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Liu, J., Fu, Z., Hellman, L. & Svärd, S. G. (2019). Cleavage specificity of recombinant Giardia intestinalis cysteine proteases: Degradation of immunoglobulins and defensins. Molecular and biochemical parasitology (Print), 227, 29-38
Open this publication in new window or tab >>Cleavage specificity of recombinant Giardia intestinalis cysteine proteases: Degradation of immunoglobulins and defensins
2019 (English)In: Molecular and biochemical parasitology (Print), ISSN 0166-6851, E-ISSN 1872-9428, Vol. 227, p. 29-38Article in journal (Refereed) Published
Abstract [en]

Giardia intestinalis is a protozoan parasite and the causative agent of giardiasis, a common diarrheal disease. Cysteine protease (CP) activities have been suggested to be involved in Giardia's pathogenesis and we have recently identified and characterized three secreted Giardia CPs; CP14019, CP16160 and CP16779. Here we have studied the cleavage specificity of these CPs using substrate phage display and recombinant protein substrates. The phage display analyses showed that CP16160 has both chymase and tryptase activity and a broad substrate specificity. This was verified using recombinant protein substrates containing different variants of the cleavage sites. Phage display analyses of CP14019 and CP16779 failed but the substrate specificity of CP14019 and CP16779 was tested using the recombinant substrates generated for CP16160. CP16160 and CP14019 showed similar substrate specificity, while CP16779 has a slightly different substrate specificity. The consensus sequence for cleavage by CP16160, obtained from phage display analyses, was used in an in silico screen of the human intestinal proteome for detection of potential targets. Immunoglobulins, including IgA and IgG and defensins (α-HD6 and β-HD1) were predicted to be targets and they were shown to be cleaved by the recombinant CPs in vitro. Our results suggest that the secreted Giardia CPs are key players in the interaction with host cells during Giardia infections since they can cleave several components of the human mucosal defense machinery.

Keywords
Cysteine protease, Defensins, Diarrhea, Immunoglobulins, Parasite, Phage display
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-372982 (URN)10.1016/j.molbiopara.2018.10.004 (DOI)000457660900006 ()30458129 (PubMedID)
Funder
Swedish Research Council, 2017-02918
Available from: 2019-01-10 Created: 2019-01-10 Last updated: 2019-03-05Bibliographically approved
Li, Z., Peirasmaki, D., Svärd, S. & Åbrink, M. (2019). Giardia excretory-secretory proteins modulate the enzymatic activities of mast cell chymase and tryptase. Molecular Immunology, 114, 535-544
Open this publication in new window or tab >>Giardia excretory-secretory proteins modulate the enzymatic activities of mast cell chymase and tryptase
2019 (English)In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 114, p. 535-544Article in journal (Refereed) Published
Abstract [en]

Background

Mast cells are involved in the host immune response controlling infection with the non-invasive intestinal protozoan parasite Giardia intestinalis. Experimental infections in rodents with G. intestinalis showed increased intestinal expression of mucosal and connective mast cell specific proteases suggesting that both mucosal and connective tissue mast cells are recruited and activated during infection. During infection Giardia excretory-secretory proteins (ESPs) with immunomodulatory capacity are released. However, studies investigating potential interactions between Giardia ESPs and the connective tissue mast cell specific serine proteases, i.e. human chymase and mouse mast cell protease (mMCP)-4 and, human and mouse tryptase (mMCP-6) remain scarce.

Results

We first investigated if soluble Giardia proteins (sGPs), which over-lap extensively in protein content with ESP fractions, from the isolates GS, WB and H3, could induce mast cell activation. sGPs induced a minor activation of bone marrow derived mucosal-like mast cells, as indicated by increased IL-6 secretion and no degranulation. Furthermore, sGPs were highly resistant to degradation by human tryptase while human chymase degraded a 65 kDa sGP and, wild-type mouse ear tissue extracts degraded several protein bands in the 10 to 75 kDa range. In striking contrast, sGPs and ESPs were found to increase the enzymatic activity of human and mouse tryptase and to reduce the activity of human and mouse chymase.

