Annals of Tropical Medicine & Parasitology, Vol. 98, No. 7, 725–731 (2004) Porcine and rodent infection with Trichinella, in the
Sierra Grande area of Río Negro province, Argentina

E. LARRIEU*, V. MOLINA†, S. ALBARRACÍN‡, S. MANCINI*, R. BIGATTI*,L. LEDESMA‡, C. CHIOSSO*, S. KRIVOKAPICH†, E. HERRERO* andE. GUARNERA† *Secretaría de Estado de Salud, Laprida 240, 8500 Viedma, ArgentinaDepartamento de Parasitología, Instituto Nacional de Enfermedades Infecciosas, AdministraciónNacional de Laboratorios e Institutos de Salud ‘Dr Carlos G. Malbrán’, Av. Vélez Sarsfield 563,1281 Buenos Aires, ArgentinaHospital Sierra Grande, Diagonal sin Nombre, 8532 Sierra Grande, Argentina Received 16 February 2004, Revised 12 May 2004,Accepted 14 May 2004 In 2000, two cases of human trichinellosis were detected in the Sierra Grande area of Río Negro province, Argentina.
As part of an investigation of the aetiology of these cases, 300 pigs slaughtered for consumption in the area between2000 and 2002 were checked for Trichinella infection, by artificial digestion of a muscle sample. Twelve (5.6%) —four (7.3%) of the 55 checked in 2000, five (4.8%) of the 105 investigated in 2001, and three (2.1%) of the 140investigated in 2002 — were found infected. Blood samples were collected from other pigs aged >6 months old,so that sera could be tested, in ELISA and by western blotting, for anti-Trichinella antibodies. Of the 181 animalschecked in the initial serological survey, 36 (19.9%) were found seropositive for Trichinella. When 35 of theseronegative pigs were re-checked 6 months later, three (8.6%) were found to have seroconverted.
Four (15.4%) of 26 local rodents, caught in Sherman-type traps, were also found positive when checked for infection by artificial digestion. It appears that about 20% of pigs in the study area are infected each year, this highlevel of transmission being sustained by a high prevalence of infection in the local rodent populations.
Trichinellosis is a zoonosis that is broadly When, in 1984, staff of the veterinary service distributed throughout the world. The caus- of the Río Negro health secretariat checked ative agents, nematodes of the genus Trich- pigs held in breeding pens in this area, so inella, are mainly to be found infecting carnivorous or omnivorous mammals, inclu- with Trichinella that the area was officially declared severely affected by trichinellosis et al., 1991; Gamble, 2000; Sequeira et al., (unpubl. obs.). This declaration prompted Health Service and the culling of every pig (>600 animals) in the area. Microscopical porcine and human trichinellosis (Larrieu, examinations (i.e. trichinoscopies) revealed that 22% of the pigs killed were infected south–eastern corner of the province, has (unpubl. obs.). Since 1985, the local pig disease, which remains locally endemic.
former size, with new introductions fromseveral regions of Argentina. In the last 20years, however, the human population has Reprint requests to: E. Larrieu.
E-mail: [email protected]; fax: +55 2920 declined (from 12,000 residents in 1985 to <6000 in 2000) as a result of the closure of 2004 The Liverpool School of Tropical MedicineDOI: 10.1179/000349804225021460 in place so that the location of each tagged pig could be updated, as necessary, and any ment. As the current level of unemployment pigs entering or leaving the area could be exceeds 50%, life for the local inhabitants is generally very difficult, with much social deprivation. The pigs in the area are owned pig owners to the effect that any pig found by small-scale producers whose primary aim seropositive for Trichinella (see below) would is the production of pork for their families be confined to its breeding pen. Breeding to eat, although some of the meat is sold or pens with seropositive swine were inspected exchanged for other goods. Little of the meat systematically by the veterinary service of the is processed into salami or ham, to extend its shelf-life. Fresh pork sausages are marketedin a few butchers’ shops.
In 2000, clinicians at the hospital serving Immunodiagnosis
the Sierra Grande region reported two cases of human trichinellosis (unpubl. obs.). The central caudal vein of each pig tagged in the all of the pigs in the two pig-breeding pens centrifugation, and then kept frozen at - subsequently identified as the geographical 20dC until they could be checked for anti- Trichinella antibodies in ELISA and western blots (Ruitemberg et al., 1975). Six to checked for Trichinella, by ‘artificial’ diges- 23 months later, some of the pigs that had tion of muscle samples (Gamble, 1998), 10% been found seronegative and two of the pigs were found to be infected (unpubl. obs.).
