Research Institute of Biopharmacy and Veterinary Drugs

Jílové u Prahy, Czech Republic

Research Institute of Biopharmacy and Veterinary Drugs

Jílové u Prahy, Czech Republic
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Trefil P.,Research Institute of Biopharmacy and Veterinary Drugs | Bakst M.R.,U.S. Department of Agriculture | Yan H.,Hunan Institute of Animal and Veterinary Science | Hejnar J.,Academy of Sciences of the Czech Republic | And 2 more authors.
Theriogenology | Year: 2010

Transplantation of male germ line cells into sterilized recipients has been used in mammals for conventional breeding as well as for transgenesis. We have previously adapted this approach for the domestic chicken and we present now an improvement of the germ cell transplantation technique by using an enriched subpopulation of c-Kit-positive spermatogonia as donor cells. Dispersed c-Kit positive testicular cells from 16 to 17 week-old pubertal donors were transplanted by injection directly into the testes of recipient males sterilized by repeated gamma irradiation. We describe the repopulation of the recipient's testes with c-Kit positive donor testicular cells, which resulted in the production of functional heterologous spermatozoa.Using manual semen collection, the first sperm production in the recipient males was observed about nine weeks after the transplantation. The full reproduction cycle was accomplished by artificial insemination of hens and hatching of chickens. © 2010 Elsevier Inc.


Trefil P.,Research Institute of Biopharmacy and Veterinary Drugs | Mucksova J.,Research Institute of Biopharmacy and Veterinary Drugs | Kalina J.,Research Institute of Biopharmacy and Veterinary Drugs
Journal of Poultry Science | Year: 2012

Transplantation of male germ cells into sterilized recipients in chicken opened a new approach for chicken transgenesis as well as for preservation of endangered bird species. We describe the post transplantation re-population of the recipient seminiferous epithelium up to the production of heterologous sperm in about 50% of transplanted males. However, it is important to precisely identify basic germinal cell populations that trigger renewal cascade of the whole process of the spermatogenesis in the testes of cockerels. Detection of the nuclear DNA content by fluorescence-activated cell sorting (FACS) and expression of specific genes in single cell suspensions of testicular tissues seems to be suitable way for selection of proper cells. An improvement in the approach to germ cell transplantation between fowl males seems to be remarkably influenced by using an enriched subpopulation of c-Kit positive spermatogonia as well as SSEA-1 positive testicular cells as donor cells. Other chicken spermatogonia specific gene markers such as gfra, stra8 or dazl can also be used for basic sper-matogonial cells identification. Chicken recombinant GFRA1 receptor protein has been prepared and was used for obtaining mouse specific polyclonal antibodies. The presence of GFRA1 protein on the cell surface was confirmed by immunocytochemical and immunohistochemical analyses. Particular identification of cellular and molecular markers unique to the germ cells together with efficient transplantation model will make testicular stem cells an indispensable tool not only for biotechnological application but can also contribute to endangered species preservation programs. © 2012, Japan Poultry Science Association.


Vrba V.,Research Institute of Biopharmacy and Veterinary Drugs | Pakandl M.,Research Institute of Biopharmacy and Veterinary Drugs
International Journal for Parasitology | Year: 2014

Coccidiosis is a disease caused by apicomplexan parasites of the genus Eimeria, which has a significant economic impact on poultry production. Multiple species infecting the turkey have been described; however, due to the general lack of unambiguous description, their identification and taxonomy is debatable. In this work, a systematic approach was taken to isolate, characterise and compare coccidian species in the turkey. Individual species were tracked according to their unique 18S ribosomal DNA sequence. The single-oocyst isolation technique and passaging of mixed species field isolates in selectively immunised birds enabled the derivation of pure species. Six distinct strains representing five eimerian species that infect the turkey were obtained. It appears highly probable that these species represent all species described in the past with the exception of Eimeria subrotunda. The species were analysed using both traditional methods and DNA sequencing. For each strain the oocyst morphology, prepatent period, gross pathology, pathogenicity, host specificity and endogenous cycle were studied. Antigenic similarity was investigated in multiple cross-immunity experiments. For identification and quantification of each individual species or strain, quantitative real-time PCR markers were also developed. Parallel characterisation of pure strains allowed comprehensive comparison with the original descriptions and assignment of correct species names. The species Eimeria meleagridis, Eimeria dispersa, Eimeria gallopavonis, Eimeria meleagrimitis and Eimeria innocua were identified. Comparison of our data with those of previous studies indicates that Eimeria adenoeides is most probably a synonym for either E. meleagridis or E. gallopavonis, or a description based on a mixture of these species, and thus nomen dubium. The species E. dispersa and E. innocua were also found to infect Bobwhite Quail. Phylogenetic reconstruction based on 18S rDNA and cytochrome c oxidase subunit I gene (COI) sequences showed that these two species form a distinct clade unrelated to other turkey coccidia and point to a polyphyletic origin of the species infecting the turkey. © 2014 The Authors.