Conclusion

Our finding suggests that Giardia ssp. via enhancement or reduction of mast cell protease activity may modulate mast cell-driven intestinal immune responses. ESP-mediated modulation of the mast cell specific proteases may also increase degradation of tight junctions, which may be beneficial for Giardia ssp. during infection.

Keywords
Mast cell, Tryptase, Chymase, Infection, Giardia ssp., Parasite, Intestine, Giardia excretory-secretory proteins (ESPs)
National Category
Immunology Infectious Medicine
Identifiers
urn:nbn:se:uu:diva-396740 (URN)10.1016/j.molimm.2019.07.024 (DOI)000490625600050 ()31518857 (PubMedID)
Funder
Swedish Research Council
Available from: 2019-11-26 Created: 2019-11-26 Last updated: 2019-11-26Bibliographically approved
Liu, J., Svärd, S. & Klotz, C. (2019). Giardia intestinalis cystatin is a potent inhibitor of papain, parasite cysteine proteases and, to a lesser extent, human cathepsin B. FEBS Letters, 593(12), 1313-1325
Open this publication in new window or tab >>Giardia intestinalis cystatin is a potent inhibitor of papain, parasite cysteine proteases and, to a lesser extent, human cathepsin B
2019 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 593, no 12, p. 1313-1325Article in journal (Refereed) Published
Abstract [en]

Cystatins are important regulators of papain-like cysteine proteases. In the protozoan parasite Giardia intestinalis, papain-like cysteine proteases play an essential role in the parasite's biology and pathogenicity. Here, we characterized a cysteine protease inhibitor of G. intestinalis that belongs to type-I-cystatins. The parasite cystatin is shown to be a strong inhibitor of papain (K-i approximate to 0.3 nm) and three parasite cysteine proteases (CP14019, CP16160 and CP16779, K-i approximate to 0.9-5.8 nm), but a weaker inhibitor of human cathepsin B (K-i approximate to 79.9 nm). The protein localizes mainly in the cytoplasm. Together, these data suggest that cystatin of G. intestinalis plays a role in the regulation of cysteine protease activities in the parasite and, possibly, in the interaction with the host.

Place, publisher, year, edition, pages
John Wiley & Sons, 2019
Keywords
cystatin, cysteine protease inhibitor, cysteine proteases, Giardia intestinalis, protozoa
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-390594 (URN)10.1002/1873-3468.13433 (DOI)000472673700005 ()31077354 (PubMedID)
Funder
Swedish Research Council, 2017-02918
Available from: 2019-08-13 Created: 2019-08-13 Last updated: 2019-08-13Bibliographically approved
Davids, B. J., Liu, C. M., Hanson, E. M., Le, C. H. Y., Ang, J., Hanevik, K., . . . Eckmann, L. (2019). Identification of Conserved Candidate Vaccine Antigens in the Surface Proteome of Giardia lamblia. Infection and Immunity, 87(6), Article ID e00219-19.
Open this publication in new window or tab >>Identification of Conserved Candidate Vaccine Antigens in the Surface Proteome of Giardia lamblia
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2019 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 87, no 6, article id e00219-19Article in journal (Refereed) Published
Abstract [en]

Giardia lamblia, one of the most common protozoal infections of the human intestine, is an important worldwide cause of diarrheal disease, malabsorption, malnutrition, delayed cognitive development in children, and protracted postinfectious syndromes. Despite its medical importance, no human vaccine is available against giardiasis. A crude veterinary vaccine has been developed, and experimental vaccines based on expression of multiple variant-specific surface proteins have been reported, but poorly defined vaccine components and excessive antigen variability are problematic for pharmaceutical vaccine production. To expand the repertoire of antigen candidates for vaccines, we reasoned that surface proteins may provide an enriched source of such antigens since key host effectors, such as secretory IgA, can directly bind to such antigens in the intestinal lumen and interfere with epithelial attachment. Here, we have applied a proteomics approach to identify 23 novel surface antigens of G. lamblia that show >90% amino acid sequence identity between the two human-pathogenic genetic assemblages (A and B) of the parasite. Surface localization of a representative subset of these proteins was confirmed by immunostaining. Four selected proteins, uridine phosphorylase-like protein-1, protein 21.1 (GL50803_ 27925), alpha 1-giardin, and alpha 11-giardin, were subsequently produced in recombinant form and shown to be immunogenic in mice and G. lamblia-infected humans and confer protection against G. lamblia infection upon intranasal immunization in rodent models of giardiasis. These results demonstrate that identification of conserved surface antigens provides a powerful approach for overcoming a key rate-limiting step in the design and construction of an effective vaccine against giardiasis.

