Relationship between exposure to Fasciola hepatica in roe deer (Capreolus capreolus) and cattle extensively reared in an endemic area.

The aim of this work is to know the prevalence of Fasciola hepatica in 301 roe deer and in 676 beef cattle kept in an endemic area. Detection of antibodies was determined in roe deer using a homemade ELISA with excretory/secretory antigens (FhES) and a recombinant protein (FhrAPS). None of the deer passed eggs by faeces and none flukes in their livers were found. The seroprevalence of F. hepatica was 29% using FhES, with significantly higher values in the oldest ones (36%). Twenty-eight percent of the samples were positive to FhrAPS. Twenty-three percent of the cows eliminated eggs of F. hepatica and the seroprevalence was 67% using FhrAPS. No relationship between the seropositivity values of deer and cattle was demonstrated. The role of wild ruminants as reservoirs of F. hepatica is discussed. We encourage the use of ELISA to know the possibility of exposure to trematodes in wild ruminants.

An approach of the laboratory to the field: assessment of the influence of cattle management on the seroprevalence of fascioliasis by using polyclonal- and recombinant-based ELISAs.

A cross-sectional study was conducted to assess the seroprevalence of fascioliasis by immunoenzymatic probes in an endemic area (northwestern Spain). Blood samples were collected from 1,034 cattle (crossbred, Rubia Gallega, and Friesian breeds), and the diagnosis of fascioliasis was carried out by determining both the occurrence of antigenemia and the presence of specific IgG antibodies against a Fasciola hepatica recombinant protein (FhrAPS). The IgG seroprevalence was 65% (95% CI, 62-68) by the FhrAPS-ELISA, and 32% (29-35) exhibited antigenemia; the lowest percentages occurred in the Friesians, and the highest percentages were found in the crossbreds. These results confirm an elevated seroprevalence of fascioliasis that is unexpected considering that most of the cattle livestock (Friesian and Rubia Gallega) receive fasciolicide treatment. The lack of adequate measures on the environment and erratic chemotherapy seem to be responsible for the fact that control of fascioliasis has not improved in the last 10 yr in the area of study.

A recombinant-based ELISA evaluating the efficacy of netobimin and albendazole in ruminants with naturally acquired fascioliasis.

The therapeutic efficacy of albendazole and netobimin in ruminants with naturally occurring fascioliasis was investigated using a recombinant-based ELISA. The variation in the IgG response against a 2.9-kDa recombinant protein (FhrAPS), termed efficacy index (EI) 1, and the egg-output changes, termed EI 2, were used to evaluate drug efficacy. The values of EI 1 ranged between 0% and 50% in sheep, and between 0% and 30% in cattle after treatment with albendazole and netobimin. Similar EI 2 values were observed in sheep receiving albendazole or netobimin, but the highest values were found in cattle treated with netobimin. The significant reduction in the IgG response to FhrAPS found in this study shows promise in terms of developing alternative methods for evaluating the efficacy of chemotherapy against Fasciola hepatica in grazing ruminants.

The addition of a new immunomodulator with the adjuvant adaptation ADAD system using fatty acid binding proteins increases the protection against Fasciola hepatica.

Fatty acid binding proteins (FABP) have shown protective immune response against Fasciola hepatica infection. We evaluated the protection induced by the Fh12 FABP from F. hepatica (Fh12) combined with the new immunomodulator the lipidic aminoalcohol OA0012 in the ADAD system in mice and sheep. In this work we introduced a lipidic aminoalcohol OA0012 as immunomodulator alone or in combination with the hydroalcoholic extract of Phlebodium pseudoaureum; PAL. Mice vaccinated with ADAD containing OA0012+Fh12 or OA0012+Qs+Fh12 had survival rates of 40-50%. Sheep ADAD-vaccinated with OA0012+Qs+Fh12 showed lower fluke recovery, less hepatic lesions and higher post-infection daily weight gain than F. hepatica infected control animals. Sheep ADAD-vaccinated with OA0012 combined PAL and Qs+Fh12 showed lower fluke recovery (42%), lower adult worms count (57%) lower faecal egg count (38%), less hepatic lesions and higher post-infection daily weight gain than F. hepatica infected control animals. Thus, the addition of a new immunomodulator of synthesis to ADAD system with FABPs increased the protection against F. hepatica.

