Understanding the relationship between Mycobacterium bovis spoligotypes from cattle in Latin American countries.

Spoligotyping is the most frequently used method for genotyping isolates of Mycobacterium bovis worldwide. In the current work, we compared spoligotypes from 1684 M. bovis isolates from Argentina (816), Brazil (412), Chile (66), Mexico (274) and Venezuela (116), obtained from cattle, humans, pigs, wild boars, farmed deer, goats, buffaloes, cats, and wild animals. A total of 269 different spoligotypes were found: 142 (8.4%) isolates presented orphan spoligotypes, whereas 1542 (91.6%) formed 113 different clusters. In cattle, SB0140 was the most representative spoligotype with 355 (24.6%) isolates, followed by SB0121 with 149 (10.3%) isolates. Clustering of spoligotypes ranged from 95.2% in Argentina to 85.3% in Mexico. Orphan spoligotypes were also variable, ranging from 23.7% in Mexico to 4.1% in Brazil. A large proportion of spoligotypes were common to the neighboring countries Argentina, Brazil and Chile. In conclusion, despite the diversity of spoligotypes found in the five countries studied, there are major patterns that predominate in these neighboring countries. These clusters may reflect a long-lasting active transmission of bovine tuberculosis or common historical origins of infection.

High frequency of Mycobacterium bovis DNA in colostra from tuberculous cattle detected by nested PCR.

We evaluated by nested PCR reaction, different cow secretions from a herd with 48% of prevalence of bovine tuberculosis (BTB), seeking to determine niches where Mycobacterium bovis could be found. Postmortem examination of 18 (75%) tuberculin reacting cows allowed demonstrates BTB-compatible lesions in six, all of them PCR positives in milk and four in colostra samples. Our results showed that up to 62% of the colostra analysed contained M. bovis DNA, whereas only 18% of milk gave a positive reaction. Moreover, in bronchoalveolar lavages from cattle with compatible lesions in lungs or lymph nodes, where macrophages account up to 90% of cells, we did not find evidences of M. bovis. Altogether, these results suggest that differences in the anti-bacterial capacity of bovine macrophages, dependent upon microenvironment and organ-specific factors, exist. Alternatively, we hypothesize that hypoxic conditions that are encountered in mammary glands macrophages could induce M. bovis entrance into a 'dormancy-like' state, and that the high number of colostra samples were M. bovis was detected, could be an indicator of reactivation during 'peripartum'.

High-level expression of NRAMP1 in peripheral blood cells and tuberculous granulomas from Mycobacterium bovis-infected bovines.

By Western blotting, we demonstrate high-level expression of NRAMP1 proteins in peripheral blood cells and granulomas of Mycobacterium bovis-infected bovines. Immunohistochemistry of granulomatous lesions showed heavily labeled epithelioid macrophages and Langhans cells. These data suggest that M. bovis infection enhances NRAMP1 expression and that active tuberculosis can occur despite this response.

Swine trichinellosis in slaughterhouses of the metropolitan area of Toluca.

In order to determine the prevalence of Trichinella spiralis infections in abattoirs of the metropolitan area of Toluca where pigs from commercial farms as well as backyard pigs are slaughtered, 539 swine diaphragm tissue samples were collected and examined by trichinoscopy and artificial digestion. Serum samples from the same animals were analyzed by ELISA using somatic and excretory/secretory antigens, and by Western blot analysis. T. spiralis muscle larvae were not found by trichinoscopy or artificial digestion. However, specific antibodies were detected by ELISA and confirmed by Western blotting in 12.4% of the serum samples examined. Analysis of risk factors showed no association of seropositive results with sex. However, significant higher risk was observed in swine seven to 12 months old and in backyard pigs, compared with pigs from commercial farms.

Detection of Trichinella infection in slaughter horses by artificial digestion, ELISA and PCR.

In this study we compared the sensitivity of molecular, serologic and parasitologic methods for diagnosis of equine trichinellosis in two abattoirs, one rural and one federal inspection type. Diaphragm muscle samples were obtained from 170 slaughter horses and examined by artificial digestion and PCR. Serum samples from these horses were also analyzed by ELISA. No Trichinella muscle larvae were detected by artificial digestion. However, specific antibodies against Trichinella were detected in 17% and 7% of the serum samples examined from the rural and the federal abattoirs respectively. By PCR, 15% and 2% of the samples from these two abattoirs were Trichinella positive.

