Periodic appearance of a predominant variant antigen type during a chronic Giardia lamblia infection in a mouse model

Giardia lamblia (syn. Giardia duodenalis, Giardia intestinalis) infections are associated with continuous antigenic variation of the parasite which is mediated by the parasite's major surface antigen, named variant surface protein. Offspring mice and corresponding mothers were infected with G. lamblia clone GS/M-83-H7 (expressing variant surface protein H7) and various parameters of this infection were assessed in a long-term follow-up investigation. Our experimentation revealed that variant surface protein H7-type trophozoites were replaced by new variant-type trophozoites during the early stage of infection (around day 8 p.i.), but the original variant-type re-emerged at at least two time-points during the later stages of infection (at days 22 and 42 p.i.). Such periods of variant surface protein H7-type trophozoite re-expansion were accompanied by transient production of intestinal IgA against variant-specific epitopes on a 314-aa N-terminal region of variant surface protein H7. At late stages of infection (between days 42 and 200 p.i.), most mice produced intestinal IgA against both variant surface protein H7 and other antigens of the parasite. At these stages, infection seemed to be resolved in most mice, but occasional reappearance of relatively high (at day 64 p.i.) or at least detectable (at days 80 and 120 p.i.) amounts of intestinal parasites indicated that G. lamblia GS/M-83-H7 infections in mice may enter into a latent chronic phase which is interrupted by sporadic breakthroughs of parasite growth.     

Molecular characterisation of a predominant antigenic region of Giardia lamblia variant surface protein H7

During infection, the intestinal protozoan parasite Giardia lamblia undergoes continuous antigenic variation which is determined by diversification of the parasite's major surface antigen, named VSP (variant surface protein). One member from this protein family, VSP H7, is expressed by G. lamblia clone GS/M-83-H7. In the present study, we characterised a highly antigenic portion of VSP H7 which is positioned inside a 130 amino acid C-terminal region of the protein. This region overlaps with a cysteine-rich motif that is rather conserved within the VSP family. Detailed molecular dissection of the antigenic portion monitored a 12 amino acid peptidyl structure which constitutes a non-conformational epitope of VSP H7. In the murine host, this epitope is recognised relatively early (before day 10 p.i.) during infection and stimulates a strong intestinal immunoglobulin A response. At late infective stages (after day 10 p.i.) this immune reaction is progressively complemented by reactions against 'late' antigenic epitopes which are also located inside the 130 amino acid antigenic portion but in closer proximity to the C-terminal end of VSP H7 than the 12 amino acid epitope. Both the high antigenicity and the conserved character suggest that the 12 amino acid epitope is a key factor within the immunological interplay between G. lamblia and the experimental murine host.     

Antigenic variation and the murine immune response to Giardia lamblia

The protozoan parasite Giardia lamblia is an important causative agent of acute or chronic diarrhoea in humans and various animals. During infection, the parasite survives the hosts reactions by undergoing continuous antigenic variation of its major surface antigen, named VSP (variant surface protein). The VSPs form a unique family of cysteine-rich proteins that are extremely heterogeneous in size. The relevance of antigenic variation for the survival in the host has been most successfully studied by performing experimental infections in a combined mother/offspring mouse system and by using the G. lamblia clone GS/M-83-H7 (human isolate) as model parasite. In-vivo antigenic variation of G. lamblia clone GS/M-83-H7 is characterised by a diversification of the intestinal parasite population into a complex mixture of different variant antigen types. It could be shown that maternally transferred lactogenic anti-VSP IgA antibodies exhibit cytotoxic activity on the Giardia variant-specific trophozoites in suckling mice, and thus express a modulatory function on the proliferative parasite population characteristics. Complementarily, in-vitro as well as in-vivo experiments in adult animals indicated that non-immunological factors such as intestinal proteases may interfere into the process of antigen variation in that they favour proliferation of those variant antigen-type populations which resist the hostile physiological conditions within the intestine. These observations suggest that an interplay between immunological and physiological factors, rather than one of these two factor alone, modulates antigenic diversification of a G. lamblia population within an experimental murine host and thus influences the survival rate and strategy of the parasite.     

Overlapping genes in parasitic protist Giardia lamblia.

