Differential Neutrophil Function and Intracellular Signaling in Dairy Cows with Specific CXCR1 Genotypes

Mastitis continues to be the most economically devastating disease affecting dairy cows worldwide. Neutrophil recruitment to the mammary gland and neutrophil functional ability once there often determine whether a bacterial infection is eliminated or becomes chronic. A slow or ineffective neutrophil attack allows bacteria to continue their assault and generate a longer lasting and potentially more damaging infection. Since neutrophils are key players in the resolution of mastitis, and a polymorphism in CXCR1 +777 (G→C) has been associated with susceptibility to mastitis, the hypothesis of this research was that different CXCR1 +777 genotypes were associated with efficiency of in vitro neutrophil activity and/or IL-8 receptor expression and intracellular signaling. Neutrophil function (migration, adhesion molecule upregulation, survival from spontaneous apoptosis, glutathione levels, reactive oxygen generation and bactericidal activity) as well as CXCR1 and CXCR2 expression and signaling were evaluated in cows with different CXCR1 +777 genotypes. Cows with a CC genotype, which have previously shown increased susceptibility to mastitis, had decreased adhesion molecule upregulation and neutrophil migration towards IL-8 compared to cows with a GG genotype. Furthermore, cows with a CC genotype displayed decreased ROS generation and, paradoxically, increased survival from spontaneous apoptosis. In addition, initial observations revealed that overall IL-8 receptor numbers tended to be lower in cows with a CC genotype compared to cows with a GG genotype. However, in the presence of a CXCR2 inhibitor (SB225002), CXCR1 affinity was about 5-fold lower in cows with a CC genotype and this may have resulted in an underestimation of receptor numbers in cows with this genotype. Additionally, intracellular calcium ([Ca++]i) release was lower in cows with a CC genotype when cells were stimulated with IL-8 but not epithelial derived neutrophil attractant-78. When neutrophils were stimulated with an optimal dose of IL-8 in the presence of SB225002, allowing only signaling through CXCR1, [Ca++]i release was lower in cows with a CC genotype, suggesting decreased CXCR1 signaling in these animals, potentially due to lower CXCR1 affinity for IL-8. This research provides evidence for neutrophil functional differences and differential CXC receptor activity and signaling in cows with specific CXCR1 genotypes. Future research aimed at better characterizing CXCR1 and CXCR2 in the bovine as well as corroboration of these findings in an in vivo model may provide prospective ways to enhance or regulate neutrophil function in dairy cows and potentially increase their resistance to mastitis and other inflammatory diseases

Dynamics of Leukocytes and Cytokines During Experimentally-Induced Streptococcus uberis Mastitis

Environmental mastitis is of increasing prevalence in well managed dairy herds throughout the world. Of the environmental pathogens, Streptococcus uberis is one of the most prevalent, accounting for a significant proportion of clinical and subclinical intramammary infections in lactating and nonlactating dairy cows and heifers. In spite of this, the pathogenesis of S. uberis mastitis is not well understood. The objective of this study was to determine dynamics of leukocytes and cytokines during experimentally-induced S. uberis mastitis. Five Jersey and 5 Holstein cows were challenged via intramammary inoculation with S. uberis into 2 uninfected mammary glands. One uninfected unchallenged mammary gland from each cow served as a negative control. Rectal temperatures, blood and milk samples were collected frequently for one week after challenge. Clinical status of mammary glands was recorded twice daily during milking. Milk samples were collected to enumerate bacteria and somatic cells, and to determine concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-8 (IL-8). Neutrophils were isolated from blood to determine expression of CD11b, CD18 and CD62 during the course of experimental infection. Sixteen of 20 challenged mammary glands developed clinical mastitis between 84 and 108 hours after intramammary challenge, and peak clinical signs of mastitis were observed at 144 hours. The number of S. uberis in milk from challenged mammary glands increased significantly (P\u3c0.05) by 48 hours after challenge, in spite of a massive leukocyte infiltration from the bloodstream, evidenced by an increase in the number of somatic cells in milk which began at 18 hours after challenge (P\u3c0.001) and remained elevated throughout the study period. Significant amounts of TNF-α, IL-1β and IL-8 in milk were detected 66 hours after challenge (P\u3c0.05). Peak concentrations of TNF-α were observed at 120 hours after challenge, preceding peak clinical signs. Concentrations of IL-1β in mammary secretions fluctuated considerably from 66 to 144 hours after challenge, but were significantly higher (P\u3c0.01) than prechallenge values and values found in milk from control mammary glands. Similar to TNF-α, peak IL-8 concentrations were observed at 120 hours after challenge. The 4 challenged mammary glands that did not become infected exhibited an increase in somatic cells and IL-1β concentrations in a pattern that appeared to be similar to that of challenged infected mammary glands, but of lesser magnitude. Expression of CD11b was upregulated at 84, 90 and 114 hours after challenge (P\u3c0.05), and expression of CD18 was increased 20% at 48 hours after challenge (P\u3c0.05). Expression of CD18 decreased sharply 96 hours after challenge and remained depressed until the end of the sampling period (P\u3c0.01). Results from this experiment demonstrated that S. uberis experimental intramammary infection induced local production of TNF-α, IL-1β and IL-8, which may play a role in the pathogenesis of S. uberis mastitis. Furthermore, an increase in somatic cells in milk occurred earlier than increases in adhesion molecule expression or cytokine production, suggesting that other mediators may be involved in initial leukocyte recruitment into the mammary gland after infection