Epidemiology of trichinellosis in Mexico, Central and South America.

Trichinella species are widely distributed throughout the world and are found in a large number of carnivorous animals, humans and incidental hosts. The data presented in this review show that Trichinella infection has been reported in both humans and animals in Mexico, Argentina and Chile since the end of the 19th century, and more recently in Bolivia. This parasitic infection is still a public health problem in countries such as Argentina and Chile. Although efforts have focused on the control and prevention of trichinellosis in these countries, there were still human cases and outbreaks of trichinellosis reported. Diagnosis of infection in animals such as pigs still includes, in many endemic areas, the use of trichinoscopy. In Argentina, however, artificial digestion has been recently introduced in slaughterhouses to detect Trichinella infection in pigs. In some endemic areas in Mexico, the use of serological assays for human trichinellosis and pig infections have resulted in improved detection. Most of the outbreaks of human trichinellosis in Mexico, Argentina and Chile have occurred as a result of the consumption of undercooked pork or pork products from animals raised under poor hygienic conditions and which are clandestinely slaughtered. In several studies, rats, dogs and cats have been found to be infected with Trichinella and may be considered a risk for transmission of the infection to pigs or other animals intended for human consumption. Another potential source of transmission of Trichinella to humans is horsemeat; however, information related to horse trichinellosis in Latin-American countries is scarce. In most cases the etiological agent of human trichinellosis in Central and South America has been reported to be Trichinella spiralis; however, only in a few cases has the parasite species been properly identified. Based on the reports available, it is clear that there is a need to carry out better controlled epidemiological studies to determine the true prevalence of the infection in this region of the world. Also, more sensitive methods of diagnosis and improvements in conditions for pig production as well as better sanitary inspection systems, are needed for controlling and preventing transmission of trichinellosis in these countries.

Hemangiopericitoma orbitario: un caso en el espacio intraconal / Orbital hemangiopericytoma: an intraconal case

Objetivo/métodos: Presentamos un caso de hemangiopericitoma orbitario en un muchacho de 14 años. Se detallan los hallazgos radiológicos de la tomografía axial computerizada (TAC), resonancia magnética nuclear (RMN), y ultrasonografía.Resultados/conclusiones: El diagnóstico por imagen mostró una masa sólida bien circunscrita de localización intraconal. Macroscópicamente el tumor era de coloración púrpura, muy vascularizado y encapsulado. El examen histológico reveló un hemangiopericitoma de grado intermedio, con hipercelularidad y moderado número de mitosis. Destacamos la rareza del hemangiopericitoma orbitario, especialmente cuando se localiza dentro del cono muscular (AU)

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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.

Serologic survey of trichinellosis in wild mammals kept in a Mexico City Zoo.

A serologic survey of Trichinella infection was carried out to determine the prevalence of this parasitosis among wild mammals kept in captivity at the Chapultepec Zoo. This was prompted by the necropsy finding of a heavy Trichinella infection in a Canadian polar bear (Ursus maritimus) that had been kept at the Zoo for more than 11 years. The parasites recovered were identified as T. nativa (T2). A serologic study based on ELISA and Western blot analysis was performed in serum samples from two polar bears (U. maritimus), six wolves (Canis lupus); nine foxes (Urocyon cinereoargenteus); seven coyotes (Canis latrans); nine jaguars (Panthera onca); ten lions (Panthera leo); 11 tigers (Panthera tigris); six panthers (Panthera pardus); eight leopards (Panthera pardus); two lynxes (Lynx rufus); five pumas (Felis concolor); one yagouaroundi (Felis yagouaroundi); and one ocelot (Felis pardalis). In these assays, 25% and 27% of the samples studied were positive using total muscle larva extract from T. nativa (T2) or T. spiralis (T1), respectively. When T. spiralis (T1) excretory/secretory products or surface/stichosomal antigens were used, 15 and 13% positivity was obtained respectively. The reactivity rates obtained among the different groups varied from 11 to 83%, wolves having the highest infection rate. Western blot analysis of positive ELISA sera showed an antigenic recognition pattern characteristic of animals infected with Trichinella.

