Besnoitia besnoiti lytic cycle in vitro and differences in invasion and intracellular proliferation among isolates

Background Bovine besnoitiosis, caused by the protozoan Besnoitia besnoiti, reduces productivity and fertility of affected herds. Besnoitiosis continues to expand in Europe and no effective control tools are currently available. Experimental models are urgently needed. Herein, we describe for the first time the kinetics of standardised in vitro models for the B. besnoiti lytic cycle. This will aid to study the pathogenesis of the disease, in the screening for vaccine targets and drugs potentially useful for the treatment of besnoitiosis. Methods We compared invasion and proliferation of one B. tarandi (from Finland) and seven B. besnoiti isolates (Bb-Spain1, Bb-Spain2, Bb-Israel, Bb-Evora03, Bb-Ger1, Bb-France, Bb-Italy2) in MARC-145 cell culture. Host cell invasion was studied at 4, 6, 8 and 24 h post infection (hpi), and proliferation characteristics were compared at 24, 48, 72, 96, 120, and 144 hpi. Results In Besnoitia spp., the key parameters that determine the sequential adhesion-invasion, proliferation and egress steps are clearly distinct from those in the related apicomplexans Toxoplasma gondii and Neospora caninum. Besnoitia spp. host cell invasion is a rather slow process, since only 50 % of parasites were found intracellular after 3–6 h of exposure to host cells, and invasion still took place after 24 h. Invasion efficacy was significantly higher for Bb-France, Bb-Evora03 and Bb-Israel. In addition, the time span for endodyogeny to take place was as long as 18–35 h. Bb-Israel and B. tarandi isolates were most prolific, as determined by the tachyzoite yield at 72 hpi. The total tachyzoite yield could not be predicted neither by invasion-related parameters (velocity and half time invasion) nor by proliferation parameters (lag phase and doubling time (dT)). The lytic cycle of Besnoitia was asynchronous as evidenced by the presence of three different plaque-forming tachyzoite categories (lysis plaques, large and small parasitophorous vacuoles). Conclusions This study provides first insights into the lytic cycle of B. besnoiti isolates and a standardised in vitro model that allows screening of drug candidates for the treatment of besnoitiosis.

Effect of calf purchase and other herd-level risk factors on mortality, unwanted early slaughter, and use of antimicrobial group treatments in Swiss veal calf operations.

The objective of this survey was to determine herd level risk factors for mortality, unwanted early slaughter, and metaphylactic application of antimicrobial group therapy in Swiss veal calves in 2013. A questionnaire regarding farm structure, farm management, mortality and antimicrobial use was sent to all farmers registered in a Swiss label program setting requirements for improved animal welfare and sustainability. Risk factors were determined by multivariable logistic regression. A total of 619 veal producers returned a useable questionnaire (response rate=28.5%), of which 40.9% only fattened their own calves (group O), 56.9% their own calves and additional purchased calves (group O&P), and 2.3% only purchased calves for fattening (group P). A total number of 19,077 calves entered the fattening units in 2013, of which 21.7%, 66.7%, and 11.6% belonged to groups O, O&P, and P, respectively. Mortality was 0% in 322 herds (52.0%), between 0% and 3% in 47 herds (7.6%), and ≥3% in 250 herds (40.4%). Significant risk factors for mortality were purchasing calves, herd size, higher incidence of BRD, and access to an outside pen. Metaphylaxis was used on 13.4% of the farms (7.9% only upon arrival, 4.4% only later in the fattening period, 1.1% upon arrival and later), in 3.2% of the herds of group O, 17.9% of those in group O&P, and 92.9% of those of group P. Application of metaphylaxis upon arrival was positively associated with purchase (OR=8.9) and herd size (OR=1.2 per 10 calves). Metaphylaxis later in the production cycle was positively associated with group size (OR=2.9) and risk of respiratory disease (OR=1.2 per 10% higher risk) and negatively with the use of individual antimicrobial treatment (OR=0.3). In many countries, purchase and a large herd size are inherently connected to veal production. The Swiss situation with large commercial but also smaller herds with little or no purchase of calves made it possible to investigate the effect of these factors on mortality and antimicrobial drug use. The results of this study show that a system where small farms raise the calves from their own herds has a substantial potential to improve animal health and reduce antimicrobial drug use.

Antimicrobial drug use and risk factors associated with treatment incidence and mortality in Swiss veal calves reared under improved welfare conditions

Ninety-one Swiss veal farms producing under a label with improved welfare standards were visited between August and December 2014 to investigate risk factors related to antimicrobial drug use and mortality. All herds consisted of own and purchased calves, with a median of 77.4% of purchased calves. The calves' mean age was 29±15days at purchasing and the fattening period lasted at average 120±28 days. The mean carcass weight was 125±12kg. A mean of 58±33 calves were fattened per farm and year, and purchased calves were bought from a mean of 20±17 farms of origin. Antimicrobial drug treatment incidence was calculated with the defined daily dose methodology. The mean treatment incidence (TIADD) was 21±15 daily doses per calf and year. The mean mortality risk was 4.1%, calves died at a mean age of 94±50 days, and the main causes of death were bovine respiratory disease (BRD, 50%) and gastro-intestinal disease (33%). Two multivariable models were constructed, for antimicrobial drug treatment incidence (53 farms) and mortality (91 farms). No quarantine, shared air space for several groups of calves, and no clinical examination upon arrival at the farm were associated with increased antimicrobial treatment incidence. Maximum group size and weight differences >100kg within a group were associated with increased mortality risk, while vaccination and beef breed were associated with decreased mortality risk. The majority of antimicrobial treatments (84.6%) were given as group treatments with oral powder fed through an automatic milk feeding system. Combination products containing chlortetracycline with tylosin and sulfadimidine or with spiramycin were used for 54.9%, and amoxicillin for 43.7% of the oral group treatments. The main indication for individual treatment was BRD (73%). The mean age at the time of treatment was 51 days, corresponding to an estimated weight of 80-100kg. Individual treatments were mainly applied through injections (88.5%), and included administration of fluoroquinolones in 38.3%, penicillines (amoxicillin or benzylpenicillin) in 25.6%, macrolides in 13.1%, tetracyclines in 12.0%, 3th and 4th generation cephalosporines in 4.7%, and florfenicol in 3.9% of the cases. The present study allowed for identifying risk factors for increased antimicrobial drug treatment and mortality. This is an important basis for future studies aiming at reducing treatment incidence and mortality in veal farms. Our results indicate that improvement is needed in the selection of drugs for the treatment of veal calves according to the principles of prudent use of antibiotics.

