Mabel Ribicich

Trichinella patagoniensis n. sp. (Nematoda), a new encapsulated species infecting carnivorous mammals in South America ☆

Until a few years ago, Trichinella spiralis was the only taxon of the genus Trichinella detected in both domestic and wild animals of South America. Recently, a new genotype, named Trichinella T12, was identified in cougars (Puma concolor) from Argentina, on the basis of molecular studies using mitochondrial and nuclear ribosomal markers. In the present study, cross-breeding experiments indicated that Trichinella T12 is reproductively isolated from all other encapsulated Trichinella spp. and suggested that it is biologically more similar to Trichinella britovi and Trichinella murrelli than to the other encapsulated species/genotypes. Biological assays revealed that the reproductive capacity index of Trichinella T12 was ~4 and >2000 times lower than those of T. spiralis in mice and rats, respectively. The reproductive capacity index of Trichinella T12 in domestic pigs ranged from 0.0 to 0.05. Larvae parasitising the muscles of carnivores were infective to mice after freezing at -5°C for 3 months, but they lost infectivity after freezing at -18°C for 1 week. The region within the rDNA, known as the expansion segment V, showed a unique sequence which differs from those of all other known Trichinella spp./genotypes. The biological, geographical and molecular data support the classification of the genotype Trichinella T12 as a new species widespread in the Neotropical region, for which we propose the name Trichinella patagoniensis n. sp.

A distinctive Western blot pattern to recognize Trichinella infections in humans and pigs

Trichinellosis is a zoonotic disease caused by parasites of the genus Trichinella, which have a cosmopolitan distribution. For diagnostic purposes, a confirmatory test for ELISA-positive human and pig sera such as Western blotting is required, due to the high number of ELISA false positive sera. The objective of this study was to identify the Trichinella-specific antigens most frequently recognized by sera from Trichinella-infected humans and pigs, so as to define a distinctive pattern of Trichinella infection in sera from infected hosts using Western blots which allow false positive sera to be distinguished from true positive sera. Using excretory/secretory antigens, 450 human sera were tested by Western blotting: 150 from persons with a confirmed diagnosis of trichinellosis and 300 from persons who did not have trichinellosis but who tested positive by ELISA (i.e., false positives). We also tested 210 pig sera: (i) 30 from pigs experimentally infected with Trichinella spiralis; (ii) 90 from naturally T. spiralis-infected pigs; and (iii) 90 from pigs not infected with Trichinella, as shown after artificial digestion of the diaphragm pillars, yet which tested positive by ELISA (i.e., false positives). All true positive sera (i.e., sera from persons with confirmed trichinellosis as well as sera from naturally and experimentally infected pigs), reacted with a three-band pattern ranging in size from 48-72kDa. A distinctive pattern for recognizing Trichinella spp. infections in humans and pigs by Western blots is defined; it shows a sensitivity of 100% and it allows sera from Trichinella-infected humans and pigs to be distinguished from sera from persons and pigs that were not infected with Trichinella spp. (100% specificity).

Trichinella infection in wild animals from endemic regions of Argentina

Natural infection with Trichinella has been described in more than 150 mammalian species. However, few reports of Trichinella infection in wild animals have come from Argentina. In this study, muscle tissue was obtained from wild animals in Argentina with the aim of evaluating the presence of Trichinella. A total of 169 muscle samples were collected to determine the presence of Trichinella larvae by artificial digestion. The 169 muscle samples originated from 12 species including 36 opossums (Didelphis albiventris), 19 armadillos (Chaetophractus villosus), 9 capybaras (Hydrocaeris hydrocaeris), 1 puma (Puma concolor), 3 grey fox (Lycalopex gymnocercus), 6 coypus (Myocastor coypus), 6 skunks (Conepatus chinga), 2 ferrets (Galictis cuja), 66 rats (Rattus norvegicus), 6 mice (Mus musculus), 12 wild boars (Sus scrofa), and 3 wild cats (Felis geoffroyi). Trichinella infection was detected in 1 puma [2 larvae per gram (LPG)], 3 wild boars (8–420 LPG), 3 armadillos (0.04–0.08 LPG), and 9 rats (0.1 to 150 LPG). Only 3 Trichinella isolates, of 1 rat and 2 wild boars from Neuquén, were identified as Trichinella spiralis by nested PCR. The presence of Trichinella infection among wild animal populations suggests a sylvatic cycle of transmission in Argentina, which can serve as a reservoir for humans and domestic animals. Further, evidence of high prevalence in rats emphasizes the need to improve pig management, mainly in small individual farms without adequate technology, to enhance the quality of feeds, and to improve veterinary services to avoid exposure of pigs to Trichinella.

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.

Assessment of the risks of rats (Rattus norvegicus) and opossums (Didelphis albiventris) in different poultry-rearing areas in Argentina

We have studied the prevalence of Trichinella spiralis, Leptospira spp. and Salmonella spp. in rats and opossums that inhabit poultry farms of Exaltacion de la Cruz, Buenos Aires, Argentina, to determine the potential sanitary risk for humans that are in contact with these animals. The study was carried out on 48 poultry farms between spring 1999 and winter 2001. The study of opossums began in winter 2000. During the study period we captured 152 Rattus norvegicus, 3 Rattus rattus, 16 Didelphis albiventris and 1 Lutreolina crassicaudata. We have registered the presence of rats and opossums in 70% and 27% of the studied farms, respectively. The percentage of farms with rats was independent of the presence or absence of pigs. We did not detect the presence of Leptospira spp. and Trichinella spiralis in any individual. We detected the presence of Salmonella Enteritidis in one Rattus norvegicus and one Didelphis albiventris. According to our results, the rats and opossums of poultry farms may not report a risk factor in the transmission of Trichinella and Leptospira under the present conditions; but the detection of Salmonella Enteritidis in rats as well as in opossums suggests the idea of applying prophylactics measurements on poultry farms.

