Rodenbeck for technical assistance and Dr
Christoph Englert (Leibniz Institute for Age Research-Fritz
Lipmann Institute, Jena, Germany) for kindly providing the
Tg(wt1b:EGFP) transgenic line. We are grateful to Dr Jens
Fahrenberg (Innovation Department, Karlsruhe Institute of
Technology, Germany) for general support. We thank Sundeep
Dhillon for critically reading the manuscript. Supporting Information Duration of drug
treatment and its transferability to the human situation is
another limitation of the pilot screen in zebrafish. In zebrafish,
glomerular filtration starts around 48 hpf and the pronephros is
fully matured at 4 dpf [58]. Hence, due to the rapid
embryogenesis of the zebrafish, future studies have to employ
different treatment periods to analyze the impact of compounds
at the different stages of nephrogenesis [42]. Table S2. Glomerular alterations in zebrafish larvae
following drug treatment. (DOCX) Table S3. Tubular angle and distance in zebrafish larvae
following drug treatment. (DOCX) Macro S1. Fiji macro to crop images and generate
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CD: 005091. 12. d’Alençon CA, Peña OA, Wittmann C, Gallardo VE, Jones RA et al. (2010) A high-throughput chemically induced inflammation assay in
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21176202. 33. Hentschel DM, Park KM, Cilenti L, Zervos AS, Drummond I et al. (2005) Acute renal failure in zebrafish: a novel system to study a
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Stages of embryonic development of the zebrafish. Dev Dyn 203:
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Automated high-throughput mapping of promoter-enhancer interactions
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Chemical modifier screen identifies HDAC inhibitors as suppressors of
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Developmental toxicity screening in zebrafish. Birth Defects Res C
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intraflagellar transport mutants disrupt cilia but do not affect hedgehog
signaling. Dev Dyn 238: 1744-1759. doi:10.1002/dvdy.21999. PubMed:
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approaches and technologies. Birth Defects Res B Dev Reprod Toxicol
92: 413-420. doi:10.1002/bdrb.20315. PubMed: 21770025. 26. Sukumaran S, Perkins BD (2009) Early defects in photoreceptor outer
segment morphogenesis in zebrafish ift57, ift88 and ift172 Intraflagellar
Transport mutants. Vision Res 49: 479-489. doi:10.1016/j.visres. 2008.12.009. PubMed: 19136023. pp
g
p
92: 413-420. doi:10.1002/bdrb.20315. PubMed: 21770025. 6. Pepperkok R, Ellenberg J (2006) High-throughput fluorescence
microscopy for systems biology. Nat Rev Mol Cell Biol 7: 690-696. doi:
10.1038/nrm1979. PubMed: 16850035. 27. Author Contributions Conceived and designed the experiments: JHW JG SG MS
PLB. Performed the experiments: SS JHW JG MS. Analyzed
the data: JHW SS MS JG. Contributed reagents/materials/
analysis tools: SG. Wrote the manuscript: JHW JG MS. Designed the orientation tool: SG JG. Generated data handling
and visualization tools: JG. Designed morpholino injections
experiments: MS PLB. Designed the study: JHW JG. Finally, for genuine high content screening, an automated
image analysis pipeline for extracted morphological features
would be highly beneficial [22,60]. Moreover, compounds
influencing
kidney
function
without
altering
pronephros
morphology cannot be identified using this pipeline. Thus,
protocols for the large scale analysis of kidney function need to PLOS ONE | www.plosone.org 11 December 2013 | Volume 8 | Issue 12 | e82137 Automated Screening Platform for Zebrafish Kidneys Supervised the study: JHW JG UL. Gave general advice and
edited the manuscript: BT. Supervised the study: JHW JG UL. Gave general advice and
edited the manuscript: BT. Automated Screening Platform for Zebrafish Kidneys Automated Screening Platform for Zebrafish Kidneys U S A 107: 17315-17320. doi:10.1073/pnas.1008209107. PubMed:
20855591. 51. Rosenbaum JL, Witman GB (2002) Intraflagellar transport. Nat Rev Mol
