Identification of Sarcocystis spp. in wild boars (Sus scrofa) from Argentina.

Sarcocystis spp. are intracellular protozoan parasites with an obligatory heteroxenous life cycle. The objective of this study is to identify Sarcocystis spp. in wild boar muscles from Argentina by light and transmission electron microscopy and molecular characterization. Muscle samples from diaphragm, tongue, masseter, intercostals, heart, and forelimbs of 240 wild boars were analyzed. Of the animals, 48.3% (116/240) were positive for sarcocysts by light microscopy, whereas 45.8% (110/240) were positive for Sarcocystis spp. by PCR targeting 18S rRNA fragment. These samples were subjected to a specific PCR for S. suihominis coxI gene, 3.6% (4/110) of which were weak positives. Unfortunately, sequence analysis was inconclusive. This could be related to a potentially low S. suihominis cyst load in the samples, or to an incomplete primer matching with the South American S. suihominis sequences. Seventeen individual sarcocysts were positive by PCR for the 18S rRNA fragment, whose sequences showed 99.75-100% identity with each other and with previously reported S. miescheriana sequences. A total of 21 cysts collected from 11 muscle samples and analyzed by TEM presented a cyst wall type compatible with S. miescheriana, and one cyst presented an ultrastructure compatible with S. suihominis. The latter came from a sample that also contained S. miescheriana cysts, indicating that the animal was co-infected. This is the first study that provides infection rates and describes and identifies morphological and molecular features of Sarcocystis spp. cysts in wild boars from South America.

Isolation of viable Toxoplasma gondii from organs and Brazilian commercial meat cuts of experimentally infected pigs.

The present study evaluated the distribution and viability of Toxoplasma gondii tissue cysts in the organs and Brazilian commercial cuts of experimentally infected pigs. The pigs were infected with 3 × 103 oocysts of the T. gondii isolate TgCkBr57 (Type BrII). Mouse bioassays were performed on the brain, retina, tongue, diaphragm, and heart as well as the following muscle cuts: loin (longissimus), coppa (longissimus, spinalis dorsi, rhomboideus), tenderloin (psoas major), outside flat (biceps femoris), topside (semimembranosus), and top sirloin (gluteus medius). Toxoplasma gondii was isolated from the coppa, heart, diaphragm, and tongue of three pigs; from the tenderloin, outside flat, and brain of two pigs; and from the top sirloin and loin of one pig. Thus, the viability of T. gondii cysts was observed in all of the organs and cuts evaluated (except for the topside and retina), demonstrating the broad distribution of this parasite in pig organs and commercial meat cuts, and the importance of this species as a source of human infection.

Survey for Toxoplasma gondii by PCR detection in meat for human consumption in Colombia.

The overall risk for toxoplasmosis in meat produced in Colombia is unknown. We analyzed by PCR assay meat samples for human consumption in two types of plants in Colombia: 120 samples from class I plants (60 samples from chicken, 30 from swine and 30 from beef) and 60 from class II plants (30 samples from beef and 30 from swine). Presence of Toxoplasma DNA was established by targeted B1 nested PCR assay. We detected 79 (43%) samples that were positive by B1 nested PCR (33 from chicken, 22 from beef, and 24 from pork). No differences were found by region or species. Eleven positive samples were confirmed by sequencing of the B1 repeated region. Some polymorphisms were detected without relation with clonal groups nor meat species. Food animals are highly exposed to Toxoplasma in Colombia. Detailed studies are needed to establish the reasons for differences in Toxoplasma prevalence between farms, regarding practices of animal food production.

Integral approach to evaluation of the pathogenic activity of Trypanosoma cruzi clones as exemplified by the Mexican strain.

Comparative histopathological study and analysis of parasite load in different muscle groups were carried out in BALB/c mice during the acute phase of Chagas disease. Activities of C104 clone of T. cruzi strain TPAP/MX/2002/Albarrada and the parental strain were compared. Panoramic 2D-microscopy imaging of sample surface was used and quantitative analysis of parasitism and pathologic damage was performed. The infection rates in various muscle groups were as follows: myocardium=abdominal muscles=lumbar muscles=femoral muscles<--diaphragm for the clone and myocardium¬abdominal muscles=lumbar muscles=femoral muscles-->diaphragm for the parental strain.

