Risk factors associated with Toxoplasma gondii seroprevalence in domestic pig farms in Argentina.

Toxoplasmosis is a worldwide parasitic zoonosis caused by Toxoplasma gondii. Pigs can become infected by consuming water or food contaminated with sporulated oocysts, or by carnivorism (like the consumption of infected rodents). In pigs most infections are asymptomatic. In certain countries, pig meat containing tissue cysts is a major source of infection for human beings. The aims of this study were to estimate the seroprevalence of toxoplasmosis and to identify which factors were related with the increase of the risk of infection in Argentina. The seroprevalence of T. gondii was determined in 240 pigs from 27 farms in the central-western area of Buenos Aires province, Argentina. Serum samples were analyzed using indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) techniques. Prevalence determined was 53.33% and 32.08% by IFAT and ELISA, respectively. Results showed that 81.5% (22/27) of the farms were seropositive to T. gondii. Seropositivity for T. gondii was related with the following risk factors (p value ≤0.05): presence of felids and rodents in the farms, feeding with waste of human food and storage of food outdoors with free access to felids and to the reservoirs when applying both serological techniques. Our results strongly suggest that the risk of infection with T. gondii in pigs is related to the outdoor/extensive type of production system with low infrastructure conditions, which allows both felids and rodents to have free access to pigs and stored food. Also, the high seroprevalence detected in the present study could indicate a potential role of pork in human infections in the region.

Humoral immune response of pigs infected with Toxocara cati.

Toxocara cati is one of the causative agents of human toxocariasis. Serological methods are used for diagnosis in paratenic hosts like humans but the humoral immune response triggered by this parasite is unknown. We characterized the humoral immune response to T. cati excretory-secretory antigens (TES) in pigs as animal model during the acute and chronic stages of infection. ELISA and Western Blot techniques were used to determine antibody response. Pigs were experimentally inoculated with 100,000 infective Toxocara cati eggs. Blood was collected at 7, 14, 21 and 28 days post-inoculation (d.p.i.) to assess the acute stage of infection and 90, 120 and 180 d. p.i. for chronic stage analysis. ELISA showed values higher than the cut-off of specific IgM and IgG at 7 d. p.i. with significant differences at 0 and 7 d. p.i. for IgM and at 14, 21 and 28 d. p.i. for IgG in the acute stage. Higher and stable levels were detected in the chronic stage. Western Blot showed bands from 102 to 38 kDa detected by specific IgM and IgG. More immunogenic bands were identified by specific IgG. In the chronic stage of infection a band near 31 kDa was the only band detected by IgM until 150 d. p.i. Specific IgG recognized bands between 102 and 31 kDa. This study demonstrates how the humoral immune response evolves in the acute and chronic stages of infection and provides evidence on the role of the pig as a paratenic host of T. cati.

A Toxocara cati eggs concentration method from cats' faeces, for experimental and diagnostic purposes.

Toxocariosis is a zoonotic parasite infection worldwide distributed, now considered a neglected disease associated to poverty. For experimental infection in animals and to develop the diagnosis in humans it is necessary to obtain large number of Toxocara spp. larval eggs. Toxocara cati eggs recovered percentage from faeces of infected cats was determined employing a novel egg concentration method. The McMaster egg counting technique and the concentration method were applied on 20 positive cats' sample faeces obtained from naturally infected cats. The mean percentage of eggs recovered by the concentration method was 24.37% higher than the count obtained by McMaster egg counting technique. The main advantage of this method is that it can be obtained a small final volume with a high number of recovered eggs and a good quality inoculum for experimental and diagnostic purposes.

Experimental infection with Toxocara cati in BALB/c mice, migratory behaviour and pathological changes.

Experimental inoculations of 1000 Toxocara cati larval eggs were carried out in 18 BALB/c mice. The T. cati eggs used for inoculation were collected from the faeces of naturally infected cats. Euthanasia was performed on two mice on days 1, 2, 3, 4, 5, 6, 14, 21 and 28 post-inoculation (p.i.). Tissue samples were taken for digestion and histopathology. Larvae were recovered from all infected mice and the average of all larvae recovered was 28.3% (95%; CI: 14.1-42.4). Maximum number was obtained from liver on days 1 and 2 p.i.; from the lung on day 2 p.i. and from the brain on day 28 p.i. In muscle, the recovery was high as from day 3 p.i., with the maximum obtained on day 28 p.i. Superficial foci of congestion and haemorrhage were macroscopically observed in the lungs between days 2 and 5 p.i. and in the brain between days 3 and 6 p.i. Microscopic lesions were observed in the liver between days 2 and 14 p.i., with periportal and subcapsule inflammatory infiltrates. In the lungs, haemorrhages and inflammatory infiltrates can be observed in the alveolar parenchyma, close to bronchioles and large blood vessels. In the brain, congestive areas without inflammatory reactions were seen. In muscle, the presence of inflammatory infiltrates and degenerated muscle can be observed surrounding a parasite larva. These same lesions were observed in myocardium and pericardium. The kidneys were congested with inflammatory infiltrates. The inflammatory cells present in all the tissues studied were lymphocytes, neutrophils and a few eosinophils. Formation of granulomas or signs of larva encapsulation were not observed. The migratory pattern of T. cati larvae in BALB/c mice and its tendency to become concentrated in the muscle reinforce the importance of the mouse as a paratenic host for the parasite's cycle in the environment.