Cell Biol 3: 813-825. doi:10.1038/nrm952. PubMed: 12415299. 41. Neto JA, Oliveira-Filho RM, Simões MJ, Soares JM Jr., Kulay L Jr. (2004) Long-term acetaminophen (paracetamol) treatment causes liver
and kidney ultra-structural changes during rat pregnancy. Clin Exp
Obstet Gynecol 31: 221-224. PubMed: 15491069. 52. Scholey JM (2003) Intraflagellar transport. Annu Rev Cell Dev Biol 19:
423-443. doi:10.1146/annurev.cellbio.19.111401.091318. PubMed:
14570576. 53. Ocbina PJ, Eggenschwiler JT, Moskowitz I, Anderson KV (2011)
Complex interactions between genes controlling trafficking in primary
cilia. Nat Genet 43: 547-553. doi:10.1038/ng.832. PubMed: 21552265. 42. Peng HC, Wang YH, Wen CC, Wang WH, Cheng CC et al. (2010)
Nephrotoxicity assessments of acetaminophen during zebrafish
embryogenesis. Comp Biochem Physiol C Toxicol Pharmacol 151:
480-486. doi:10.1016/j.cbpc.2010.02.004. PubMed: 20170747. 54. de Vries J, Yntema JL, van Die CE, Crama N, Cornelissen EA et al. (2010) Jeune syndrome: description of 13 cases and a proposal for
follow-up
protocol. Eur
J
Pediatr
169:
77-88. doi:10.1007/
s00431-009-0991-3. PubMed: 19430947. 43. Shrim A, Berger H, Kingdom J, Hamoudi A, Shah PS et al. (2005)
Prolonged exposure to angiotensin-converting enzyme inhibitors during
pregnancy. Fetal toxicity could be reversible. Can Fam Physician 51:
1335-1337. PubMed: 16250418. 55. Friedland-Little JM, Hoffmann AD, Ocbina PJ, Peterson MA, Bosman
JD et al. (2011) A novel murine allele of Intraflagellar Transport Protein
172 causes a syndrome including VACTERL-like features with
hydrocephalus. Hum Mol Genet 20: 3725-3737. doi:10.1093/hmg/
ddr241. PubMed: 21653639. 44. Martinovic J, Benachi A, Laurent N, Daikha-Dahmane F, Gubler MC
(2001) Fetal toxic effects and angiotensin-II-receptor antagonists. Lancet 358: 241-242. doi:10.1016/S0140-6736(01)05426-5. PubMed:
11480433. 56. Sun Z, Amsterdam A, Pazour GJ, Cole DG, Miller MS et al. (2004) A
genetic screen in zebrafish identifies cilia genes as a principal cause of
cystic kidney. Development 131: 4085-4093. doi:10.1242/dev.01240. PubMed: 15269167. 45. Pryde PG, Sedman AB, Nugent CE, Barr M Jr. (1993) Angiotensin-
converting enzyme inhibitor fetopathy. J Am Soc Nephrol 3: 1575-1582. PubMed: 8507813 57. Sabaliauskas NA, Foutz CA, Mest JR, Budgeon LR, Sidor AT et al. (2006) High-throughput zebrafish histology. Methods 39: 246-254. doi:
10.1016/j.ymeth.2006.03.001. PubMed: 16870470. 46. Quan A (2006) Fetopathy associated with exposure to angiotensin
converting enzyme inhibitors and angiotensin receptor antagonists. Early Hum Dev 82: 23-28. doi:10.1016/j.earlhumdev.2005.11.001. PubMed: 16427219. 58. References (2012) Mechanism-based urinary biomarkers to identify the potential for
aminoglycoside-induced nephrotoxicity in premature neonates: a proof-
of-concept study. PLOS ONE 7: e43809. doi:10.1371/journal.pone. 0043809. PubMed: 22937100. 14. Kokel D, Bryan J, Laggner C, White R, Cheung CY et al. (2010) Rapid
behavior-based identification of neuroactive small molecules in the
zebrafish. Nat Chem Biol 6: 231-237. doi:10.1038/nchembio.307. PubMed: 20081854. 35. Kent AL, Maxwell LE, Koina ME, Falk MC, Willenborg D et al. (2007)
Renal glomeruli and tubular injury following indomethacin, ibuprofen,
and gentamicin exposure in a neonatal rat model. Pediatr Res 62:
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Glomerular nephrotoxicity of aminoglycosides. Toxicol Appl Pharmacol
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and amikacin-induced nephrotoxicity in patients with normal baseline
renal function. Fundam Clin Pharmacol 23: 515-520. doi:10.1111/j. 1472-8206.2009.00702.x. PubMed: 19709328. 17. Drummond IA (2005) Kidney development and disease in the zebrafish. J Am Soc Nephrol 16: 299-304. doi:10.1681/ASN.2004090754. PubMed: 15647335. 18. Drummond IA, Davidson AJ (2010) Zebrafish kidney development. Methods
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mitochondria, and cell death mechanisms in drug-induced liver injury:
lessons learned from acetaminophen hepatotoxicity. Drug Metab Rev
44: 88-106. doi:10.3109/03602532.2011.602688. PubMed: 22229890. 19. Perner B, Englert C, Bollig F (2007) The Wilms tumor genes wt1a and