Immunohistochemical identification of Toxoplasma gondii in tissues from Modified Agglutination Test positive sheep.

Toxoplasma gondii is a zoonotic agent of great importance in veterinary and public health. The aim of this study was to identify T. gondii by IHC (immunohistochemistry) in different sheep tissues and to determine if an association exists between the results obtained by this method and those obtained by the Modified Agglutination Test (MAT). Tissue specimens of twenty-six sheep seroreactive for T. gondii were selected for histopathological evaluation. The presence of T. gondii was investigated in brain, liver and heart samples by IHC and a possible anti-T. gondii antibody cross reactions with other parasites. McNemar's, Chi-square and Fisher's Exact Tests were applied for the statistical analysis of the results. The analysed tissues showed at least one of the following histopathological changes: mild-to-moderate congestion, focal polymorphonuclear inflammatory infiltrate and multifocal or focal mononuclear inflammatory infiltrate. Sarcocystis spp. were identified in the histological sections from both the heart and diaphragm tissues of 88.5% (23/26) of the animals. A total of 46.2% (12/26) of the T. gondii seroreactive sheep was also positive for T. gondii by IHC in at least one organ (brain, liver or heart). The liver IHC-positivity for T. gondii was statistically equivalent to the global individual IHC-positivity, according to McNemar's test. In addition, IHC allowed the detection of T. gondii in infected animals regardless of the titration observed in the MAT. The statistical difference observed between the three organs when comparing the low titration group, suggested that the heart might be the most suitable organ to detect T. gondii infection by IHC. The IHC results in this study revealed that almost half of MAT positive animals could serve as potential sources of infection for humans because bradyzoites were identified in different tissues, regardless of the MAT titration.

Is Gnathostoma turgidum an annual parasite of opossums? Drastic seasonal changes of infection in Didelphis virginiana in Mexico.

Gnathostoma turgidum is a nematode that parasitizes the stomach of opossums, Didelphis virginiana. Despite its wide distribution in the Americas, its natural life cycle is poorly understood. Recently, we found an endemic area for G. turgidum infection in Sinaloa, Mexico (Diaz-Camacho et al., 2009). Based on sporadic surveys for several years, the prevalence was apparently high in summer and extremely low in winter. To confirm that this is really a seasonal variance, we conducted a longitudinal survey on G. turgidum infection in opossums from November 2007 to November 2008. The results showed amazing seasonal changes in the prevalence, with synchronized migration and maturation of worms in opossums. Between February and March, many juvenile worms, with occasional AL3, were found in the liver, but no worms were found in the stomach. Mature adult worms began to appear in the stomach around April and rapidly increased in number toward July, when all worms resided in the stomach. Then, the worms disappeared almost completely by November. These results suggest that G. turgidum is an annual parasite of the opossum, D. virginiana, in Mexico.

Seroprevalence and isolation of Toxoplasma gondii from sheep from São Paulo state, Brazil.

Sheep are important in the epidemiology of Toxoplasma gondii infection but little is known of ovine toxoplasmosis in Brazil. Antibodies to T. gondii were assayed in sera of 495 sheep from 36 counties of São Paulo State, Brazil, using the modified agglutination test (MAT titer > or =1:25) and found in 120 (24.2%). Samples of brain, heart, and diaphragm of 82 seropositive sheep were pooled, digested in pepsin, and bioassayed in mice. Toxoplasma gondii was isolated from tissue homogenates of 16 sheep and the isolates were designated TgShBr1-16. Six of the 16 T. gondii isolates killed 100% of infected mice. Results indicate that asymptomatic sheep can harbor mouse-virulent T. gondii, and hence they can serve as a source of infection for humans.

Application of Giemsa stain for easy detection of Trichinella spiralis muscle larvae.

