Immunology of experimental and natural human hookworm infection.

Human hookworm infection is one amongst the most prevalent of the neglected tropical diseases. An informative experimental animal model, that is, one that parallels a human infection, is not available for the study of human hookworm infection. Much of our current understanding of the human immune response during hookworm infection relies on the studies from experimental infection of hookworm-naïve individuals or the natural infections from individuals residing in hookworm-endemic areas. The experimental human infections tend to be acute, dose-controlled infections, often with a low larval inoculum so that they are well tolerated by human volunteers. Natural hookworm infections usually occur in areas where hookworm transmission is constant and infection is chronic. In cases where there has been drug administration in an endemic area, re-infection often occurs quickly even amongst those who were treated. Hence, although many of the characteristics of experimental and natural hookworm infection differ, both models have elements in common: mainly an intense Th2 response with the production of total and specific IgE as well as elevated levels of eosinophilia, IL-5, IL-10 and TNF. While hookworm infection affects millions of individuals worldwide, much of the human immunology of this infection still needs to be studied and understood.

Anaemia in advanced chronic fasciolosis.

The association between fasciolosis-induced anaemia and related factors has been quantified in a rodent model. Haematological parameters were analysed in Wistar rats at 20 and 60 weeks post-infection (p.i.). Pigment stones and bile specimens were collected. Serum IgG1, IgG2a and IgE were determined in rat serum samples. Cytokine levels have been correlated with haematological parameters. The screening for gastrointestinal bleeding was carried out. Bacteriological bile cultures revealed viable bacteria in 53.8% of specimens at 60 weeks p.i. The results show that the type of anaemia in fasciolosis might be considered a biomarker of the chronicity period of the disease, changing from normocytic to macrocytic in the early chronic period (20 weeks p.i.) and to microcytic in the advanced chronic period (60 weeks p.i.). Likewise, changing from normochromic in the early chronic period to hypochromic in the advanced chronic period. Multivariate analysis suggested an association between anaemia and the following factors: fluke burden, eggs per gram of faeces, body area of parasite, presence of blood in faeces, IgG1 and eosinophil levels, and % of splenic weight. Of all variables analysed, the fluke burden is the one which presents the highest anaemia risk, even exceeding the variable presence of blood in faeces. The development of anaemia appears to be complex and may involve multiple mechanisms. However, to the mechanisms that until now explain Fascioliosis-related anaemia (compensatory increase in erythrocyte production and a continuous drain on iron stores resulting from the parasites' blood-sucking activities) the following causes ought to be added: haemolysis of red blood cells, the general effects of inflammation on erythropoiesis, concomitant parasitic and bacterial infections and pre-morbid nutritional abnormalities. Extrapolation to human fasciolosis is discussed. The results of the rodent model lead to the assumption that a high risk of anaemia in subjects with a heavy parasitic burden in human hyperendemic areas of fasciolosis is to be expected.

Identification of genotypes of Giardia intestinalis of human isolates in Egypt.

In Egypt, the genotyping study of Giardia intestinalis in human is limited. To determine the prevalence of G. intestinalis, faecal samples were collected from Egypt. Samples were concentrated using density gradient centrifugation. The samples were subjected to PCR and DNA sequence analysis for TPI gene. Prevalence of Giardia infection was 34.6% of 52 examined. DNA sequence showed that the Assemblage B was the most prevalent (80%) genotype, the 15% of the positive samples belonged to Assemblage E, and the 5% of them belonged to Assemblage A. Certainly, both genotypes A and B are highly common in human worldwide. However, up to now, Assemblage E had not been known to be infectious for humans. Therefore, this is the first time that Assemblage E is reported in human. However, further analyses of a second locus are required to confirm this result. The extent to which Giardia-infected cattle in Egypt might pose a risk of human infection is unknown.

First phenotypic description of Fasciola hepatica/Fasciola gigantica intermediate forms from the human endemic area of the Nile Delta, Egypt.

Fasciola gigantica is the main fasciolid species in Africa; however, F. hepatica and F. gigantica overlap in some countries. Egypt deserves mentioning because of the emerging situation of human fascioliasis in the Nile Delta area. The morphometric characteristics of fasciolid adults infecting the main livestock species present in the Nile Delta human endemic area are analyzed through a computer image analysis system (CIAS) on the basis of standardized measurements known to be useful for the differentiation of both fasciolid species. This is the first time that such a study is performed in an African country and, therefore, the results are compared to (i) F. hepatica (European Mediterranean area) and F. gigantica (Burkina Faso) standard populations, i.e. geographical areas where both species do not co-exist, and (ii) F. hepatica and F. gigantica populations from geographical areas where both species do co-exist, including the presence of intermediate forms (Iran). Results indicate the presence of F. hepatica, F. gigantica and intermediate forms (Fasciola sp.) in Egypt for the first time, and demonstrate the usefulness of CIAS for the phenotypic characterization of liver fluke adults from a concrete fascioliasis endemic area. Body roundness, body length over body width, and distance between the ventral sucker and the posterior end of the body provide useful tools for studying inter- and intraspecific morphological diversity in Fasciola adults. The application of these markers to specimens from geographical areas where F. hepatica and F. gigantica co-exist, such as in Egypt and Iran, suggest a strong population-level variation in Fasciola adult morphology.

Phenotypic analysis of adults of Fasciola hepatica, Fasciola gigantica and intermediate forms from the endemic region of Gilan, Iran.