Coxiella burnetii Infections in Small Ruminants and Humans in Switzerland

The recent Q fever epidemic in the Netherlands raised concerns about the potential risk of outbreaks in other European countries. In Switzerland, the prevalence of Q fever in animals and humans has not been studied in recent years. In this study, we describe the current situation with respect to Coxiella (C.) burnetii infections in small ruminants and humans in Switzerland, as a basis for future epidemiological investigations and public health risk assessments. Specific objectives of this cross-sectional study were to (i) estimate the seroprevalence of C. burnetii in sheep and goats, (ii) quantify the amount of bacteria shed during abortion and (iii) analyse temporal trends in human C. burnetii infections. The seroprevalence of C. burnetii in small ruminants was determined by commercial ELISA from a representative sample of 100 sheep flocks and 72 goat herds. Herd-level seroprevalence was 5.0% (95% CI: 1.6-11.3) for sheep and 11.1% (95% CI: 4.9-20.7) for goats. Animal-level seroprevalence was 1.8% (95% CI: 0.8-3.4) for sheep and 3.4% (95% CI: 1.7-6) for goats. The quantification of C. burnetii in 97 ovine and caprine abortion samples by real-time PCR indicated shedding of >10(4) bacteria/g in 13.4% of all samples tested. To our knowledge, this is the first study reporting C. burnetii quantities in a large number of small ruminant abortion samples. Annual human Q fever serology data were provided by five major Swiss laboratories. Overall, seroprevalence in humans ranged between 1.7% and 3.5% from 2007 to 2011, and no temporal trends were observed. Interestingly, the two laboratories with significantly higher seroprevalences are located in the regions with the largest goat populations as well as, for one laboratory, with the highest livestock density in Switzerland. However, a direct link between animal and human infection data could not be established in this study.

[Evaluation of risk factors for Mastitis-Metritis-Agalactia in pig farms in Switzerland].

Mastitis-Metritis-Agalactia (MMA), also known as postpartum dysgalactia syndrome (PPDS) is the most important disease complex in sows after birth. The present study compared 30 MMA problem herds (over 12% of farrowing sows affected) with 30 control farms (less than 10% of farrowing sows affected) to identify risk factors and treatment incidence. Important risk factors identified were in gilts the integration into the herd after the first farrowing, in gestating sows firm fecal consistency as well as in lactating sows soiled troughs, a low flow rate (<2 liters per minute) in drinking nipples and a high prevalence of lameness. The treatment incidence was also significantly different between the two groups. The MMA prevalence could be reduced through optimization of husbandry, feeding and management, which could essentially diminish the use of antibiotics.

Longitudinal study on the colonisation and transmission of methicillin-resistant Staphylococcus aureus in pig farms.

Knowledge about the dynamics of methicillin-resistant Staphylococcus aureus (MRSA) in pigs lacks detail at the level of individual animal. The aim of our study was therefore to determine the colonisation status of MRSA in individual pigs from birth to slaughter in order to gain a better understanding of substantial factors involved in transmission. Two farrow-to-finish and two grow-to-finish herds were included in the study. A total of 1728 nasal swabs from 390 pigs and 592 environmental wipes were collected at 11 different time points. Intermittent colonisation throughout the entire production cycle was conspicuous in the tracking of MRSA in individual pigs. Almost all pigs from a MRSA-positive herd changed MRSA status several times, which implies that pigs are transiently rather than permanently colonised. We highly recommend the definition of MRSA status at herd level rather that at the level of the individual pig when considering prevention measures against MRSA. Therefore, to avoid the further spread of MRSA in countries with moderate prevalence, such as in Switzerland, defining farms as MRSA positive or MRSA negative and allowing the trade of pigs only within herds of the same status seems feasible. This will also be important for combating the further dissemination of livestock-associated (LA)-MRSA into healthcare facilities and the community via humans who have close contact with animals.

Observations of muskoxen and caribou in the high Arctic

Summary: Summer daily activity and movement patterns and habitat selectivity by Peary caribou (Rangifer tarandus pearyi) and muskoxen (Ovibos moschatus) were studied at two sites in Canada's High Arctic. Caribou showed a greater mobility and broader selection of habitat than muskoxen. Muskoxen fed more than they rested in contrast to the greater amount of time spent resting than feeding by caribou. The sedge-producing hydric-meadow vegetalion type was highly selected for by muskoxen at both study areas; caribou selected against the hydric-meadow type and preferred the polar desert and mesic-meadow types. Caribou displayed a greater variety in plant species selection than muskoxen, favouring willow (Salix arctica), grasses, forbs, and the flowers of vascular plants- Muskoxen feci extensively on sedges in the hydric-meadow. It is suggested the abundance and distribution of sedge-producing meadows may control the regional abundance and distribution of muskoxen. Winter climate is probably the ultimate factor controlling densities of muskoxen and caribou in the High Arctic.

