Maria Angeles Gomez Morales

Cryptosporidium parvum at Different Developmental Stages Modulates Host Cell Apoptosis In Vitro

ABSTRACT We studied apoptosis in a human ileocecal adenocarcinoma tumor cell line (HCT-8) infected with Cryptosporidium parvum, from 2 to 72 h postinfection (h.p.i.). At 2 h.p.i., the percentage of annexin V-positive cells in the cell culture had increased to 10% compared to 2.5% in noninfected control culture; sorted infected cells expressed mRNA of FasL, the active form of caspase 3, and high caspase 3 activity, whereas the noninfected neighboring cells sorted from the same culture showed no signs of apoptosis. At 24 h.p.i., the percentages of early (annexin V positive) and late (DNA fragment) apoptotic cells were 13 and 2%, respectively, in the entire cell culture, and these percentages were not statistically significant in comparison with those from noninfected control cultures. At this time, sorted infected cells expressed the inactive form of caspase 3, a low caspase 3 activity, and the antiapoptotic protein Bcl-2. Noninfected cells sorted from the same culture showed expression of the active form of caspase 3, a moderate caspase 3 activity, and no Bcl-2 expression. At 48 h.p.i., the percentages of early and late apoptotic cells and caspase 3 activity had increased in the total cell culture, and both sorted infected and noninfected cells showed the active form of caspase 3. These results show that C. parvum, depending on its developmental stage, can inhibit (at the trophozoite stage) or promote (at the sporozoite and merozoite stages) host cell apoptosis, suggesting that it is able to interact with and regulate the host-cell gene expression.

Clinical aspects, diagnosis and treatment of trichinellosis.

Trichinellosis, the human disease induced by worms of the genus Trichinella, is caused by the consumption of raw or undercooked meat of various types of animals and has a worldwide prevalence of approximately eleven million. Since there are no pathognomonic signs or symptoms, clinical diagnosis is difficult and the only reliable diagnostic methods are serodiagnosis and muscle biopsy. Treatment consists of benzimidazoles and glucocorticosteroids, yet in order for these drugs to be effective, they must be administered before the end of the acute stage; thus early diagnosis is fundamental. To aid in the recognition and treatment of trichinellosis, an overall description of its clinical aspects, diagnosis and treatment has been prepared.

Opisthorchis felineus, an emerging infection in Italy and its implication for the European Union.

The liver fluke Opisthorchis felineus is one of the few zoonotic trematodes that circulates in the European Union (EU). It is transmitted from freshwater snails to fish and then to fish-eating mammals, including humans, in which it causes opisthorchiasis. In the 20th century, the majority of infections in humans have been reported in Eastern Europe (e.g., Belarus, Russia, and Ukraine) and Asia (Siberia). In EU in the last fifty years, the parasite has been detected in humans of Germany and Greece, and in red foxes, polecats, cats, dogs, fish and mollusks of Germany, Italy, Poland, Portugal and Spain. In Italy, four individual cases and eight outbreaks of opisthorchiasis were reported from 2003 to 2011, for a total of 211 confirmed infections in humans. All infected persons had consumed raw fillets of tench (Tinca tinca) fished from two lakes in central Italy, but some of infected people were tourists who developed the disease in their respective home-countries. In the past decade, it has become increasingly popular to consume raw marinated fillets of fish. The objective of this review is to show how a change in human food habits have caused and increased the transmission of O. felineus, which has probably been circulating in the EU yet in a silent form for many years.

Human illnesses caused by Opisthorchis felineus flukes, Italy.

We report 2 outbreaks of Opisthorchis felineus infection caused by the consumption of tench filets (Tinca tinca) from a lake in Italy. Of the 22 infected persons, 10 (45.4%) were asymptomatic. When present, symptoms (fever, nausea, abdominal pain, and myalgias) were mild. Eosinophilia occurred in all infected persons.

Evaluation of ELISA and Western Blot Analysis using three antigens to detect anti-Trichinella IgG in horses.