(confined to their pens) that had been found evaluate the general prevalence of Trichinellainfection in the pigs and rodents of the Sierra   Grande area, and so gain some insight into The larval excretory–secretory products the epidemiology of trichinellosis in this and used as antigens for the serological tests were prepared from T. spiralis ISS643 that hadbeen maintained in CF1 mice (Su et al.,1990). Larvae were recovered from infected murine muscle (from mouse carcasses thathad been skinned, eviscerated and ground) Study Area
by digestion with 1% pepsin in 1% HCl for 3 h at 37dC. They were washed in Dulbecco’s steppe and is characterized by low rainfall tures, and very low winter temperatures. The (500 µg/ml), and then incubated, for 18 h at with 10 m N-(2-hydroxyethyl)piperazine-Np-(2-ethanesulphonic acid) (HEPES), 2 Pig Census and Control
m glutamine, 1 m pyruvate, penicillin In October 2000, all the pigs in the Sierra (50 U/ml) and streptomycin (50 µg/ml). The aged. The older animals (those aged >6 rane. The filtrate was concentrated under tags, each tag being placed in a left ear.
Subsequently, a monitoring system was put retention. The protein concentration in a sample of the concentrated filtrate was esti- (phosphate-buffered saline, pH 7.2, contain- mated by the method of Bradford (1976).
The remainder of the concentrated filtrate, being cut into 2-mm-wide strips. Each strip used as the antigen source for the serological was then shaken for 1 h at room temperature tests, was maintained frozen at -20dC until with a test serum diluted 1:100 in blocking solution. After three, 10-min rinses withwash buffer, the strips were shaken for 1 h atroom temperature with the same peroxidase conjugate as used for the ELISA, then rinsed Flat-bottomed, polyvinyl, microtitre plates twice, for 10 min each, with washing buffer, and once, for 10 min, with pure phosphate- buffered saline (PBS). Finally, a substrate buffer, pH 9.6, overnight at 4dC. Each well buffer (phosphate-buffered saline, pH 7.2, antigens, of 49, 52 and 54 kDa, were consid- were then blocked with blocking buffer (3% ered blot-positive for Trichinella (Su et al., albumin in wash buffer) for 30 min at 37dC.
After three more rinses with wash buffer,100 µl of a test serum, diluted 1:250 inblocking buffer, were added to each well and Pig Parasitology and Muscle Digestion
the plates incubated 30 min at 37dC. Then a horseradish-peroxidase conjugate of rabbit collected, post-mortem, from each pigslaughtered for consumption between 2000 blocking buffer was left in the wells for subsample of each sample was digested with 30 min at 37dC. After a final three washes, pepsin/HCl (as used for the antigen prepara- tion) before being checked under a micro- H2O2 in citrate–phosphate buffer (pH 5) scope for Trichinella larvae. The samples that were added as substrate. After incubation for 5 min in a dark box, the optical densities Malbrán parasitology laboratory in Buenos Aires, for confirmation and for typing a few 450 nm, in an automated microplate reader.
To establish a threshold for seropositivity, the pigs found ELISA-positive (which were Trichinella, by the digestion of muscle samples (see below), were run in the ELISA; no larvae were found in a 10-g subsample, the value calculated by adding three .. to ever-larger samples were used until larvae the mean OD for these sera was used as the    The proteins in a sample of the extract of excretory–secretory antigens were separated described by Zarlenga et al. (1999) was used to identify the Trichinella in each sample to species level. DNA was extracted from single transblotted onto nitrocellulose membrane larvae, each larva being placed in 5 µl lysis at a constant voltage for 1 h. The membrane buffer [10 m Tris (pH 8.3), 50 m KCl, NP-40, 0.01% gelatin, and 0.2 mg protein- up and digested with pepsin/HCl to release ase K/ml], overlaid with a drop of mineral any Trichinella larvae present. One of the oil, and then heated, first for 90 min at 65dC larvae released was identified to species and then for 15 min at 90dC, before being Each 50-µl reaction mixture for the first- Infection in the Human Population
A serum sample was collected from each of extract, 5 µl 10xPCR buffer, 0.4 µl of a the 112 patients who presented at the Sierra 2.5 m solution of each deoxynucleotide Grande hospital over a 7-day period in 2001, triphosphate, 0.6 U Taq polymerase, and0.05 µ of each primer (5p-TCT TGG TGG for the treatment or diagnosis of conditionsother than trichinosis (no symptomatic cases of human trichinosis were detected in the AAC GC). After denaturation for 3 min at94dC, the DNA was amplified for 25 cycles, area in 2001 or 2002). These sera weretested for anti-Trichinella antibodies, using each cycle consisting of 1 min at 94dC, 1 min at 55dC, and 2 min at 72dC, before a finalextension for 10 min at 72dC.