Potential connections (least-cost paths) between existing and potential bison habitat patches in the Carpathian Mountains

Habitat connectivity is important for the survival of species that occupy habitat patches too small to sustain an isolated population. A prominent example of such a species is the European bison (Bison bonasus), occurring only in small, isolated herds, and whose survival will depend on establishing larger, well-connected populations. Our goal here was to assess habitat connectivity of European bison in the Carpathians. We used an existing bison habitat suitability map and data on dispersal barriers to derive cost surfaces, representing the ability of bison to move across the landscape, and to delineate potential connections (as least-cost paths) between currently occupied and potential habitat patches. Graph theory tools were then employed to evaluate the connectivity of all potential habitat patches and their relative importance in the network. Our analysis showed that existing bison herds in Ukraine are isolated. However, we identified several groups of well-connected habitat patches in the Carpathians which could host a large population of European bison. Our analysis also located important dispersal corridors connecting existing herds, and several promising locations for future reintroductions (especially in the Eastern Carpathians) that should have a high priority for conservation efforts. In general, our approach indicates the most important elements within a landscape mosaic for providing and maintaining the overall connectivity of different habitat networks and thus offers a robust and powerful tool for conservation planning.

Tab.1: Total estimated muskox numbers and quotas in management zones of the Northwest Territories and Nunavut

In view of the drastic growth in the Canadian Inuit population, the rising costs of living, the missing job and income alternatives and the high unemployment rate in the arctic, efforts are being made to make use of the muskox populations in order to provide additional sources of food and/or revenue. The present paper attempts to review the course of muskox utilization in the Canadian Arctic and to tentatively assess its present as weIl as its future economic importance. Starting with the pre-European status of muskoxen in Canada, the drastic reduction in numbers resulting from the combined efforts of hide traders, whalers and expedition parties in the 19th and early 20th centuries, the impact of the legal protection and the recovery since 1917 are being described. Establishing muskox farms with semi-domesticated herds failed in Canada in the 1970's. Since 1969, though, increasing numbers of animals have been allotted to many Inuit communities, and despite the fact that most of the animals were primarily used for subsistence purposes, some communities could reserve part of their quotas for trophy (sport) hunters. While controlled sustainable subsistence and trophy hunts may eventually be carried out over the whole muskox range, including recently colonized northern Quebec, commercial harvesting for meat, hides and wool, introduced in 1981, will at least for some time be restricted to Banks and Victoria islands which at present show 78 % of the Canadian muskox population and 94 % of the overall quota.

Integration of socioeconomic and genetic aspects involved in the conservation of animal genetic resources = Integración de aspectos socioeconómicos y genéticos involucrados en la conservación de recursos genéticos animales

