First evidence of systemic infection and specific cytotoxic T lymphocyte immune response evoked by oral infection with recombinant Salmonella enterica serovar Albany-Ovalbumin in C57BL/6 mice.

Our group isolated Salmonella enterica serovar Albany from food and feces of wild captive carnivores in a zoo from northwestern Mexico. This serovar was also associated with the death of an ocelot (Leopardus pardalis) in the same zoo. Another group associated S. Albany with the death of a human patient. It is due to this zoonotic potential that the in vivo study of the host-S. Albany relationship is critical. The recombinant S. Albany-Ovalbumin (rSAO) strain was used to analyze a murine oral infection and its specific cytotoxic T lymphocyte (CTL) response. Our results have shown for the first time that rSAO establishes a systemic infection and evokes epitope-specific lysis with a Th1-like cytokine profile in vivo.

The plasmacytoid dendritic cells evoke Salmonella-specific CTL effector response / Las células dendríticas plasmacitoides evocan la respuesta efectora de los linfocitos T citotóxicos específicos para Salmonella

Introduction: The immunological role of plasmacytoid dendritic cells (pDC) in bacterial infections such as Salmonella has been poorly documented. Therefore, we analyzed the effector function of these cells by presenting cytotoxic T lymphocytes (CTL) with Salmonella Typhimurium antigens. Objective: To analyze the Salmonella-specific CTL response evoked by pDCs. Materials and methods: We used plasmacytoid dendritic cells stained with carboxyfluorescein succinimidyl ester (CFSE) and pulsed with OmpC73, Salmonella Kb restricted epitopes or S. Typhimurium as targets for cytotoxicity assays. Results: Specific lysis was shown to be statistically significant in pDC + OmpC73 for all effector:target ratios (p≤0.05). For pDC + S. Typhimurium, statistical significance was only observed at a 1:100 ratio (p≤0.05) using OmpC73. Conclusion: Plasmacytoid dendritic cells evoke CTL response during S. Typhimurium infection.

Introducción. La función inmunológica de las células dendríticas plasmacitoides durante las infecciones bacterianas, como la de Salmonella spp., es poco conocida. En ese contexto, se analizó su función efectora para presentar antígenos de Salmonella Typhimurium ante linfocitos T citotóxicos. Objetivo. Analizar la respuesta de los linfocitos T citotóxicos específicos para Salmonella evocada por las células dendríticas plasmacitoides. Materiales y métodos. Se usaron células dendríticas plasmacitoides marcadas con éster de succinimidil-carboxifluoresceína, pulsadas con el epítopo de Salmonella OmpC73 Kb restringido o infectadas con S. Typhimurium como blanco en ensayos de citotoxicidad. Resultados. La lisis específica tuvo significación estadística usando células dendríticas plasmacitoides positivas pulsadas con OmpC73 en todas las relaciones de células efectoras y blanco (E:B) (p≤0,05); en cuanto a las células dendríticas plasmacitoides positivas para S. Typhimurium, solo se observó significación estadística en la relación de 1:100 (p≤0,05) usando las células efectoras OmpC73. Conclusión. Las células dendríticas plasmacitoides pueden evocar la respuesta de los linfocitos T citotóxicos durante la infección con S. Typhimurium.

Las células dendríticas plasmacitoides evocan la respuesta efectora de los linfocitos T citotóxicos específicos para Salmonella / The plasmacytoid dendritic cells evoke Salmonella-specific CTL effector response

Resumen Introducción. La función inmunológica de las células dendríticas plasmacitoides durante las infecciones bacterianas, como la de Salmonella spp., es poco conocida. En ese contexto, se analizó su función efectora para presentar antígenos de Salmonella Typhimurium ante linfocitos T citotóxicos. Objetivo. Analizar la respuesta de los linfocitos T citotóxicos específicos para Salmonella evocada por las células dendríticas plasmacitoides. Materiales y métodos. Se usaron células dendríticas plasmacitoides marcadas con éster de succinimidil-carboxifluoresceína, pulsadas con el epítopo de Salmonella OmpC73 Kb- restringido o infectadas con S. Typhimurium como blanco en ensayos de citotoxicidad. Resultados. La lisis específica tuvo significación estadística usando células dendríticas plasmacitoides positivas pulsadas con OmpC73 en todas las relaciones de células efectoras y blanco (E:B) (p≤0,05); en cuanto a las células dendríticas plasmacitoides positivas para S. Typhimurium, solo se observó significación estadística en la relación de 1:100 (p≤0,05) usando las células efectoras OmpC73. Conclusión. Las células dendríticas plasmacitoides pueden evocar la respuesta de los linfocitos T citotóxicos durante la infección con S. Typhimurium.