wt1b control different steps during formation of the zebrafish
pronephros. Dev Biol 309: 87-96. doi:10.1016/j.ydbio.2007.06.022. PubMed: 17651719. 20. Westerfield M (2000) The zebrafish book. A guide for the laboratory
use of zebrafish (Danio rerio), 4th edition. University of Oregon Press,
Eugene, OR. 40. North TE, Babu IR, Vedder LM, Lord AM, Wishnok JS et al. (2010)
PGE2-regulated wnt signaling and N-acetylcysteine are synergistically
hepatoprotective in zebrafish acetaminophen injury. Proc Natl Acad Sci 12 PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e82137 Automated Screening Platform for Zebrafish Kidneys Automated Screening Platform for Zebrafish Kidneys Kramer-Zucker AG, Wiessner S, Jensen AM, Drummond IA (2005)
Organization of the pronephric filtration apparatus in zebrafish requires
Nephrin, Podocin and the FERM domain protein Mosaic eyes. Dev Biol
285: 316-329. doi:10.1016/j.ydbio.2005.06.038. PubMed: 16102746. 47. Coleman CM, Minor JJ, Burt LE, Thornhill BA, Forbes MS et al. (2007)
Angiotensin AT1-receptor inhibition exacerbates renal injury resulting
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Zebrafish: a model system for the study of vertebrate renal
development, function, and pathophysiology. Curr Opin Nephrol
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20:
416-424. doi:10.1097/MNH.0b013e3283477797. PubMed: 21519251. 48. Friberg P, Sundelin B, Bohman SO, Bobik A, Nilsson H et al. (1994)
Renin-angiotensin system in neonatal rats: induction of a renal
abnormality in response to ACE inhibition or angiotensin II antagonism. Kidney Int 45: 485-492. doi:10.1038/ki.1994.63. PubMed: 8164437. 60. Vogt A, Cholewinski A, Shen X, Nelson SG, Lazo JS et al. (2009)
Automated image-based phenotypic analysis in zebrafish embryos. Dev Dyn 238: 656-663. doi:10.1002/dvdy.21892. PubMed: 19235725. 49. Loria A, Reverte V, Salazar F, Saez F, Llinas MT et al. (2007) Changes
in renal hemodynamics and excretory function induced by a reduction
of ANG II effects during renal development. Am J Physiol Regul Integr
Comp Physiol 293: R695-R700. doi:10.1152/ajpregu.00191.2007. PubMed: 17491111. 61. Rider SA, Tucker CS, del-Pozo J, Rose KN, MacRae CA et al. (2012)
Techniques for the in vivo assessment of cardio-renal function in
zebrafish (Danio rerio) larvae. J Physiol 590: 1803-1809. PubMed:
22331420. 50. Drukker A (2002) The adverse renal effects of prostaglandin-synthesis
inhibition in the fetus and the newborn. Paediatr Child Health 7:
538-543. PubMed: 20046466. PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 13 December 2013 | Volume 8 | Issue 12 | e82137
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NOTES
ON
MISCELLANEOUS
ENDOPARASITES.
By
T.
Harvey
Jounston,
M.A.,
D.Sc.,
University,
({
Read
before
the
Royal
F.L.8.,
Biology
Queensland,
25th
Dept.,
Brisbane.
Society
of
November,
1918).
CESTODA.
Acanthotaenia
Last
region
year
this
the
intestine
of
gallardt
cestode
a
Johnston.
was
found
infesting
black
snake,
Pseudechts
at
riacus,
killed
addition
to
this
found
just
under
the
peritoneum
company
with
A.
gallardi
there
were
In
individuals
phalus)
by
of
‘
of
sp.
intestine,
Munro
Hull
parasite,
larval
cestodes
a
strongyle,
distributed
while
many
Mr.
reported
from
inalamb
this
from
in
sp.)
body
cavity.
numerous
isolated
(
Kalice-
part
of
the
stomach
harboured
been
previously
had
Queensland.
trigonophora
St.
&
Hass.
1918,
was
found
in
January
It
had
not
been
Eumundi.
(Sparganum
greater
entozoa
host
tapeworm
at
and
No
Moniezia
This
csophagus
porphyIn
Diaphanocephalus
the
mid-
Eumundi.
the
were
throughout
the
Physaloptera.
of
the
in
numbers
previously
reported
Queensland.
Multiceps
Dr.
Dodd
(1918,
p.
parasites
of
live
stock
statement
that
“‘
not
J
have
the
multiceps
502)
in
Leske.
a
recent
address
referred.
to
the
the
condition
Gid
is
not
common
been
able
obtain
any
records
cases
of
occurrence
known
as
Cenurus
cases
)
of
so-called
to
of
parasite
on
and
endo-
made
in
N.S.W.”
of
authentic
worm,
generally
the
gid
bladder
cerebralis,
in
any
part
of
Australia,
gid
traceable
to
other
causes.
being
a
all
210
NOTES
Since
the
Dr.
parasite
information.
ON
MISCELLANEOUS
ENDOPARASITES.