The application of Giemsa technique to stain compressed diaphragm samples obtained from rodents experimentally infected with Trichinella spiralis is described. Diaphragm samples from rats heavily infected with 20 muscle larvae per gram of body weight (20 ML/gbw) were cut into several pieces and stained with Giemsa; on the other hand, whole diaphragms from slightly infected mice (1 ML/gbw) were also stained with Giemsa. Besides, muscle samples were also stained with Giemsa. Observation at 10 x magnification revealed that both ML and nurse cells (NC) look as bluish structures clearly contrasting with the pinkish color of the non-infected muscle fibers. NC in the diaphragms of mice could be easily observed at naked eye as blue points contrasting with the pink surrounding areas formed by the non-infected muscle fibers. Among NC observed in the diaphragms of rats infected with 20 ML/gbw, 4.4% was multiple infection. These findings were confirmed in sectioned and hematoxylin-eosin stained specimens. This data could be usefulness for a rapid diagnosis of trichinellosis in post-mortem mammals without magnification procedures.

Porcine and rodent infection with Trichinella, in the Sierra Grande area of Rio Negro province, Argentina.

In 2000, two cases of human trichinellosis were detected in the Sierra Grande area of Rio Negro province, Argentina. As part of an investigation of the aetiology of these cases, 300 pigs slaughtered for consumption in the area between 2000 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 140 investigated 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 animals checked in the initial serological survey, 36 (19.9%) were found seropositive for Trichinella. When 35 of the seronegative 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 high level of transmission being sustained by a high prevalence of infection in the local rodent populations.

Skeletal muscle regeneration and Trypanosoma cruzi-induced myositis in rats.

Although Chagas' disease is known to provoke severe acute myositis, information on muscle regeneration is missing. The current paper shows that during T. cruzi infection in rats, skeletal muscle parasitism and the consequent inflammatory process are higher in muscle with a high proportion of type-I myofibres (soleus and diaphragm). Immunohistochemistry showed an acute inflammatory process characterized by ED1+ and ED2+ macrophages, CD8+ lymphocytes, and NK cells. Parasite-nest rupture provoked segmental degeneration of myofibres followed by regeneration. These phenomena were observed at both light and transmission electron microscopy levels. Myofibre regeneration involved activation of satellite cells assessed by the expression of MyoD, a muscle-specific transcription factor. Ultrastructural evidence of fusion of myoblast-like cells with the intact segment of degenerating fibres has been provided. At the chronic phase no signs of fibrosis were found, but sparse and small inflammatory foci were found. Our results argue against the relevant participation of autoimmunity phenomena in both acute and chronic phases and furnish a new view for explaining histopathological findings in human patient muscles.

Trypanosoma cruzi: mixture of two populations can modify virulence and tissue tropism in rat.

In rats, CL-Brener clone caused high mortality, severe acute myocarditis, and myositis that subsided completely in surviving animals. Accordingly, no parasite kDNA could be amplified in several organs after 4 months. The monoclonal JG strain caused null mortality, acute predominantly focal myocarditis, discrete and focal myositis, and a chronic phase with sparse inflammatory foci. Double infection with both Trypanosoma cruzi populations turned mortality very low or null. At the end of the acute phase, the heart exhibited only JG strain kDNA (LSSP-PCR), while skeletal muscles and rectum exhibited only CL-Brener kDNA. Molecular and histopathological findings were accordant. In double infection chronic phase, JG strain remains in heart and appeared in organs previously parasitized by CL-Brener clone. Understanding the virulence and histotropism shifts now described could be important to clarify the variable clinical course and epidemiological peculiarities of Chagas' disease.

Trypanosoma cruzi: role of host genetic background in the differential tissue distribution of parasite clonal populations.

Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, has quite a variable clinical presentation, ranging from asymptomatic to severe chronic cardiac and/or gastrointestinal disease. The reason for that is not completely understood, but both parasite and host genetic traits are certainly involved. Recently, we have demonstrated clinically and experimentally that the genetic variability of T. cruzi is one of the determinants of the pattern of tissue involvement in Chagas' disease. We then decided to turn our attention to the role of host genetic background. To study this, we compared the infection of four lineages of mice [three inbred (BALB/c, DBA-2, and c57Black/6) and one outbred (Swiss)] with two T. cruzi clonal populations, the Col1.7G2 clone and the JG monoclonal strain. The tissue distribution of T. cruzi strains was identical for BALB/c and DBA-2 mice, but very different in C57BL/6 (H-2(b)) and outbred Swiss mice. This result clearly demonstrates the importance of host genetic aspects in the process. Since BALB/c and DBA-2 have the same H-2 haplotype (H-2(d)) and C57BL/6 does not (H-2(b)), it is possible that MHC variability may be involved in influencing the tissue distribution of involvement in experimental Chagas' disease of the mouse.