Neospora caninum

Neospora caninum es un protozoo parásito que causa la neosporosis, una enfermedad global, cuyo efecto negativo sobre la eficiencia reproductiva se manifiesta por las grandes pérdidas económicas que causa en la ganadería bovina. En el bovino provoca abortos y frecuentemente produce el nacimiento de animales congénitamente infectados. En el perro genera diversos cuadros neuromusculares.Facultad de Ciencias Veterinaria

Serologic profiles for Sarcocystis sp. and Neospora caninum and productive performance in naturally infected beef calves

Sarcocystis sp. and Neospora caninum infections affect cattle worldwide causing important economic losses. The objective of the present study was to trace serologic profiles for Sarcocystis sp. and N. caninum in naturally infected beef calves and analyze their relationship with transmission routes and productive performance. Samples were collected in two cow-calf operations located in Buenos Aires province, Argentina. In farm 1, 43 calves were bled and weighed three times. In farm 2, 69 calves were bled and weighed six times. Sarcocystis sp. and N. caninum immunofluorescence antibody test (IFAT) titers were averaged for each sampling point in order to trace serologic profiles for each infection. Categories were created to evaluate differences in daily weight gain. For S. cruzi antigen, animals were separated in a low-titer (>200) and high-titer group (>200); for N. caninum, animals were grouped as infected and uninfected. Sarcocystis sp. antibody titer as well as the number of infected animals increased gradually over time in both farms. In farm 2 the low-titer group had significantly higher daily weight gain than the high-titer group. For N. caninum 44% (farm 1) and 65% (farm 2) of calves were considered infected, and the serological profile was horizontal or decreasing over time. However, seroprevalence increased in both farms and vertical and horizontal transmission frequency were estimated between 18.5%-29% and 22-25.5%, respectively. No differences were detected in daily weight gain between N. caninum groups from both farms. This is the first report of serological profiles for Sarcocystis sp. and N. caninum by IFAT in naturally infected beef calves and their relationship to different transmission routes and productive performance.Fil: Moré, Gastón Andrés. Universidad Nacional de La Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Bacigalupe, Diana. Universidad Nacional de La Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Basso, Walter Ubaldo. Universidad Nacional de La Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Rambeaud, Magdalena. Universidad Nacional de La Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Venturini, María Cecilia. Universidad Nacional de La Plata; ArgentinaFil: Venturini, Lucila Maria. Universidad Nacional de La Plata; Argentin

Evaluación y comparación de pruebas serológicas para la detección de la neosporosis bovina en Argentina

