Optimization and application of a high-resolution melting protocol in the characterization of avian infectious laryngotracheitis virus.

A previous sequence analysis of a US5 gene fragment of infectious laryngotracheitis virus (ILTV) performed in an Argentinian epidemiological study allowed to differentiate between wild and vaccine strains. This analysis also defined five ILTV haplotypes with specific variations at positions 461, 484, 832, 878 and 894 of the US5 gene. This characterization of viral strains may also be accomplished using the High-Resolution Melting Analysis (HRMA), which has been described as an effective, fast and sensitive method to detect mutations in PCR products. In the present study, an HRM protocol was developed with the aim of characterizing the circulating ILTV strains in Argentina. The specificity of this tool was confirmed in different DNA diluents, without interference from heterologous DNA or other cellular metabolites. Additionally, the salt concentration in the elution buffer used for DNA extraction did not alter the curve profiles. Higher concentrations of DNA (Ct≅26.0) displayed well-defined curve profiles, whereas lower concentrations (Ct≅32.5) exhibited more heterogeneous curves. The HRMA showed 97.49% concordance with the reference technique, i.e., sequencing. The HRM protocol has the capability to perform DNA amplification prior to its characterization. Thus, eventually this technique may be used simultaneously as a diagnostic tool. This advantage implies a significant reduction in the time and effort involved in sample processing.

Genetic and antigenic heterogeneity of infectious bronchitis virus in South America: implications for control programmes.

Infectious bronchitis virus (IBV) is a persistent sanitary problem for the South American poultry industry despite extensive vaccination. The IBV single-stranded RNA genome has high rates of mutation and recombination that generate a notorious virus variability. Since most IBV vaccines are type-specific, there is a need for constant surveillance of the circulating lineages and knowledge about their genetic and antigenic properties. Here we present an integrative analysis that provides the pattern of genetic variation of the South American IBV strains and information about their antigenic characteristics. The genetic analysis was performed using the S1 complete coding sequences of all available South American strains, including newly obtained Argentine and Uruguayan field samples. Our phylogenetic and phylodynamic analyses evidence that three main lineages (GI-1, GI-11 and GI-16) are extensively circulating in South American flocks. Strains of the GI-1 lineage (Massachusetts-type) were detected in Argentina, Brazil, Chile and Colombia. The GI-11 lineage is an exclusively South American lineage that emerged in the 1950s, and is the predominant lineage in Brazil and Uruguay at present. The GI-16 lineage emerged around 1979, and is currently circulating in most South American territories (Argentina, Chile, Uruguay, Colombia and Peru). The virus cross-neutralization test performed here reveals very low antigenic relatedness between GI-11 and GI-16 lineages (i.e. they are different serotypes). The results of this study extend our knowledge about the present and past IBV variability in South America and provide relevant elements to improve the control programmes by considering the genetic and antigenic attributes of IBV.

Different regulation of cortisol and corticosterone in the subterranean rodent Ctenomys talarum: Responses to dexamethasone, angiotensin II, potassium, and diet.

When harmful environmental stimuli occur, glucocorticoids (GCs), cortisol and corticosterone are currently used to evaluate stress status in vertebrates, since their secretions are primarily associated to an increased activity of the hypothalamic-pituitaryadrenal (HPA) axis. To advance in our comprehension about GCs regulation, we evaluated the subterranean rodent Ctenomys talarum to assess cortisol and corticosterone response to (1) the negative feedback of the HPA axis using the dexamethasone (DEX) suppression test, (2) angiotensin II (Ang II), (3) potassium (K+) intake, and (4) different diets (vegetables, grasses, acute fasting). Concomitantly, several indicators of individual condition (body mass, neutrophil to lymphocyte ratio, blood glucose, triglycerides and hematocrit) were measured for diet treatments. Results confirm the effect of DEX on cortisol and corticosterone in recently captured animals in the field but not on corticosterone in captive animals. Data suggest that Ang II is capable of stimulating corticosterone, but not cortisol, secretion. Neither cortisol nor corticosterone were responsive to K+ intake. Cortisol levels increased in animals fed with grasses in comparison to those fed with vegetables while corticosterone levels were unaffected by diet type. Moreover, only cortisol responded to fasting. Overall, these results confirm that cortisol and corticosterone are not interchangeable hormones in C. talarum.

Expanding the actions of cortisol and corticosterone in wild vertebrates: A necessary step to overcome the emerging challenges.