Dodd’s
remarks
seem
he
stated
that
he
had
not
personally
Gid
in
State,
but
had
been
informed.
in
Melbourne.
it
had
been
He
went
on
to
statement
can
be
case
of
say,
state
causal
seen
and
though
proof
parasite
has
all
that
State
he
had
in
this
continent.*
believed
that
gid
were
probably
due
the
list
hydatids
of
for
the
in
the
by
Dr.
Dodd,
cannot
at
given
we
the
It
must
known
first
time.
as
as
occurring
Pall.
the
in
cavity.
affirm
actually
pigs
to
present
entozoa
in
persons.
as.
Australia.
of
In.
well
in
hydatigena
case.
cranial
multiceps
presence
tenuicollis)
noted
for
evidence
occasional
sheep
and
Multiceps
the
of
Gid,
Tenia
now
numbers
collected,
off
is
large
and
view
(Cystieercus
examined
He
conditions
In
The
demonstrated.
that
Hchinococcus
struck
because
possible
of
be
not.
quite
presence
of
N.S.W.,
been
mistaken
occurrence
in
exist
is
was
cases
have
not
he
the
it
the
I
that
before
not
that
of
which
necessary
a
some
the
is
encountered
confused
of
occasionally
having
agreed
other
asking
conclusive,
does
without
had
that
as
Gid
he
letter
there
that
that
later
that
accepted
mentioned
a
further
reply
that
over
for
I
a
the
that:
N.S.W.,
with
to
imply
in
In
wrote
to
occurs
met
prepared
would
bladder
south-eastern
worm
stage
Queensland
*In the same paper (p. 506) Dr. Dodd refers to the presence of
Trichostrongylus axei (Strongylus gracilis) and other stomach worms in
Australian sheep and cattle. (See also Dodd, Ann. Rep. Dept. Agric. Q’land,
1909, p. 93-4, where he calls it Str. gracilis). Cobbold (Parasites, etc., 1879,
p. 283) gave the name Strongylus axei to certain minute nematodes occurring in the mucous membrane of the stomach of donkeys. Railliet and
Henry (C. R. Soe. Biol., 66, 1909, p. 87) placed S. gracilis McFad. and 8.
extenuatus Raill. as synonyms of T'richo. axei Cobbold. Ransom in his
fine account of the nematodes occurring in the alimentary tract of sheep,
cattle, etc. (Bull. 127, U.S. Dept. Agric., B.A.I., 1911, p. 94) referred to.
S. gracilis McF. as a synonym of Railliet’s Tricho, extenuatus, making no
mention of Tr. axe: as a parasite of stock—apparently not accepting
Railliet’s views. Leiper (Jour. London Sch. Trop. Med., 1 (1), 1911, p. 25)
in his check list, mentioned T'r. axe7 as a parasite of Equide and (l.c. 1
(2), 1912, p. 116) queried its presence in cattle. In my census of Queensland endoparasites (1916) I referred to Dodd’s record of Sir. gracilis as
Tr. extenuatus Raill, following Ransom (1911) rather than Railliet (1909).
Tn view of these remarks 7'r. axei cannot yet be added to our known helminth fauna unless it be admitted that the two names Str. axei Cobbold
and S. gracilis McFadyean (not Leuckt ‘are synonymous,
BY
T.
HARVEY
Dipylidium
caninum
the
with
which
to
of
maturity
it
may
be
of
interest
this
cestode
as
well
as
egg-bearing
in
a
puppy
specimens
of
canis,
full
in
(Sydney,
September,
211
some
can
L.
Asevidence
reach
rapidity
JOHNSTON.
mention
the
entozoa
that
I
nematode
only
six
found
Toxascaris
weeks
old
1909).
TREMATODA.
Bird
In
some
of
reference
was
made
under
the
my
broad
submitted
Johnston,
them,
along
Host.
Aig
Numenius
cyanopus
Sydney
others,
as
SEW
of
in
his
Australian
certain
were
J.
They
of
flukes
8.
Birds
presence
of
e.g.
Prof.
are
the
parasites
names:
The
Australian
of
generic
etc.
of
lists
to
stomum,
with
Trematodes.
trematodes
Hchinostomum,
to
birds
Mono-
my
friend,
University,
who
described
paper
the
Trematodes
on
(1916).
follows
mes"
:—
|
2,
|
Locality.
|
Q.
|
Echinostoma J., 1912, 1916,—=
| Himasthla harrisoni S.J.J.
Himantopus leucocephalus | South Australia, Monostomum J., 1910.
1912,—Hematotrephus
adelphus 8. J. J.
Charadrius
dominicus
Sydney
Echinostoma
J.,
1910,
=
|
Acanthoparyphium
spinulosum 8. J. J,
Herodias
timoriensis
Hidsvold,
Q.
|
Hchinosioma
J.,
1912
=
_ Echinoparyphium oxyurum
Ibis
molucca
Chenopsis
Micreca
Gladstone,
Parasite.
