Occurrence of Giardia duodenalis infection in chinchillas (Chincilla lanigera) from Italian breeding facilities.

The present work investigated the occurrence of Giardia infection in Chinchilla lanigera reared in three Italian breeding facilities and determined their role as potential zoonotic reservoir. One hundred and four fecal samples were tested for the presence of Giardia spp. cysts using a Direct Fluorescent Assay (DFA). A high positivity rate (39.4%) was found despite all animals were asymptomatic at the time of sampling. Thirty-one positive samples were genetically characterized by sequence analysis of the ITS1-5.8S-ITS2 region of the Giardia ribosomal DNA. Assemblages B (29 isolates) and C (two isolates) were identified. These results showed that Giardia infection can be common in chinchillas, thus spurring further molecular epizootiological studies of the infection to assess the zoonotic potential or host specificity of their isolates, to determine the source of infections, to identify the routes of transmission, and to control the infection among animal populations.

Prevalence of Anaplasma phagocytophilum in fallow deer (Dama dama) and feeding ticks from an Italy preserve.

Up to date, information concerning the Anaplasma phagocytophilum infection in fallow deer is scant, therefore, to verify its prevalence in these ungulates serological and PCR screenings were performed on blood of 72 fallow deer hunted in a Central-Northern Italian preserve. Molecular analyses were also performed on 90 ticks removed from the animals. A. phagocytophilum infection in fallow deer was confirmed in 20 out 72 by IFA assay and in 11 out 72 by PCR. The sequence obtained revealed a complete genetic homology among the blood samples and strong degrees of homology with other European isolates. Considering the 90 ticks collected we found that 7.3% of Ixodes ricinus harboured A. phagocytophilum specific DNA. The data obtained confirmed that fallow deer can be a competent host for A. phagocytophilum and, therefore, that may represent a biological reservoir playing an important role in the epidemiological scenarios of the infection, in the geographical areas where is widespread.

Seroprevalence and risk factors for Toxoplasma gondii infection on finishing swine reared in the Umbria region, central Italy.

Toxoplasma gondii is a worldwide zoonotic protozoan parasite and pork is considered the major meat source of Toxoplasma infection in humans. To determine the prevalence of infection of Toxoplasma gondii in pigs reared in the Umbria Region (central Italy), blood samples of 960 pigs from 10 different farms (96 for each farm) were randomly collected and tested for antibodies (IgG) against T. gondii using an IFA assay. Sera were screened at 1/16 titrr and the endpoint titre was determined. Farm management questionnaires were completed and used to develop descriptive statistics on the tested farms as well as to determine measures of association for risk factors for the presence of T. gondii-seropositive pigs. A total of 155 seropositive pigs (16.14%) were identified; within herds prevalence ranged from 8.33 to 25%. The statistical analysis identified all-in-all-out housing and cleaning method as risk factors for Toxoplasma infection.

Pathological changes caused by Anoplocephala perfoliata in the equine ileocecal junction.

Gastrointestinal motility disorders represent a significant cause of morbidity and mortality in horses. Previously regarded as a non-pathogenic tapeworm, Anoplocephala perfoliata has been recently associated with equine colic. In this study, pathological changes related to A. perfoliata at the ileocecal junction were investigated in 31 slaughtered horses. Our results showed a significant relationship between parasitic burden and grading of histopathological lesions in both the mucosa and submucosa. Moreover, in infested horses, hypertrophy of the circular muscle layer was determined. Finally, an enteric nervous system (ENS) evaluation showed injury to intestinal nervous elements in horses with moderate to high parasitism. In summary, our results on the ENS support a correlation between colic and A. perfoliata infestion in the horse.

Epidemiological survey on equine cryptosporidium and giardia infections in Italy and molecular characterization of isolates.