We conducted a review of scientific articles published between 2000 and 2014 and evaluated how frequently various aspects of cortisol and corticosterone (CORT) actions have been considered in studies on wild vertebrates. Results show that (1) the notion that CORT are stress-responsive hormones is central in our theoretical frameworks and it is reflected by the fact that several articles refer to CORT as "stress hormones". (2) The large majority of studies do not contemplate the possibility of decrease and no change in CORT levels in response to chronic stressors. (3) Our ideas about CORT actions on energy balance are slanted towards the mobilization of energy, though there are several studies considering -and empirically addressing- CORT's orexigenic actions, particularly in birds. (4) The roles of CORT in mineral-water balance, though widely documented in the biomedical area, are virtually ignored in the literature about wild vertebrates, with the exception of studies in fish. (5) Adrenocorticotropic hormone (ACTH) independent regulation of CORT secretion is also very scarcely considered. (6) The preparative, permissive, suppressive and stimulatory actions of CORT, as described by Sapolsky et al. (2000), are not currently considered by the large majority of authors. We include an extension of the Preparative Hypothesis, proposing that the priming effects of baseline and stress-induced CORT levels increase the threshold of severity necessary for subsequent stimuli to become stressors. Studies on animal ecology and conservation require integration with novel aspects of CORT actions and perspectives developed in other research areas.

Cortisol and corticosterone exhibit different seasonal variation and responses to acute stress and captivity in tuco-tucos (Ctenomys talarum).

In this work we aimed to evaluate variations in plasma glucocorticoids (GCs, cortisol and corticosterone) levels throughout an annual cycle in free-living male tuco-tucos (Ctenomys talarum) and compare their responses to acute and chronic stressors (trapping, manipulation, immobilization, confinement in a novel environment, transference to captivity). In addition, we used leukocyte profiles to allow discrimination between basal and stress-induced seasonal changes in GC concentrations. Our results showed that cortisol and corticosterone are differently affected by environmental stimuli in C. talarum. Both hormones showed different patterns of variation in the field and responses to captivity. Moreover, only cortisol was responsive to acute stressors. Leukocyte profiles indicated that animals were unstressed in the field and therefore, that we were able to measure basal, stress-independent, fluctuations in GC levels. GC concentrations were low in comparison to values frequently reported for other mammals. Our results suggest differentiated physiological roles for cortisol and corticosterone in our study species and further emphasize the complexity of GC physiology in wild mammals.

Decreased glucose tolerance but normal blood glucose levels in the field in the caviomorph rodent Ctenomys talarum: the role of stress and physical activity.

Hystricomorph rodents have a divergent insulin molecule with only 1-10% of the biological activity in comparison to other mammalian species. In this study, we used the subterranean rodent Ctenomys talarum as a model and performed blood glucose tolerance tests (GTTs) with trained and untrained individuals to evaluate blood glucose regulation and the possible role of physical activity as a compensatory mechanism. Additionally, we evaluated the variations in blood glucose during acute and chronic stress and gathered data in the field to evaluate natural-occurring variations in blood glucose levels. The GTTs showed that C. talarum have a diminished capacity of regulating blood glucose levels in comparison to other mammals and suggest that unexplored differences in the compensatory mechanisms, insulin structure and/or glucose transporters exist within species of hystricomorph rodents. However, blood glucose levels in the field stayed within the normal mammalian range. Physical activity did not prove to be a compensatory mechanism for blood glucose regulation. The individuals did not display important increases in blood glucose after acute stressors and managed to adequately regulate blood glucose during chronic stress. We suggest that the species may not face a selective pressure favoring a more tightly, mammalian like, capacity of regulating blood glucose levels.

Development of RT-qPCR assays for the specific identification of two major genotypes of avian infectious bronchitis virus.

Infectious bronchitis virus (Gammacoronavirus, Coronaviridae) is a genetically variable RNA virus (27.6kb) that causes one of the most persistent respiratory disease in poultry. The virus is classified in genotypes with different epidemiological relevance and clinical implications. The present study reports the development and validation of specific RT-qPCR assays for the detection of two major IBV genotypes: South America I (SAI) and Asia/South America II (A/SAII). The SAI genotype is an exclusive and widespread South American lineage while the A/SAII genotype is distributed in Asia, Europe and South America. Both identification assays employ TaqMan probes that hybridize with unique sequences in the spike glycoprotein gene. The assays successfully detected all the assessed strains belonging to both genotypes, showing high specificity and absence of cross-reactivity. Using serial dilutions of in vitro-transcribed RNA we obtained acceptable determination coefficients, PCR efficiencies and relatively small intra- and inter-assay variability. The assays demonstrated a wide dynamic range between 10(1)-10(7) and 10(2)-10(7) RNA copies/reaction for SAI and A/SAII strains, respectively. The possibility to characterize a large number of samples in a rapid, sensitive and reproducible way makes these techniques suitable tools for routine testing, IBV control, and epidemiological research in poultry.