:
|
Eidsvold,
Q.
atrata
Victoria
fascinans
Eidsvold,
Dolichopera
This
by
trematode
Macalpine
was
(1891,
p.
S a .J. & Patagifer fraternus
S: JL di.
|
Hchinostoma
J.,
1912
=
| Patagifer acuminatus S. J. J
Monostomum
J.,
1910,
1912
_ = Ayptiasmus magnus 8. J.J.
Q.
Echinostoma
J.,
1912
=
Echinoparyphium
harveyanum 8. J. J.
macalpini
originally
40)
superba
in
Victoria.
(which
named)
in
his
he
and
in
as
hosts,
1918
the
gave
tiger
a
genus
description
snake
described
from
Denisonia
Nicoll.
the
Nicoll
(but
not
named)
copper-headed
snake,
included
Dolichopera
of
Noteckis
the
the
(1914,
species
scutatus
species
p.
343),
mentioning
(Victoria),
the
7
dl
be
NOTES
-copperheaded
unnamed
ON
MISCELLANEOUS
snake,
snake
Denisonia
collected
by
superba
Dr.
J.
Island
(1918a).
Quite
recently
he
parasite
and
published
2
figure
(1918b,
p.
ENDOPARASITES.
(Victoria)
B.
and
Cleland*
referred
on
Flinders
briefly
illustrating
an
to
its
the
anatomy
374).
I
obtained
D.
macalpini
from
the
Denisonia
superba
and
the
black
snake
Pseudechis
poi
phy-
riacus
in
Sydney
district,
recording
its
presence
under
the
name
Hemiurus
parasite
of
in
N.S.W.
I
snakes
seems
Australia.
Echinochasmus
tenuicollis
described
by
cormorant,
have
trematodes
sp.
Eastern
Recently
a
Apoblema)
venomous
distributed
from
(
J.
as
from
of
(1910a,
1911).
This
to
fairly
widely
S.
be
J.
Jnstn.
Johnston,
Phalacrocorazx
identified
collected
S.
csophagus
(1916,
p.
melanoleucus,
belonging
to
this
the
same
host
206)
Tuggerah,
species
species
a
few
near
Long-
reach,
Thompson
River,
by
Miss
M.
J.
Bancroft
(August,
1918).
In
specimens
the
vitellaria
extend
further
of
the
ventral
sucker,
relatively
more
remote
my
forwards,
reaching
while
the
male
and
female
glands
from
the
hinder
end
of
animal,
being
situated
the
original
figure.
ventral
sucker
Some
human
the
than
time
blood
posterior
shown
spp.
ago
reference
made
flukes
being
troops
from
(Johnston,
1916,
p.
was
in
possessed
ional
contain
terminal
spines
in
addition.
Thus
at
present
in
In
1917
urine
returned
to
examination,
ova.
Most
specimens
haematobium
Bilh.)
Bilharzia
(S.
mansoni
both
species
State.
It
may
Sambon)
of
human
is
not
species,
presumably
8.
haematobium
has
become
West
summary
Australia.
in
his
Major
article
on
me
from
for
these
in
by
countries
(S.
origin,
Australia
neighbouring
conditions
African
of
and
certain
South
likelihood
the
Egypt
eggs
this
to
the
into
submitted
to
lateral-spined
was
nearer
introduced
37).
Queensland,
found
in
are
Schistosomum
returning
soldiers
the
edge
but
oceas-
were
present
blood
unlikely
become
fluke
that
under
endemic.
One
on
account
established
in
Cherry
gave
Bilharziosis
are
an_
of
one
its
locality
interesting
(1917).
*Dr. Cleland has informed me that the snake was Notechis seulatus.
BY
Capt.
early
Lawton
clinical
(1917,
stools
a
number
Hospital.
In
addition
in
cases,
some
cols,
HARVEY
recently
published
an
of
the
serious
disease
21).
He
found
of
Australian
features
mansoni
of
T.
p.
to
All
account
the
by
S.
ova
in
caused
lateral-spined
Military
the
faces
were
found,
other
parasites
such
intesttnalts,
Lamblia
men
subsequently
the
contain
soldiers
of
Cairo
to
these
21S:
a
Trichomonas
hominis.
JOHNSTON.
ova,
were
in
as
Hntamoeba
and
Blastocystis
discharged
to-
Australia.
NEMATODA.
Dictyocaulus
Dr.
In
1893
T.
L.
Strongylus
on
Diseases
Stock
micrurus
Mehlis,
in
sheep
occurrence
in
horses
in
Sydney,
using
the:
almost
certainly
D.
arnfields
from
the
Brisbane)
and
their
parasite
(Sydney
(Queensland).
is
and
S.
micrurus
(=D.
and
D.
viiparus)
lung
worms,
horses.
Perrie
filaria
from
sheep-
is
lung
worm
goat’s
lung
the
calves.