Cryptosporidium and Giardia are two of the most common enteric pathogens of domestic and wild animals and humans. However, little is known on the prevalence, clinical manifestations and economic and zoonotic significance of these infections in horses. This study was undertaken to investigate the prevalence, excretion patterns and risk factors related to the faecal shedding of Cryptosporidium oocysts and Giardia cysts in horses and the zoonotic potential of species/genotypes isolated. The survey was performed on 120 foals and 30 broodmares reared in five Italian farms. Foals were divided in four homogeneous groups of 30 animals each (age classes: 0-2, 2-4, 4-8, >8 weeks). Three sequential faecal samples were collected from each animal and analysed by three techniques: direct fluorescent antibody test (DFA), faecal flotation (FF) and stained faecal smears (SFS). The DFA results showed a prevalence of 8% for Cryptosporidium and of 13.33% for Giardia; the prevalence values obtained by FF and SFS were lower and in poor agreement with DFA results. Giardia and Cryptosporidium infections were more common in foals (23.33% and 26.66% respectively) and higher excretions were observed in the youngest foals. Distribution of Cryptosporidium prevalence was statistically related to farms (P < 0.01), age of animals (P < 0.01), but was unrelated to the presence of diarrhoea. In the case of Giardia, the prevalence was only related to age (P < 0.01). Pattern sheddings were related to intestinal diseases and horse age (P < 0.01). Risk factors for shedding included residence farms and age older than 8 weeks for both parasites. All DFA-positive faecal samples were submitted to DNA extraction and PCR to determine Giardia and Cryptosporidium species/genotypes. Sequence analysis of the COWP gene of Cryptosporidium and of the SSU-rRNA gene of Giardia revealed that they were identical to each other and identified Cryptosporidium parvum and Giardia duodenalis assemblage E. The potential role of infected horses in zoonotic transmission of Cryptosporidium was supported by the findings of this study.

Strategies for successful vaccination against hepatocellular carcinoma.

Current therapies against hepatocellular carcinoma (HCC) are not curative in the majority of patients. In the past, immunotherapy approaches aimed to non-specifically stimulate immune response were quite ineffective. New treatments based on stimulation of specific anti-tumor immune response are currently proposed and appear more promising. Tumor-specific antigens identified in HCC demonstrated immunogenicity both in preclinical and clinical trials. Effectiveness in animal studies raised interest in the clinical applicability of non-specific adoptive immunotherapy that prevented disease recurrence after tumor resection. Dendritic cell (DC)-based tumor vaccines achieved encouraging results, and cellular vaccines based on DCs have already entered clinical trials. Preventive and therapeutic DNA vaccination have been proposed, all based on tumor-associated antigens (TAAs), either modified or not, an example being alpha-fetoprotein (AFP). The concomitant expression of co-stimulatory molecules and cytokines was used to increase tumor immunogenicity. Syngeneic or nude mice models indicated that immunotherapy for HCC could stimulate an anti-tumor T-cell response leading to clinical benefit devoid of significant toxicity. The use of DNA-based vaccination raises exciting possibilities in preventing HCC in high-risk individuals such as those with cirrhosis. Novel immunotherapy strategies may contribute in the future to prevention and treatment of HCC.

Specific IgG4 response directed against the 45-kDa glycoprotein in trichinellosis: a re-evaluation of patients 15 years after infection.

The aim of this study was to evaluate the humoral immune response in late human trichinellosis with particular attention to the presence of IgG4 antibodies directed against the Trichinella-45-kDa glycoprotein (gp). This study re-evaluates subjects 15 years after they were involved in a trichinellosis outbreak that occurred in Central Italy following the consumption of raw boar meat infected with Trichinella britovi. The results show that ELISA tests using the E/S antigen identified five IgM- and eight IgG-positive patients and no IgA-positive patients. Tests using immunoblot (IB) with E/S antigens identified three IgM-, five IgA-, seven- IgG1- and three IgG4-positive sera. When the purified 45-kDa gp was used as an antigen, the IB revealed that six of the ten sera tested were positive for IgG4. Sera were also evaluated with a commercial kit, revealing that 11 of 12 patients had a highly sensitive reactivity against Trichinella proteins (64 and 44-43 kDa). In conclusion, humoral immune response against Trichinella is still present in these patients 15 years after the initial infection, including an IgG4 response directed to the 45-kDa gp.

Re-evaluation of patients involved in a trichinellosis outbreak caused by Trichinella britovi 15 years after infection.

This study re-evaluates 13 out of 48 subjects involved in a trichinellosis outbreak that occurred in Central Italy (Umbria Region) in 1988 resulting from the consumption of raw boar meat harboring Trichinella britovi. During the outbreak, 28 of 48 serologically positive subjects were asymptomatic, whereas 20 subjects presented one or more clinical signs including but not limited to fever, myalgia, periorbital oedema and conjunctivitis. Several patients were hospitalized with severe clinical signs requiring treatment with mebendazole and corticosteroids. Upon re-evaluation of 13 patients, none presented clinical signs; however, three still had increased CPK or LDH serum levels with some signs of electromyographic changes. In this study, enzyme immunoassays (EIA) were used to test the 13 positive sera for reactivity with T. britovi antigens using both excretory/secretory (E/S) antigens and a synthetic antigen composed of beta-tyvelose conjugated to bovine serum albumin. Western blots (WB) were also carried out using a commercial kit. Studies using EIA with E/S antigen identified five positive sera; however, using beta-tyvelose as antigen, only one positive sample was identified. Nearly all sera reacted positively with one or more Trichinella antigens when analyzed by WB, in particular to the 45 k Da beta-tyvelose containing glycoprotein. Results indicate that T. britovi, though less pathogenic than other Trichinella species, is clearly capable of inducing sustainable sequelae.