Abstract Introduction: The immunological role of plasmacytoid dendritic cells (pDC) in bacterial infections such as Salmonella has been poorly documented. Therefore, we analyzed the effector function of these cells by presenting cytotoxic T lymphocytes (CTL) with Salmonella Typhimurium antigens. Objective: To analyze the Salmonella-specific CTL response evoked by pDCs. Materials and methods: We used plasmacytoid dendritic cells stained with carboxyfluorescein succinimidyl ester (CFSE) and pulsed with OmpC73, Salmonella Kb- restricted epitopes or S. Typhimurium as targets for cytotoxicity assays. Results: Specific lysis was shown to be statistically significant in pDC + OmpC73 for all effector:target ratios (p≤0.05). For pDC + S. Typhimurium, statistical significance was only observed at a 1:100 ratio (p≤0.05) using OmpC73. Conclusion: Plasmacytoid dendritic cells evoke CTL response during S. Typhimurium infection.

First report of autochthonous canine leishmaniasis caused by Leishmania (L.) mexicana in Sinaloa, Mexico.

Leishmaniasis is a neglected tropical disease caused by different species of protozoan parasites of the genus Leishmania. Dogs have been proven as primary hosts of the parasite. Cases of cutaneous leishmaniasis in humans caused by Leishmania mexicana have been reported in Sinaloa; however, the vectors and hosts involved in the epidemiology of the parasite in northwestern Mexico are still unknown. Given the public health implications of this parasite's domestic hosts regarding the permanence and transmission of the disease to humans, the objective of the present study was to detect and determine the species of Leishmania that caused the first three cases of autochthonous canine leishmaniasis in the state of Sinaloa, Mexico. Three domestic dogs showing symptoms similar to canine leishmaniasis were identified, including chronic eye inflammation, corneal opacity, ocular exudate, emaciation and hyporexia. DNA was extracted from venous blood of the infected animals using a commercial kit. The internal transcribed spacer (ITS-1) of ribosomal DNA (rDNA) was amplified by specific primers for Leishmania from the extracted DNA, and the PCR products were digested with the restriction enzyme HaeIII. In addition, PCR products were subjected to automated sequencing. Molecular analysis showed that the infecting species was L. mexicana. This is the first report of autochthonous canine leishmaniasis caused by L. mexicana in Sinaloa, Mexico. Further studies are required to identify the species that serve as vectors and other wild and domestic hosts of the parasite, as well as to determine if there are more species of Leishmania circulating in Sinaloa.

High frequency of HPV16 European variant E350G among Mexican women from Sinaloa.

BACKGROUND & OBJECTIVES: Human papillomavirus (HPV) infections play a crucial role in the aetiology of cervical cancer (CC), and HPV16 is the primary viral genotype associated with CC. A number of variants of the HPV16 E6 gene are involved in the progression of CC, differing in their prevalence and biological and biochemical properties. This study was designed to determine the frequency of HPV types 16/18 and to identify the presence of HPV16 E6-variants in asymptomatic Mexican women. METHODS: A total of 189 cervical Pap smears were collected from women attending public health services in three different cities in Sinaloa, Mexico. Viral DNA was identified by amplification of E6 viral gene fragments using polymerase chain reaction (PCR). Identification of variants was done by sequencing a DNA fragment (321bp) of the HPV16 E6 gene. RESULTS: More than half of the women tested were HPV-positive (52.38%), with HPV16 being the most frequent genotype (21.16%), followed by HPV18 (8.99%). Sequence analysis of the E6-HPV16 PCR products showed that in all cases, the viruses corresponded to European variants. It was further observed that the E350G intra-variant was the most common (>76%). INTERPRETATION & CONCLUSIONS: This study showed a predominance of European lineage variants of HPV16 among asymptomatic women from Sinaloa, Mexico, predominantly with of the E350G variant. This variant has been shown to be associated with an increased risk of early development of CC. The use of molecular identification of carcinogenic HPV and Pap test screening may be a good strategy for monitoring women to prevent CC.