Neospora caninum is a protozoan parasite that causes abortion and important economic losses in cattle worldwide. The accurate diagnosis of neosporosis is essential for management and control measures. The aims of this study were: i) to evaluate the performance of an in-house enzyme-linked immunosorbent assay based on the 38 kDa native antigen (p38-ELISA) to diagnose bovine neosporosis in Argentina using a well- characterized local sera panel from experimentally infected and naturally exposed cattle and ii) to compare the diagnostic performance and agreement of three N. caninum serological tests: p38-ELISA, indirect fluorescence antibody test (IFAT) and immunoblotting (IB) using the same sera panel. Serum samples testing either positive or negative by IFAT and IB were considered “Relative Standards of Comparison” (RSC) and used for p38-ELISA evaluation. Receiver operating characteristics analysis revealed that p38-ELISA was highly accurate (area under the curve= 0.982) according to RSC with a cut-off index of 0.0905. Relative sensitivity and specificity of p38-ELISA were 97.8 % and 99.5 %, respectively and agreement between RSC and p38-ELISA was almost perfect (k= 0.97). The evaluation and performance comparison of serological tests were performed according to the definition of gold standard based on the decision of the “majority of tests”. All tests displayed high sensitivity and specificity values (greater than 95 %); and excellent agreement. This study describes the accurate performance of p38-ELISA evaluated locally and the highly accurate diagnostic performance of the studied tests for the detection of anti-N.caninum antibodies in cattle from Argentina.Neospora caninum es un parásito protozoo responsable de abortos y pérdidas económicas en bovinos. La realización de un diagnóstico serológico preciso y con resultados comparables obtenidos por diferentes pruebas contribuye al manejo de este problema y a encarar medidas de control. Los objetivos del presente trabajo fueron los siguientes: 1) evaluar en Argentina una prueba de enzimoinmunoensayo in-house con el antígeno nativo de 38 kDa de N. caninum (ELISA-p38) para el diagnóstico de la neosporosis bovina, utilizando un panel de sueros locales bien caracterizados, procedentes de bovinos infectados de modo experimental o naturalmente expuestos; 2) comparar el desempeno˜ y establecer el nivel de concordancia de tres pruebas serológicas para la detección de N. caninum, ELISA-p38, inmunofluorescencia indirecta (IFI) e inmunoblot (IB), con el mismo panel de sueros. Los sueros que resultaron positivos o negativos a IFI e IB fueron considerados como estándares relativos de comparación (ERC) para evaluar la prueba de ELISA-p38. El análisis de característica operativa del receptor determinó que la prueba de ELISA-p38 fue altamente precisa (área bajo la curva= 0,982) usando el punto de corte 0,0905. La sensibilidad y especificidad relativa del ELISA-p38 fue 97,8 % y 99,5 %, respectivamente, con una concordancia casi perfecta (k= 0,97) respecto del ERC. La comparación del desempeno˜ de las pruebas se realizó usando como gold standard el criterio de la decisión de la ‘‘mayoría de las pruebas’’. Las pruebas exhibieron altos valores de sensibilidad y especificidad (mayores del 95 %) y excelente concordancia. Este trabajo describe un buen desempeno˜ de la prueba de ELISA-p38 evaluada localmente y adecuada performance diagnóstica de las pruebas serológicas analizadas para la detección de anticuerpos anti N. caninum en bovinos de Argentina.Facultad de Ciencias Veterinaria

Evolución en la metodología del curso de Inmunobiología Animal Básica e impacto de la incorporación de nuevas herramientas didácticas en el desempeño de los estudiantes

El curso de Inmunobiología Animal Básica (IAB) se encuentra ubicado con 70 hs, en el primer cuatrimestre del segundo año de la carrera de Ciencias Veterinarias de la Universidad Nacional de La Plata. Tiene dos bandas horarias, con 5 horas presenciales por semana /alumno, agrupados en 5 comisiones por turno. La metodología de enseñanza que estamos utilizando tiene como finalidad estimular en los estudiantes hábitos de reflexión, para permitir el desarrollo de capacidad crítica e interés por la investigación (Resolución 1034/05), capacitándolos para comprender y resolver casos problema o situaciones problemáticas que se plantean al principio y en la mitad del curso, posibilitando la integración de los temas tratados.Eje 1: La enseñanza universitaria en el contexto actual: transformaciones y propuestas. Reflexiones y experiencias en la enseñanza de la Medicina Veterinaria y la Agronomía.Secretaría de Asuntos Académico

<i>Cryptosporidium varanii</i> infection in leopard geckos (<i>Eublepharis macularius</i>) in Argentina

Cryptosporidiosis is observed in reptiles with high morbidity and considerable mortality. The objective of this study was to achieve the molecular identification of Cryptosporidium spp. in pet leopard geckos (Eublepharis macularius) from a breeder colony in Buenos Aires, Argentina. Oocysts comparable to those of Cryptosporidium spp. were detected in three geckos with a history of diarrhea, anorexia and cachexia. Molecular identification methods confirmed the presence of Cryptosporidium varanii (syn. C. saurophilum). This agent was considered to be the primary cause of the observed clinical disease. This is the first description of C. varanii infection in pet reptiles in Argentina.Facultad de Ciencias VeterinariasInstituto de Genética Veterinari

Relación entre Anatomía I y desempeño de los estudiantes en el curso de Inmunobiología Animal Básica (IAB)

El curso de IAB pertenece al segundo año del plan de estudios 406 de la carrera de Ciencias Veterinarias de la UNLP. El curso se aprueba por promoción o con una evaluación final integradora (EFI). Hasta el año 2014, como pre-requisito para cursar IAB, los estudiantes necesitaban haber aprobado las actividades presenciales obligatorias de las asignaturas de primer año (APOs) pero no necesariamente las de Anatomía I. El objetivo propuesto fue determinar si el haber integrado los contenidos de Anatomía I previamente influía en el desempeño de los estudiantes para aprobar o promocionar IAB, con el fin de proponer su inclusión como correlativa.Facultad de Ciencias Veterinaria

An Ibero-American inter-laboratory trial to evaluate serological tests for the detection of anti-<i>Neospora caninum</i> antibodies in cattle