Prevalence of canine dirofilariosis in the City of Buenos Aires and its outskirts (Argentina)

During the past 10 years, the prevalence of canine dirofilariosis in the City of Buenos Aires and its outskirts, particularly in the northern and southern areas, has increased significantly. In the present work, studies were carried out in dogs living in the city and in its northern, western and southern outskirts from 1997 to 2001. For this purpose, 782 blood samples were collected and analyzed to determine circulating antigen, processed with the Witness Merial antigen test. The samples resulted in negative tests for subjects who lived in the city; however, 17.7 and 23.5% of the tests were positive from the northern and southern outskirts, respectively. When analyzed by sex, positive results were distributed as follows: 62.5% males and 37.5% females (P<0.05). No significant statistical difference was found on comparing purebred and cross-breds (P<0.05). It is interesting to point out the geographical distribution of the disease, which confirms that ecological factors such as water currents, abundant vegetation and the existence of mosquitoes all year round, are important for the biological cycle of Dirofilaria immitis.

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</=0.01) from day 14 to 42 p.i. Western blots run on day 7 p.i. with serum from the 2000 eggs group showed two polypeptide bands of 70 and 55kDa. These same bands were observed for both infected groups from day 15 p.i. until the end of the experiment. Eosinophil counts were significantly higher (p</=0.05) for both inoculated groups between weeks 2 and 7 p.i. as compared with the control group. No significant changes were observed for other blood parameters. The pig is a useful animal model since T. canis induces an early immunological response and ELISA can detect the presence of these antibodies against T. canis L(2)/L(3) ES antigens in animals inoculated with low doses of Toxocara eggs.

Experimental infection with Trichinella T12 in domestic cats

Trichinella spiralis has been documented in wild animals in Argentina, including puma, armadillos, rats and wild boars. In 2008, molecular analysis identified Trichinella T12 from a naturally infected puma (Puma concolor) from Patagonia. The aim of the present work was to study the relationship between the infectivity and pathology of Trichinella T12 in the puma and in domestic cats, and the possible risks that may be present for transmission among these animals. Two cats (A and B) were orally-infected with 3300 and 1850 Trichinella T12 muscle larvae, respectively; one additional cat was used as a control. During the 54 days post-infection, a daily examination was performed which included monitoring body temperature, and cardiac and respiration rates; the animals were then euthanized. Hematological studies included hematocrit (%), hemoglobin (g/dl), and white cell, neutrophil, lymphocyte and eosinophil counts. Blood biochemistry included urea, creatinine, AST, ALT, CK, LDH and ALP. An ELISA assay was also performed. At necropsy, organs (liver, spleen, brain, cerebellum and kidney), nails and muscle samples were obtained for histopathology studies and artificial digestion. The muscles that were studied included the diaphragm, massetter, cutaneous, temporal, intercostals, lumbar, tongue, limbs, neck and tail. Clinical signs, such as anorexia, diarrhea, vomiting, shaggy hair, decay and muscle pain, were observed in both cats. The eosinophil counts were elevated in both cats A and B. Trichinella larvae were recovered from all of the muscles analyzed where the histopathology showed larvae in several muscles without degenerative reaction. Neither larvae nor lesions were observed in non-muscular organs. Cat A had a maximum of 246 larvae per gram (lpg) in the temporal muscle and a minimum of 80 lpg in the tongue, while cat B had a maximum of 65 lpg in muscles of the leg and a minimum of 10 lpg in tail muscles. This study represents the first record of experimental infection of cats with Trichinella T12.

Larval development of Aelurostrongylus abstrusus in experimentally infected Rumina decollata snails

Aelurostrongylus abstrusus is a lungworm distributed worldwide that affects wild and domestic cats, causing bronchopneumonia of varying intensity. Snails serve as intermediate hosts. The aim of the present study was to assess the larval development of A. abstrusus in R. decollata snails and to investigate its potential as an intermediate host. For this purpose, first-stage larvae (L1) of A. abstrusus were obtained from the faeces of naturally infected cats. Doses of 500 L1/snail were given to 24 R. decollata snails, placed on the soil of the breeder chamber, and maintained under laboratory conditions. Three snails were killed at 8, 10, 12, 16, 22, 26, 45 and 55 days post-infection (dpi), and the muscular foot and visceral body were separately digested by an artificial digestion technique. The morphometric parameters of different larval stages were recorded. The mean number of larvae reaching the infective stage at the end of the study (L3) was 262 larvae/snail. The greatest development to L3 was recorded from days16 to 55 pi, during which the isolation was maximum. A. Abstrusus L3 were isolated from the viscera, but isolation from the snail foot was significantly higher. Our results showed for the first time the ability of A. Abstrusus larvae to develop in R. decollata, thus serving as a potential intermediate host.

Trichinella spiralis in a South American sea lion (Otaria flavescens) from Patagonia, Argentina

Trichinella spp. from a sylvatic cycle has been found in several animal species such as pumas (Puma concolor), armadillos (Chaetophractus villosus), rats (Rattus norvegicus), and wild boars (Sus scrofa) in Argentina. Moreover, Trichinella infection has been detected in a wide range of marine mammals around the world, including polar bears (Ursus maritimus) and walruses (Odobenus rosmarus). Until the present time, Trichinella spp. infection has not been detected in marine mammals of South America. Samples from four South American sea lions (Otaria flavescens) found dead in Rio Negro, Argentina, were analyzed by artificial digestion, and in the case of one animal, Trichinella larvae were identified at the species level by nested multiplex PCR as Trichinella spiralis. This is the first report of a Trichinella species infecting marine mammals from South America.