Tungiasis: a case report

This is a case report of a patient who developed a nodule in one foot, which upon biopsy, was diagnosed as tungiasis, a cutaneous infestation caused by a human flea. The natural life cycle, clinical and pathological expressions are discussed.

Reduction in the perinatal HIV transmission: the experience at the Maternal Infant Studies Center and Gamma Projects at the University of Puerto Rico School of Medicine

The AIDS pandemic had a significant impact in Puerto Rico, especially among the heterosexual populations, in particular women. Women are one of the fastest growing risk groups with HIV/AIDS in the USA and constitute about half of the AIDS cases in the world. During the past 10 years Puerto Rico has ranked among the top 5 jurisdictions in the United States in AIDS cases rates, among men, women and children. In 1987 a universal prenatal HIV screening program was implemented in the University Hospital catchment area consisting of approximately 5,000 deliveries per year. Because of the early identification of pregnant women living with HIV, access to lifesaving clinical research and the implementation of multiple strategies and comprehensive care, the perinatal HIV transmission has been reduced to zero since 1997, with a blip of one case in 2002, and none since then. The availability and access to clinical and behavioral research has been one of the key elements for this success story. The programs involved and responsible for this spectacular outcome, namely the Maternal Infant Studies Center (CEMI-Spanish Acronym) and Gamma Projects at the University of Puerto Rico School of Medicine are described. The cost savings impact of stopping mother-infant perinatal HIV-1 transmission has been calculated to be approximately $34 to $58 million dollars in 10 years. The impact of the effectiveness of these programs in having healthy uninfected infants, prolonging and improving the quality of life of those living with HIV, and providing hope to families affected by this epidemic is incalculable.

Immunodiagnosis of current fasciolosis in sheep naturally exposed to Fasciola hepatica by using a 2.9 kDa recombinant protein.

A 2.9 kDa recombinant-Fasciola hepatica protein (FhrAPS) was employed to estimate the prevalence of fasciolosis in sheep maintained under field conditions. For this purpose, 340 samples with known status in relation to fasciolosis by using a direct-ELISA and the coprological sedimentation were used. These samples were analysed by using an indirect-ELISA (iELISA) and the FhrAPS recombinant protein and excretory/secretory antigens (FhES) of this trematode. Current fasciolosis (CF) was named when results were positive to antigenemia and/or coprology. Out of 198 sheep with current fasciolosis, 68% were positive to the FhrAPS-ELISA test and 53% to the FhES. We observed 14% of the CF-neg sheep were positive to the FhrAPS, whereas this percentage was 52% with the FhES. A significant correlation between FhrAPS and current fasciolosis was obtained (r2=0.513, p=0.001). We concluded that the FhrAPS provides a more suitable antigen than FhES for developing field trials to know the prevalence of early and current fasciolosis.

Progress in the development of Fasciola hepatica vaccine using recombinant fatty acid binding protein with the adjuvant adaptation system ADAD.

Fatty acid binding proteins (FABP) have been designed as a potential vaccine against fasciolosis. In this work, the immunoprophylaxis of the recombinant Fh15 FABP from F. hepatica (Fh15) in adjuvant/immunomodulator ADAD system was evaluated using mice and sheep challenged with F. hepatica. The ADAD system combines the Fh15 antigen with an immunomodulator (hydroalcoholic extract of Polypodium leucotomos; PAL) and/or an adjuvant (saponins of Quillaja saponaria; Qs) in a water/oil emulsion (30/70) with a non-mineral oil (Montanide). All the infected control mice died by 41-48 days post-infection. The mice vaccinated with ADAD only with PAL+Fh15 present a survival rate of 40-50% and those vaccinated with ADAD containing PAL+Qs+Fh15 had a survival rate of 50-62.5%. IgG1 antibodies were lower in surviving mice in comparison with non-surviving mice. The sheep vaccinated with ADAD PAL+Qs+Fh15 showed lower fluke recovery (43%), less hepatic lesions and higher post-infection daily weight gain than F. hepatica infected control animals. Thus, the ADAD system using recombinant fatty acid binding proteins from F. hepatica could be a good option to develop vaccines against F. hepatica.