El sector ganadero está siendo gradualmente dominado por sistemas intensivos y especializados en los que los factores de producción están controlados y en los que los caracteres productivos son los criterios principales para la selección de especies y razas. Entretanto, muchos de los bienes y servicios que tradicionalmente suministraba el ganado, tales como los fertilizantes, la tracción animal o materias primas para la elaboración vestimenta y calzado están siendo reemplazados por productos industriales. Como consecuencia de ambos cambios, las razas seleccionadas intensivamente, las cuales están estrechamente ligadas a sistemas agrícolas de alta producción y altos insumos, han desplazado a muchas razas autóctonas, en las que la selección prácticamente ha cesado o es muy poco intensa. Actualmente existe una mayor conciencia social sobre la situación de las razas autóctonas y muchas funciones del ganado que previamente habían sido ignoradas están siendo reconocidas. Desde hace algunas décadas, se ha aceptado internacionalmente que las razas de ganado cumplen funciones económicas, socio-culturales, medioambientales y de seguridad alimentaria. Por ello, diferentes organismos internacionales han reconocido que la disminución de los recursos genéticos de animales domésticos (RGADs) es un problema grave y han recomendado su conservación. Aun así, la conservación de RGADs es un tema controvertido por la dificultad de valorar las funciones del ganado. Esta valoración es compleja debido que los RGADs tiene una doble naturaleza privada - pública. Como algunos economistas han subrayado, el ganado es un bien privado, sin embargo debido a algunas de sus funciones, también es un bien público. De esta forma, el aumento del conocimiento sobre valor de cada una de sus funciones facilitaría la toma de decisiones en relación a su conservación y desarrollo. Sin embargo, esta valoración es controvertida puesto que la importancia relativa de las funciones del ganado varía en función del momento, del lugar, de las especies y de las razas. El sector ganadero, debido a sus múltiples funciones, está influenciado por factores técnicos, medioambientales, sociales, culturales y políticos que están interrelacionados y que engloban a una enorme variedad de actores y procesos. Al igual que las funciones del ganado, los factores que afectan a su conservación y desarrollo están fuertemente condicionados por localización geográfica. Asimismo, estos factores pueden ser muy heterogéneos incluso dentro de una misma raza. Por otro lado, es razonable pensar que el ganadero es el actor principal de la conservación de razas locales. Actualmente, las razas locales están siendo Integration of socioeconomic and genetic aspects involved in the conservation of animal genetic resources 5 explotadas por ganaderos muy diversos bajo sistemas de producción también muy diferentes. Por todo ello, es de vital importancia comprender y evaluar el impacto que tienen las motivaciones, y el proceso de toma de decisiones de los ganaderos en la estructura genética de las razas. En esta tesis doctoral exploramos diferentes aspectos sociales, económicos y genéticos involucrados en la conservación de razas locales de ganado vacuno en Europa, como ejemplo de RGADs, esperando contribuir al entendimiento científico de este complejo tema. Nuestro objetivo es conseguir una visión global de los procesos subyacentes en la conservación y desarrollo de estas razas. Pretendemos ilustrar como se pueden utilizar métodos cuantitativos en el diseño y establecimiento de estrategias de conservación y desarrollo de RGADs objetivas y adecuadas. En primer lugar, exploramos el valor económico total (VET) del ganado analizando sus componentes públicos fuera de mercado usando como caso de estudio la raza vacuna Alistana-Sanabresa (AS). El VET de cualquier bien está formado por componentes de uso y de no-uso. Estos últimos incluyen el valor de opción, el valor de herencia y el valor de existencia. En el caso del ganado local, el valor de uso directo proviene de sus productos. Los valores de uso indirecto están relacionados con el papel que cumple las razas en el mantenimiento de los paisajes y cultura rural. El valor de opción se refiere a su futuro uso potencial y el valor de herencia al uso potencial de las generaciones venideras. Finalmente, el valor de existencia está relacionado con el bienestar que produce a la gente saber que existe un recurso específico. Nuestro objetivo fue determinar la importancia relativa que tienen los componentes fuera de mercado sobre el VET de la raza AS. Para ello evaluamos la voluntad de la gente a pagar por la conservación de la AS mediante experimentos de elección (EEs) a través de encuestas. Estos experimentos permiten valorar individualmente los distintos componentes del VET de cualquier bien. Los resultados los analizamos mediante de uso de modelos aleatorios logit. Encontramos que las funciones públicas de la raza AS tienen un valor significativo. Sus valores más importantes son el valor de uso indirecto como elemento cultural Zamorano y el valor de existencia (ambos representaron el 80% de VET). Además observamos que el valor que gente da a las funciones públicas de la razas de ganado dependen de sus características socioeconómicas. Los factores que condicionaron la voluntad a pagar para la conservación de la raza AS fueron el lugar de residencia (ciudad o pueblo), el haber visto animales de la raza o haber consumido sus productos y la actitud de los encuestados ante los conflictos entre el desarrollo económico y el medioambiente. Por otro lado, encontramos que no todo el mundo tiene una visión completa e integrada de todas las funciones públicas de la raza AS. Por este motivo, los programas o actividades de concienciación sobre su estado deberían hacer hincapié en este aspecto. La existencia de valores públicos de la raza AS implica que los ganaderos deberían recibir compensaciones económicas como pago por las funciones públicas que cumple su raza local. Las compensaciones asegurarían un tamaño de población que permitiría que la raza AS siga realizando estas funciones. Un mecanismo para ello podría ser el desarrollo del turismo rural relacionado con la raza. Esto aumentaría el valor de uso privado mientras que supondría un elemento añadido a las estrategias de conservación y desarrollo. No obstante, los ganaderos deben analizar cómo aprovechar los nichos de mercado existentes, así como mejorar la calidad de los productos de la raza prestando especial atención al etiquetado de los mismos. Una vez evaluada la importancia de las funciones públicas de las razas locales de ganado, analizamos la diversidad de factores técnicos, económicos y sociales de la producción de razas locales de ganado vacuno existente en Europa. Con este fin analizamos el caso de quince razas locales de ocho países en el contexto de un proyecto de colaboración internacional. Investigamos las diferencias entre los países para determinar los factores comunes clave que afectan a la viabilidad de las razas locales. Para ello entrevistamos mediante cuestionarios a un total de 355 ganaderos en las quince razas. Como indicador de viabilidad usamos los planes de los ganaderos de variación del tamaño de las ganaderías. Los cuestionarios incluían diferentes aspectos económicos, técnicos y sociales con potencial influencia en las dinámicas demográficas de las razas locales. Los datos recogidos los analizamos mediante distintas técnicas estadísticas multivariantes como el análisis discriminante y la regresión logística. Encontramos que los factores que afectan a la viabilidad de las razas locales en Europa son muy heterogéneos. Un resultado reseñable fue que los ganaderos de algunos países no consideran que la explotación de su raza tenga un alto valor social. Este hecho vuelve a poner de manifiesto la importancia de desarrollar programas Europeos de concienciación sobre la importancia de las funciones que cumplen las razas locales. Además los países analizados presentaron una alta variabilidad en cuanto a la importancia de los mercados locales en la distribución de los productos y en cuanto al porcentaje en propiedad del total de los pastos usados en las explotaciones. Este estudio reflejó la variabilidad de los sistemas y medios de producción (en el sentido socioeconómico, técnico y ecológico) que existe en Europa. Por ello hay que ser cautos en la implementación de las políticas comunes en los diferentes países. También encontramos que la variabilidad dentro de los países puede ser elevada debido a las diferencias entre razas, lo que implica que las políticas nacionales deber ser suficientemente flexibles para adaptarse a las peculiaridades de cada una de las razas. Por otro lado, encontramos una serie de factores comunes a la viabilidad de las razas en los distintos países; la edad de los ganaderos, la colaboración entre ellos y la apreciación