Barkway C.P.,Royal Veterinary College | Pocock R.L.,Royal Veterinary College | Vrba V.,Research Institute of Biopharmacy and Veterinary Drugs | Blake D.P.,Royal Veterinary College
Journal of Visualized Experiments | Year: 2015

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm’s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable. © JoVE 2006-2015. All Rights Reserved.


PubMed | Royal Veterinary College and Research Institute of Biopharmacy and Veterinary Drugs
Type: | Journal: Journal of visualized experiments : JoVE | Year: 2015

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farms anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.


PubMed | University of Guelph and Research Institute of Biopharmacy and Veterinary Drugs
Type: Journal Article | Journal: Mitochondrial DNA. Part A, DNA mapping, sequencing, and analysis | Year: 2016

The complete mitochondrial genome of Eimeria innocua KR strain (Eimeriidae, Coccidia, Apicomplexa) was sequenced. This coccidium infects turkeys (Meleagris gallopavo), Bobwhite quails (Colinus virginianus), and Grey partridges (Perdix perdix). Genome organization and gene contents were comparable with other Eimeria spp. infecting galliform birds. The circular-mapping mt genome of E. innocua is 6247bp in length with three protein-coding genes (cox1, cox3, and cytb), 19 gene fragments encoding large subunit (LSU) rRNA and 14 gene fragments encoding small subunit (SSU) rRNA. Like other Apicomplexa, no tRNA was encoded. The mitochondrial genome of E. innocua confirms its close phylogenetic affinities to Eimeria dispersa.


PubMed | Research Institute of Biopharmacy and Veterinary Drugs
Type: Journal Article | Journal: International journal for parasitology | Year: 2014

Coccidiosis is a disease caused by apicomplexan parasites of the genus Eimeria, which has a significant economic impact on poultry production. Multiple species infecting the turkey have been described; however, due to the general lack of unambiguous description, their identification and taxonomy is debatable. In this work, a systematic approach was taken to isolate, characterise and compare coccidian species in the turkey. Individual species were tracked according to their unique 18S ribosomal DNA sequence. The single-oocyst isolation technique and passaging of mixed species field isolates in selectively immunised birds enabled the derivation of pure species. Six distinct strains representing five eimerian species that infect the turkey were obtained. It appears highly probable that these species represent all species described in the past with the exception of Eimeria subrotunda. The species were analysed using both traditional methods and DNA sequencing. For each strain the oocyst morphology, prepatent period, gross pathology, pathogenicity, host specificity and endogenous cycle were studied. Antigenic similarity was investigated in multiple cross-immunity experiments. For identification and quantification of each individual species or strain, quantitative real-time PCR markers were also developed. Parallel characterisation of pure strains allowed comprehensive comparison with the original descriptions and assignment of correct species names. The species Eimeria meleagridis, Eimeria dispersa, Eimeria gallopavonis, Eimeria meleagrimitis and Eimeria innocua were identified. Comparison of our data with those of previous studies indicates that Eimeria adenoeides is most probably a synonym for either E. meleagridis or E. gallopavonis, or a description based on a mixture of these species, and thus nomen dubium. The species E. dispersa and E. innocua were also found to infect Bobwhite Quail. Phylogenetic reconstruction based on 18S rDNA and cytochrome c oxidase subunit I gene (COI) sequences showed that these two species form a distinct clade unrelated to other turkey coccidia and point to a polyphyletic origin of the species infecting the turkey.