A 2.9 kDa Fasciola hepatica-recombinant protein based ELISA test for the detection of current-ovine fasciolosis trickle infected.

The suitability of an enzyme linked immunosorbent assay (ELISA) test with a 2.9 kDa Fasciola hepatica-recombinant protein (FhrAPS) for diagnosing early and current-ovine fasciolosis was analyzed, and compared to that obtained by using a direct ELISA for detecting F. hepatica-circulating FhES antigens and to the coprological sedimentation for fluke egg quantitation. Fourteen Gallega autochthonous breed sheep were experimentally infected with metacercariae by a trickle system (small repetitive infections) and divided into two groups: G-I represented a primary infection for 34 weeks; G-R, animals with primary infection and reinfected 18 w.a.p.i. Seven sheep were left uninfected as the control group (G-C). Serum IgG antibody values against the FhrAPS rose rapidly by 1st w.a.p.i. in all infected sheep. Antibody levels in those with primary infection (G-I, G-C) peaked at 10 weeks, diminishing slightly and levelling from 16 to 34 weeks. Those with primary infection reinfected at 18 weeks had a rebound effect with the highest values observed. Circulating F. hepatica-ES antigens were detected by the 1st w.a.p.i. in all infected groups peaking at 6 weeks, decreasing rapidly to uninfected control values by 10 weeks of infection. Faecal egg-output started 11 weeks after primary infection. An increase in the IgG antibody as well as antigen responses to the FhrAPS and to anti-FhES from the 18 w.a.p.i. was recorded in G-T and G-R after the challenge infection. Antibody levels remained high whereas antigenemia values diminished after 6 weeks. A positive significant correlation between the IgG response against the FhrAPS and the F. hepatica circulating antigens (r2 = 0.428, p = 0.001) was obtained. In conclusion, our standardized diagnostic ELISA for fasciolosis based on the detection of IgG responses to the FhrAPS would be a valuable tool to diagnosis early and current F. hepatica-infections in sheep.

Fasciola antigens as vaccines against fascioliasis and schistosomiasis.

Fascioliasis is an important trematode infection of herbivores worldwide with increasing evidence of prevalence as a disease of humans. Vaccination studies with purified native and recombinant Fasciola antigens suggest that this approach to diminished morbidity and mortality and reduced transmission is a realistic goal. Among the major potential vaccine candidates are fatty acid binding protein (FABP), cysteine (cathepsin) proteases, haemoglobulin, leucine aminopeptidase, and a saposin-like protein. In the case of Fasciola hepatica FABP, cross-reaction and cross-protection against Schistosoma mansoni is an important feature. In addition to protective effects with significant worm burden reductions, some vaccine candidates also have anti-fecundity (smaller flukes), anti-pathology (less liver lesions), and anti-embryonation effects. Optimism is tempered by the fact that fascioliasis in humans is an orphan disease and in need of governmental and foundation support.

Isolation, identification and expression of a Fasciola hepatica cDNA encoding a 2.9-kDa recombinant protein for the diagnosis of ovine fasciolosis.

A 400-bp Fasciola hepatica cDNA clone was isolated from an expression library by immunological screening using rat sera taken 2 weeks after experimental infection. The nucleotide sequence of the cDNA revealed the presence of an open reading frame of 78 bp which encoded a 25 amino acid polypeptide with a predicted molecular weight of 2.9 kDa. This polypeptide was expressed in bacteria as a GST-fusion protein and used for the production of specific antigen. The 2.9 kDa recombinant protein (APS) was evaluated against sera from experimentally infected sheep using an indirect ELISA, and the results were compared to those obtained using F. hepatica excretory/secretory products (ESP). The pattern of IgG was very similar both against the recombinant and the native proteins, increasing early following the infection. After treatment with triclabendazole, the IgG response against the APS seroreverted to negative values, whereas it remained elevated against the ESP. We conclude that this recombinant protein could be used in diagnostic assays for the identification of recently infected sheep.