Optimization and application of a high-resolution melting protocol in the characterization of avian infectious laryngotracheitis virus

Resumen En un estudio epidemiológico realizado previamente en Argentina, se analizó la secuencia de un fragmento del gen US5 del virus de la laringotraqueítis infecciosa (ILTV), lo que permitió diferenciar las cepas de campo de las vacunales. También esto permitió definir cinco haplotipos del ILTV, con variaciones específicas en las posiciones 461, 484, 832, 878 y 894 del gen US5. La caracterización de las cepas virales también puede lograrse mediante el análisis de la disociación de alta resolución o high-resolution melting analysis (HRMA), descripto como un método efectivo, rápido y sensible para detectar mutaciones en productos de PCR. En el presente estudio se desarrolló un protocolo de disociación de alta resolución con el objetivo de caracterizar cepas del ILTV circulantes en Argentina. Para ello,se confirmó la especificidad de esta herramienta en diferentes diluyentes del ADN de las muestras, sin observarse interferencias en presencia de ADN heterólogo u otros metabolitos celulares. Asimismo, la concentración de sales en el buffer de elución utilizado durante la extracción de ADN no alteró los perfiles de las curvas. Se obtuvieron perfiles bien definidos con concentraciones de ADN más elevadas (Ct = 26.0), mientras que concentraciones más bajas presentaron curvas heterogéneas (Ct = 32.5). El HRMA mostró una concordancia del 97.49% con la técnica de referencia, la secuenciación. El protocolo de disociación de alta resolución amplifica el ADN antes de su caracterización, por lo que esta técnica podría ser eventualmente utilizada para confirmar la presencia del ILTV y, al mismo tiempo, distinguir haplotipos, optimizando su valor como herramienta de diagnóstico. Esta característica implica una reducción significativa en el tiempo dedicado al procesamiento de muestras.

Differential responses of cortisol and corticosterone to adrenocorticotropic hormone (ACTH) in a subterranean rodent (Ctenomys talarum).

We aimed to evaluate the responses of cortisol, corticosterone, and blood glucose to adrenocorticotropic hormone (ACTH) in males and females of the subterranean rodent Ctenomys talarum and addressed interannual variations in the plasma levels of both hormones. The most important results indicate that: (1) cortisol positively responds to the ACTH signal but corticosterone does not, even though corticosterone levels were higher than cortisol concentrations, (2) plasma corticosterone concentrations in free-living animals were 20 times higher compared to values reported for the same population during previous annual periods and, as cortisol levels were similar, this resulted in much lower cortisol/corticosterone ratios, (3) cortisol and corticosterone differentiated in their relative proportions in plasma in free-living males and females. These results indicate that cortisol and corticosterone are differentially regulated in our study species and emphasize that a remarkable temporal variation in the relative proportions of these hormones may occur in natural populations. Therefore, the conclusions regarding the presence of cortisol and corticosterone in plasma of wild animals may differ substantially depending on the moment when the study is conducted. Recent data indicate that cortisol and corticosterone are not interchangeable hormones in species of free-living vertebrates. We suggest that, in addition to the classical roles of glucocorticoids (GCs), it is crucial that other physiological functions be kept in mind when interpreting GC data from wild species.

Validation of a radioimmunoassay for measuring testosterone concentrations in plasma samples of the subterranean rodent Ctenomys talarum: outstandingly elevated levels in the wild and the effect of captivity.

We validated the Coat-a-Count radioimmunoassay (RIA) kit for measuring testosterone in plasma samples of the subterranean rodent Ctenomys talarum and evaluated testosterone levels in free-living and captive individuals. The performance of the assay was evaluated by the assessment of parallelism, accuracy and precision. Moreover, the high specificity of the assay antibody was confirmed by high-pressure liquid chromatography with ultraviolet detector, followed by electrospray ionization tandem mass spectrometry. Results indicated that plasma samples have to be treated with heat and diluted before the RIA for the optimization of the assay. Plasma testosterone concentrations in free-living animals were outstandingly elevated (up to 329 ng/mL), which are among the highest ever reported for mammals. On the other hand, captivity produced a 14-fold decrease in plasma testosterone concentrations, emphasizing that very significant changes in the endocrine milieu may occur in wild animals kept under laboratory conditions. Our results place tuco-tucos as an interesting model for the study of androgen regulation in mammals, suggesting that target tissues may have low sensitivity to the testosterone signal and agree with a scenario of elevated levels of sex hormone-binding globulin in plasma.