Bacteriophage cocktail for biocontrol of Escherichia coli O157:H7: Stability and potential allergenicity study.

Escherichia coli O157:H7 has become a global public health and a food safety problem. Despite the implementation of control strategies that guarantee the safety in various products, outbreaks persist and new alternatives are necessary to reduce this pathogen along the food chain. Recently, our group isolated and characterised lytic bacteriophages against E. coli O157:H7 with potential to be used as biocontrol agents in food. To this end, phages need certain requirements to allow their manufacture and application. The aim of this study was to determine the physical stability and allergenic potential of free and microencapsulated (ME) bacteriophage cocktails against E. coli O157:H7. In vitro and in vivo studies were performed to determine phage survival under different pH, gastrointestinal conditions, temperature and UV light intensities. Results showed that the stability of ME phages was significantly (P<0.05) higher than free phages after ultraviolet irradiation, pH conditions between 3 to 7, and exposure to temperatures between at -80°C and 70°C. Both formulations were highly sensitive to very low pH in simulated gastric fluid, but stable in bile salts. In vivo studies in mice confirmed these phages passed through the gastrointestinal tract and were excreted in faeces. In silico, full-length alignment analysis showed that all phage proteins were negative for allergenic potential, but different predicting criteria classified seven phage proteins with a very low probability to be an allergen. In conclusion, these data demonstrated that microencapsulation provided a greater stability to phage formulation under stress conditions and assure a more suitable commercial formulation for the biological control of E. coli O157:H7.

Prevalence of Salmonella enterica serovar Albany in captive zoo wild animals in the Culiacán Zoo in Mexico.

Salmonellosis is an important zoonotic disease but little is known about the role that free-living animals play as carriers of this pathogen. Moreover, the primary route of infection in the wild needs to be elucidated. The aim of this study was to determine the source and the route of transmission of Salmonella enterica serovar Albany (S. Albany) infection in captive zoo wild animals in the Culiacán Zoo. A total of 267 samples were analyzed including 220 fecal samples from zoo animals, 15 fecal samples from rodents, 5 pooled samples each of two insects (Musca domestica and Periplaneta americana), and 22 samples of animal feed. We detected S. Albany in 28 (10.5%) of the samples analyzed, including in samples from raw chicken meat. Characterization of isolates was performed by serotyping and pulsed-field gel electrophoresis. All isolates shared a single pulsed-field gel electrophoresis profile, indicating a possible common origin. These data suggest that the infected meat consumed by the wild felines was the primary source of infection in this zoo. It is likely that the pathogen was shed in the feces and disseminated by insects and rats to other locations in the zoo.

Identification of immunodominant peptides from Gnathostoma binucleatum.

Gnathostomiasis is now recognized as a zoonosis with a worldwide distribution. In the Americas, it is caused by the third-stage larvae of Gnathostoma binucleatum and in Asia mainly by G. spinigerum. The availability and preparation of specific antigens are among the main obstacles for developing reliable immunodiagnostic tests. In this study, six immunodominant peptides were identified and characterized from G. binucleatum, somatic antigens (AgS: 24, 32, and 40 kDa) and excretory-secretory antigens (AgES: 42, 44, and 56 kDa) by two-dimensional immunoblot analysis. Among those immunodominant peptides, two AgS spots were characterized by mass spectrometric analysis (32 kDa; pI 6.3 and 6.5) and identified as type 1 galectins. In accordance with this finding, a fraction of AgS exhibited affinity to lactose and displayed a 100% specificity and sensitivity for the diagnosis of human gnathostomiasis.

Excreción fecal de Salmonella Albany, su aislamiento en la ración alimenticia y repercusión en el estado de salud de un ocelote (Leopardus pardalis) en cautiverio / Fecal excretion of Salmonella Albany, its isolation in the diet and health repercussion on an ocelot (Leopardus pardalis) in captivity

