Impact of concurrent pregnancy and lactation on maternal nestbuilding, estradiol and progesterone concentrations in rabbits

[EN] We evaluated the impact of concurrent pregnancy and lactation on: nest-building (i.e., digging, straw-carrying, hair-pulling), food intake, milk output, body weight, and the concentration of estradiol and progesterone in blood. Digging was lower in pregnant-lactating (PL) rabbits, compared with pregnant-only (PO) does, on 21-23 d (52±64 vs. 104±86 g, respectively; mean±SD; P<0.05). Straw-carrying was also reduced in PL does on 24-26 d (9±27 vs. 79±94 g; P<0.005), 27-29 (27±56 vs. 99±77 g; P<0.005), and in the total amount of material introduced into the nest box (132±167 vs. 286±217 g; P<0.02). Hair-pulling was expressed by practically all animals. Food intake declined in PO does on the three days preceding parturition (P<0.01) and increased markedly during lactation; this increase was much larger in PL than in lactating-only (LO) rabbits (P<0.01). Milk output was similar between PL and LO does during the first 21 d of lactation but a marked decline in this parameter occurred in PL does from then until 30 d. The differences in nest-building between PL and PO rabbits may be related to the concentrations of estradiol and progesterone on specific days of pregnancy. PL does showed significantly higher estradiol levels than PO animals on pregnancy 1 d (33±13 vs. 23±4 pg/mL; P<0.02) and 21 (34±19 vs. 24±6 pg/mL; P<0.05) and also higher levels of progesterone on pregnancy 1 d (4±5 vs. 1±2 ng/mL; P<0.05). However, PL rabbits had lower levels of progesterone on 7 d (6±3 vs. 9±2 ng/mL; P<0.02) and 14 d (8±3 vs. 11±3 ng/mL; P<0.005) than PO does. Our results indicate that the unique endocrine milieu of PL rabbits has a direct bearing on specific behavioral and physiological phenomena that impact productivity on the farm.González-Mariscal, G.; Gallegos, J.; Sierra-Ramírez, A.; Garza Flores, J. (2009). Impact of concurrent pregnancy and lactation on maternal nestbuilding, estradiol and progesterone concentrations in rabbits. World Rabbit Science. 17(3):145-152. doi:10.4995/wrs.2009.65414515217

Development of in vitro systems to study IFN signalling in gilthead seabream (Sparus aurata)

Type I interferon (IFN I) triggers specific signalling pathways leading to the activation of the innate immune defence of vertebrates against viral infections. In contrats, type II IFN (IFN II) is generally accepted to be part of the adaptive response. Among IFN I-stimulated genes, those coding the Mx proteins play a main role due to the direct antiviral activity of these proteins. The study of Mx genes in gilthead seabream, one of the most important species in the Mediterranean aquaculture, is especially interesting, as this species displays a high natural resistance to viral diseases, and behaves as asymptomatic carrier and/or reservoir of several viruses, such as viral nervous necrosis virus (VNNV), infectious pancreatic necrosis virus (IPNV), and viral haemorrhagic septicaemia virus (VHSV), which are pathogenic to other fish species. Three Mx genes (Mx1, Mx2, and Mx3) have been identified in S. aurata, showing the three proteins a wide spectrum of antiviral activity. The structure of the three promoters (pMx1, pMx2 and pMx3) has been disclosed, and their response to IFN I, IPNV and VHSV indicated a clear induction of the three promoters, with some differences in the kinetics and magnitude of the response. Several studies evidenced the important role of Mx transcription regulation on virus-host interaction: i) Mx promoters can respond to both IFN I and IFN II, thus Mx might be the link between innate and adaptive immunity; ii) Mx activation is blocked by several viruses, thus Mx transcription is the target of their IFN I antagonistic activity; and iii) A fish cell line modified with the promoter of a fish Mx gene was used to measure viraemia in serum with high sensitivity. Therefore, assessing the regulatory mechanisms controlling the transcription of fish Mx genes could significantly contribute to both, understanding virus-host interactions, and designing strategies to control viral infections. In our case, this approach can also give light to understand the successful antiviral strategies developed by gilthead seabream in nature. Thus, the purpose of the present work was to develop three stable transgenic cell lines expressing the firefly luciferase gene under the control of the gilthead seabream Mx promoters. These in vitro systems were established and their response to poly I:C, and to two viral infections was characterized. In the case of IPNV, a clear antagonistic activity was observed for pMx2, as the activity of the promoter was 78.53% lower, however, this effect was not observed for pMx1 and pMx3. When cells were infected with VHSV, no changes in the promoters’ activity were detected, thus indicating that seabream Mx promoters are not targeted by VHSV antagonistic activity. These results confirm the specificity of the interactions between each virus/promoter combination, and support the use of the three cell lines developed as useful tools to characterize virus-host interactions in this species. Further studies aimed at the identification of the molecular mechanisms behind our observations will allow us to get more insight into this complex system.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Differential induction of the gilthead seabream Mx1, Mx2 and Mx3 promoters by IPNV and VHSV