Place, publisher, year, edition, pages
AMER SOC MICROBIOLOGY, 2019
Keywords
giardiasis, immunization, surface antigens, vaccines
National Category
Infectious Medicine
Identifiers
urn:nbn:se:uu:diva-385959 (URN)10.1128/IAI.00219-19 (DOI)000468521100017 ()30962402 (PubMedID)
Funder
NIH (National Institute of Health), AI112594
Available from: 2019-06-19 Created: 2019-06-19 Last updated: 2019-06-19Bibliographically approved
Stairs, C. W., Kokla, A., Astvaldsson, A., Jerlström-Hultqvist, J., Svärd, S. & Ettema, T. J. G. (2019). Oxygen induces the expression of invasion and stress response genes in the anaerobic salmon parasite Spironucleus salmonicida. BMC Biology, 17(1), Article ID 19.
Open this publication in new window or tab >>Oxygen induces the expression of invasion and stress response genes in the anaerobic salmon parasite Spironucleus salmonicida
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2019 (English)In: BMC Biology, ISSN 1741-7007, E-ISSN 1741-7007, Vol. 17, no 1, article id 19Article in journal (Refereed) Published
Abstract [en]

Background: Spironucleus salmonicida is an anaerobic parasite that can cause systemic infections in Atlantic salmon. Unlike other diplomonad parasites, such as the human pathogen Giardia intestinalis, Spironucleus species can infiltrate the blood stream of their hosts eventually colonizing organs, skin and gills. How this presumed anaerobe can persist and invade oxygenated tissues, despite having a strictly anaerobic metabolism, remains elusive.

Results: To investigate how S. salmonicida response to oxygen stress, we performed RNAseq transcriptomic analyses of cells grown in the presence of oxygen or antioxidant-free medium. We found that over 20% of the transcriptome is differentially regulated in oxygen (1705 genes) and antioxidant-depleted (2280 genes) conditions. These differentially regulated transcripts encode proteins related to anaerobic metabolism, cysteine and Fe-S cluster biosynthesis, as well as a large number of proteins of unknown function. S. salmonicida does not encode genes involved in the classical elements of oxygen metabolism (e.g., catalases, superoxide dismutase, glutathione biosynthesis, oxidative phosphorylation). Instead, we found that genes encoding bacterial-like oxidoreductases were upregulated in response to oxygen stress. Phylogenetic analysis revealed some of these oxygen-responsive genes (e.g., nadh oxidase, rubrerythrin, superoxide reductase) are rare in eukaryotes and likely derived from lateral gene transfer (LGT) events into diplomonads from prokaryotes. Unexpectedly, we observed that many host evasion- and invasion-related genes were also upregulated under oxidative stress suggesting that oxygen might be an important signal for pathogenesis.

Conclusion: While oxygen is toxic for related organisms, such as G. intestinalis, we find that oxygen is likely a gene induction signal for host invasion- and evasion-related pathways in S. salmonicida. These data provide the first molecular evidence for how S. salmonicida could tolerate oxic host environments and demonstrate how LGT can have a profound impact on the biology of anaerobic parasites.

Keywords
Anaerobiosis, Diplomonads, Giardia, Lateral gene transfer, Oxygen stress, Parasitology, Protist, RNAseq, Spironucleosis, Spironucleus
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-378917 (URN)10.1186/s12915-019-0634-8 (DOI)000459961200001 ()
Funder
EU, European Research Council, 310039-PUZZLE_CELLSwedish Foundation for Strategic Research Swedish Research Council, 2015-04959Swedish Research Council Formas, 2016-00539
Available from: 2019-03-12 Created: 2019-03-12 Last updated: 2019-03-22Bibliographically approved
Astvaldsson, A., Hultenby, K., Svärd, S. & Jerlström-Hultqvist, J. (2019). Proximity Staining using Enzymatic Protein Tagging in Diplomonads. mSphere, 4(2), Article ID e00153-19.
Open this publication in new window or tab >>Proximity Staining using Enzymatic Protein Tagging in Diplomonads
2019 (English)In: mSphere, E-ISSN 2379-5042, Vol. 4, no 2, article id e00153-19Article in journal (Refereed) Published
Abstract [en]