Synthetocaulus
Dr.
Cleland
parasitised
by
nematodes
identified
as
S.
extremely
brief
account
of
the
had
not
been
me.
J.
capillarts
B.
in
forwarded
goats
in
collected
a
dozen
by
have
braziliense
specimens
rat,
kindly
man,
Director
of
the
Queensland
rattus.
This
the
only
occasion
reported
from
Z£.
H.
p. 96).
From
fowl
though
in
the
to
to
reported
as.
retusa
occur
the
in
were
from
Mr.
H.
Long-
Museum,
as
by
which
I
Brisbane
A.
H.
Hpimys
braziliense
have
recorded
and
Sydney
its.
(1918,
Raill.
alimentary
previously
strongyle
1918)
on
rattus.
Capillaria
Brisbane—not
known
available
May,
determined
in
of
tiny
(Brisbane,
norvegicus
walls
an
Travassos.
this
a
in
Taylor
of
of
presence
Only
is
previously
B.
been
provisionally
District).
nematode
B.
is
a
been
(Sydney
Miss
intestine
of
Australia.
Heligmosomum
About
Muller.
portion
which
capillaris
Lungworms
occurring
has
of
Queensland,
occurrence
name.
the
in
the
same
of
report
recorded
to
horse
his
Cobbold.
Bancroft
referred
The
in
arnfieldt
canal
recorded
Sydney
and
of
from
the
domestic
Queensland.
Melbourne.
214
NOTES
ON
MISCELLANEOUS
Pneumonema
This
Tiliqua
the
tilique
was
collected
recently
scincoides
on
Facing
Island
recorded
having
been
vegicus.
In
(Port
tetraptera
from
found
in
Queensland
Curtis)
and
in
Mus
first
time,
and
in
Epimys
nor-
musculus
only
one
worm,
collected.
This
constitutes
the
first
the
nematode
in
(1916)
mentioned
by
Dr.
Hall
(collected
by
Miss
B.
agrees
in
all
given
by
Hall.
particulars
the
indebted
to
a
a
rat
Taylor,
Sept.,
with
the
account
nasuta
G.
female,
of
the
is
not
which
host.
B.
Miss
mature
record
brown
as
Cheilospirura
am
of
the
case
I
lungs
for
latter
of
the
Nitzsch.
the
presence
of
from
District.
Oxyuris
Now
~
Johnston.
worm
Brisbane
was
ENDOPARASITES.
The
specimen
1918,
Brisbane)
of
the
species
Rud.
Y.
James,
B.Sc.
for
specimens
a
domestic
this
worm
collected
from
the
urodeum
of
fowl
at
West
Burleigh,
1918—not
previously
recorded
this
State.
Habronema
The
occurrerice
Australian
horses
of
musce
the
has
and
history
has
been
The
fully
followed
He
did
Ransom
(1913).
recently
published
an
have
tumours
Habronema
granulomata
of
horses
to
which
of
three
species
horse
these
re-examined
my
have
microstoma
appears
to
Sydney
the
larve
belonged.
material
labelled
found
be
the
that
many
years
ago
by
Mr.
A.
S.
Le
Soeuf,
Habronema
was
collected
numbers.
Spiroptera
musce
Specimens
small
**
in
H.
in
Habronema
form
Brisbane,
on
the
of
although
commoner
in
habronemiasis.”’
as
and
present
Australia
cancer”
“‘
the
horses,
South
of
care-
in
and
form
Queens-
of
Victoria
another
and
in
in
account
be
microstoma’’
collected
interesting
or
“‘“swamp
stage
to
know
the
I
certain
believed
not
infesting
life
by
Territory
been
N.S.W.
1912).
He
has
in
(Johnston,
(1916).
its
flies
land
larval
though
certain
in
by
in
larval
present
recorded,
musce
the
be
caused
H.
since
to
of
of
inferred,
reported
occurrence
stage
been
be
Northern
adult
yet
could
Bull
Carter.
not
presence
from
in
material
also
Melbourne
re-examination
were
all
found
recorded
by
H.
BY
T.
to
belong
must
be
and
occurring
in
N.S.W.
Dr.
J.
B.
the
in
nodules
as
of
evidently
musce
nor
H.
added
to
H.
in
from
(Sydney
District).
fied
as
recorded
215
as
previously
known
entozoa
*
cavities
in
dense
horses
in
West
refer
to
H.
has,
as
of
fibrous
Australia.
as
I
tissue
in
of
his
Neither
4H.
been
noted
Both
megastomat.
far
Spiroptera
know,
State.
Dr.
the
of
presence
Gongylonema
tumours
list
the
or
that
year,
the
recorded
microstoma
occurring
Last
microstoma
Queensland.