Type I interferon (IFN I) system triggers specific signalling pathways leading to the activation of the innate immune defence of vertebrates against viral infections. The complex expression regulation of Interferon Stimulated Genes (ISGs) is responsible for the control of the IFN I response. Hence, one of the key issues in understanding virus-host interactions relies on the knowledge of the regulatory mechanisms governing ISGs expression. Among ISGs, the Mx proteins play a main role due to their direct antiviral activity. The study of Mx genes in the farmed fish gilthead seabream is especially interesting, since this species displays an unusually high natural resistance to viral diseases, and behaves as an asymptomatic carrier and/or reservoir of several viruses, such as infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV), pathogenic to other fish species. Three independent Mx genes (Mx1, Mx2, and Mx3) have been identified in this species, showing the three proteins a wide spectrum of antiviral activity. The structure of the three promoters (pMx1, pMx2 and pMx3) has been disclosed, and their response to poly I:C characterized in RTG-2 cells, where a clear induction of the three promoters, although with some differences in the kinetics and magnitude of the response, was observed. To further analyse these promoters, the response of pMx1, pMx2 and pMx3 to two viral infections has been evaluated in the present study. For that purpose, RTG-2 cells were transiently transfected with plasmids containing each promoter driving the luciferase gene, and subsequently inoculated with either IPNV or VHSV. Although the three promoters were induced by IPNV and VHSV, several differences were recorded. In general, the response was stronger in cells inoculated with VHSV compared to IPNV-inoculated cells, and the fold induction was higher for pMx2. These results highlight the specific regulation that controls the activity of each promoter, and support the idea that a complex interaction between host cells, specific Mx promoters, and viruses, is the responsible of the final outcome of a viral infection, in terms of Mx induction. The authors want to thank Dr. C. P. Dopazo (University of Santiago de Compostela, Spain) for supplying the VHSV isolate used in this work.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