Regulation of interleukin-6 expression in porcine immune cells.

Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates the immune response, acute-phase reaction, and hematopoiesis. As a first step in studying the actions of IL-6 in pigs, the regulation of IL-6 expression was examined in various swine cells, including a fibroblast cell line, peripheral blood mononuclear cells (PBMC), and alveolar macrophages. IL-6 expression in transformed swine testicular (TST) fibroblasts was enhanced by TNF and IL-1 beta and to a lesser extent by poly(I).(C) and LPS. IL-6 was induced in porcine PBMC by either LPS or PHA; however, the combination of LPS plus PHA resulted in maximal IL-6 expression. Furthermore, in PBMC cells separated by adherence, LPS was a more potent inducer than PHA in adherent cells, whereas PHA was more potent in nonadherent cells. Alveolar macrophages collected from different pigs could be divided into low and high responders with respect to IL-6 induction by LPS. IL-6 mRNA induction by LPS could be detected in only 6 of 20 donor animals. Other inflammatory cytokines (IL-8, IL-beta, and TNF) were readily induced by LPS in alveolar macrophages from both low and high responders. Treatment of low-responder alveolar macrophages with conditioned medium containing IFN-gamma did not significantly alter the capacity of these macrophages to synthesize IL-6 mRNA in response to LPS. Comparison of IL-6 production capacity by the cell types in this study revealed the following order: PBMC = high-responder alveolar macrophages >> TST.cells > low-responder alveolar macrophages. Thus, PBMC appear to be quantitatively the most significant source of IL-6 in swine on a per cell basis.

Detection of Trichinella spiralis muscle larvae in naturally infected horses.

Human trichinellosis outbreaks related to horsemeat consumption have been reported in France and Italy in recent years. In order to determine if Trichinella is present in horses slaughtered at an abattoir in the State of Mexico, diaphragm muscle tissue samples (22-37 g) from 80 horses were examined by artificial digestion. Four of these samples had larvae that were characterized as Trichinella sp. by morphological criteria and as Trichinella spiralis by the polymerase chain reaction.

Detection of circulating Trichinella spiralis muscle larva antigens in serum samples of experimentally and naturally infected swine.

A follow up study was carried out to determine the kinetics of appearance of surface/stichosomal (S/S) components, recently included in the TSL-1 group of Trichinella spiralis muscle larva (ML), in serum samples from 13 experimentally infected pigs. Detection of circulating antigens in these animals was done by a sandwich enzyme-linked immunosorbent assay (ELISA) using T. spiralis specific rabbit polyclonal immunoglobulins to capture free antigens and monoclonal antibody NIM-M1 to recognize S/S antigens. The assay developed was able to detect as little as 35 ng ml-1 of S/S components added to normal pig serum. Antigenemia was observed in 54% of the experimentally infected swine with two peaks of appearance, one early at 1-4 weeks post-infection (pi) and one late at 10-14 weeks pi. Specific antibodies against S/S components were demonstrated in serum samples from all experimentally infected pigs starting at 3-4 weeks pi. Free antigen was also detected in serum samples from naturally infected backyard pigs with a sensitivity of 56% compared with 94% when antibody production was determined using purified S/S components in an ELISA.

Fasciola hepatica: characterization of somatic and excretory-secretory antigens of adult flukes recognized by infected sheep.

The immune response of sheep to somatic components and excretory/secretory products of adult Fasciola hepatica, was studied during an experimental infection. Antibodies against adult fluke somatic and excretory/secretory antigens were detected by thin layer immunoassay from the second week post infection. Similarly, the results of Western blot analysis showed specific recognition of several components as early as two weeks after infection. However, an increase in the number and intensity of bands with time of infection was observed in the patterns of antigenic recognition. Most notorious was the strong reactivity of all infected sheep sera towards components of 20-23 kDa in the somatic preparation and components of 23-27 kDa in the excretory/secretory products of adult F. hepatica, specially noticeable after the sixth week post-infection. Since these polypeptides were recognized by all infected animals, they could play an important role in the diagnosis of sheep fascioliasis.