Vrba V.,Research Institute of Biopharmacy and Veterinary Drugs | Pakandl M.,Research Institute of Biopharmacy and Veterinary Drugs
Veterinary Parasitology | Year: 2015

Protozoan parasites of the Eimeria genus have undergone extensive speciation and are now represented by a myriad of species that are specialised to different hosts. These species are highly host-specific and usually parasitise single host species, with only few reported exceptions. Doubts regarding the strict host specificity were frequent in the original literature describing coccidia parasitising domestic turkeys. The availability of pure characterised lines of turkey and chicken Eimeria species along with the recently developed quantitative PCR identification of these species allowed to investigate the issue of host specificity using well-controlled cross-transmission experiments. Seven species of gallinaceous birds (. Gallus gallus, Meleagris gallopavo, Alectoris rufa, Perdix perdix, Phasianus colchicus, Numida meleagris and Colinus virginianus) were inoculated with six species and strains of turkey Eimeria and six species of chicken coccidia and production of oocysts was monitored. Turkey Eimeria species E. dispersa, E. innocua and E. meleagridis could complete their development in the hosts from different genera or even different families. Comparison of phylogenetic positions of these Eimeria species according to 18S rDNA and COI showed that the phylogeny cannot explain the observed patterns of host specificity. These findings suggest that the adaptation of Eimeria parasites to foreign hosts is possible and might play a significant role in the evolution and diversification of this genus. © 2015 Elsevier B.V.


PubMed | Research Institute of Biopharmacy and Veterinary Drugs
Type: Journal Article | Journal: Animal reproduction science | Year: 2013

The identification, enrichment and subsequent isolation of spermatogonial stem cells (SSCs) are integral to the success of SCC transplants between fertile donor and sterilized recipient males. In birds generally and particularly in chicken, SSC-specific has yet to be identified. The receptor for glial cell-derived neurotrophic factor (GDNF), i.e. GDNF family receptor alpha-1 (GFR1), has been identified as a potential marker for different mouse spermatogonial subtypes. In the present study, we characterized the chicken cGFR1 receptor and compared its predicted amino-acid sequence with mouse, rat and human GFR1 proteins. Using specific polyclonal mouse anti-cGFR1 serum, a total of 2.8% cells were recognized as cGFR1-positive among isolated testicular cells recovered from sexually mature cockerels. The percentages of cGFR1-positive testicular cells with haploid, diploid, tetraploid and SP DNA content were 1.6%, 2.5%, 39.3% and 76.8%, respectively. The presence of cGFR1 protein on the surfaces of all cells of the seminiferous epithelium was confirmed by immunocytochemical and immunohistochemical analyses. Tissue specificity of cGFR1 mRNA expression was significantly higher in adult testes compared to brain tissue which itself was several times higher than tissues prepared from the spleen, liver and heart. No expression was observed in muscular tissue. At last, we demonstrated the successful repopulation of sterilized recipients testes with transplanted cGFR1-positive donor testicular cells. Recipient males subsequently produced functional heterologous spermatozoa capable of fertilizing an ovum and obtaining chicks with donor cell genotypes.


PubMed | Research Institute of Biopharmacy and Veterinary Drugs
Type: Journal Article | Journal: Veterinary parasitology | Year: 2015

Protozoan parasites of the Eimeria genus have undergone extensive speciation and are now represented by a myriad of species that are specialised to different hosts. These species are highly host-specific and usually parasitise single host species, with only few reported exceptions. Doubts regarding the strict host specificity were frequent in the original literature describing coccidia parasitising domestic turkeys. The availability of pure characterised lines of turkey and chicken Eimeria species along with the recently developed quantitative PCR identification of these species allowed to investigate the issue of host specificity using well-controlled cross-transmission experiments. Seven species of gallinaceous birds (Gallus gallus, Meleagris gallopavo, Alectoris rufa, Perdix perdix, Phasianus colchicus, Numida meleagris and Colinus virginianus) were inoculated with six species and strains of turkey Eimeria and six species of chicken coccidia and production of oocysts was monitored. Turkey Eimeria species E. dispersa, E. innocua and E. meleagridis could complete their development in the hosts from different genera or even different families. Comparison of phylogenetic positions of these Eimeria species according to 18S rDNA and COI showed that the phylogeny cannot explain the observed patterns of host specificity. These findings suggest that the adaptation of Eimeria parasites to foreign hosts is possible and might play a significant role in the evolution and diversification of this genus.

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