Fascioliasis is an important human and animal disease caused by Fasciola hepatica and Fasciola gigantica. In Iran, the distribution of these two species overlaps in most areas, including the northern human endemic province of Gilan where both fasciolids are simultaneously found in individual cattle and buffaloes. A phenotypic study of fasciolid adult flukes from naturally infected bovines from Gilan was carried out by means of an exhaustive morphometric analysis using traditional microscopic measurements and an allometric model. The Iranian fasciolids were compared to F. hepatica and F. gigantica standard populations, i.e. from geographical areas where both species do not co-exist (Bolivia and Burkina Faso, respectively). Although morphometric values somewhat overlapped, there were clear differences in allometric growth. The allometric function was adjusted to 25 pairs of variables. Results obtained revealed that Iranian F. hepatica-like specimens are larger than the F. hepatica standard and Iranian F. gigantica-like specimens are longer and narrower than the F. gigantica standard, but with smaller body area. Measurements which permit a specific differentiation in allopatric populations (distance between ventral sucker and posterior end of the body; ratio between body length and body width) overlap in the specimens from Gilan, thus proving the presence of intermediate forms. When compared to the standard populations, the different Iranian fasciolid morphs show greater differences in F. gigantica-like specimens than in F. hepatica-like specimens. This study shows that simple, traditional microscopic measurements may be sufficient for the morphometric characterisation of fasciolids, even in areas where intermediate forms are present.

Crowding effect on adult growth, pre-patent period and egg shedding of Fasciola hepatica.

Fascioliasis pathogenesis depends on fluke burden. In human hyperendemic zones, individual infection intensities reach very high levels and the majority of infected subjects should be in the advanced chronic phase. The rat model offers a useful approach for pathological research in the advanced chronic period. The influence of infection intensity per rat on fluke development, pre-patent period and egg shedding (eggs/g faeces/worm) was analysed in 3 groups (I: 1-3 worms/rat; II: 4-6; III: 7-9). Ontogenetic trajectories of fluke body measures followed a logistic model. Results showed that when the burden increases, the maximum values of fluke measures decrease. The crowding effect is manifest when fluke measures approximate their maximums in the advanced chronic stage. The pre-patent period and egg production decrease when the burden increases. This means that measurements of eggs per gramme of faeces tend to underestimate the fluke burden. The present study demonstrates how to quantify the fascioliasis experimental rat model crowding effect on adult growth, pre-patent period and egg production. This quantification may be of great interest in epidemiological studies and in experimental research on the in vivo actions of different anthelminthic drugs and vaccines, pathology, immunology and resistance studies.

Phenotypic comparison of allopatric populations of Fasciola hepatica and Fasciola gigantica from European and African bovines using a computer image analysis system (CIAS).

The quantification of the different sizes and shapes of Fasciola hepatica and Fasciola gigantica from bovines has been achieved for the first time in natural allopatric populations. Linear measurements, areas and ratios of gravid adults and eggs of F. hepatica (from France and Spain) and F. gigantica (from Burkina Faso) were analysed using a computer image analysis system and an allometric model: (y2m - y2)/y2 = c[(y1m - y1)/y1](b), where y1 = body area or body length, y2 = one of the measurements analysed, y1m, y2m = maximum values towards which y1 and y2, respectively, tend and c, b = constants. All the measurements overlap in the two fasciolids, apart from the distance between the ventral sucker and the posterior end of the body, body roundness and body length/body width ratio. The results obtained may be useful in Fasciola species identification in countries where both species coexist.

Human and animal fascioliasis in Mazandaran province, northern Iran.

The fascioliasis situation in humans and livestock of Iranian Mazandaran is analysed for the first time. Coprological studies showed 7.3% and 25.4% global prevalences in sheep and cattle, respectively. Studies in slaughterhouses indicate that sheep and cattle may be the main reservoir species, buffaloes may play local roles in the transmission, and goats and horses probably only participate sporadically. Morphometric studies by computer image analysis showed that forms intermediate between Fasciola hepatica and F. gigantica appear in addition to both species. A total of 107 infected humans were diagnosed during the 1999-2002 period, which suggests that fascioliasis may be widespread. The absence of differences in human fascioliasis among gender and age groups differs from other human endemic areas. Both human and animal infections show marked differences between western and eastern Mazandaran. Traditions in herbal condiments for human consumption, methods of animal husbandry and annual rainfall may explain the higher prevalences in western Mazandaran.

Differential sensitivity of Chinese hamster V79 and Chinese hamster ovary (CHO) cells in the in vitro micronucleus screening assay.

Both the V79 and CHO cell lines are routinely used in the in vitro MN screening assay for the detection of possible genotoxicants. The CHO cell line is the predominant cell line currently used in the genetic toxicology testing industry. However, some laboratories routinely utilize the V79 cell line since the in vitro MN screening assay was initially developed using V79 cells. Our laboratory has historically used the CHO cell line. Therefore, our laboratory was interested in comparing the two cell lines with regard to possible similarities or differences in MN induction sensitivity after exposure to cyclophosphamide (CPA) and mitomycin C (MMC), the two standard positive control chemicals routinely used in this assay. Three exposure conditions in the presence of CPA and MMC were examined in both cell lines. Replicate cultures of CHO cells in McCoy's 5A and V79 cells in both McCoy's 5A and E-MEM were established and treated with 5 microg CPA/ml (4h exposure with S9), 0.5 microg MMC (4h exposure without S9) and 0.5 microg MMC (24h exposure without S9). A total of 400 cytochalasin B-blocked binucleated cells and 200 consecutive cells were analyzed from each culture for MN and cell cycle kinetics, respectively. Analysis of the data demonstrated that CHO cells were up to approximately five-fold more sensitive to the induction of CPA- and MMC-induced MN than V79 cells. Both cell lines exhibited similar average generation times among identical exposure groups. Therefore, the difference in MN sensitivity cannot be attributed to possible differences in cell cycle kinetics and is possibly related to inherent cellular differences in the processing of and/or repair of CPA- and MMC-induced damage by V79 and CHO cells.