The parasitic protist Giardia lamblia lacks mitochondria and peroxisomes, as well as many typical membrane-bound organella characteristics of higher eukaryotic cells, together with extremely economized usage of DNA sequence, as demonstrated by the lack of introns. We describe here the presence of overlapping genes in G. lamblia, in which a part of the protein coding sequence of one mRNA exists in a region corresponding to the 3′-noncoding region of another mRNA transcribed from a gene on the opposite strand. Recently we isolated 13 kinesin-related cDNAs from G. lamblia. Nine of these cDNAs contain long 3′-noncoding sequences in which long open reading frames (ORFs) exist (in the remaining four cDNAs, the lengths of the 3′-noncoding sequences are very short). The predicted amino acid sequences of these ORFs were subjected to a search for homologies with sequences in databases. The amino acid sequences of the six ORFs exhibited significant sequence similarities with known sequences. These lines of evidence suggest the frequent occurrence of gene overlap in Giardial genome.     

Prevalence and genotyping of human isolates of Giardia duodenalis from Albania

Microscopical and PCR-based techniques were performed in order to investigate the prevalence of infection and the genotypes of Giardia duodenalis from 125 stool samples collected from children living in the urban and the rural areas of Tirana (Albania) and hospitalized with acute gastroenteritis. 7 out of 125 samples resulted positive for Giardia at the microscopic examination (5.6%). In 50 selected samples including the 7 samples positive for Giardia by microscopy, 3 and 15 additional positive samples were detected by immunofluorescence and PCR, respectively. Seasonality appeared as an important parameter to be evaluated in order to better understand the prevalence of infection. Sequence analysis revealed both human Assemblage A and B. This result represents the first data on G. duodenalis genotypes in Albania.     

Recent insights into the mucosal reactions associated with Giardia lamblia infections

Giardia lamblia is an intestinal protozoan parasite infecting humans and various other mammalian hosts. The most important clinical signs of giardiasis are diarrhoea and malabsorption. Giardia lamblia is able to undergo continuous antigenic variation of its major surface antigen, named VSP (variant surface protein). While intestinal antibodies, and more specifically anti-VSP IgA antibodies, were proven to be involved in modulating antigenic variation of the parasite the participation of the local antibody response in control of the parasite infection is still controversial. Conversely, previous studies based on experimental infections in mice showed that cellular immune mechanisms are essential for elimination of the parasite from its intestinal habitat. Furthermore, recent data indicated that inflammatory mast cells have a potential to directly, or indirectly, interfere in duodenal growth of G. lamblia trophozoites. However, this finding was challenged by other reports, which did not find a correlation between intestinal inflammation and resistance to infection. Since intestinal infiltration of inflammatory cells and/or CD8+T-cells were demonstrated to coincide with villus-shortening and crypt hyperplasia immunological reactions were considered to be a potential factor of pathogenesis in giardiasis. The contribution of physiological factors to pathogenesis was essentially assessed in vitro by co-cultivation of G. lamblia trophozoites with epithelial cell lines. By using this in vitro model, molecular (through surface lectins) and mechanical (through ventral disk) adhesion of trophozoites to the epithelium was shown to be crucial for increased epithelial permeability. This phenomenon as well as other Giardia-induced intestinal abnormalities such as loss of intestinal brush border surface area, villus flattening, inhibition of disaccharidase activities, and eventually also overgrowth of the enteric bacterial flora seem to be involved in the pathophysiology of giardiasis. However, it remains to be elucidated whether at least part of these pathological effects are causatively linked to the clinical manifestation of the disease.     

Antigenic variation of Giardia lamblia in experimental human infections

To determine if Giardia surface Ag vary in human infections volunteers were inoculated enterally with trophozoites of uncloned GS/M-85 and in later experiments with two clones derived from GS/M. The surface Ag of trophozoites reisolated from 6/6 volunteers differed from the inoculum. To determine if the surface Ag of trophozoites derived from clones would also change, volunteers were inoculated with two clones, B6 or H7. B6 possesses a 200-kDa surface Ag recognized by mAb 3F6 and H7 has a 72-kDa surface Ag recognized by mAb G10/4. One of thirteen B6 and four of four H7-inoculated volunteers became infected. Analysis of Giardia obtained on day 22 from the intestines of the four H7-infected volunteers and cultures derived from these trophozoites revealed loss of the initial major surface Ag as determined by surface IFA using mAb, surface radiolabeling and loss of cytotoxicity to mAb, and Western blots. Loss of the 72-kDa Ag began after day 14 and was practically complete by day 22. The 200-kDa surface Ag was almost totally absent from the surface of Giardia isolated from the single B6-infected volunteer. Serum surface-reactive antibodies, as measured by IFA and cytotoxicity to H7 and the day 22 isolates, showed high levels of antibodies to H7, primarily to the 72-kDa surface Ag, but negligible or low levels of late-appearing antibodies to the day 22 isolates. These studies document antigenic variation of Giardia in human infections and show that humoral responses are in part isolate-specific.     