social de las funciones culturales, medioambientales y sociales del ganado local. El envejecimiento de los ganaderos de razas locales no es solo un problema de falta de transferencia generacional, sino que también puede suponer una actitud más negativa hacia la inversión en las actividades ganaderas y en una menor capacidad de adaptación a los cambios del sector. La capacidad de adaptación de los ganaderos es un factor crucial en la viabilidad de las razas locales. Las estrategias y políticas de conservación comunes deben incluir las variables comunes a la viabilidad de las razas manteniendo flexibilidad suficiente para adaptarse a las especificidades nacionales. Estas estrategias y políticas deberían ir más allá de compensación económica a los ganaderos de razas locales por la menor productividad de sus razas. Las herramientas para la toma de decisiones ayudan a generar una visión amplia de la conservación y desarrollo de las razas locales. Estas herramientas abordan el diseño de estrategias de conservación y desarrollo de forma sistemática y estructurada. En la tercera parte de la tesis usamos una de estas herramientas, el análisis DAFO (Debilidades, Amenazas, Fortalezas y Oportunidades), con este propósito, reconociendo que la conservación de RGADs depende de los ganaderos. Desarrollamos un análisis DAFO cuantitativo y lo aplicamos a trece razas locales de ganado vacuno de seis países europeos en el contexto del proyecto de colaboración mencionado anteriormente. El método tiene cuatro pasos: 1) la definición del sistema; 2) la identificación y agrupación de los factores influyentes; 3) la cuantificación de la importancia de dichos factores y 4) la identificación y priorización de estrategias. Identificamos los factores utilizando multitud de agentes (multi-stakeholder appproach). Una vez determinados los factores se agruparon en una estructura de tres niveles. La importancia relativa de los cada uno de los factores para cada raza fue determinada por grupos de expertos en RGADs de los países integrados en el citado proyecto. Finalmente, desarrollamos un proceso de cuantificación para identificar y priorizar estrategias. La estructura de agrupación de factores permitió analizar el problema de la conservación desde el nivel general hasta el concreto. La unión de análisis específicos de cada una de las razas en un análisis DAFO común permitió evaluar la adecuación de las estrategias a cada caso concreto. Identificamos un total de 99 factores. El análisis reveló que mientras los factores menos importantes son muy consistentes entre razas, los factores y estrategias más relevantes son muy heterogéneos. La idoneidad de las estrategias fue mayor a medida que estas se hacían más generales. A pesar de dicha heterogeneidad, los factores influyentes y estrategias más importantes estaban ligados a aspectos positivos (fortalezas y oportunidades) lo que implica que el futuro de estas razas es prometedor. Los resultados de nuestro análisis también confirmaron la gran relevancia del valor cultural de estas razas. Las factores internos (fortalezas y debilidades) más importantes estaban relacionadas con los sistemas de producción y los ganaderos. Las oportunidades más relevantes estaban relacionadas con el desarrollo y marketing de nuevos productos mientras que las amenazas más importantes se encontraron a la hora de vender los productos actuales. Este resultado implica que sería fructífero trabajar en la motivación y colaboración entre ganaderos así como, en la mejora de sus capacidades. Concluimos que las políticas comunes europeas deberían centrarse en aspectos generales y ser los suficientemente flexibles para adaptarse a las singularidades de los países y las razas. Como ya se ha mencionado, los ganaderos juegan un papel esencial en la conservación y desarrollo de las razas autóctonas. Por ello es relevante entender que implicación puede tener la heterogeneidad de los mismos en la viabilidad de una raza. En la cuarta parte de la tesis hemos identificado tipos de ganaderos con el fin de entender cómo la relación entre la variabilidad de sus características socioeconómicas, los perfiles de las ganaderías y las dinámicas de las mismas. El análisis se ha realizado en un contexto sociológico, aplicando los conceptos de capital cultural y económico. Las tipologías se han determinado en función de factores socioeconómicos y culturales indicadores del capital cultural y capital económico de un individuo. Nuestro objetivo era estudiar si la tipología socioeconómica de los ganaderos afecta al perfil de su ganadería y a las decisiones que toman. Entrevistamos a 85 ganaderos de la raza Avileña-Negra Ibérica (ANI) y utilizamos los resultados de dichas entrevistas para ilustrar y testar el proceso. Definimos los tipos de ganaderos utilizando un análisis de clúster jerarquizado con un grupo de variables canónicas que se obtuvieron en función de cinco factores socioeconómicos: el nivel de educación del ganadero, el año en que empezó a ser ganadero de ANI, el porcentaje de los ingresos familiares que aporta la ganadería, el porcentaje de propiedad de la tierra de la explotación y la edad del ganadero. La tipología de los ganaderos de ANI resultó ser más compleja que en el pasado. Los resultados indicaron que los tipos de ganaderos variaban en muchos aspectos socioeconómicos y en los perfiles de sus Integration of socioeconomic and genetic aspects involved in the conservation of animal genetic resources 9 ganaderías. Los tipos de ganaderos determinados toman diferentes decisiones en relación a la modificación del tamaño de su ganadería y a sus objetivos de selección. Por otro lado, reaccionaron de forma diferente ante un hipotético escenario de reducción de las compensaciones económicas que les planteamos. En este estudio hemos visto que el capital cultural y el económico interactúan y hemos explicado como lo hacen en los distintos tipos de ganaderos. Por ejemplo, los ganaderos que poseían un mayor capital económico, capital cultural formal y capital cultural adquirido sobre la raza, eran los ganaderos cuyos animales tenían una mayor demanda por parte de otros ganaderos, lo cual podría responder a su mayor prestigio social dentro de la raza. Uno de los elementos claves para el futuro de la raza es si este prestigio responde a una superioridad genética de las animales. Esto ocurriría si los ganaderos utilizaran las herramientas que tienen a su disposición a la hora de seleccionar animales. Los tipos de ganaderos identificados mostraron también claras diferencias en sus formas de colaboración y en su reacción a una hipotética variación de las compensaciones económicas. Aunque algunos tipos de ganaderos mostraron un bajo nivel de dependencia a estas compensaciones, la mayoría se manifestaron altamente dependientes. Por ello cualquier cambio drástico en la política de ayudas puede comprometer el desarrollo de las razas autóctonas. La adaptación las políticas de compensaciones económicas a la heterogeneidad de los ganaderos podría aumentar la eficacia de las mismas por lo que sería interesante explorar posibilidades a este respecto. Concluimos destacando la necesidad de desarrollar políticas que tengan en cuenta la heterogeneidad de los ganaderos. Finalmente abordamos el estudio de la estructura genética de poblaciones ganaderas. Las decisiones de los ganaderos en relación a la selección de sementales y su número de descendientes configuran la estructura demográfica y genética de las razas. En la actualidad existe un interés renovado por estudiar las estructuras poblacionales debido a la influencia potencial de su estratificación sobre la predicción de valores genómicos y/o los análisis de asociación a genoma completo. Utilizamos dos métodos distintos, un algoritmo de clústeres basados en teoría de grafos (GCA) y un algoritmo de clustering bayesiano (STRUCTURE) para estudiar la estructura genética de la raza ANI. Prestamos especial atención al efecto de la presencia de parientes cercanos en la población y de la diferenciación genética entre subpoblaciones sobre el análisis de la estructura de la población. En primer lugar evaluamos el comportamiento de los dos algoritmos en poblaciones simuladas para posteriormente analizar los genotipos para 17 microsatélites de 13343 animales de 57 ganaderías distintas de raza ANI. La ANI es un ejemplo de raza con relaciones complejas. Por otro lado, utilizamos el archivo de pedigrí de la raza para estudiar el flujo de genes, calculando, entre otras cosas, la contribución de cada ganadería a la constitución genética de la raza. En el caso de las poblaciones simuladas, cuando el FST entre subpoblaciones fue suficientemente alto, ambos algoritmos, GCA y STRUCTURE, identificaron la misma estructura genética independientemente de que existieran o no relaciones familiares. Por el contrario, cuando el grado de diferenciación entre poblaciones fue bajo, el STRUCTURE