A flow cytometry based method for studying embryogenesis and immune reactivity to embryogenic stages in filarial parasites
Identifieur interne : 002008 ( Ncbi/Merge ); précédent : 002007; suivant : 002009A flow cytometry based method for studying embryogenesis and immune reactivity to embryogenic stages in filarial parasites
Auteurs : Bikash Ranjan Sahu [Inde] ; Alok Das Mohapatra [Inde] ; Arindam Majumder [Inde] ; Pradip K. Das [Inde] ; Balachandran Ravindran [Inde]Source :
- Filaria Journal [ 1475-2883 ] ; 2005.
Abstract
In the absence of intermediate animal hosts, the process of embryogenesis leading to fecundity of adult female filarial worms is very critical for persistence of these obligate parasites in human communities. Embryogenesis in adult female filarial parasites involves fertilization of eggs or oocytes by sperms and their subsequent development into motile microfilariae inside the uterine cavity of worms. Development of assays for monitoring embryogenesis in adult female worms is a critical requirement in filariasis research – filarial worms are known to harbour endosymbionts such as
Adult female filarial worms of bovine filarial parasites,
The forward and side scatter for intrauterine stages delineated 3 distinct populations labeled as R1, R2 and R3. The three populations were sorted and identified to be a) fully stretched microfilariae, b) early and c) late developmental stages of eggs respectively. Lectins such as Wheat Germ agglutinin or Concanavalin-A were found to bind strongly to egg stages and less prominently to intra-uterine microfilariae. Similarly the binding of antibodies in filarial sera to the three intra-uterine stages could also be precisely quantified.
The manuscript reports a novel flow cytometry based method to monitor progression of embryogenesis in adult filarial worms. Apart from relative quantification of different intra uterine developmental stages, the assay allows quantitative binding of lectins and antibodies to each of the intrauterine stages. It may now be possible to quantify levels of antibodies in infected and immune hosts to monitor anti-fecundity immunity in filariasis – the assay can thus be used as a powerful tool for drug development and in immunological studies in human and experimental filariasis.
Url:
DOI: 10.1186/1475-2883-4-11
PubMed: 16274474
PubMed Central: 1291383
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<front><div type="abstract" xml:lang="en"><sec><title>Background</title>
<p>In the absence of intermediate animal hosts, the process of embryogenesis leading to fecundity of adult female filarial worms is very critical for persistence of these obligate parasites in human communities. Embryogenesis in adult female filarial parasites involves fertilization of eggs or oocytes by sperms and their subsequent development into motile microfilariae inside the uterine cavity of worms. Development of assays for monitoring embryogenesis in adult female worms is a critical requirement in filariasis research – filarial worms are known to harbour endosymbionts such as <italic>Wolbachia </italic>
which play a significant role in fecundity. Tetracycline or doxycycline treatment of the infected hosts effectively eliminates the endosymbionts resulting in inhibition of embryogenesis in female worms. Currently, inhibition of embryogenesis in adult filarial worms can be monitored only by microscopic examination of <italic>in vitro </italic>
harvested intrauterine stages.</p>
</sec>
<sec sec-type="methods"><title>Methods</title>
<p>Adult female filarial worms of bovine filarial parasites, <italic>Setaria digitata </italic>
were collected from the peritoneum of infected animals and intrauterine stages were harvested in culture medium and were analyzed for forward and side scatter by flowcytometry using a BD FACS Calibur. Different populations were gated, sorted and identified by phase microscopy. Binding of biotinylated lectins to intra uterine stages was monitored using FITC labeled Avidin and monitored by flow cytometry of gated populations. Similarly, binding of antibodies in human filarial sera to intrauterine stages was monitored using FITC labeled anti-human immunoglobulins.</p>
</sec>
<sec><title>Results</title>
<p>The forward and side scatter for intrauterine stages delineated 3 distinct populations labeled as R1, R2 and R3. The three populations were sorted and identified to be a) fully stretched microfilariae, b) early and c) late developmental stages of eggs respectively. Lectins such as Wheat Germ agglutinin or Concanavalin-A were found to bind strongly to egg stages and less prominently to intra-uterine microfilariae. Similarly the binding of antibodies in filarial sera to the three intra-uterine stages could also be precisely quantified.</p>
</sec>
<sec><title>Conclusion</title>
<p>The manuscript reports a novel flow cytometry based method to monitor progression of embryogenesis in adult filarial worms. Apart from relative quantification of different intra uterine developmental stages, the assay allows quantitative binding of lectins and antibodies to each of the intrauterine stages. It may now be possible to quantify levels of antibodies in infected and immune hosts to monitor anti-fecundity immunity in filariasis – the assay can thus be used as a powerful tool for drug development and in immunological studies in human and experimental filariasis.</p>
</sec>
</div>
</front>
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<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Filaria J</journal-id>
<journal-title>Filaria Journal</journal-title>
<issn pub-type="epub">1475-2883</issn>
<publisher><publisher-name>BioMed Central</publisher-name>
<publisher-loc>London</publisher-loc>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">16274474</article-id>
<article-id pub-id-type="pmc">1291383</article-id>
<article-id pub-id-type="publisher-id">1475-2883-4-11</article-id>
<article-id pub-id-type="doi">10.1186/1475-2883-4-11</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Research</subject>
</subj-group>
</article-categories>