[Problems and limitations of conventional and innovative methods for the diagnosis of Toxoplasmosis in humans and animals]. / Problematiche e limiti dei metodi convenzionali ed innovativi nella diagnosi di Toxoplasmosi nell'uomo e negli animali.

Diagnosis of toxoplasmosis is useful for human and animal health. Several techniques are employed for the diagnosis in feline and canine population. Coprological tests for the detection of oocysts in cat faeces are of little significance owing to short patency (15 days). Histological examinations of biological samples show a lack of reliability when the animals are infected with few parasites; the mouse inoculation is the most reliable method even if the detection of cysts in mice brain require 40 days. However tachyzoites of virulent strains can be isolated from peritoneal exudate 3-4 days after inoculation. Samples inoculation in cell cultures (VERO, human fibroblasts) requires specialized laboratories and fails if non viable parasites are present due to tissutal autolysis. Serological tests are the most used diagnostic methods; Dye test and IFAT that require intact tachyzoites are more sensitive and specific compared to IHA, LA, ELISA because, during the infection, the first significant increase of IgM and IgG antibodies was observed against cuticolar antigens. A PCR to identify T. gondii DNA in canine and feline biological samples was developed. The B1 PCR performed on blood samples was less sensitive than when it was performed on other biological fluids requiring 100 tachyzoites, instead of 10. Aqueous humor PCR results could be negative if the infection is low grade or is restricted to the posterior segment or the animal was previously treated with anti-Toxoplasma drugs. SNC disease may be also difficult to diagnose because an high serum IgG titer may be associated with locally production or leakage from serum through a compromised blood-CSF barrier. AB1 PCR was successfully applied for the diagnosis of Toxoplasma abortion in ewes requiring only 10 parasites in placental cotyledon samples; the test compared with mouse inoculation showed similar sensitivity. Discrepancies may have been due to a low and focal distribution of parasites in the tissues or to the presence of non viable parasites if the tissues are autolysed. In regard to diagnostic methods adaptable to slaughter testing, several serological tests have been studied (IFAT, ELISA, IHA) for detection of IgG in sheep, pigs, cattle using also recombinant antigens (gene fragments H4 and H11) to lack the cross reactivity. The problem is the antibodies fall to near background levels as the infection became chronic (6-10 months p.-i.). A highly sensitive and specific method (Toxo Taq Man) has been developed to detect and quantitate T. gondii burden in animal tissue samples (0.1 pg of T. gondii genomic DNA, which is equivalent to 1 bradyzoite) using T. gondii ITS1-derived primers and a fluorogenic probe via Real-Time PCR. This assay is compatible with automation technology for potential slaughterhouse use. The diagnosis of acute infection in human pregnancy is difficult since IgM antibodies can be detected for a very long time after the acute phase; an IgA increase is of more diagnostic value because can be detected only for 6-7 months while the short kinetics of IgE can be useful only to date the infection precisely. In addition an IgG seroconversion is essential for the diagnosis. Among the most reliable tests, IgG avidity test is useful when a single serum sample, in the first months of gestation, is available, but low avidity results may persist for as long as 1 year. For this purpose a panel of serologic tests must be performed (ELISA, EIA, ISAGA, IgG avidity, IFAT, Dye test) for IgM, IgA, IgG and IgE. The serological diagnosis of prenatal infection is difficult since maternal IgG are passively transferred in utero to the foetus and caution must be exercised in interpretation of IgM or IgA results. A technique of Western blots of paired maternal and baby sera for evidencing different bands in the blots of two sera was developed for this purpose (specificity 97-100%, sensitivity 96-98%). The most reliable methods for prenatal diagnosis are PCR, mouse inoculation and cultural techniques performed on amniotic fluid, foetal blood and peripheral maternal blood in pregnants serologically positive. PCR (targets B1, SAG-1, rDNA) with amniotic fluid performed from 18 weeks of gestation is more sensitive and more rapid than conventional diagnostic procedures. PCR has been successfully used to diagnose Toxoplasma encephalitis in immunocompromised patients (cerebral biopsy is the only diagnostic method) and in ocular toxoplasmosis. In this evenience it is useful the study of IgG, IgM, IgA profile of paired serum and aqueous humor (Western blots).