The diplomonads are a group of understudied eukaryotic flagellates whose most prominent member is the human pathogen Giardia intestinalis. Methods commonly used in other eukaryotic model systems often require special optimization in diplomonads due to the highly derived character of their cell biology. We have optimized a proximity labeling protocol using pea ascorbate peroxidase (APEX) as a reporter for transmission electron microscopy (TEM), to enable study of ultrastructural cellular details in diplomonads. Currently available TEM-compatible tags requires light-induced activation (1, 2) or are inactive in many cellular compartments (3) while ascorbate peroxidase has not been shown to have those limitations. Here we have optimized the in vivo activity of two versions of pea ascorbate peroxidase (APXW41F and APEX) using the diplomonad fish parasite Spironucleus salmonicida, a relative of G. intestinalis. We exploited the well-known peroxidase substrates, Amplex UltraRed and 3,3’-diaminobenzidine (DAB), to validate the activity of the two tags and argue that APEX is the most stable version to use in Spironucleus salmonicida. Next, we fused APEX to proteins with established localization to evaluate the activity of APEX in different cellular compartments of the diplomonad cell and used Amplex UltraRed as well as antibodies along with super-resolution microscopy to confirm the protein-APEX localization. The ultrastructural details of protein-APEX fusions were determined by TEM and we observed marker activity in all cellular compartments tested when using the DAB substrate. Finally, we show that the optimized conditions established for S. salmonicida can be used in the related diplomonad G. intestinalis.

National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-379669 (URN)
Funder
Swedish Research Council Formas, 2016-00539
Available from: 2019-03-19 Created: 2019-03-19 Last updated: 2019-03-20
Astvaldsson, A., Hultenby, K., Svärd, S. G. & Jerlström-Hultqvist, J. (2019). Proximity Staining Using Enzymatic Protein Tagging in Diplomonads. MSPHERE, 4(2), Article ID e00153-19.
Open this publication in new window or tab >>Proximity Staining Using Enzymatic Protein Tagging in Diplomonads
2019 (English)In: MSPHERE, ISSN 2379-5042, Vol. 4, no 2, article id e00153-19Article in journal (Refereed) Published
Abstract [en]

The diplomonads are a group of understudied eukaryotic flagellates whose most prominent member is the human pathogen Giardia intestinalis. Methods commonly used in other eukaryotic model systems often require special optimization in diplomonads due to the highly derived character of their cell biology. We have optimized a proximity labeling protocol using pea ascorbate peroxidase (APEX) as a reporter for transmission electron microscopy (TEM) to enable the study of ultrastructural cellular details in diplomonads. Currently available TEM-compatible tags require light-induced activation (1, 2) or are inactive in many cellular compartments (3), while ascorbate peroxidase has not been shown to have those limitations. Here, we have optimized the in vivo activities of two versions of pea ascorbate peroxidase (APX(W41F) and APEX) using the diplomonad fish parasite Spironucleus salmonicida, a relative of G. intestinalis. We exploited the well-known peroxidase substrates, Amplex UltraRed and 3,3'-diaminobenzidine (DAB), to validate the activity of the two tags and argue that APEX is the most stable version to use in Spironucleus salmonicida. Next, we fused APEX to proteins with established localization to evaluate the activity of APEX in different cellular compartments of the diplomonad cell and used Amplex UltraRed as well as antibodies along with superresolution microscopy to confirm the protein-APEX localization. The ultrastructural details of protein-APEX fusions were determined by TEM, and we observed marker activity in all cellular compartments tested when using the DAB substrate. Finally, we show that the optimized conditions established for S. salmonicida can be used in the related diplomonad G. intestinalis. IMPORTANCE The function of many proteins is intrinsically related to their cellular location. Novel methods for ascertainment of the ultrastructural location of proteins have been introduced in recent years, but their implementation in protists has so far not been readily realized. Here, we present an optimized proximity labeling protocol using the APEX system in the salmon pathogen Spironucleus salmonicida. This protocol was also applicable to the human pathogen Giardia intestinalis. Both organisms required extraneous addition of hemin to the growth medium to enable detectable peroxidase activity. Further, we saw no inherent limitation in labeling efficiency coupled to the cellular compartment, as evident with some other proximity labeling systems. We anticipate that the APEX proximity labeling system might offer a great resource to establish the ultrastructural localization of proteins across genetically tractable protists but might require organism-specific labeling conditions.