Detection of Trichinella infection in slaughter horses by ELISA and western blot analysis.

In order to determine the presence of Trichinella infections in horses slaughtered at an abattoir in Mexico, 147 serum samples were examined by two immunoenzymatic methods. Specific antibodies were detected by ELISA in 7% of the serum samples at a dilution 1:400 and in 10% at lower dilutions (1:20, 1:40) using Trichinella spiralis muscle larvae (ML) excretory/secretory (E/S) products. Serum samples from four naturally infected horses (confirmed by direct methods) gave negative O.D. values in an ELISA at a 1:400 dilution and only two of them were positive at a 1:20 and 1:40 dilutions. Serum samples from experimentally infected horses reacted by Western blotting with ML components with molecular weights of 47, 52, 59, 67, 72 and 105 kDa which correspond to the TSL-1 antigens. Serum samples from the four naturally infected horses and from the abattoir horses that were positive in ELISA using E/S antigens recognized several ML components, some of them reacted with all the TSL-1 antigens mentioned above and others recognized preferentially two or three of these molecules. Since the serologic assays may not offer the sensitivity required in the diagnosis of horses trichinellosis and the direct methods had not always been useful in the detection of larvae in horsemeat related to trichinellosis outbreaks in Europe, it is proposed that additional assays are performed to determine Trichinella infection in horses. These can include detection of parasite antigens by ELISA and Dot ELISA or PCR, which in turn may also help to determine the presence of the parasite in early and late infections of horses.

Diagnosis of porcine trichinellosis: parasitological and immunoserological tests in pigs from endemic areas of Argentina.

In order to compare the reliability of serological and parasitological techniques for the diagnosis of porcine trichinellosis from endemic areas in Argentina, 116 pigs were studied: 61 animals from two separate outbreaks and 55 from a small abattoir. Direct diagnostic techniques included trichinoscopy and the artificial digestion method. Indirect diagnostic tests used in this study were the enzyme-linked immunosorbent assay (ELISA), employing the excretory-secretory products of muscle larvae (ML) as antigen, and the indirect immunofluorescence assay using as antigen ML in suspension (IIF-susp), cryostat sections of infected rat muscle or of free ML (IIF-slide). The percentage of parasitologically positive pigs was invariably lower than that of serologically positive animals (IIF-slide), even when digestion studies were carried out individually with a greater amount of muscle sample than required by current regulations. Close correlation was found between IIF using as antigen tissue sections and IIF using free ML sections, while IIF-susp proved unsuitable for diagnosis since this assay presented a high percentage of false negative results (20%). The IIF-slide technique proved positive in all parasitologically positive animals. ELISA rendered a lower percentage of positive reactions than IIF-slide, especially when worm burden was low. Since most parasitologically positive animals rendered at least two positive serological tests (two variations of IIF or IIF plus ELISA), those negative by digestion and positive by two serological methods were strongly suspected of having trichinellosis. Upon studying swine from a abattoir it was found that 9% of the pigs were positive when assayed by two serological techniques, but Trichinella spiralis infection could not be parasitologically confirmed. To sum up, serological methods may be used for screening all pigs and positive findings should be tested by the digestion method by analysing a greater quantity of pork than that required by current regulations, above all in areas with reported clinical trichinellosis in humans, to ensure that the pork is safe for human consumption.

Frecuencia de triquinosis humana en Concepción y Arauco, Chile: estudios en personas autopsiadas en el Servicio Médico Legal de Concepción: junio 1996 a marzo 1997 / Frequency of human trichinosis in Concepción and Arauco, Chile: study in autopsied persons at the Servicio Médico Legal, Concepción: june 1996 to march 1997

The frequency of trichinosis was determined in 300 dead persons, residents in the provinces of Concepción and Arauco, and requiring necropsy at the Servicio Medico Legal of Concepción. Diaphragm muscle samples were taken from june 1996 to march 1997. The diagnosis was made by trichinoscopy and artificial digestion techniques. Five samples (1.67 percent) were positive. There was not differences between sexes. Positive cases were found mainly among 30 to 49 years old people belonging to middle-lower class. Both diagnostic techniques showed diferent sensitivity as a consequence of the parasitic load and on the contrary, the artificial digestion was less sensitiva when the cyts were fully calcified

[Frequency of human trichinosis in Concepción and Arauco, Chile: study in autopsied persons at the Concepción Medico-Legal Service. June 1996-March 1997]. / Frecuencia de triquinosis humana en Concepción y Arauco, Chile: estudio en personas autopsiadas en el Servicio Médico Legal de Concepción. Junio 1996-Marzo 1997.