Cleland}
stomach
records
to
JOHNSTON.
me.
musce
microstoma
HARVEY
ingluvicola
Cleland
forwarded
some
small
crop
gizzard
of
have
been
walls
of
The
causal
G.
ingluvicola,
which,
as
far
as
only
from
locality
in
the
United
a
the
Ransom.
and
parasites
I
worm
a
know,
chicken
indenti-
has
States
been
(Ransom,
1904).
ACANTHOCEPHALA.
*
This
Centrorhynchus
asturinus
parasite,
infests
novaehollandiae
as
a
Gm.,
Gigantorhynchus
Centrorhynchus,
the
summarised
by
three
long
tubular
ones
as
I
which
shown
have
specimens
hawk,
Baza
gum,
near
but
is
my
the
now
Cleave
as
of
glands
The
to
the
genus
have
been
has
only
male
several
smaller
(1913,
pl.
17,
41).
figure
to
the
J.
B.
subcristata
Gd.,
in
December,
well
described
of
belonging
as
Astur
instead
Dr.
N.S.W.,
was
which
by
Casino,
hawk,
transferred
(1916).
original
white
Queensland,
characters
cement
identified
collected
North
chief
Van
in
in
Johnston.
as
Cleland
a
fig.
species
from
a
a
1916
at
number
in
few
crested
Mummul-
the
collec-
*My attention has recently been drawn to a reference in the Scientific
Australian (June 1918), where it stated that a paper by Mr. G. F. Hill,
dealing with the life histories of Habrone na musce, +4. microstoma and H.
megastoma, had been read before the May meeting (1918), of the Heyal
Society of Victoria.
TCleland. Diseases of Animals, etc., Bull 33, Dept. Agric.
Australia, 1909, p. 3; Trypanosomiasis, etc. Bull. 34, 1909, p. 15.
West
{Desmond (Journ. Agric. Ind. South Australia, 7, 1904, p. 569) referred
to the presence of Sclerostomum hypostomum in tumours in the stomach
of South Australian horses. The reference should be H. megastoma.
216
NOTES
ON
tion
of
ville,
obtained
in
North
Astur
clarus
(A.
cinereus
A.
the
MISCELLANEOUS
Australian
Brienl,
the
opportunity
to
The
found
Institute
in
of
boobook
District—T.H.J.,
rhynchus
sp.
riacus
presence
in
my
specimens
already
referred
district.
It
intestine
though
specimens,
for
this
is
as
been
Bancroft
also
a
typical
to
having
as
frequents
the
generally
more
been
in
the.
Centro-
Pseudechis
poi
phy-
the
first
from
the
black
snake
in
the
Eumundi
killed
three
time
quarters
abundant
hirundinaceus
the
for
been
lower
me
Johnston.
in
taken
Dr.
having
Dr.
recorded
having
thank
by
parasite
now
Goshawk,
giving
rotundocapitatus
of
Hormorhynchus
to
mentioned
is
Towns—
gray
desire
his
1912)
Queensland
the
Institute,
(collected
Echinorhynchus
The
I
was
Medicine,
from
the
which
Kidsvold
Tropical
Vieill).
examine
Ninox
of
Queensland
dircctor
echinorhynch
ENDOPARASITES.
in
of
;
the
the
rectum.
(Gigantorhynchus
gigas
Goze).
This
is
met
Queensiand—not
with
occasionally
previously
in
reported
pigs
in
from
south-eastern.
this
State.
*
LINGUATULIDA.
Porocephalus
A
black
snake
teretiusculus
Pseudechis
porphyriacus
—referred
to
earlier
in
sitised
the
above
pentastome,
the
lung,
while
the
small
males
organ.
This
constitutes
in
by
the
extremity
of
of
the
now
the
presence
known
Pseudechis
and
tiger
snake
known
Queensland
to
D.
infest
the
Notechis
and
I
to
the
islands
larval
denticulatum,
is
stage,
now
the
Australian
(Blue
of
Strait.
record.
which
is.
snakes—
from
Mountains,
Australia
the
Diemenia.
taken
Western
generally
at
superba,
specimens
para-
distributed.
first
following
Bass
be
found
entozoon,
serrata
definitely
being
were
Kumundi
to
the
have
from
Tinguatula
The
females
of
scutatus
extends
from
found
Denisonia
reticulata.
range
paper—was
Queensland
porphyriacus,
textilis
Its
in
this
Baird.
to
the
N.S.W.).
Southerr-
¥rol.
known
recorded
as
as
Pentastomum.
oceurring
in
the
mesenteric
glands
occasionally
found
BY
T.
of
cattle
in
HARVEY
in
JOHNSTON.
2G
specimens
being
Queensland,
animals
killed
in
the
Brisbane
the
Brisbane
district
abattoirs.
ACARIDA.
Cytodites
This
in
the
mite
is
found
sacs
of
the
air
mange
opportunity
to
(Sarcoptes
muris,
domestic
on
Epimys
Sydney
by
I
rattus
on
‘Dr:
record
rattus
J.-B:
Cleland.*)
have
already
in
N.S.W.
and
Z£.