Evaluation of the risk of transmission of Trichinella in pork production systems in Argentina.

Recently, there has been interest in programs that certify pork production practices that minimize the risk of exposure of pigs to Trichinella spiralis. Certification might be useful for reducing the risk of human trichinellosis from pork in Argentina, but more information is needed on pig production practices and sources of Trichinella infection in Argentinian pigs. In this study, 21 pig farms were assessed for Trichinella infection including some farms using total and partial confinement management, and others with pigs raised exclusively outdoors. A total of 3224 muscle samples were collected from pigs raised on these farms and tested to determine the presence of T. spiralis larvae by artificial digestion. Serum samples from the same 3224 pigs were tested for antibodies to T. spiralis by ELISA. For each farm, a questionnaire was completed summarizing information about management factors and this information was used to assess risk factors for exposure of T. spiralis. Based on the results, pigs raised outdoors were more likely to be infected than pigs raised in total or partial confinement (p< or =0.05). Pigs fed waste products containing meat were 12.5 times more likely to be infected than pigs not fed waste containing meat (p<0.01). The role played by rats in transmission of Trichinella is unclear; however, on farms with evidence of wild animals and access of pigs to wildlife carcasses, the prevalence of Trichinella infection was significantly higher. All pigs raised under good hygienic and sanitary conditions were negative for Trichinella infection by both artificial digestion and ELISA.

Clinical, haematological, biochemical and economic impacts of Trichinella spiralis infection in pigs.

The purpose of this work was to assess the clinical, haematological and biochemical responses of pigs experimentally inoculated with Trichinella spiralis. Groups of three pigs were inoculated per os with 100, 500 and 5000 T. spiralis muscle larvae, two pigs were used as control. Clinical evaluation of disease in pigs included daily examination, rectal temperature measurements and cardiac and respiration rates. Haematological studies included: hematocrit (%), hemoglobin (g/dl), and white cell, neutrophil, lymphocyte and eosinophil counts. Blood biochemistry included: bun (mg/dl), creatinine (mg/dl), AST (UI/l), ALT (UI/l), CPK (UI/l) and ALP (UI/l). No significant differences were observed in rectal temperature and in cardiac and respiration rates between inoculated animals and the control group (p> or =0.05). Significant differences were detected (p< or =0.05) in the values of % hemoglobin, and eosinophils, as well as in the values of CK, ALP, AST and ALT. The variations observed in some cases were related to the number of T. spiralis larvae inoculated and varied with the number of days post-infection. Inoculated pigs showed significant differences (p< or =0.05) in weight gain when compared with uninoculated controls. This study has clinical, haematological, and enzyme alterations in Trichinella infected pigs provides a better understanding of acute and chronic trichinellosis in pigs.

Prevalence of Toxocara cati and other parasites in cats' faeces collected from the open spaces of public institutions: Buenos Aires, Argentina.

Toxocarosis is a worldwide parasitic infection that affects both cats and dogs. Toxocara cati (Schrank, 1788) syn. Toxocara mystax (Zeder, 1800) prevalence was studied in faeces from stray cats collected from the open spaces of public institutions of Buenos Aires city, both building and surrounding open spaces are fenced off. Of the 465 samples obtained from March to June of 2005, 58.3% were found to have parasite eggs. The following parasites were identified from the 271 positive samples: T. cati (61.2%), Cystoisospora spp. (20.3%), Trichuris spp. (17.0%), Toxascaris leonina (15.1%), Ancylostoma spp. (14%) and Aelurostrongylus abstrusus (2.6%). T. cati prevalence was 35.7% (95% confidence interval: 31.2-40.1), with a 42.2% single isolations. The most frequent combination was T. cati and Cystoisospora spp. (9%). More than half the areas studied showed over 40% prevalence. Seventy-one percent of the collected samples were fresh with a variable moist consistency and 29% were older with a dry consistency. A statistically significant association was found between sample consistency and presence of parasites (chi2 = 10.81; p = 0.001) as also between sample consistency and presence of T. cati (chi2 = 11.27; p = 0.0007). Moist consistencies were significantly different from the rest: consistency (wet or dry) versus parasites (z = 1.95; p = 0.02) (95% confidence interval: 0.004-0.203); consistency (wet or dry) versus T. cati (z = 3.25; p = 0.0006) (95% confidence interval: 0.075-0.254). The cat population that inhabits these public green spaces contaminates the environment, thus transforming them into dangerous spaces with a variable rate for the human population that spends time in these places.