New Zealand White rabbits show non-selective nursing in various types of nests

[EN] We investigated the capacity of New Zealand White female rabbits to nurse their litter in a nestdifferent from their own (i.e., from another female, in a box containing synthetic or male hair, or in a new boxcontaining part of the original nest material). In females that nursed in their own nest across lactation days 1-3 (one nest-condition) the mere addition of any of the above boxes (without pups) across days 4, 5, 6, 7, or8 provoked an increase in the latency to enter their own nest for nursing (0.05±0 vs. 2±1 min; P<0.01; Group1A). In the two-nest condition females took a longer time to enter any box for nursing than they did to enter theirown. These differences were significant only for the synthetic nest (4±1.5 min; P<0.05). When the two-nestcondition began on lactation day 1 (Group 1B) the latency to enter significantly increased only with the malenest (8±4 vs. 1±0 min, own nest; P<0.05). The time inside the nest box devoted to nursing was practically thesame under all conditions and within the normal duration (ca. 3 min). Milk production rose steadily across thedays of observation, regardless of the type of nest-box the litter was placed in. We also explored the effectof using alien kits, rather than the female's own, on the rabbit's capacity to nurse them in any of the above nesttypes (Group 2). The only differences between Group 2 and Group 1B were: a) on day 1 females nursing analien litter produced less milk (48±5 g) than the two groups nursing their own (69±5, 75±8 g; P<0.02); b) thelatency to enter the male nest was smaller in Group 2 (2±2 vs. 8±4 min; P<0.05). These results show that,although able to distinguish among different nest types, rabbits can nurse their own or alien kits in severalenvironments. These findings can be useful in small farm rabbit husbandry practices for facing the problemsof kit death or nest deterioration in early lactation.González-Mariscal, G.; Gallegos, J. (2007). New Zealand White rabbits show non-selective nursing in various types of nests. World Rabbit Science. 15(3). doi:10.4995/wrs.2007.592SWORD15

Caracterización de los promotores de las proteínas antivirales Mx1, Mx2 y Mx3 de dorada (Sparus aurata)

La dorada es una especie de gran interés para la Acuicultura del Sur de Europa. Las notables pérdidas en la acuicultura moderna debidas a patologías de distinta índole, han suscitado entre los científicos la necesidad de conocer las características de las interacciones patógeno-hospedador. La falta de tratamientos efectivos contra infecciones víricas, ha motivado el desarrollo de nuevas técnicas y aplicaciones biotecnológicas como medida preventiva. Para ello, el estudio de las proteínas antivirales Mx en peces resulta interesante, sobre todo en especies que se comportan como portadores asintomáticos de diversos virus, como es el caso de la dorada (Sparus aurata). Las proteínas Mx tienes propiedades antivirales y han sido estudiadas en otras especies con resultados prometedores. Además el cDNA de las proteínas Mx1, Mx2 y Mx3, han sido previamente clonados y caracterizados por nuestro grupo de investigación, lo cual ha motivado el estudio de sus promotores con el fin de comprender como se regulan estas interacciones patógeno-hospedador. El objetivo de este trabajo consistió en la caracterización estructural y funcional de las proteína antivirales Mx1, Mx2 y Mx3 de Sparus aurata. Para la caracterización estructural fue necesario la clonación y secuenciación de las regiónes 5’ reguladoras mediante la estrategia de Genome Walking, el análisis filogenético entre las secuencias obtenidas, el análisis comparativo con promotores de Mx de otras especies de peces y vetebrados superiores, la determinación de los motivos reguladores más relevantes que lo componen mediante el estudio bibliográfico. El análisis funcional se llevó a cabo mediante: la