The
parasite
sets
up
sometimes
the
other
;
the
presence
Raill.)
true
in
norvegicus
and
referred
in
to
a
warty
of
Knemidocoptes
mutans
domestic
in
of
I
take
Notedres
Brisbane
and
the
muris
Melbourne
in
Launceston
Adelaide
on
“rats’’
(collected
occurrence
in
#.
norvegicus
in
Perth,
West
Australia.
of
the
tail,
ears
causes
~~
scaly-leg’’ bE in
its
condition
the
entozoa,
;
alexandrinus
parts
fowl
Miégnin.
not
E.
#.
fowl.
are
alepis
and
in
muris
mites
S.
Viz.
occasionally
Notedres
Though
nudus
and
and
head.
Rob.
Brisbane.
BIBLIOGRAPHY.
1916 Bull, L. B. A granulomatous affliction of the horse, Habronemie
granulomata. Journ. Comp. Path. Therap., 29, (3). (Reprint
15pp.)
1917 Cherry, T., Bilharziosis, ete. Pamphlet, Defence Dept. Commonwealth of Australia.
1918 Dodd, S. Some observations on the internal parasites of live
stock. Agr. Gaz. N.S.W., 1918, pp. 497-507.
1916
Hall, M. Nematode parasites
Mus., 50, pp. 1-258.
of
Rodents,
etc.
Proc.
U.S.
Nat.
1910 a Johnston, T. H. (Notes and Exhibits) P.R.S. N.S.W., 44, p. xviii.
6b — —— On Australian Avian Entozoa. P.R.S. N.S.W., 44,
pp. 84-122.
911 ————— Census of Australian Entozoa. P.R.S.Q., 23, pp. 233249.
“Dr Cleland has recently referred to the presence of the parasite in
Sydney rats. P.R.S. N.S.W 1918, p. 110.
218
NOTES
ON
MISCELLANEOUS
ENDOPARASITES,
1912 a ————— The internal parasites recorded from Australian Birds,
“Emu,” Oct., 1912, pp. 105-112.
1912 ) —————_ Notes on some Entozoa. P.R.S.Q., 24, pp. 63-91.
1913
———— Report on Cestoda and Acanthocephala.
Inst. Trop. Med., 1911 (1913), pp. 75-06.
Rept.
Aust.
1916 ———— Census of the endoparasites recorded as oceurring in
Queensland, etc. P.R.S.Q., 28, pp. 31-79.
1918 ————— Notes on certain entozoa of rats and mice, etc., P.R.S.Q.
30 (3), pp. 53-78.
1916
Johnston,
8.
J.
Ongthe
Trematodes
of
Australian
Birds,
P.R.S.,
N.S.W., 50, pp. 187-261.
1917 Lawton, F. B. Schistosomum mansoni. Early clinical features of
the disease. In pamphlet ‘‘ Bilharziosis.”” See under T. Cherry.
1891 Macalpine, D. Remarks on a fluke parasite in the copperhead
snake.
P.R.S.
Vict.,
3,
pp.
40-45.
1914 Nicoll, W. Trematode parasites of North Queensland. 1. Parasitology, 6 (4), pp. 333-350.
1918 a —————
290-3.
Dolichopera macalpimi, etc. Parasitol., 10 (2), pp.
1918 6 —_——— The trematode parasites of North Queensland, No. iv.
l.c., 10 (3), pp. 368-374.
1904 Ransom B. A new nematode (Gongylonema ingluvicola) parasitic
in the crop of chickens. U.S.D.A., B.A.L., Cire. No. 64, 3 pp.
1913 -————— [he life history of Habronema musce, ete. U.S.D.A.,
B.A.1., Bull. 163, 36 pp.
1916 Van Cleave, H. Acanthocephala of the genera Centrorhynchus and
Mediorhynchus, ete. Trans. Amer. Micr. Soc., 35, pp. 221-232.
Johnston, T. Harvey. 1918. "Notes on miscellaneous endoparasites." The
Proceedings of the Royal Society of Queensland 30, 209–218.
https://doi.org/10.5962/p.91011.
View This Item Online: https://www.biodiversitylibrary.org/item/49266
DOI: https://doi.org/10.5962/p.91011
Permalink: https://www.biodiversitylibrary.org/partpdf/91011
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|
Various open science
| null |
None
|
None
|
English
|
Spoken
| 8,023
| 16,153
| "D Dale HH, Feldberg W, Vogt M. Release of acetylcholine at voluntary motor nerve endings. J. Physio(...TRUNCATED)
| 9,556
|
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Size of the auto-converted Parquet files (First 5GB):
2.5 GB
Number of rows (First 5GB):
96,860
Estimated number of rows:
283,842