Keywords
APEX, DAB, Giardia, proximity labeling, Spironucleus salmonicida
National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-383485 (URN)10.1128/mSphereDirect.00153-19 (DOI)000465544000005 ()30894436 (PubMedID)
Funder
Swedish Research Council Formas
Available from: 2019-05-16 Created: 2019-05-16 Last updated: 2019-05-16Bibliographically approved
Ankarklev, J., Lebbad, M., Einarsson, E., Franzen, O., Ahola, H., Troell, K. & Svärd, S. (2018). A novel high-resolution multilocus sequence typing of Giardia intestinalis Assemblage A isolates reveals zoonotic transmission, clonal outbreaks and recombination. Infection, Genetics and Evolution, 60, 7-16
Open this publication in new window or tab >>A novel high-resolution multilocus sequence typing of Giardia intestinalis Assemblage A isolates reveals zoonotic transmission, clonal outbreaks and recombination
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2018 (English)In: Infection, Genetics and Evolution, ISSN 1567-1348, E-ISSN 1567-7257, Vol. 60, p. 7-16Article in journal (Refereed) Published
Abstract [en]

Molecular epidemiology and genotyping studies of the parasitic protozoan Giardia intestinalis have proven difficult due to multiple factors, such as low discriminatory power in the commonly used genotyping loci, which has hampered molecular analyses of outbreak sources, zoonotic transmission and virulence types. Here we have focused on assemblage A Giardia and developed a high-resolution assemblage-specific multilocus sequence typing (MLST) method. Analyses of sequenced G. intestinalis assemblage A genomes from different sub-assemblages identified a set of six genetic loci with high genetic variability. DNA samples from both humans (n = 44) and animals (n = 18) that harbored Giardia assemblage A infections, were PCR amplified (557-700 bp products) and sequenced at the six novel genetic loci. Bioinformatic analyses showed five to ten-fold higher levels of polymorphic sites than what was previously found among assemblage A samples using the classic genotyping loci. Phylogenetically, a division of two major clusters in assemblage A became apparent, separating samples of human and animal origin. A subset of human samples (n = 9) from a documented Giardia outbreak in a Swedish day-care center, showed full complementarity at nine genetic loci (the six new and the standard BG, TPI and GDH loci), strongly suggesting one source of infection. Furthermore, three samples of human origin displayed MLST profiles that were phylogenetically more closely related to MLST profiles from animal derived samples, suggesting zoonotic transmission. These new genotyping loci enabled us to detect events of recombination between different assemblage A isolates but also between assemblage A and E isolates. In summary, we present a novel and expanded MLST strategy with significantly improved sensitivity for molecular analyses of virulence types, zoonotic potential and source tracking for assemblage A Giardia.

Place, publisher, year, edition, pages
ELSEVIER SCIENCE BV, 2018
Keywords
Parasite, Diarrhea, Recombination, Zoonosis, Transmission
National Category
Infectious Medicine
Identifiers
urn:nbn:se:uu:diva-353095 (URN)10.1016/j.meegid.2018.02.012 (DOI)000428317200002 ()29438742 (PubMedID)
Funder
Swedish Research Council, 2012-03364
Available from: 2018-06-20 Created: 2018-06-20 Last updated: 2018-06-20Bibliographically approved
Emery, S. J., Baker, L., Ansell, B. R. E., Mirzaei, M., Haynes, P. A., McConville, M. J., . . . Jex, A. R. (2018). Differential protein expression and post-translational modifications in metronidazole-resistant Giardia duodenalis. GigaScience, 7(4)
Open this publication in new window or tab >>Differential protein expression and post-translational modifications in metronidazole-resistant Giardia duodenalis
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2018 (English)In: GigaScience, ISSN 2047-217X, E-ISSN 2047-217X, Vol. 7, no 4Article in journal (Refereed) Published
Abstract [en]