Immune response at birth, long-term immune memory and 2 years follow-up after in-utero anti-HBV DNA immunization.

Infections occurring at the end of pregnancy, during birth or by breastfeeding are responsible for the high toll of death among first-week infants. In-utero DNA immunization has demonstrated the effectiveness in inducing specific immunity in newborns. A major contribution to infant immunization would be achieved if a vaccine proved able to be protective as early as at the birth, preventing the typical 'first-week infections'. To establish its potential for use in humans, in-utero DNA vaccination efficiency has to be evaluated for short- and long-term safety, protection at delivery, efficacy of boosts in adults and effective window/s for modulation of immune response during pregnancy, in an animal model suitable with human development. Here we show that a single intramuscular in-utero anti-HBV DNA immunization at two-thirds of pig gestation produces, at birth, antibody titers considered protective in humans. The boost of antibody titers in every animal following recall at 4 and 10 months demonstrates the establishment of immune memory. The safety of in-utero fetus manipulation is guaranteed by short-term (no fetus loss, lack of local alterations, at-term spontaneous delivery, breastfeeding) and long-term (2 years) monitoring. Treatment of fetuses closer to delivery results in immune ignorance without induction of tolerance. This result highlights the repercussion of selecting the appropriate time point when this approach is used to deliver therapeutic genes. All these findings illustrate the relevance of naked DNA-based vaccination technology in therapeutic efforts aimed to prevent the high toll of death among first-week infants.

Neospora caninum infection and congenital transmission: serological and parasitological study of cows up to the fourth gestation.

In this paper, a parasitological and serological study performed in three cows up to the fourth gestation is reported in order to clarify the extent of vertical propagation and, secondly, in which period of gestation the recrudescence of previous infection occurs. The cows selected for the study delivered healthy but congenitally infected calves in first pregnancy. The parasite was found, by biological tests in Swiss mice, in all the placentas of the three cows examined, during the three subsequent gestations, at calving. The parasite was found, at slaughtering, in the brains of all nine calves born clinically healthy from the three cows as well. The serological profile, performed at monthly intervals on serum of the cows, showed that IgG and IgM increased in the third trimester of gestation; this rise of antibodies was constantly observed during the three gestations and in all three cows. In the calves, the IgG titres increase after colostrum consumption and an IgM peak at birth, were indicative of a late infection. These findings, along with negative results obtained by a serological study conducted simultaneously on 38 cows housed in the same stable as the experimental animals and the negative results obtained in isolating parasite or antibodies from farm dogs, suggest that N. caninum infection can be maintained over several bovine generations and that recrudescing persistent infection, rather than a new infection, explains the Neospora infection of calves.

Canine filariosis in Umbria: an update of the occurrence one year after the first observation of autochthonous foci.

Following the first observation of two autochthonous foci of canine filariosis occurred in Umbria region in the year 2001, a survey on prevalence and risk factors was conducted 12 months later to better understand the actual entity of the Dirofilaria problem in Umbria region. Blood samples were collected between January and December 2002 from 2406 dogs living in a total of 7 towns located in the identified areas at risk. Blood samples were tested by a modified Knott's technique to evaluate the microfilaraemia and, by a commercial ELISA kit, to detect in the sera adult antigens of D. immitis. The results were subject to statistical analysis. A total of 439 dogs were found to be infected. The true prevalence (LC 95%) was of 18%. Microfilariae of D. immitis were detected in 286 dogs (13%) while 112 dogs (6%) showed only microfilariae of D. repens and 41 dogs (1.6%) microfilariae of both D. immitis and D. repens. The prevalence ratio (PR) for each species of Dirofilaria (LC 95%) calculated in association with different risk factors (age, sex, use, outdoor night status, position, living together with other dogs, breed) and the statistical significance between the risk factors and the presence/absence of the infection, evaluated for each species of Dirofilaria, are discussed.

Retrospective ultramicroscopic investigation on naturally cryptosporidial-infected commercial turkey poults.