We carried out an inter-laboratory trial to compare the serological tests commonly used for the detection of specific Neospora caninum antibodies in cattle in Ibero- American countries. A total of eight laboratories participated from the following countries: Argentina (n = 4), Brazil (n = 1), Peru (n = 1), Mexico (n = 1), and Spain (n = 1). A blind panel of well-characterized cattle sera (n = 143) and sera representative of the target population (n = 351) was tested by seven in-house indirect fluorescent antibody tests (IFATs 1–7) and three enzyme-linked immunosorbent assays (ELISAs 1–3; two in-house and one commercial). Diagnostic performance of the serological tests was calculated and compared according to the following criteria: (1) the BPre-test information,^ which uses previous epidemiological and serological data; (2) the BMajority of tests,^ which classifies a serumas positive or negative according to the results obtained by most tests evaluated. Unexpectedly, six tests showed either sensitivity (Se) or specificity (Sp) values lower than 90%. In contrast, the best tests in terms of Se, Sp, and area under the ROC curve (AUC) values were IFAT 1 and optimized ELISA 1 and ELISA 2. We evaluated a high number of IFATs, which are the most widely used tests in Ibero-America. The significant discordances observed among the tests regardless of the criteria employed hinder control programs and urge the use of a common test or with similar performances to either the optimized IFAT 1 and ELISAs 1 and 2.Facultad de Ciencias Veterinaria

Evaluación y comparación de pruebas serológicas para la detección de la neosporosis bovina en Argentina

Neospora caninum is a protozoan parasite that causes abortion and important economic losses in cattle worldwide. The accurate diagnosis of neosporosis is essential for management and control measures. The aims of this study were: i) to evaluate the performance of an in-house enzyme-linked immunosorbent assay based on the 38 kDa native antigen (p38-ELISA) to diagnose bovine neosporosis in Argentina using a well- characterized local sera panel from experimentally infected and naturally exposed cattle and ii) to compare the diagnostic performance and agreement of three N. caninum serological tests: p38-ELISA, indirect fluorescence antibody test (IFAT) and immunoblotting (IB) using the same sera panel. Serum samples testing either positive or negative by IFAT and IB were considered “Relative Standards of Comparison” (RSC) and used for p38-ELISA evaluation. Receiver operating characteristics analysis revealed that p38-ELISA was highly accurate (area under the curve= 0.982) according to RSC with a cut-off index of 0.0905. Relative sensitivity and specificity of p38-ELISA were 97.8 % and 99.5 %, respectively and agreement between RSC and p38-ELISA was almost perfect (k= 0.97). The evaluation and performance comparison of serological tests were performed according to the definition of gold standard based on the decision of the “majority of tests”. All tests displayed high sensitivity and specificity values (greater than 95 %); and excellent agreement. This study describes the accurate performance of p38-ELISA evaluated locally and the highly accurate diagnostic performance of the studied tests for the detection of anti-N.caninum antibodies in cattle from Argentina.Neospora caninum es un parásito protozoo responsable de abortos y pérdidas económicas en bovinos. La realización de un diagnóstico serológico preciso y con resultados comparables obtenidos por diferentes pruebas contribuye al manejo de este problema y a encarar medidas de control. Los objetivos del presente trabajo fueron los siguientes: 1) evaluar en Argentina una prueba de enzimoinmunoensayo in-house con el antígeno nativo de 38 kDa de N. caninum (ELISA-p38) para el diagnóstico de la neosporosis bovina, utilizando un panel de sueros locales bien caracterizados, procedentes de bovinos infectados de modo experimental o naturalmente expuestos; 2) comparar el desempeno˜ y establecer el nivel de concordancia de tres pruebas serológicas para la detección de N. caninum, ELISA-p38, inmunofluorescencia indirecta (IFI) e inmunoblot (IB), con el mismo panel de sueros. Los sueros que resultaron positivos o negativos a IFI e IB fueron considerados como estándares relativos de comparación (ERC) para evaluar la prueba de ELISA-p38. El análisis de característica operativa del receptor determinó que la prueba de ELISA-p38 fue altamente precisa (área bajo la curva= 0,982) usando el punto de corte 0,0905. La sensibilidad y especificidad relativa del ELISA-p38 fue 97,8 % y 99,5 %, respectivamente, con una concordancia casi perfecta (k= 0,97) respecto del ERC. La comparación del desempeno˜ de las pruebas se realizó usando como gold standard el criterio de la decisión de la ‘‘mayoría de las pruebas’’. Las pruebas exhibieron altos valores de sensibilidad y especificidad (mayores del 95 %) y excelente concordancia. Este trabajo describe un buen desempeno˜ de la prueba de ELISA-p38 evaluada localmente y adecuada performance diagnóstica de las pruebas serológicas analizadas para la detección de anticuerpos anti N. caninum en bovinos de Argentina.Facultad de Ciencias Veterinaria