Vaccination of mice and sheep with Fh12 FABP from Fasciola hepatica using the new adjuvant/immunomodulator system ADAD.

We evaluate the ability of a Fasciola hepatica FABP native antigen (Fh12) with a new vaccination system called ADAD to protect mice and sheep against an experimental F. hepatica infection. The vaccination protocol consists of a set of two injections. The first injection contains a micelle in which two components are included, saponin from Quillaja saponaria (Qs) and/or Anapsos (A) a Polypodium leucotomos hydroalcoholic extract, both emulsified in a non-mineral oil (Montanide) in a water/oil emulsion (30/70). This is subcutaneously injected to achieve the "adaptation" of the immune system to subsequent stimuli. The second injection contains in addition the Fh12 antigen. Two different experiments were carried out using two mouse strains (BALB/c and CD-1). Mice vaccinated with Qs+A+Fh12 presented a survival rate of 40%, when compared with control groups. Furthermore, we evaluated the efficiency of the vaccination in sheep against an experimental F. hepatica challenge. The vaccinated sheep presented lower fluke recovery (24.5%), number of eggs in bile fluid (58.1%) and faeces (40.3%) than control groups. The recovered flukes were shorter (32.7%), immature (34.0%) and with lower body mass (31.6%) than non-complete vaccinated sheep. Thus, the new ADAD system could be a good alternative for future vaccination experiments against fasciolosis.

Virologic risk factors for vertical transmission of HIV type 1 in Puerto Rico.

HIV-1 vertical transmission in Puerto Rico has decreased significantly due to the implementation of antiviral therapy. Several studies have shown that the phenotype of the HIV-1 isolates initially recovered from infected infants has generally been one that replicates rapidly, infects macrophages, and preferentially use the CCR5 coreceptor. Our hypothesis is that viral genotypic and phenotypic differences exist between HIV-1 nontransmitter and transmitter mothers. Viral DNA samples and virus isolates were analyzed from a Puerto Rican perinatal population. Heteroduplex tracking assay (HTA) was performed on DNA samples to detect env V3 evolutionary variants and the extent of heterogeneity within each sample. HIV-1 C2-V3 variants were cloned from each patient to study sequence variation among the groups. Differences in replication kinetics of viral isolates in macrophage and GHOST CCR5 cells were analyzed by use of repeated measures linear regression analysis. HTA analysis showed that only two nontransmitter patient samples showed the presence of evolutionary variants. Phylogenetic analysis between maternal-infant pairs showed that transmission of a single maternal variant occurred, with the exception of one sample pair. When evaluating amino acid sequences from cloned PCR products, nontransmitting mothers appear to have a higher number of distinct sequences than both the transmitting mothers (p = 0.0410) and the infected infants (p = 0.0315). Analysis of replication kinetics indicated that transmitters showed faster replication kinetics in GHOST CCR5 cell cultures at 12 days postinfection (p = 0.0434) and 15 days postinfection (p = 0.0181). In conclusion, viral homogeneity and rapid replication kinetics were correlated with vertical transmission.

Isolation and immunological characterization of fatty acid binding protein isoforms from Fasciola hepatica.