The frequency of trichinosis was determined in 300 dead persons, residents in the provinces of Concepción and Arauco, and requiring necropsy at the Servicio Medico Legal of Concepción. Diaphragm muscle samples were taken from June 1996 to March 1997. The diagnosis was made by trichinoscopy and artificial digestion techniques. Five samples (1.67%) were positive. There was not differences between sexes. Positive cases were found mainly among 30 to 49 years old people belonging to middle-lower class. Both diagnostic techniques showed different sensitivity as a consequence of the parasitic load and on the contrary, the artificial digestion was less sensitive when the cyts were fully calcified.

Tendencia actual de la epidemiología de la triquinosis humana en Chile / Present trends of human trichinosis epidemilogy in Chile

Surveillance of the epidemiology of human triquinosis in Chile has been maintained during the last 30 years by the Department of Parasitology. Incidence and prevalence have been followed-up by analysing Ministry of Health annual reports and periodical phototrichinoscopic examination of diaphragm samples from individuals autopsied at the Santiago Medico Legal Service. A decrease has been observed on: incidence from 1.4 per 100.000 in the 1960s to 0.7 in the 1980s down to 0.3 in 1993 and 1994 and to 0.5 in 1995, and prevalence from 3.4 percent 2.8 percent, 2.0 percent and 0.8 percent in 1972, 1982, 1992 and 1997 respectively. A predominance of the prevalence in the groups of more age, with the observation of calcified Trichinella spiralis larvae should be indicating a decline of new infections in the general population. This decrease of frequence of human trichinosis in Chile is possible due to the interaction of two main factors, efficacy and higher implementation of control measures and significant advances in porcine technology with a resulting provision to consumers of pork from young animals practically trichinosis free

[Current trends of the epidemiology of human trichinosis in Chile]. / Tendencia actual de la epidemiología de la triquinosis humana en Chile.

Surveillance of the epidemiology of human trichinosis in Chile has been maintained during the last 30 years by the Department of Parasitology. Incidence and prevalence have been followed-up by analysing Ministry of Health annual reports and periodical phototrichinoscopic examination of diaphragm samples from individuals autopsied at the Santiago Medico Legal Service. A decrease has been observed on: incidence from 1.4 per 100,000 in the 1960s to 0.7 in the 1980s down to 0.3 in 1993 and 1994 and to 0.5 in 1995, and prevalence from 3.4% to 2.8%, 2.0% and 0.8% in 1972, 1982, 1992 and 1997 respectively. A predominance of the prevalence in the groups of more age, with the observation of calcified Trichinella spiralis larvae should be indicating a decline of new infections in the general population. This decrease of frequency of human trichinosis in Chile is possible due to the interaction of two main factors, efficacy and higher implementation of control measures and significant advances in porcine technology with a resulting provision to consumers of pork from young animals practically trichinosis free.

Sarcocystis sp. en el diafragma de un gato doméstico de Valdivia, Chile / Sarcocystis sp. in a domestic cat diaphragm at Valdivia, Chile

During 1987, diaphragm of 24 domestic cats from the city of Valdivia, Chile, were examined. In one (3,7 percent) cat infection by cysts of Sarcocystis sp. was observed. This is the first report of feline muscular infection by Sarcocystis sp. in Chile

[Sarcocystis sp. in the diaphragm of a domestic cat (Felis catus) from Valdivia, Chile]. / Sarcocystis sp. en el diafragma de un gato doméstico (Felis catus) de Valdivia, Chile.

During 1987, diaphragms of 24 domestic cats from the city of Valdivia, Chile, were examined. In one (3.7%) cat infection by cysts of Sarcocystis sp. was observed. This is the first report of feline muscular infection by Sarcocystis sp. in Chile.