Toxocara canis infections in a pig model: immunological, haematological and blood biochemistry responses.

The immunological, haematological and enzymatic responses to the inoculation in pigs of 100,000 embryonated eggs of Toxocara canis were studied. Fifteen females were inoculated and three remained as controls. Haematological values were analysed from day 7 p.i. until day 126 p.i. In the inoculated group, white blood cells were raised on day 14 p.i. and eosinophil values on days 7, 14, 21, 35 and 49 p.i. showing significant differences compared with controls (P < 0.05). Absolute eosinophil counts (per ml) presented two rises, the first on days 7, 14 and 21 p.i. and the second on days 35 and 49 p.i. Blood biochemistry was maintained within normal values. Serological examination by ELISA to determine antibody levels against Toxocara canis L2/L3 excretory-secretory (ES) antigens showed values higher than the positive cut-off (1:32) from day 7 p.i. and until the end of the study on day 126 p.i., presenting two peaks: one on day 28 p.i. and the second covering days 49 to 56 p.i. Western blots of sera of inoculated animals presented, from day 7 p.i., two polypeptide bands of 55 and 70 kDa MW and, from day 56 p.i., an additional band of 120 kDa MW, all of which persisted until the end of the study. Immunological responses were sustained over time. No direct correlation was observed between the rise in eosinophils and antibody titres. To validate the conclusions, more studies are required on the polypeptide bands.

Toxocara canis in experimentally infected pigs: migratory pattern and tissue lesions.

Fifteen Yorkshire female pigs were inoculated with 100,000 infective T. canis eggs. Three animals were used as uninfected controls. Groups of three infected pigs were euthanized by accepted methods on days 7, 14, 21, 28 and 126 p.i., respectively. Larvae were recovered from all animals included in each group slaughtered on days 7 and 14 p.i.; on day 21 p.i. from two pigs, on day 28 p.i. from one, and no larvae were found on day 126 p.i. Differences in the mean number of larvae per gram in lymph nodes, liver and lungs between slaughter days, were significant for livers on day 7 p.i. and for lungs on day 14 p.i. (P < 0.10). The decrease over time was significant in all the organs that previously had larvae. Larvae were not found in the other organs and tissues analysed. Macroscopical lesions were found in the liver, lungs and lymph nodes on days 7, 14, 21, and 28 p.i. The entire surface of the liver was covered with small white spots on day 7 p.i., on days 14 and 21 p.i. the spots were distinctly nodular and, in some places, individual lesions were confluent. Lesions had apparently started to heal on days 28 and 126 p.i. appearance was normal. Lymph nodes were enlarged and oedematous during the first 4 weeks and the lungs had small areas of consolidation visible all over the surface, but by day 126 p.i., no visible lesions could be seen. Microscopical lesions were observed in the liver on day 7 p.i., with a largely periportal hepatitis. Numerous eosinophils and lymphocytes were present. The typical granulomatous reaction was observed on days 14 and 21 p.i. with a central necrotic core and a narrow region of fibroblastic tissue. By day 28 p.i. lesions had almost disappeared and the number of eosinophils was fewer. There were fewer leukocytes and the fibrous tissue had disappeared from the liver on day 126 p.i. For the first 3 weeks, pictures of the lymph nodes and the lungs were characterised by the formation of a granuloma. In the center of the granuloma larvae were observed. The majority of the lesions had healed by day 126 p.i.

Immunological and hematological response in experimental Toxocara canis-infected pigs.

The relationship between the immunological and hematological response to infection was studied in pigs inoculated experimentally with Toxocara canis. Two groups of four pigs were infected with doses of 1000 and 2000 infective eggs, respectively. Two uninfected animals were used as negative controls. Blood samples were collected from each pig once a week. Serological examination by ELISA to determine antibody levels against T. canis L2/L3 excretory-secretory (ES) antigens showed values higher than the positive cut-off point (1:32) for both the infected groups. These values increased from day 7 p.i. and remained high during the experimental period until day 56. Significant differences were recorded for the two inoculating doses (p