reaction mixture contained 2.5 µl of the first- round amplification products, 5 µl 10xPCRbuffer, 0.4 µl of a 2.5 m solution of each Pig Census
deoxynucleotide triphosphate, 1.25 U Taq During the 2000 census just 27 pig-breeding pens were located. Together, these pens held 105 older animals that were tagged). Each 94dC, the reaction was cycled 40 times, each cycle of 1 min at 94dC, 1 min at 60dC, and 2 min at 72dC, before a final extension for town, 20 (74.1%) were on the outskirts of this town (100–1000 m from urban areas), rated by electrophoresis in 2.5% (w/v) agar- 5–20 km from the town, on sheep farms or ose gel and visualized, by trans-illumination with ultra-violet light, after staining withethidium bromide. Reference isolates ofthe muscle larvae of T. spiralis (ISS599), Porcine Infection
T. nativa (ISS532), T. britovi (ISS447),  T. pseudospiralis (ISS13), T. murrelli (ISS103) and Trichinella T6 (ISS34) were months, and 27 (28.4%) of these sampleswere found positive in the ELISA (18) or by Rodent Collection and Parasitology
western blotting only (nine). Four (9.1%) Wild rodents in the study area were caught alive, using Sherman-type traps set in the production in 2001 were found seropositive, pig-breeding pens (661 trap-nights) or in the municipal rubbish dump (441 trap-nights).
Each rodent caught was killed, skinned and anti-Trichinella antibodies, three by ELISA eviscerated. Each carcass was then chopped Table). Overall, 181 pigs were investigated serological investigations had begun), four by serology and 36 (19.9%) found seroposi- (7.3%) were found to contain Trichinella tion and five (4.8%) were found infected.
the animals aged >6-months–<2 years to Twenty-three of the 105 pigs investigated by 18.4% in those aged 2–3 years and 27.3% in digestion in 2001 were old enough to have the pigs that were >3 years of age (x2 for been tested in an earlier serological survey.
trend; P<0.01). Gender also appeared to 15.3% of the female pigs investigated and at slaughter in 2001 (including the other were piglets aged <6 months and had not re-tested, three (8.6%) were found seroposi- tive by ELISA and/or western blotting. Two the initial screening were still seropositive, Worryingly, at least one seropositive pig in the study area — 14 (50%) of the pens infected with Trichinella larvae, the parasite in urban or peri-urban areas and one (25%) of those in rural areas. The small sample size meant that the likelihood of finding aseropositive pig in an urban/peri-urban breeding pen was not significantly higher Overall 26 rodents were caught, the level of rural pen (odds ratio=3.2; 95% confidence interval=0.23–92; P>0.32).
higher in the pig-breeding pens than in therubbish dump (2.9 v. 1.6) and higher in the     seen in 2000 (the rodent collections being made after all the pigs from these two pens Detection of Trichinella infection in pigs from the Sierra Grande area of the Argentinian province of Río Negro, pens that still held pigs (5.7 v. 2.3).
the present study are probably more sensi- tive methods of detecting Trichinella (43%) of the seven caught in the dump and infection than either trichinoscopy or muscle digestion, the seroprevalences and serocon- pens — were found infected with Trichinella versions recorded in 2000–2002 are worry- ing. Similarly, even though the sample wassmall, the prevalence recorded in the rodents(15.4%) is also cause for concern, indicating Identification of Trichinella Isolates
that, as observed in other regions of the Two isolates of muscular larvae from pigs world (Loufty et al., 1999; Theodoropoulos slaughtered in 2002 and one from a rodent et al., 2003), a ‘reservoir’ of Trichinella may (Mus musculus) were investigated by nested pattern characteristic of T. spiralis (data not study area (as indicated by the 2.7 rodentscaught/100 trap-nights) appear much lower Human Infection
than those recorded, by Larrieu et al. (2002), Twelve (10.7%) of the 112 patients tested elsewhere in Río Negro province (12–35 rodents/100 trap-nights). The size of the rodent population in the Sierra Grande areais probably severely limited by the scarcewater and food supplies.