Association between diarrhoea and shedding of group A and atypical groups B to E rotaviruses in suckling pigs.

The frequency of shedding of rotavirus in faeces of diarrhoeic piglets was studied in 2 farms. In farm I, where 82% of the litters had diarrhoea, group A rotavirus was detected in 52/117 (44%) faeces of pigs with diarrhoea while atypical groups B to E rotaviruses were detected in 2/117 (2%) faeces of diarrhoeic piglets that came from litters where group A rotavirus had also been found. In farm II where the morbidity due to diarrhoeas was lower (57% of the litters had diarrhoea), 8/141 (6%) faeces of piglets with diarrhoea had group A rotavirus and 4/141 (3%) had groups B to E atypical rotaviruses. It was concluded that group A and groups B to E rotaviruses can coexist in the same farm but group A rotavirus seems to induce more diarrhoeas in piglets.

Trichinella spiralis: recognition of muscle larva antigens during experimental infection of swine and its potential use in diagnosis.

Longitudinal studies with Trichinella spiralis experimentally infected pigs were carried out to identify muscle larva antigens recognized during infection. This was approached using Western blot analysis and ELISA assays. Immunoblots of sera from experimentally infected pigs using total parasite extracts revealed five principal parasite antigens throughout infection. A similar pattern of antigen recognition was given by sera from backyard pigs in areas of Mexico, some of them endemic for Trichinella. Four of the five antigens recognized (MW 47, 52, 67, and 72 kDa) corresponded to surface/stichosomal antigens purified by monoclonal antibody NIM-M1. In addition, Western blots of excretions-secretions of muscle larva contained three (MW 52, 67, and 72 kDa) of the four surface/stichosomal components recognized by NIM-M1. Affinity-purified surface/stichosomal components, total soluble extracts, and excretory-secretory antigens of muscle larva were then evaluated in ELISA for detection of T. spiralis infections in experimentally infected, noninfected control, and 295 backyard pigs. These assays showed that purified surface/stichosomal components and excretory-secretory antigens increased the specificity of ELISA. These results suggest that muscle larva components purified by monoclonal antibody NIM-M1 are the major antigens recognized during infection of pigs with T. spiralis and therefore potentially useful for diagnosis of swine trichinellosis.

Cerebrospinal nematodiasis in prairie dogs from a research facility.

Baylisascaris spp larvae were determined to cause CNS disease in 3 prairie dogs maintained at a research facility. Clinical signs consisted of ataxia, stumbling, and head tilt, which progressed to severe torticollis, paddling in lateral recumbency, and loss of the righting reflex. Gross lesions were not found at necropsy. Microscopically, the cerebellar white matter and medulla oblongata were most severely affected and had numerous sections of large ascarid larvae. Our findings indicated that natural infections of Baylisacaris spp larvae can cause CNS disease in prairie dogs. Also, Baylisascaris spp larval infection should be considered in differential diagnoses of CNS disease in prairie dogs.

Interference by hemolysis with various methods for total calcium and its correction by trichloroacetic acid precipitation.

We evaluated the influence of hemolysis on total serum calcium as determined with the Du Pont aca, SMA 12/60, Ektachem 400, Corning 940 EGTA titrator, and the Beckman Astra 8, comparing results with those obtained by atomic absorption spectroscopy. We find that hemoglobin does not influence calcium measurements with the SMA 12/60, Ektachem 400, or Beckman Astra 8. The presence of hemoglobin exceeding 2 to 3 g/L caused falsely high results with the aca and falsely low results with the Corning 940 titrator. Similar interferences may be observed with other titrating or colorimetric procedures that involve direct reaction of the sample with o-cresolphthalein complexone or calcein. Upon removal of the hemoglobin by precipitation with trichloroacetic acid, the values obtained with the aca and the Corning 940 EGTA titrator were similar to those measured by atomic absorption. With nonhemolyzed serum samples the acid treatment had little or no effect on the aca procedure but resulted in a positive bias of approximately 10% with the EGTA procedure. Thus we recommend this trichloroacetic acid procedure for measuring calcium in hemolyzed samples with the aca and, with certain reservations, with the EGTA titrator.