The morphometric characteristics and the ultramicroscopic findings of Cryptosporidium spp. at various stages of their life cycle in the intestinal and bursal epithelial cells of naturally infected 30-day-old commercial turkeys are reported. Small, sporulated oocysts, observed in the small intestinal content after flotation, were identified as Cryptosporidium meleagridis on the basis of morphometric characteristics (round in shape and 4.5-5.0 microm in size) and the small intestinal localization. Light section examinations revealed the presence of the protozoon in multiple organs, but its prevalence was highest in the intestinal and bursal epithelial cells. Ultramicroscopic studies on ileum and bursal samples showed the presence of all the life cycle stages in the microvillar brush epithelial cells in both the organs examined. On the basis of the comparison of the morphology and the sizes of the microorganisms parasitizing the ileum and the bursa, hypotheses are considered on the possible species involved.

The use of a synthetic antigen for the serological diagnosis of human trichinellosis.

Hosts infected with Trichinella produce antibodies specific for an epitope common to the TSL-1 family antigens. This epitope contained uncommon terminal 3, 6-dideoxy-D-arabinohexose (so called tyvelose) residues. The disaccharide moiety was synthesized and an immunodiagnostic assay was developed, which was specific and sensitive in swine trichinellosis. We aimed to verify the specificity and sensitivity of this immunodiagnostic test in human trichinellosis. 15 sera from normal subjects, 12 from patients with other parasitic diseases and 50 from trichinellosis patients were tested. Indirect enzyme linked immunosorbent assay (ELISA) for specific IgG and an amplified ELISA for specific IgE were performed using beta-tyvelose-GalNAc-bovine serum albumin (BSA) disaccharide conjugate or T. spiralis muscle larvae excretory/secretory (E/S) products, as antigens. Neither control sera nor other parasitic infection sera resulted positive both for IgG and IgE when synthetic or E/S antigens were used. In trichinellosis patient sera, specific IgG were present in 100% of cases, irrespective of the antigen used, but whereas specific IgE were detected in 78% using E/S antigens, a 100% positivity rate was obtained, using the beta-tyvelose-BSA conjugate.

Experimental trichinellosis in fallow-deer (Dama dama L.).

Herbivora can play a very important role in spreading trichinellosis, as showed by the massive epidemics in man, caused by the consumption of horse meat in the last years. In this context, the present study has been undertaken to verify, through an experimental infection, the susceptibility, together with other biological parameters, of fallow-deer to Trichinella infection. The four animals, 8-9 months of age and 18-25 Kg body weight, were orally infected with low doses of Trichinella britovi and T. pseudospiralis (2,000 larvae/animal). After day 30 p.i., the animals were necropsied and, using artificial digestion methods, larval burden of Trichinella in muscle tissues was determined. Histopathological, serological (IgG monoclonal blocking ELISA) and biochemical data were assessed during the experiment. The results showed the susceptibility of fallow-deer to T. britovi and T. pseudospiralis infection; under the same inoculum size, the number of larvae/g was higher in group infected with T. britovi. The animals showed a higher immunological response to T. pseudospiralis infection. The results are discussed.

Trichinella spp. in ostrich meat: a public health risk?

In the present work the biological behaviour of T. spiralis and T. pseudospiralis in ostriches is reported. Oral infections were performed in eight ostriches with two infective doses (10,000 and 80,000 larvae) for each species of Trichinella. On day 0, 30 and 60 p.i. blood samples were collected to assay the serum changes concerning specific muscle enzyme activities and total proteins. The immunological study, to determine specific IgG in sera, was conducted employing a monoclonal blocking ELISA. From the carcasses of sacrificed animals, samples of various muscle tissues were examined by the digestion method and by standard histopathologic procedures. The study showed a low susceptibility of the ostriches to T. pseudospiralis; preferential sites of larval distribution were muscle tissues of the legs. T. spiralis could be found in muscle tissues only when a high number of larvae were inoculated. Immunological reactivity was found only in animals infected with higher doses of T. pseudospiralis.

Susceptibility of nutria (Myocastor coypus) to Trichinella infection: biological aspects.

Experimental infections with three different species of Trichinella in nutria in order to evaluate the susceptibility and the role of these rodents in the spreading of parasitosis in nature were carried out. The nutria is present in many italian wet areas and its distribution is expanding. The nutria meat is utilized as food in different countries and is retained responsible for trichinellosis in man. Two groups of ten animals were infected per os with 500 and 5,000 (n. 10) infective larvae of T. britovi; an additional study was arranged with two groups of animals infected with 5,000 larvae of T. spiralis and T. pseudospiralis, respectively. After 45 days, all animals were slaughtered and samples of different muscles were processed by standard artificial digestion and by routine histological methods. Serological investigations (specific IgG) have been carried out on sera samples by employing a monoclonal blocking ELISA. The animals showed a significant susceptibility to the infection with all species of tested Trichinella and immunological reactivity. Data obtained are discussed.