identificó la estructura familiar mientras que GCA no permitió obtener ningún resultado concluyente. El GCA resultó ser un algoritmo más rápido y eficiente para de inferir la estructura genética en poblaciones con relaciones complejas. Este algoritmo también puede ser usado para reducir el número de clústeres a testar con el STRUTURE. En cuanto al análisis de la población de ANI, ambos algoritmos describieron la misma estructura, lo cual sugiere que los resultados son robustos. Se identificaron tres subpoblaciones diferenciadas que pudieran corresponderse con tres linajes distintos. Estos linajes estarían directamente relacionados con las ganaderías que han tenido una mayor contribución a la constitución genética de la raza. Por otro lado, hay un conjunto muy numeroso de individuos con una mezcla de orígenes. La información molecular describe una estructura estratificada de la población que se corresponde con la evolución demográfica de la raza. Es esencial analizar en mayor profundidad la composición de este último grupo de animales para determinar cómo afecta a la variabilidad genética de la población de ANI. SUMMARY Summary Livestock sector is gradually dominated by intensive and specialized systems where the production environment is controlled and the production traits are the main criteria for the selection of species and breeds. In the meantime, the traditional use of domestic animals for draught work, clothes and manure has been replaced by industrial products. As a consequence of both these changes, the intensively selected breeds closely linked with high-input highoutput production systems have displaced many native breeds where the selection has practically ceased or been very mild. People are now more aware of the state of endangerment among the native breeds and the previously ignored values of livestock are gaining recognition. For some decades now, the economic, socio-cultural, environmental and food security function of livestock breeds have been accepted worldwide and their loss has been recognized as a major problem. Therefore, the conservation of farm animal genetic resources (FAnGR) has been recommended. The conservation of FAnGR is controversial due to the complexity of the evaluation of its functions. This evaluation is difficult due to the nature of FAnGR both as private and public good. As some economists have highlighted, livestock animals are private goods, however, they are also public goods by their functions. Therefore, there is a need to increase the knowledge about the value of all livestock functions since to support the decision-making for the sustainable conservation and breeding of livestock. This is not straightforward since the relative importance of livestock functions depends on time, place, species and breed. Since livestock play a variety of roles, their production is driven by interrelated and everchanging economic, technical, environmental, social, cultural and political elements involving an enormous range of stakeholders. Not only FAnGR functions but also the importance of factors affecting the development and conservation of FAnGR can be very different across geographical areas. Furthermore, heterogeneity can be found even within breeds. Local breeds are nowadays raised by highly diverse farmers in equally diverse farms. It is quite reasonable to think that farmer is the major actor in the in situ conservation of livestock breeds. Thus, there is a need to understand the farmers’ motivations, decision making processes and the impact of their decisions on the genetic structure of breeds. In this PhD thesis we explore different social, economic and genetic aspects involved in the conservation of local cattle breeds, i.e. FAnGR, in Europe seeking to contribute to the scientific understanding of this complex issue. We aim to achieve a comprehensive view of the processes involved in the conservation and development of local cattle breeds and have made special efforts in discussing the implications of the research results in this respect. The final outcome of the thesis is to illustrate how quantitative methods can be exploited in designing and establishing sound strategies and programmes for the conservation and development of local livestock breeds. Firstly we explored the public non-market attributes of the total economic value (TEV) of livestock, using the Spanish Alistana-Sanabresa (AS) cattle breed as a case study. Total economic value of any good comprises both use and non-use components, where the latter include option, bequest and existence values. For livestock, the direct use values are mainly stemming from production outputs. Indirect use values relate to the role of livestock as a maintainer of rural culture and landscape. The option value is related to the potential use of livestock, the bequest values relate to the value associated with the inheritance of the resources to future generation and the existence values relate to the utility perceived by people from knowing that specific resources exist. We aimed to determine the relative importance of the non-market components of the TEV of the AS breed, the socio-economic variables that influence how people value the different components of TEV and to assess the implications of the Spanish national conservation strategy for the AS breed. To do so, we used a choice experiment (CE) approach and applied the technique to assess people’s willingness to pay (WTP) for the conservation of AS breed. The use of CE allows the valuation of the individual components of TEV for a given good. We analysed the choice data using a random parameter logit (RPL) model. AS breed was found to have a significant public good value. Its most important values were related to the indirect use value due to the maintenance of Zamorian culture and the existence value (both represent over 80% of its TEV). There were several socioeconomic variables influencing people’s valuation of the public service of the breed. In the case of AS breed, the place of living (city or rural area), having seen animals of the breed, having eaten breed products and the respondents’ attitude towards economic development – environment conflicts do influence people’s WTP for AS conservation. We also found that people do not have a complete picture of all the functions and roles that AS breed as AnGR. Therefore, the actions for increasing awareness of AS should go to that direction. The farmers will need incentives to exploit some of the public goods values and maintain the breed population size at socially desirable levels. One such mechanism could be related to the development of agritourism, which would enhance the private good value and provide an important addition to the conservation and utilisation strategy. However, the farmers need a serious evaluation on how to invest in niche product development or how to improve product quality and brand recognition. Using the understanding on the importance of the public function of local cattle we tried to depict the current diversity regarding technical, economic and social factors found in local cattle farming across Europe. To do so we focused in an international collaborative project on the case of fifteen local cattle breeds in eight European countries. We investigated the variation among the countries to detect the common key elements, which affect the viability of local breeds. We surveyed with interviews a total of 355 farms across the fifteen breeds. We used the planned herd size changes by the farmer as an indicator of breed viability. The questionnaire included several economic, technical and social aspects with potential influence on breeds’ demographic trends. We analysed the data using multivariate statistical techniques, such as discriminat analysis and logistic regression. The factors affecting a local breed’s viability were highly heterogeneous across Europe. In some countries, farmers did not recognise any high social value attached to keeping a local cattle breed. Hence there is a need to develop communication programmes across EU countries making people aware about the diversity and importance of values associated to raising local breeds. The countries were also very variable regarding the importance of local markets and the percentage of farm land owned by the farmers. Despite the country specificities, there were also common factors affecting the breed viability across Europe. The factors were from different grounds, from social, such as the age of the farmer and the social appreciation of their work, to technicalorganizational, such as the farmers’ attitude to collaborating with each other. The heterogeneity found reflects the variation in breeding systems and production environment (in the socioeconomic, technical and ecological sense) present in Europe. Therefore, caution should be taken in implementing common policies at the country level. Variability