Histopathology and cellular response in Enteromyxum leei (Myxozoa) infections of Diplodus puntazzo (Teleostei)

Enteromyxum leei is an intestinal parasite responsible for serious outbreaks in Mediterranean sharpsnout sea bream Diplodus puntazzo. E. leei infection was experimentally transmitted to healthy D. puntazzo (R) by cohabitation with infected donor fish. Haematological changes and histopathological damage were evaluated in relation to the course of infection. The prevalence of infection in R fish was 100% from day 10 post-exposure (p.e.) onwards, and the infection intensity and histopathological damage increased progressively. Different developmental stages were found in the infected intestines, including proliferative (stages 1–3) and sporogonic (stages 4 and 5) stages. Intestinal damage consisted of vacuolation, necrosis, detachment and sloughing of epithelium, and was correlated with the progression of the infection and with the development of the parasite. Sporogonic stages appeared from day 20 p.e. onwards. Initially, D. puntazzo seems to counteract the infection through the increase in leucocyte numbers, respiratory burst activity, haematopoietic activity and MMC. Two types of eosinophilic granular cells (EGC1 and EGC2) were detected in the intestinal epithelium and lamina propria. EGC1 numbers decreased with the progression of infection, whereas an increase in EGC2 occurred, mainly in the lamina propria. The involvement of the cellular immunity in the response of D. puntazzo to E. leei was demonstrated. The depletion of this response at a certain point of the infection could contribute to the high virulence of this myxozoan in this fish species.     

Host immunity and pathogen strain contribute to intestinal disaccharidase impairment following gut infection

Infection or other inflammatory insults in the small intestine often result in reduced disaccharidase enzyme levels. Using a mouse model of giardiasis, we examined the role of host immunity and pathogen virulence in mediating disaccharidase deficiency postinfection (p.i.). C57BL/6J mice were infected with two strains, WB and GS, of the human parasite Giardia duodenalis. The levels of sucrase, maltase, and lactase decreased in wild-type mice p.i. with the GS strain but not with the WB strain. Both CD4-deficient and SCID mice failed to eliminate the infection and did not exhibit disaccharidase deficiency. β(2)-Microglobulin knockout animals controlled infections similar to wild-type mice but exhibited no decrease in disaccharidase activity. Analysis of cytokine production by spleen and mesenteric lymph node cells showed production of IL-4, IL-10, IL-13, IL-17, IL-22, TNF-α, and IFN-γ p.i. with both WB and GS, with IFN-γ being the dominant cytokine for both parasite strains. Mesenteric lymph node cells produced lower levels of cytokines compared with splenocytes in response to parasite extract, although the overall pattern was similar. These data suggest that T cell responses mediate parasite clearance whereas also contributing to pathogenesis. They also demonstrate that differences in pathogen strain can also determine the outcome of infection and further our understanding of the clinical variation seen in human giardiasis.     

Efficacy of a Parainfluenza Virus 5 (PIV5)-Based H7N9 Vaccine in Mice and Guinea Pigs: Antibody Titer towards HA Was Not a Good Indicator for Protection

H7N9 has caused fatal infections in humans. A safe and effective vaccine is the best way to prevent large-scale outbreaks in the human population. Parainfluenza virus 5 (PIV5), an avirulent paramyxovirus, is a promising vaccine vector. In this work, we generated a recombinant PIV5 expressing the HA gene of H7N9 (PIV5-H7) and tested its efficacy against infection with influenza virus A/Anhui/1/2013 (H7N9) in mice and guinea pigs. PIV5-H7 protected the mice against lethal H7N9 challenge. Interestingly, the protection did not require antibody since PIV5-H7 protected JhD mice that do not produce antibody against lethal H7N9 challenge. Furthermore, transfer of anti-H7 serum did not protect mice against H7N9 challenge. PIV5-H7 generated high HAI titers in guinea pigs, however it did not protect against H7N9 infection or transmission. Intriguingly, immunization of guinea pigs with PIV5-H7 and PIV5 expressing NP of influenza A virus H5N1 (PIV5-NP) conferred protection against H7N9 infection and transmission. Thus, we have obtained a H7N9 vaccine that protected both mice and guinea pigs against lethal H7N9 challenge and infection respectively.     