A combination of molecular sieving chromatography and 2-step preparative isoelectric focusing showed that native Fh12, a fatty acid-binding protein isolated from Fasciola hepatica adult worms, is a protein complex of at least 8 isoforms with identical molecular mass but different isoelectric points. Using enzyme-linked immunosorbent assay (ELISA) and inhibition ELISA assays, immunological differences were observed between native (nFh12) and a recombinant molecule denoted rFh15 that was obtained after screening a cDNA library from F. hepatica adult worms with an anti-Fh12 monospecific polyclonal antibody. It was confirmed that in infected rabbits, antibodies to nFh12 appear by the second week postinfection, whereas antibodies to rFh15 appear much later, by 6 wk postinfection. Four acidic forms (Fh12(1-4)) showed more immunological identity with rFh15 than with nFh12, based on the observation that they inhibited ELISA activity by nearly 50% when they were added to the anti-rFh15 polyclonal antibody at 20 microg/ml of protein concentration. Moreover, the Fh12(1-4) isoforms were poorly reactive with sera from rabbits 2-4 wk postinfection. However, the 2 acidic forms, denoted Fh12(5) and Fh12(6), and the neutral/basic forms, denoted Fh12(7) and Fh12(8), showed more immunological identity with the native nFh12 molecule than with the recombinant rFh15 because they were highly reactive with sera of rabbits with early 2-wk F. hepatica infection and inhibited ELISA activity nearly 50% when they were quantitatively added to the anti-nFh12 polyclonal antibody. These results suggest that rFh15 could be one of the acidic forms of nFh12, and that it, in fact, may be one of the less immunogenic or immunoprotective members, or both, of the nFh12 protein complex.

Immunoprophylaxis against Fasciola hepatica in rabbits using a recombinant Fh15 fatty acid-binding protein.

Previous studies of ours have demonstrated that a recombinant protein (Fh15) related to fatty acid-binding proteins did not induce significant protection in rabbits challenged 2 or 4 wk postimmunization over nonimmunized controls. In the current study, rabbits were immunized with Fh15 and challenged with Fasciola hepatica metacercariae 12 and 20 wk later. In the current study in which longer lag periods for challenge infection after the second immunization were used, worm burden reductions compared to adjuvant controls were a significant 43% and 76%, respectively. Importantly, rabbits immunized with Fh15 had significant numbers of immature flukes, 66% in the 12-wk period and 84% in the 20-wk lag period as compared to controls. In addition, liver lesions were clearly diminished in the vaccinated rabbits. Enzyme-linked immunosorbent assay absorbance values showed that immunized rabbits developed high antibody levels to Fh15 from 8 wk after the first immunization and did not increase after challenge. These results suggest that a recombinant F. hepatica molecule related to fatty acid-binding proteins induces protective (worm burden reductions), anti-fecundity (immature flukes), and anti-pathology (less liver lesions) effects in rabbits and may serve as a model for the immunoprophylaxis of fascioliasis.

Vaccination of sheep against Fasciola hepatica with homologous fatty acid binding proteins.

The current study was designed to test the immunoprophylactic properties of native (nFh12) and recombinant (rFh15) antigens from Fasciola hepatica in sheep subsequently infected with the fluke. Thirty lambs were divided into six groups according to various patterns of immunisation and times of infection and necropsy. The antigens were emulsified in Freund's adjuvant. Levels of specific anti-nFh12 and anti-rFh15 antibodies rose rapidly by 2 weeks after the first immunisation and were always significantly higher in immunised-infected sheep than in control-infected sheep. On completion of the trial there was no difference in fluke burden between groups vaccinated with either of the antigens and non-immunised controls. However, worm size and faecal egg counts were significantly diminished in the sheep vaccinated with either of the antigens, suggesting an anti-fecundity effect. This is the first report of experimental vaccination of sheep against F. hepatica with purified native and recombinant antigens related to fatty acid binding proteins.

Identification of fatty acid molecules in a Fasciola hepatica immunoprophylactic fatty acid-binding protein.