We assessed a serological method for detecting Trichinella infection in horses, specifically, an ELISA using three antigens to detect anti-Trichinella IgG (i.e. a synthetic tyvelose glycan-BSA (stg-BSA) antigen, an excretory/secretory (ES) antigen, and a crude worm extract (CWE) antigen). Serum samples were collected from 2502 horses (433 live horses from Romania and 2069 horses slaughtered in Italy and originating from Italy, Poland, Romania, and Serbia). Serum samples were also taken from horses experimentally infected with different doses of T. spiralis and T. murrelli larvae, as controls. The cut-off value of ELISA was determined on serum samples from 330 horses from Trichinella-free regions of Italy, which were also examined by artificial digestion of preferential-muscle samples. In the experimentally infected horses, the stg-BSA and ES antigens were less sensitive than the CWE antigen. Trichinella spiralis showed a higher immunogenicity than T. murrelli, and the IgG immunoresponse was dose-dependent. The kinetics of anti-Trichinella IgG were similar among all experimentally infected horses. No circulating antibodies were detected 4-5 months after experimental infection, although these horses still harbored infective larvae. Depending on the antigen used, for 4-7 of the 330 horses from Trichinella-free areas, the optical density (OD) of the serum sample was higher than the cut-off value, yet these samples were negative when subjected to Western Blot. Similar results were obtained for the 1739 horses slaughtered in Italy (originating from Italy, Poland, Romania, and Serbia) and the 433 live Romanian horses. Of the 4 horses with muscle larvae, only one was positive by ELISA and Western Blot. Because the anti-Trichinella IgG remain circulating for only a short period of time, whereas the larvae remain infective for longer periods, serology cannot be used for either diagnosing Trichinella infection in horses or estimating the prevalence of infection. Artificial digestion of at least 5 g of preferential-muscle tissue continues to be the method of choice at the slaughterhouse for preventing equine-borne trichinellosis in humans.

Cryptosporidium: different behaviour in calves of isolates of human origin

The behaviour in calves of 3 Cryptosporidium human isolates was analysed in comparison with a bovine isolate. Twenty-four neonatal calves were infected. An isolate from a patient infected with human immunodeficiency virus (HIV) and showing mild cryptosporidiosis caused severe diarrhoea with a high production of oocysts in neonatal calves, as did a bovine isolate (group 1). Two human isolates, obtained from HIV patients with severe cryptosporidiosis, caused mild diarrhoea with low oocyst production in neonatal calves (group 2). The difference between the 2 groups in numbers of oocysts shed in calves was statistically significant (P = 0.005), as was the duration of oocyst shedding (P = 0.0004). Oocysts of group 2 isolates were less resistant to storage in 2% potassium dichromate at 4 degrees C than were oocysts of group 1. The biological and epidemiological implications are discussed.

Increased CD8+-T-Cell Expression and a Type 2 Cytokine Pattern during the Muscular Phase of Trichinella Infection in Humans

ABSTRACT Cell-mediated immunity during the muscular phase of Trichinella infection in humans was studied. Cell proliferation, the phenotypic changes in the T-cell population, and expression and production of cytokines were examined by using peripheral blood mononuclear cells (PBMC) collected at different times postinfection from 10 individuals who had acquired Trichinella spiralis and five individuals who had acquired Trichinella britovi in two distinct outbreaks. T. spiralis and T. britovi crude worm extracts induced proliferation of PBMC from T. spiralis- and T. britovi-infected donors. Cytokine gene expression showed a predominant type 2 pattern for the entire period of infection studied, although gamma interferon (IFN-γ) was expressed. Interleukin-2 (IL-2), IL-5, IL-10, and IFN-γ production was found in PBMC of all donors. There was a good correspondence between the cytokine expression and production patterns. Changes in PBMC composition, with a trend toward an increase in CD8+ lymphocyte counts, were observed.