Giardia duodenalis: direct experimental evidence for the absence of a glycosylphosphatidylinositol anchor in a variant surface protein

The trophozoites of Giardia duodenalis express variant surface proteins (VSPs) that cover the entire surface of the cell and can be altered by antigenic variation. In the present study, a VSP (VSPH7) expressed by the Giardia GS isolate was purified using Triton-X-114 extraction/phase partitioning and a combination of column chromatography methods. The purified VSP was typed by mass spectrometric fingerprint mapping and peptide sequencing and found to share 58-99.8% peptide identity with the VSPH7 protein sequence previously deduced from the cloned cDNA. Carbohydrate compositional analyses consistently showed the presence of galactose in the VSP preparations but a direct association of carbohydrate with the VSPH7 could not be established. Analysis of the C-terminal part of the purified VSPH7 by off-blot myo-inositol analysis provided for the first time direct experimental evidence that this protein is not modified via a GPI lipid.     

Frequency of variant antigens in Giardia lamblia.

Giardia lamblia undergoes antigenic variation. The rate of antigenic variation and the size of the variant antigen repertoire were estimated in clones of Giardia lamblia which reexpresses surface variant antigens that are characteristics of its parent. Calculations were based on determinations of the number of trophozoites expressing defined or nondefined epitopes as well as the total number of trophozoites in newly established clones. The rate of appearance of variant antigens containing defined epitopes was expressed as the number of generations until the first trophozoite expressing a defined epitope appeared. In clones of isolate WB, tested because their major surface variant antigens were largely nondefined, variants expressing epitopes recognized by Mabs 6E7 or 3F6 appeared after approximately 12 generations. Variants expressing epitopes recognized by Mab 5C1 appeared at about 13 generations, significantly greater than for the other epitopes. The rate of antigenic variation was studied in another isolate, GS/M, whose surface epitope repertoire differs from that of isolate WB. A single epitope recognized by Mab G10/4 was tested. Trophozoites reexpressing this epitope first appeared after about 6.5 generations, significantly less than in WB. Therefore, the single epitope studied in isolate GS/M is reexpressed much more frequently than those of WB. In isolate WB, the epitopes recognized by Mab 6E7 and 3F6 tended to appear at the same time. The median number of variant antigens in WB was estimated to lie between 20.5 and 184.     

Differential cellular response of resistant and susceptible rodents to the early stages of infection of Taenia taeniaeformis

Taenia taeniaeformis infection course in C3H/He and BALB/c mice (susceptible and resistant respectively to T. taeniaeformis) was compared for 10 days post-infection (p.i.). Light microscopic observation of early organisms in BALB/c revealed host cell infiltration as early as 2 days p.i. By day 4 p.i. the larvae presented an inconspicuous acellular halo and were surrounded by a dense mass of leukocytes, mainly eosinophils. The amorphous substance reacted positive for sulphated acid mucopolysaccharides. By day 6 p.i. the parasite-host interface was filled by leukocytes and fibroblasts, staining with alcian blue was limited to small areas and the parasites appeared disrupted and lightly stained by haematoxylin-eosin. At day 10 p.i. the parasite had been destroyed by the host. In rats and C3H/He mice, the parasites at day 2 p.i. were 10 μm in diameter, bounded by a larger amorphous acellular halo that reacted positive to alcian blue stain, an indication of the presence of acidic muco-polysaccharides. By day 4 p.i. the clear halo became larger and reacted strongly positive for acid mucopolysaccharides particularly close to the organism. By day 6 p.i. alcian blue positive material appeared more diffuse, covering a broad area well inside the host tissue. By this time a layer of inflammatory cells were often present at some distance from the parasite tegument. By day 10 p.i. the larvae continued growing and increasing the number of tegumental cells. The presence of sulfated acid mucopoly-saccharides around the larvae probably serves as a protective barrier for the parasite by triggering the alternative pathway of complement, consuming C3 and C5, thus preventing cell attachment and humoral antibody attack. The early destruction of the parasite by cell defense mechanism implies a major role of cellular immunity in T. taeniaeformis elimination.     

Mebendazole and the development of Hymenolepis nana in mice

Normally in mice the intra-villus phase of Hymenolepis nana life cycle is completed by the fifth day of infection. However, when the mice were fed continuously on a mixture of 1.5 g Telmin (166.7 mg of mebendazole/g) per kg of food (Purina Lab Chow) from day 1 (24 h) post infection (p.i.), this intramucosal phase was prolonged for varying periods up to day 12 p.i. Mebendazole expelled successfully all lumen dwelling stages of the parasite, but, when treatment was terminated, cysticercoids still in the villi mucosa were able to recover from the drug's effect on their development, excyst, leave the villi and grow to fully gravid adults in the intestinal lumen.     