Fasciola hepatica adult flukes have a native protein complex denoted nFh12 and consisting of fatty acid binding proteins that comprise at least 8 isoforms. It is a potent immunogen because in several animal hosts it induces an early antibody response to F. hepatica infection. It is also a potent cross-protective immunogen because it induces a protective immune response in mice to challenge infection with Schistosoma mansoni cercariae. The gene encoding this protein has been cloned and sequenced. It produces a polypeptide of 132 amino acids with a predicted molecular mass of 14.7 kDa and is denoted rFh15. It also has a significant homology to a 14-kDa S. mansoni fatty acid binding protein (Sm14). In the present study, nFh12 was delipidated with charcoal treatment and then studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Additionally, a lipid analysis of nFh12 was undertaken using gas chromatography-mass spectrometry to demonstrate that the nFh12 protein complex is, in fact, a complex of fatty acid binding proteins. Five long-chain saturated and unsaturated fatty acids were detected. The most abundant were palmitic acid (38%), stearic acid (24%), and oleic acid (13%). These fatty acid molecules do not have covalent bonds attached to the protein molecule. Because both nFh12 and Sm14 protect mice against challenge infection with F. hepatica and S. mansoni, it is possible that they have common protective epitopes in which fatty acids could be involved. Further studies are in progress to determine the chemical nature of these potential common epitopes.

Seroprevalence of Schistosoma mansoni in Puerto Ricans with inflammatory bowel disease.

BACKGROUND: The etiology of Inflammatory Bowel Diseases, Crohn's disease (CD) and ulcerative colitis (UC), is unknown. These diseases have a higher incidence in industrialized countries and their pathogenesis involves an over-reaction of the immune system. A genetic factor is believed to predispose to the development of chronic inflammation in response to an unidentified stimulus. Exposure to infections in childhood may modulate future immune responses. Parasitosis, particularly Schistosomiasis, stimulate Th2 immune responses. It has been hypothesized that the absence of these parasitic infections, as seen in economically developed countries, favors a Th1 response that may result in the clinical appearance of Crohn's disease later in life. OBJECTIVE: To determine the prevalence of Schistosoma mansoni antibodies in Puerto Ricans with Inflammatory Bowel Disease and controls. METHODS: Serum from 92 Puerto Ricans with IBD and 106 controls was screened for S. mansoni adult microsomal antigens (MAMA) using the FAST:ELISA assay. Those positive were confirmed with an enzyme-linked immunoelectrotransfer blot test. RESULTS: Seven serum samples (3 UC and 4 controls) were positive for S. mansoni antibodies. There was no significant difference between groups in gender, municipality of origin or seroprevalence of Schistosomiasis. The control group was slightly older than the IBD group. CONCLUSIONS: Our study did not demonstrate an inverse relation between Schistosomiasis and IBD. However, the decreasing prevalence of Schistosomiasis in the general population of Puerto Rico may account for this result.

Characterization of HIV isolates from Puerto Rican maternal-infant pairs reveal predominance of non-syncytium inducing (NSI) variants with CCR5 genotype.

In this study, HIV-1 variants from a cohort of forty-eight Puerto Rican pregnant women and their 50 infants (one had triplets), were isolated and characterized, in order to determine the type of HIV-1 variants that are predominantly transmitted. All were enrolled in the prenatal AIDS Clinical Trials Group (ACTG) and received anti-retroviral therapy. Fifteen of the 50 infants (30%) were positive by V3 PCR suggesting that they harbored a copy of the HIV envelope gene. Three of 50 infants (6%) were HIV-1 culture and PCR positive, indicating active infection. HIV-positive cultures were obtained from 32 of the 48 mothers. Sixty two percent of the isolates (20/32) were macrophage-tropic and non-syncytium inducing, three percent (1/32) had dual tropism, and thirty four percent (11/32) were non-syncytium inducing and did not grow in macrophages. Phenotype and genotype of the HIV variants from the three infected infants revealed the presence of macrophage-tropic and non-syncytium-inducing strains. Genotype analysis of the HIV env V3 loop revealed the presence of specific amino acids that are predictive of CCR5 usage. Sequence analysis of the HIV pol gene from the three infected infants indicated that vertical transmission was not caused by the presence of antiviral resistance mutations. These results indicate that mothers undergoing antiretroviral treatment at different stages of the disease and with different viral loads transmit predominantly macrophage-tropic/non-syncytium inducing/CCR5 variants to their infants.