Cryptosporidium parvum-Specific CD4 Th1 Cells from Sensitized Donors Responding to Both Fractionated and Recombinant Antigenic Proteins

ABSTRACT T-cell-mediated immunity plays a central role in the host response to Cryptosporidium parvum. Human T-cell clones (TCC) were isolated from peripheral blood mononuclear cells of five healthy donors with prior cryptosporidiosis by use of a C. parvum crude extract, two antigen fractions obtained by ion-exchange chromatography (IEC1 and IEC2), and two recombinant peptides (SA35 and SA40) from C. parvum sporozoites. The T-cell lines derived from the one recently infected donor had a higher proportion (26 to 38%) of T cells exhibiting the γ/δ T-cell receptor (γ/δ-TCR) than those from donors who had recovered from cryptosporidiosis several years earlier, suggesting that the γ/δ T-cell population is involved in the early stage of the infection. The specific TCC had the α/β-TCR, had the phenotype CD45RO+ CD4+ CD8−, and were characterized by either hyperproduction of gamma interferon (IFN-γ) alone, with a Th1 profile, or IFN-γ hyperproduction together with interleukin-4 (IL-4) or IL-5 production, with a Th0 profile. SA35, SA40, IEC1, and IEC2 may be considered good targets of the cellular response against C. parvum and may play a role in maintaining the T-cell-mediated memory response to this parasite. Furthermore, the SA35 and SA40 peptides may be regarded as immunodominant antigens involved in the maintenance of the T-cell response in healthy C. parvum-sensitized persons.

The birth of a Trichinella britovi focus on the Mediterranean island of Sardinia (Italy)

For 60 years, the islands of the Mediterranean basin were considered to be Trichinella-free. In April 2005, an outbreak of human trichinellosis due to the consumption of infected pork involved 11 persons in the villages of Orgosolo and Lanusei (Nuoro province) on the island of Sardinia (Italy). We conducted an investigation to identify free-range and backyard pigs and other humans with Trichinella infection in the area of the 2005 outbreak. We also tested wild animals from various parts of Sardinia. In December 2005, eight persons were found to have been infected, and in May 2007 there was a single case of infection. The sources of all infections were domestic pigs. Artificial digestion of muscle samples from 681 pigs (325 free-range and 356 backyard pigs) revealed Trichinella sp. larvae in four sows (1.2%). All larvae, including those from the consumed pork products, were identified as Trichinella britovi. All infected pigs originated from the Orgosolo municipality. None of the 6188 wild boars (Sus scrofa) or 13 red foxes (Vulpes vulpes) examined were positive for Trichinella sp., suggesting that this parasite is restricted to free-range pigs. The origin of infected animals on Sardinia remains to be determined, although it could be related to the presence of T. britovi-infected animals on the island of Corsica (France).

Development of an ELISA to detect the humoral immune response to Trichinella zimbabwensis in Nile crocodiles (Crocodylus niloticus)

Crocodiles are known reservoir hosts of Trichinella papuae and Trichinella zimbabwensis, two zoonotic parasites that also infect mammals. Since commercial crocodile farming represents a key source of income in several countries, it is important to monitor this nematode infection in both farmed crocodiles and in breeding stocks which are frequently introduced from the wild. For this purpose, an indirect ELISA was developed to detect the anti-Trichinella immune response in crocodile sera. New Zealand rabbits were immunized with pooled sera from non-infected farmed crocodiles in the presence of Freunds complete adjuvant. The anti-crocodile serum was then conjugated with horseradish peroxidase. Serum samples from four Nile crocodiles (Crocodylus niloticus) experimentally infected with T. zimbabwensis and from four uninfected crocodiles were used to set up the ELISA. The larval burden per gram of muscle tissue was determined by muscle biopsy. The test was performed on serum samples from an additional 15 experimentally infected crocodiles as well as eight wild Nile crocodiles. Among the 19 experimentally infected crocodiles, seroconversion was observed in 11 animals. The highest antibody response was observed six weeks post infection (p.i.), but in most of these animals, antibodies were not detectable after six weeks p.i. even though live larvae were present in the muscles up to six months p.i.