could also be rather high within countries due to breed specificities. Therefore, the national policies should be flexible to adapt to the specificities. The variables significantly associated with breed viability should be positively incorporated in the conservation strategies, and considered in developing common and/or national policies. The strategy preparation and policy planning should go beyond the provision of a general economic support to compensate farmers for the lower profitability of local breeds. Of particular interest is the observation that the opportunity for farmer collaboration and the appreciation by the society of the cultural, environmental and social role of local cattle farming were positively associated with the breed survival. In addition, farmer's high age is not only a problem of poor generation transfer but it is also a problem because it might lead to a lower attitude to investing in farming activities and to a lower ability to adapt to environment changes. The farmers’ adaptation capability may be a key point for the viability of local breeds. Decision making tools can help to get a comprehensive view on the conservation and development of local breeds. It allows us to use a systematic and structured approach for identifying and prioritizing conservation and development strategies. We used SWOT (Strengths, Weaknesses Opportunities and Threats) analysis for this purpose and recognized that many conservation and development projects rely on farmers. We developed a quantified SWOT method and applied it in the aforementioned collaborative research to a set of thirteen cattle breeds in six European countries. The method has four steps: definition of the system, identification and grouping of the driving factors, quantification of the importance of driving factors and identification and prioritization of the strategies. The factors were determined following a multi-stakeholder approach and grouped with a three level structure. FAnGR expert groups ranked the factors and a quantification process was implemented to identify and prioritize strategies. The structure of the SWOT analysis allowed analyzing the conservation problem from general down to specific perspectives. Joining breed specific analyses into a common SWOT analysis permitted comparison of breed cases across countries. We identified 99 driving factors across breeds. The across breed analysis revealed that irrelevant factors were consistent. There was high heterogeneity among the most relevant factors and strategies. The strategies increased eligibility as they lost specificity. Although the situation was very heterogeneous, the most promising factors and strategies were linked to the positive aspects (Strengths and Opportunities). Therefore, the future of the studied local breed is promising. The results of our analysis also confirmed the high relevance of the cultural value of the breeds. The most important internal factors (strengths and weaknesses) were related farmers and production systems. The most important opportunities were found in developing and marketing new products, while the most relevant threats were found in selling the current conventional products. In this regard, it should be fruitful to work on farmers’ motivation, collaboration, and capacity building. We conclude that European policies should focus on general aspects and be flexible enough to be adapted to the country and breed specificities. As mentioned, farmers have a key role in the conservation and development of a local cattle breed. Therefore, it is very relevant to understand the implications of farmer heterogeneity within a breed for its viability. In the fourth part of the thesis, we developed a general farmer typology to help analyzing the relations between farmer features and farm profiles, herd dynamics and farmers’ decision making. In the analysis we applied and used the sociological framework of economic and cultural capital and studied how the determined farmer types were linked to farm profiles and breeding decisions, among others. The typology was based on measurable socioeconomic factors indicating the economic and cultural capital of farmers. A group of 85 farmers raising the Spanish Avileña-Negra Ibérica (ANI) local cattle breed was used to illustrate and test the procedure. The farmer types were defined by a hierarchical cluster analysis with a set of canonical variables derived from the following five the socioeconomic factors: the formal educational level of the farmer, the year the farmer started keeping the ANI breed, the percentage of the total family income covered by the farm, the percentage of the total farm land owned by the farmer and the farmer’s age. The present ANI farmer types were much more complex than what they were in the past. We found that the farmer types differed in many socioeconomic aspects and in the farms profile. Furthermore, the types also differentiate farmers with respect to decisions about changing the farm size, breeding aims and stated reactions towards hypothetical subsidy variation. We have verified that economic and cultural capitals are not independent and further showed how they are interacting in the different farmer types. The farmers related to the types with high economic, institutionalized and embodied cultural capitals had a higher demand of breeding animals from others farmers of the breed, which may be related to the higher social prestige within the breed. One of the key implications of this finding for the future of the breed is whether or not the prestige of farmers is related to genetic superiority of their animals, what is to say, that it is related with a sound use of tools that farmers have available to make selection decisions. The farmer types differed in the form of collaboration and in the reactions to the hypothetical variation in subsidies. There were farmers with low dependency on subsidies, while most of them are highly dependent on subsidies. Therefore, any drastic change in the subsidy programme might have influence on the development of local breeds. The adaptation of these programme to the farmers’ heterogeneity might increase its efficacy, thus it would be interesting to explore ways of doing it. We conclude highlighting the need to have a variety of policies, which take into account the heterogeneity among the farmers. To finish we dealt with the genetic structure of livestock populations. Farmers’ decisions on the breeding animals and their progeny numbers shape the demographic and genetic structure of the breeds. Nowadays there is a renovated interest in studying the population structure since it can bias the prediction of genomic breeding values and genome wide association studies. We determined the genetic structure of ANI breed using two different methods, a graphical clustering algorithm (GCA) and a Bayesian clustering algorithm (STRUCTURE) were used. We paid particular attention to the influence that the presence of closely related individuals and the genetic differentiation of subpopulations may have on the inferences about the population structure. We first evaluated the performance of the algorithms in simulated populations. Then we inferred the genetic structure of the Spanish cattle breed ANI analysing a data set of 13343 animals (genotyped for 17 microsatellites) from 57 herds. ANI breed is an example of a population with complex relationships. We used the herdbook to study the gene flow, estimation among other things, the contribution of different herds to the genetic composition of the ANI breed. For the simulated scenarios, when FST among subpopulations was sufficiently high, both algorithms consistently inferred the correct structure regardless of the presence of related individuals. However, when the genetic differentiation among subpopulations was low, STRUCTURE identified the family based structure while GCA did not provide any consistent picture. The GCA was a fast and efficient method to infer genetic structure to determine the hidden core structure of a population with complex history and relationships. GCA could also be used to narrow down the number of clusters to be tested by STRUCTURE. Both, STRUCTURE and GCA describe a similar structure for the ANI breed suggesting that the results are robust. ANI population was found to have three genetically differentiated clusters that could correspond to three genetic lineages. These are directly related to the herds with a major contribution to the breed. In addition, ANI breed has also a large pool made of individuals with an admixture of origins. The genetic structure of ANI, assessed by molecular information, shows a stratification that corresponds to the demographic evolution of the breed. It will be of great importance to learn more about the composition of the pool and study how it is related to the existing genetic variability of the breed.