construcción de distintas construcciones plasmídicas, de los promotores completos y con algunas deleciones de los elementos reguladores más relevantes, en un vector (pGL4.22Luc-Puro2CP) que contiene gen de la luciferasa como reporter para la medición de la actividad de estos promotores; la determinación de la actividad antiviral in vitro mediante inmunoestimulación con poli l:C e infección vírica experimental, en células de trucha arcoíris RTG-2 (Rainbow trout) transfectadas con cada una de las construcciones realizadas mediante electroporación, para lo que fue necesario la optimización del el proceso de nucleofección a fin de obtener la mayor eficiencia; y el establecimiento de células estables seleccionadas expresando las construcciones reporter/promotor, para el estudio del posible efecto antagonista de los virus IPNV o Virus de la Necrosis Pancreática Infecciosa (genotipo Sp), y el VHSV o Virus de la septicemia hemorrágica viral (cedido por el Dr C.P. Dopazo, de la Universidad de Santiago de Compostela) en la ruta del interferón. Para ello se realizó un ensayo de protección celular, en el que las células se estimularon primero con poli I:C, y a continuación se inocularon con cada uno de los dos virus. Las secuencias de los promotores completos presentaron 943, 1188 y 1226 nucleótidos para Mx1, Mx2 y Mx3, respectivamente. Estas secuencias se depositaron en la base de datos Genebank, con números de acceso JQ392566, JQ392567 y JQ392568. Presenta la estructura típica de promotores de genes inducidos por IFN-α/β en los que la presencias de secuencias diana ISRE (Elemento de Respuesta a Interferon o Interferon Stimulated Response Ratio) son responsables de la activación de estos genes antivirales como respuesta a una infección. La secuencia que contenía este elemento estaba muy conservada en los tres promotores. Otras dianas de elementos reguladores como secuencias GAS de respuesta a IFN-γ e IL-6 fueron localizadas, así como elementos de gran similitud a las ISRE denominadas ISRE-like, y que contenían alguna mutación puntual respecto a la canónica. Destacó la ausencia de caja TATA, salvo en Mx2, lo cual indujo a pensar el diferentes sitios de inicio de las trasncripción o en promotores alternativos. Todos estos motivos fueron encontrados además en secuencias intrónicas, destacando un número muy elevado sobre todo en el intrón 1, lo cual hizo pensar en la posible participación de este intrón en la regulación de estos genes. Funcionalmente, los tres promotores respondían a la estimulación con poli I:C e infecciones víricas con IPNV y VHSV aunque con diferentes rangos de inducción, destacando la elevada actividad de inducción de Mx2 frente a los otros dos. Las características de la inducción de cada uno fueron dosis-dependiente, mostrándose mayores valores de inducción con 10 µg/mL en cuanto a la inducción con poli I:C, y de 0.01 MOI y 0.1 MOI para IPNV y VHSV respectivamente. La menor inducción observada a mayor concentración de virus IPNV fue un indicio del posible efecto antagonista del virus en esta ruta. Se observó a demás una dependencia temporal en ambos casos, siendo la respuesta más rápida por inducción con poli I:C (máximo 24 h) que con virus (máximo 72 h). La cinética de inducción de cada promotor resultó ser similar para los tres, siendo más elevado para Mx2. En el caso de IPNV, se detectó un claro efecto antagonista del virus sobre la actividad de pMx2, ya que, en todos los casos, la presencia del virus hizo disminuir de forma significativa la estimulación de este promotor causada por poli I:C. La capacidad del IPNV de interferir con la cascada de señalización de IFN, siendo la transcripción de Mx una de las dianas de actuación de este virus. En el caso de la dorada, es altamente probable que la diversidad funcional detectada en los promotores de sus tres genes Mx, juegue un papel fundamental en la exitosa estrategia antiviral desarrollada por esta especie