<title-group><article-title>A flow cytometry based method for studying embryogenesis and immune reactivity to embryogenic stages in filarial parasites</article-title>
</title-group>
<contrib-group><contrib id="A1" contrib-type="author"><name><surname>Sahu</surname>
<given-names>Bikash Ranjan</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>bikash_sahu@rediffmail.com</email>
</contrib>
<contrib id="A2" contrib-type="author"><name><surname>Mohapatra</surname>
<given-names>Alok Das</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>Aloklight@rediffmail.com</email>
</contrib>
<contrib id="A3" contrib-type="author"><name><surname>Majumder</surname>
<given-names>Arindam</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>marindum@rediffmail.com</email>
</contrib>
<contrib id="A4" contrib-type="author"><name><surname>Das</surname>
<given-names>Pradip K</given-names>
</name>
<xref ref-type="aff" rid="I2">2</xref>
<email>dasp@cal2.vsnl.net.in</email>
</contrib>
<contrib id="A5" corresp="yes" contrib-type="author"><name><surname>Ravindran</surname>
<given-names>Balachandran</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>balaravi@sancharnet.in</email>
</contrib>
</contrib-group>
<aff id="I1"><label>1</label>
Division of Immunology, Regional Medical Research Centre, Indian Council of Medical Research, Chandrasekarpur, Bhubaneswar, 751023, India</aff>
<aff id="I2"><label>2</label>
Division of Microbiology, National Institute of Cholera and Enteric Diseases, Kolkata, 70000, India</aff>
<pub-date pub-type="collection"><year>2005</year>
</pub-date>
<pub-date pub-type="epub"><day>7</day>
<month>11</month>
<year>2005</year>
</pub-date>
<volume>4</volume>
<fpage>11</fpage>
<lpage>11</lpage>
<ext-link ext-link-type="uri" xlink:href="http://www.filariajournal.com/content/4/1/11"></ext-link>
<history><date date-type="received"><day>18</day>
<month>4</month>
<year>2005</year>
</date>
<date date-type="accepted"><day>7</day>
<month>11</month>
<year>2005</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2005 Sahu et al; licensee BioMed Central Ltd.</copyright-statement>
<copyright-year>2005</copyright-year>
<copyright-holder>Sahu et al; licensee BioMed Central Ltd.</copyright-holder>
<license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/2.0"><p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/2.0"></ext-link>
), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>
<pmc-comment>
Sahu
Ranjan
Bikash
bikash_sahu@rediffmail.com
A flow cytometry based method for studying embryogenesis and immune reactivity to embryogenic stages in filarial parasites
2005 Filaria Journal 4(1): 11-. (2005) 1475-2883(2005)4:1<11> urn:ISSN:1475-2883 </pmc-comment>
</license>
</permissions>
<abstract><sec><title>Background</title>
<p>In the absence of intermediate animal hosts, the process of embryogenesis leading to fecundity of adult female filarial worms is very critical for persistence of these obligate parasites in human communities. Embryogenesis in adult female filarial parasites involves fertilization of eggs or oocytes by sperms and their subsequent development into motile microfilariae inside the uterine cavity of worms. Development of assays for monitoring embryogenesis in adult female worms is a critical requirement in filariasis research – filarial worms are known to harbour endosymbionts such as <italic>Wolbachia </italic>
which play a significant role in fecundity. Tetracycline or doxycycline treatment of the infected hosts effectively eliminates the endosymbionts resulting in inhibition of embryogenesis in female worms. Currently, inhibition of embryogenesis in adult filarial worms can be monitored only by microscopic examination of <italic>in vitro </italic>
harvested intrauterine stages.</p>
</sec>
<sec sec-type="methods"><title>Methods</title>
<p>Adult female filarial worms of bovine filarial parasites, <italic>Setaria digitata </italic>
were collected from the peritoneum of infected animals and intrauterine stages were harvested in culture medium and were analyzed for forward and side scatter by flowcytometry using a BD FACS Calibur. Different populations were gated, sorted and identified by phase microscopy. Binding of biotinylated lectins to intra uterine stages was monitored using FITC labeled Avidin and monitored by flow cytometry of gated populations. Similarly, binding of antibodies in human filarial sera to intrauterine stages was monitored using FITC labeled anti-human immunoglobulins.</p>
</sec>
<sec><title>Results</title>
<p>The forward and side scatter for intrauterine stages delineated 3 distinct populations labeled as R1, R2 and R3. The three populations were sorted and identified to be a) fully stretched microfilariae, b) early and c) late developmental stages of eggs respectively. Lectins such as Wheat Germ agglutinin or Concanavalin-A were found to bind strongly to egg stages and less prominently to intra-uterine microfilariae. Similarly the binding of antibodies in filarial sera to the three intra-uterine stages could also be precisely quantified.</p>
</sec>
<sec><title>Conclusion</title>
<p>The manuscript reports a novel flow cytometry based method to monitor progression of embryogenesis in adult filarial worms. Apart from relative quantification of different intra uterine developmental stages, the assay allows quantitative binding of lectins and antibodies to each of the intrauterine stages. It may now be possible to quantify levels of antibodies in infected and immune hosts to monitor anti-fecundity immunity in filariasis – the assay can thus be used as a powerful tool for drug development and in immunological studies in human and experimental filariasis.</p>
</sec>
</abstract>
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<affiliations><list><country><li>Inde</li>
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<name sortKey="Das, Pradip K" sort="Das, Pradip K" uniqKey="Das P" first="Pradip K" last="Das">Pradip K. Das</name>
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<name sortKey="Ravindran, Balachandran" sort="Ravindran, Balachandran" uniqKey="Ravindran B" first="Balachandran" last="Ravindran">Balachandran Ravindran</name>
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