Background: Metronidazole (Mtz) is the frontline drug treatment for multiple anaerobic pathogens, including the gastrointestinal protist, Giardia duodenalis. However, treatment failure is common and linked to in vivo drug resistance. In Giardia, in vitro drug-resistant lines allow controlled experimental interrogation of resistance mechanisms in isogenic cultures. However, resistance-associated changes are inconsistent between lines, phenotypic data are incomplete, and resistance is rarely genetically fixed, highlighted by reversion to sensitivity after drug selection ceases or via passage through the life cycle. Comprehensive quantitative approaches are required to resolve isolate variability, fully define Mtz resistance phenotypes, and explore the role of post-translational modifications therein. Findings: We performed quantitative proteomics to describe differentially expressed proteins in 3 seminal Mtz-resistant lines compared to their isogenic, Mtz-susceptible, parental line. We also probed changes in post-translational modifications including protein acetylation, methylation, ubiquitination, and phosphorylation via immunoblotting. We quantified more than 1,000 proteins in each genotype, recording substantial genotypic variation in differentially expressed proteins between isotypes. Our data confirm substantial changes in the antioxidant network, glycolysis, and electron transport and indicate links between protein acetylation and Mtz resistance, including cross-resistance to deacetylase inhibitor trichostatin A in Mtz-resistant lines. Finally, we performed the first controlled, longitudinal study of Mtz resistance stability, monitoring lines after cessation of drug selection, revealing isolate-dependent phenotypic plasticity. Conclusions: Our data demonstrate understanding that Mtz resistance must be broadened to post-transcriptional and post-translational responses and that Mtz resistance is polygenic, driven by isolate-dependent variation, and is correlated with changes in protein acetylation networks.

Place, publisher, year, edition, pages
OXFORD UNIV PRESS, 2018
Keywords
Giardia duodenalis, quantitative proteomics, metronidazole, drug resistance, protein post-translational modifications
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-356813 (URN)10.1093/gigascience/giy024 (DOI)000433062900001 ()29688452 (PubMedID)
Available from: 2018-08-16 Created: 2018-08-16 Last updated: 2018-08-16Bibliographically approved
Caccio, S. M., Lalle, M. & Svärd, S. (2018). Host specificity in the Giardia duodenalis species complex. Infection, Genetics and Evolution, 66, 335-345
Open this publication in new window or tab >>Host specificity in the Giardia duodenalis species complex
2018 (English)In: Infection, Genetics and Evolution, ISSN 1567-1348, E-ISSN 1567-7257, Vol. 66, p. 335-345Article, review/survey (Refereed) Published
Abstract [en]

Giardia duodenalis is a unicellular flagellated parasite that infects the gastrointestinal tract of a wide range of mammalian species, including humans. Investigations of protein and DNA polymorphisms revealed that G. duodenalis should be considered as a species complex, whose members, despite being morphologically indistinguishable, can be classified into eight groups, or Assemblages, separated by large genetic distances. Assemblages display various degree of host specificity, with Assemblages A and B occurring in humans and many other hosts, Assemblage C and D in canids, Assemblage E in hoofed animals, Assemblage F in cats, Assemblage G in rodents, and Assemblage H in pinnipeds. The factors determining host specificity are only partially understood, and clearly involve both the host and the parasite. Here, we review the results of in vitro and in vivo experiments, and clinical observations to highlight relevant biological and genetic differences between Assemblages, with a focus on human infection.

Keywords
Giardia duodenalis, Host specificity, Host-parasite interactions, In vivo models, Genomics
National Category
Infectious Medicine
Identifiers
urn:nbn:se:uu:diva-373081 (URN)10.1016/j.meegid.2017.12.001 (DOI)000452589900043 ()29225147 (PubMedID)
Funder
Swedish Research Council
Available from: 2019-01-11 Created: 2019-01-11 Last updated: 2019-01-11Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-7392-1746

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