Relationship between degree of cellular differentiation in colorectal cancer and topographical distribution.

Objetivos: intentar establecer la relación existente entre el grado diferenciación celular del cáncer colon y su distribución topográfica: en 215 pacientes diagnosticados de cáncer colorrectal entre los años 1997 y 2000. Material y métodos: se estudiaron de forma prospectiva 215 pacientes (129 hombres y 86 mujeres) de edades comprendidas entre 23 y 84 años, con edad media de 64 años. En todos se realizó colonoscopia completa con varias tomas de biopsia. En los casos de estenosis tumoral con imposibilidad para sobrepasar la lesión se realizó enema opaco. Los estudios de extensión incluyeron TAC y ecografía abdominal, hemograma, perfil bioquímico completo y marcadores tumorales Ca 19-9 y alfafetoproteina). La distribución topográfica de los cánceres colorrectales fue la siguiente: recto 78 (35%), sigrna 66 (31%), descendente 21 (10%), transverso 12 (6%), ascendente 19 (9%), ciego 11 (5%), y anorrectal 8 (4%). Resultados: siendo el objetivo de nuestro estudio el establecer la relación entre el asentamiento tumoral en el colon y su grado de diferenciación celular encontramos: a) bien diferenciados 101/215 (47%); b) moderadamente diferenciados 98/215 y c) pobremente diferenciados 16/215 (7 El cáncer bien diferenciado lo encontramos en 49% de los hombres y en el 43% de las mujeres, el moderadamente diferenciado fue del 43% entre los hombres y del 49% entre las mujeres, el pobremente diferenciado fue del 7,5% entre los hombres y de 7,2% entre las mujeres. Según su distribución: en el colon izquierdo,80 adenocarcinomas eran bien diferenciados, 77 moderadamente diferenciados y 8 pobremente diferenciados; en el colon transverso; 7 adenocarcinomas eran bien diferenciados 3 moderadamente diferenciados y 2 pobremente diferenciados, en el colon derecho 11 adenocarcinomas eran bien diferenciados, 15 moderadamente diferenciados y 4 pobremente diferenciados. De los 8 cánceres recto-anales, 3 eran bien diferenciados, 3 moderadamente diferenciados y 2 pobremente diferenciados, habiendo observado que dicho grado de diferenciación no tiene un significado estadístico de relación con la distribución topográfica del tumor. Según la clasificación fueron más frecuentes en los estadios, los bien diferenciados (101/215) fueron más frecuentes en los estadios BI (32,6%) y C2 (20, los moderadamente diferenciados (98/215) lo fueron en los estadios Bi y C2 el de los estadios C2 fueron tumores pobremente diferenciados. No apreciamos diferencias estadísticamente significativas en la distribución de los grados de diferenciación estadios (p—ns). Conclusiones: nuestros resultados, no hemos observado que el grado de diferenciación celular del cáncer colorrectal se relacione con su localización inicial en el colon y es, independiente del sexo y de la edad. En cuanto a su posible relación con la estadios de Dukes y Astler-Coller tampoco hemos demostrarla.To demonstrate the relationship between degree of cellular differentiation in colorectal cancer and topographical distribution in 215 patients diagnosed with colorectal cancer from 1997 to 2000. MATERIAL AND METHODS: 215 patients (129 men and 86 women) were studied prospectively with a mean age of 64 years (range: 23-84 years). In all patients we performed a full colonoscopy with several biopsies (in patients with colon stenosis we used barium enema), radiographic studies (CT, abdominal ultrasounds), and laboratory tests for serum tumour markers (CEA, Ca 19-9, alpha-fetoprotein). The topographic location of colorectal cancer was: rectum 35%, sigmoid colon 31%, descending colon 10%, transverse colon 6%, ascending colon 9%, caecum 5%, and we included anorectal cancer 4%. RESULTS: According to histological differentiation we found: A) well-differentiated tumours 101/215 (47%); B) moderately-differentiated tumours 98/215 (45.5%), and C) poorly-differentiated tumours 16/215 (7.5%). We found no significant association among histological differentiation, topographic location, stage according to the Astler-Coller classification, sex or age (p = ns). The prevalence of well-differentiated tumours in men was 49% and 43% in women; of moderately-differentiated cancers in men was 43%, and 49% in women; for poorly-differentiated tumours in men was 7.5%, and 7.2% in women. Regarding tumour location, 165 cancers were found in the left colon: 80 were well differentiated, 77 moderately differentiated and 8 poorly differentiated. In the transverse colon we found 12 tumours: 7 well differentiated, 3 moderately differentiated and 2 poorly differentiated. 30 cancers were localized in the right colon: 11 well differentiated, 15 moderately differentiated and 4 poorly differentiated. In the anorectum 8 tumours were found: 3 well differentiated, 3 moderately differentiated and 2 poorly differentiated. According to staging classification, well differentiated tumours (101/215) were more common in Dukes' C2 (20.7%) and B1 (32.6%), moderately differentiated cancers (98/215) were in B1 (28.5%) and C2 (20.4%), and poorly differentiated tumours (16) were more common in Dukes' C2 (25%), without differences among other stages (p = ns). CONCLUSIONS: According to our results we have found that histological differentiation of colorectal cancer has no association with topographic location, and it is independent of sex or age. We have not found any relationship either between histological differentiation and stage in the Astler-Coller classification, but well differentiated cancers were more common at any location, age or sex