Salmonella enterica serotypes are 99% responsible for salmonellosis in human and animals, especially Salmonella enterica serovar Albany that has been identified in chicken carcass representing a risk for human and animal health. Salmonella enterica serovar Albany was isolated from the feces of a male ocelot (Leopardus pardalis), at the zoo in Culiacan, Sinaloa, Mexico, and from raw chicken (feline's diet). The pulsed-field gel electrophoresis pattern (PFGE) generated by Xba I enzyme was identical in both isolates, indicating that the source of infection was the raw chicken. Five months after having isolated the bacteria from the feces, a post mortem study was carried out on the feline. Macroscopically, severe hemorrhagic enterocolitis and renal fibrosis was observed and microscopically, there was evidence of severe mononuclear lymphocytic infiltration in the ileum, as well as necrosis of intestinal villi and crypts, besides severe multifocal interstitial nephritis and fibrosis in both kidneys. The invA gene was amplified from intestinal samples confirming an infection by Salmonella. The microbiologic, molecular and histopathology diagnoses suggest that death of the feline was caused by ingestion of raw chicken contaminated with Salmonella enterica serovar Albany. This clinical case highlights the importance of persistent fecal Salmonella shedding animals and describes the molecular epidemiological relationships of isolates from feces and food, which allowed to find the primary source of infection.

Los serotipos de Salmonella especie enterica son los responsables del 99% de las salmonelosis en humanos y animales, en particular, Salmonella enterica serovariedad Albany se ha identificado en canales de pollo, por lo que representa un riesgo para la salud humana y animal. Se aisló Salmonella enterica serovariedad Albany a partir de heces de un ocelote macho (Leopardus pardalis), cautivo en el zoológico de Culiacán, Sinaloa, México, y de pollo crudo (alimento del felino). El patrón por electroforesis en campo pulsado (PFGE) con la enzima Xba I fue idéntico en ambos aislados, lo que indica que la fuente de infección fue el pollo crudo. Cinco meses después de haber aislado las bacterias de las heces, se realizó estudio post mortem del felino anteriormente mencionado, y se observó macroscópicamente: enterocolitis hemorrágica severa y fibrosis renal; y microscópicamente: necrosis de vellosidades y de criptas e infiltrado mononuclear linfocitario severo en íleon, además nefritis intersticial severa multifocal y fibrosis en riñón. A partir de muestras intestinales se amplificó el gen invA que confirma la infección por Salmonella. Los diagnósticos microbiológico, molecular e histopatológico sugieren que la muerte del felino se debió a la infección causada por la ingesta de pollo crudo contaminado con Salmonella enterica serovariedad Albany. Este caso clínico confirma la importancia que tienen los animales que excretan Salmonella vía fecal y describe la relación epidemiológica-molecular de los aislamientos obtenidos de heces y alimento, lo que permitió esclarecer la fuente primaria de infección.

[Reaction of Leishmania (Leishmania) mexicana antigens by sera of patients with cutaneous leishmaniasis from Sinaloa, Mexico]. / Reacción de antígenos de Leishmania (Leishmania) mexicana con sueros de pacientes con leishmaniosis cutánea de Sinaloa, México.

OBJECTIVE: To detect Leishmania mexicana antigens reacting with sera of patients with cutaneous leishmaniasis (CL). MATERIAL AND METHODS: A crude extract of L. mexicana was used as antigen for 2-D Western blot using sera from 5 patients with CL and controls from Sinaloa, Mexico during 2008. RESULTS: Five antigens were detected in the five infected patients analyzed; their molecular weights and isoelectric points were: 26 kDa (pI 7.8), 27 kDa (pI 8.1), 28 kDa (pI 8.6), 29 kDa (pI 8.5) and 31 kDa (pI 9.0). CONCLUSION: New potentially immunodominant L. mexicana antigens were detected, suggesting that this parasite could be the species responsible for human infection in Sinaloa.

Reacción de antígenos de Leishmania (Leishmania) mexicana con sueros de pacientes con leishmaniosis cutánea de Sinaloa, México / Reaction of Leishmania (Leishmania) mexicana antigens by sera of patients with cutaneous leishmaniasis from Sinaloa, Mexico

OBJETIVO: Detectar los antígenos de Leishmania (Leishmania) mexicana que reaccionan con sueros de pacientes con leishmaniosis cutánea (LC) de Sinaloa, México. MATERIAL Y MÉTODOS: Un extracto crudo de L. (L.) mexicana fue usado como antígeno para Western blots 2-D empleando sueros de cinco pacientes con LC y controles originarios de Sinaloa, México, durante el 2008. RESULTADOS: Cinco antígenos fueron detectados sólo por los sueros de los cinco pacientes estudiados; estos son: 26 kDa (pI 7.8), 27 kDa (pI 8.1), 28 kDa (pI 8.6), 29 kDa (pI 8.5) y 31 kDa (pI 9.0). CONCLUSIONES: Se detectaron nuevos antígenos de L. (L.) mexicana potencialmente inmunodominantes, lo que sugiere a este parásito como el agente causal de la LC en Sinaloa.