Eimeria pragensis infection alters the gut microenvironment to favor extrinsic shiga toxin-producing Escherichia coli O157:H7 colonization in mice

We examined the effects of Eimeria pragensis infection on intestinal peristalsis, goblet cell proliferation and intestinal flora in C57BL/6 mice. Intestinal peristalsis was evaluated by radiography using barium at 7 days post-infection (p.i.). The intestinal peristalsis of E. pragensis-infected mice was significantly suppressed compared with uninfected control mice. Twenty-three mice were divided into 5 groups of 4 or 5 mice each; 2 groups of mice were infected with E. pragensis and the others were kept uninfected. At 7 days p.i., E. pragensis-infected and -uninfected mice were sacrificed to examine goblet cell numbers in the intestines, and significant decreases were observed only in the infected mice. Shiga toxin-producing Escherichia coli (STEC) O157:H7 was inoculated orally in mice both infected and uninfected with E. pragensis at 7 days p.i., with the remaining mice used as uninoculated controls. When mice were sacrificed at 2 days after STEC inoculation, STEC was only detected in the intestines of E. pragensis-infected mice. Colonization of STEC was also confirmed by immunohistochemistry on the surface of epithelial cells in concurrently infected/inoculated mice. Also, an overgrowth of residential E. coli was observed only in E. pragensis-infected mice. These results suggest that E. pragensis induces the suppression of intestinal peristalsis and modifies the intestinal environment to facilitate artificially introduced STEC colonization and multiplication, in addition to residential E. coli overgrowth.     

Oral lactoferrin treatment resolves amoebic intracecal infection in C3H/HeJ mice

Entamoeba histolytica is a protozoan parasite that causes amoebiasis, an illness that affects many people around the world. We have previously reported that lactoferrin is able to kill E. histolytica in in vitro cultures. The aim of the present study was to evaluate the therapeutic effect of orally administered bovine lactoferrin in the control of intestinal amoebiasis of susceptible C3H/HeJ mice. The results showed that 20 mg lactoferrin/kg orally administered each day for 1 week was able to eliminate the infection in 63% of the mice, since neither trophozoites nor evidence of epithelial damage and (or) swelling were found in tissue sections of the cecum. The rest of the treated animals (37%) showed a decrease in trophozoite numbers and mucus secreted to the lumen, as compared with untreated and infected mice (p < 0.05). By immunohistochemistry, the profile of secreted cytokines in the cecum revealed that infected but untreated animals showed a mixed Th1/regulatory cytokines profile, whereas the cecum of mice treated (cured) showed a Th2 cytokine profile (IL-4) and expression of the multifunctional IL-6. In addition, cytokines and increasing cecal production of total IgA antibodies were found associated with little inflammation and disease control observed in the cecum of lactoferrin-treated animals. These results suggest that oral administration of lactoferrin can control intestinal amoebic infection probably by killing amoebas or favoring their removal and reestablish the antiinflammatory intestinal environment.     

Giardia lamblia: a new target for miltefosine

Giardia lamblia, the causative agent of giardiasis, is an intestinal infection with worldwide distribution and high rates of prevalence. Increased resistance of the parasite and the side effects of the reference drugs employed in the treatment of giardiasis make it necessary to seek new therapeutic agents. Therefore,the aim of this study was to examine the activity of hexadecylphosphocholine (miltefosine), a membrane active alkylphospholipid, that is licensed as an antileishmanial agent against giardiasis. The efficacy of miltefosine was evaluated both in vitro and in vivo in Swiss albino mice. Results of the in vitro testing revealed susceptibility of G. lamblia trophozoites to miltefosine with the following effective concentrations:EC50s of between 20 and 40 lM, and EC90s of between 20 and 80 lM. Immediate total lysis of the organisms was achieved by 100 lM. In vivo testing showed that oral administration of miltefosine,in a daily dose regimen course of 20 mg/kg for three successive days, to infected mice resulted in total elimination of the parasite from the intestine and amelioration of intestinal pathology. Scanning and transmission electron microscopy studies revealed that miltefosine induced severe morphological alterations to G. lamblia trophozoites, mainly at the level of cell membrane and adhesive disc. In conclusion,we believe that this is the first study highlighting G. lamblia as a possible new target for miltefosine.