changes seen in the seroprevalences among Although there are no large-scale producers of pork in Río Negro province, there have seronegative to seropositive) of several pigs been recurrent, small outbreaks of porcine over just 6 months, the detection of seroposi- and human trichinellosis in the region for many years. The porcine disease is consid- breeding pens and in urban, peri-urban and rural settings indicate wide-spread transmis- appears to be the worst affected area in the sion at substantial levels. In terms of the province. Among the risk factors that prob- local epidemiology of porcine trichinellosis, ably contribute to this situation are the the high prevalence of infection recorded substandard facilities for pig rearing [staff of the local veterinary service only consider one the municipal dump may be very significant.
of the 27 breeding pens in the Sierra Grande area to have ‘adequate’ facilities (unpubl.
seropositive pigs that were found seroposi- obs.)], the feeding of pigs with swill collected the subsistence economy of the area, with its re-infected between the two periods of serol- hygiene and housing. It is clear from the present results that, in 2000–2002, the become seronegative for at least a few years.
seroprevalence of Trichinella infection in Given the relatively high seroprevalence of infection in the local pigs, it is perhaps trichinoscopy, among the pigs slaughtered trichinellosis are not detected. The results of the human serology in the present study (although not based on an unbiased, cross- Larrieu, E. (1981). Evolución de la triquinosis en la sectional sample) indicate that human infec- Provincia de Río Negro. Gaceta Veterinaria, 364,
tion with Trichinella is much more common Larrieu, E., Cantoni, G., Herrero, H., Cavagion, L., Alvarez, E., Garcia Cachau, M., Bruni, M., human trichinellosis (no local cases detected Labanchi, J. Albarracín, S., Mancini, S. Arellano, A.
& Padula, P. (2002). Vigilancia epidemiológica residents also rarely if ever eat pork, since de hantavirus en roedores y síndrome pulmonar almost all of the meat produced in the small por hantavirus en poblaciones humanas del sur de
la Argentina. Revista Brasileira de Epidemiologia, 6,
number of pig-breeding pens is eaten by the pig owners or their families. To minimize the Loufty, N., Awad, O., El Masry, M. & Kandil, G.
risk of human infection, perhaps sera from (1999). Study on rodent infestation in Alexandria and prevalence of Trichinella spiralis infection among should be tested in ELISA or western blots them. Journal of the Egyptian Society of Parasitology, 29, 897–908.
Rodríguez Aruzco, J., Gonzales, A., Sacornani, C. & digestion and can easily be used to investi- Cubero, A. (1991). Estudio epidemiológico y clínico gate many samples at one time), so that the de un brote de triquinosis en niños. Santa Rosa, La seropositives can be discarded (Geerts et al., Pampa, 1987. Archivos Argentinos de Pediatría, 89,
2002). Such screening could form the basis of an effective system of surveillance and Ruitemberg, E., Ljungstrom, I., Steeremberg, P. & Buys, J. (1975). Application of immunofluorescense control although, given the rodent infec- and immunoenzyme methods in the serodiagnosis of tions, such a system would probably have to Trichinella spiralis infection. Annals of the New York Academy of Sciences, 254, 293–303.
Sequeira, A., Molina, V., Montefellano, M., Bolpe, J. & Guarnera, E. (2000). Genetic diversity amongisolates of Trichinella spiralis from the province of Buenos Aires, Argentina. Journal of Helminthology, 74,
Bradford, M. (1976). Rapid and sensitive method Su, X. & Prestwood, A. (1990). Isolation of Trichinella for the quantitation of microgram quantities of specific antigens for diagnosis by gradient monoclonal protein utilizing the principle of protein-dye binding.
Gamble, H. R. (1998). Sensitivity of artificial digestion Theodoropoulos, G., Styliara, M., Petrakos, M. & and enzyme immunoassay methos of inspection Kapel, C. (2003). Effect of fox, pig, sheep and poultry bile on the establishment of domestic and sylvatic Gamble, H. R. (2000). Biology and epidemiology of Trichinella. Investigacion Veterinaria, 2, 75–76.
Zarlenga, D. S., Chute, M. B., Martin, A. & Kapel, Geerts, S., de Bordgrave, J., Dorny, P. & Brandt, C. M. (1999). A multiplex PCR for unequivocal dif- J. (2002). Trichinellosis: old facts and new develop- ferentiation of all encapsulated and non-encapsulated ments. Verhandelingen — Koninklijke Academie voor Geneeskunde van Belgie, 64, 233–250.


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