OBJECTIVE: To detect Leishmania mexicana antigens reacting with sera of patients with cutaneous leishmaniasis (CL). MATERIAL AND METHODS: A crude extract of L. mexicana was used as antigen for 2-D Western blot using sera from 5 patients with CL and controls from Sinaloa, Mexico during 2008. RESULTS: Five antigens were detected in the five infected patients analyzed; their molecular weights and isoelectric points were: 26 kDa (pI 7.8), 27 kDa (pI 8.1), 28 kDa (pI 8.6), 29 kDa (pI 8.5) and 31 kDa (pI 9.0). CONCLUSION: New potentially immunodominant L. mexicana antigens were detected, suggesting that this parasite could be the species responsible for human infection in Sinaloa.

[Cutaneous leishmaniasis caused by Leishmania mexicana in Durango, Mexico: first clinical case report]. / Leishmaniasis cutánea causada por Leishmania mexicana en Durango, Mexico. Informe del primer caso clínico.

BACKGROUND: Leishmanisis is a parasitic diseases caused by intracellular protozoan of Leishmania genus. These parasites are transmitted by the bite of phlebotomine flies. Leishmanises are classified in different clinic variants: cutaneous localized or diffuse, mucocutaneous and visceral. In Mexico, the leishmanisis are distributed in several states, however Durango was considered free of leishmaniasis. CLINICAL CASE: A 9 year old male patient with an ulcerated pruriginous node of circular shape, 13 x 18 mm diameter, localized in the back of the right arm with 6 months progression. The patient was a permanent resident of Durango, Mexico. Histopathology evidenced macrophages infected with amastigotes. The PCR-RFLP result was consistent with Leishmania mexicana. Treatment with glucantime was satisfactory. CONCLUSIONS: Here we report the first clinical case of leishmanisis cutaneous localized caused by Leishmania mexicana from Durango, Mexico in a 9 years old male, confirming the increasing propagation of this protozoan parasite in Mexico.

[Tissular cestodiasis: role of helper type 1 and 2 t-lymphocytes]. / Cestodiasis tisulares: participación de los linfocitos T cooperadores 1 y 2.

Cysticercosis and hydatidosis are parasitic diseases caused by larvae of Taenia solium and Echinococcus sp., respectively. Parasitic diseases are useful models for in vivo studies of effector functions of T helper cell subsets 1 and 2, (called Th1 and Th2 on the basis of the pattern of cytokines they produce). The polarization to Th1 or Th2 is related to protective or permissive immune responses in these diseases. The role of Th1 and Th2 lymphocytes in tissue cestodiasis (cysticercosis and hydatidosis) has been studied in experimentally infected mice and in human patients; study results suggest that in individuals with cysticercosis or hydatidosis, the cellular immune response is polarized to Th2, while individuals in whom parasites are killed or their growth is limited, have an immune cellular response that is polarized towards Th1. The English version of this paper is available at: http://www.insp.mx/salud/index.html.

Cestodiasis tisulares: participación de los linfocitos T cooperadores 1 y 2 / Tissular cestodiasis: role of helper type 1 and 2 t-lymphocytes

Cysticercosis and hydatidosis are parasitic diseases caused by larvae of Taenia solium and Echinococcus sp., respectively. Parasitic diseases are useful models for in vivo studies of effector functions of T helper cell subsets 1 and 2, (called Th1 and Th2 on the basis of the pattern of cytokines they produce). The polarization to Th1 or Th2 is related to protective or permissive immune responses in these diseases. The role of Th1 and Th2 lymphocytes in tissue cestodiasis (cysticercosis and hydatidosis) has been studied in experimentally infected mice and in human patients; study results suggest that in individuals with cysticercosis or hydatidosis, the cellular immune response is polarized to Th2, while individuals in whom parasites are killed or their growth is limited, have an immune cellular response that is polarized towards Th1.