A novel virus in swine is closely related to the human hepatitis E virus

A novel virus, designated swine hepatitis E virus (swine HEV), was identified in pigs. Swine HEV crossreacts with antibody to the human HEV capsid antigen. Swine HEV is a ubiquitous agent and the majority of swine ≥3 months of age in herds from the midwestern United States were seropositive. Young pigs naturally infected by swine HEV were clinically normal but had microscopic evidence of hepatitis, and developed viremia prior to seroconversion. The entire ORFs 2 and 3 were amplified by reverse transcription–PCR from sera of naturally infected pigs. The putative capsid gene (ORF2) of swine HEV shared about 79–80% sequence identity at the nucleotide level and 90–92% identity at the amino acid level with human HEV strains. The small ORF3 of swine HEV had 83–85% nucleotide sequence identity and 77–82% amino acid identity with human HEV strains. Phylogenetic analyses showed that swine HEV is closely related to, but distinct from, human HEV strains. The discovery of swine HEV not only has implications for HEV vaccine development, diagnosis, and biology, but also raises a potential public health concern for zoonosis or xenozoonosis following xenotransplantation with pig organs.

Endometrial response toward bovine herpesvirus 4 infection

Metriti ed endometriti sono le patologie maggiormente responsabili delle perdite economiche negli allevamenti bovini da latte, specialmente nel periodo successivo al parto. Mentre le metriti coinvolgono e si sviluppano in tutto l’utero e sono caratterizzate dalla presenza di sintomi sistemici, le endometriti consistono in una infiammazione che riguarda il solo endometrio, con la presenza di perdite purulente, distruzione della superficie epiteliale, congestione vascolare, edema stromale ed accumulo di linfociti e plasmacellule. Queste patologie, inoltre, possono causare, disfunzione ovarica, con conseguente infertilità e riduzione sia dell’efficienza riproduttiva della vacca sia della produzione stessa di latte. Nonostante queste malattie siano, nella maggior parte dei casi, correlate all’instaurarsi di infezioni batteriche, che possono subentrare nell’utero direttamente durante il parto, il ruolo di alcuni virus nello sviluppo di queste patologie è stato recentemente approfondito e la correlazione tra l’ Herpesvirus Bovino 4 e l’insorgere di metriti ed endometriti è stata dimostrata. L’ Herpesvirus Bovino 4 (BoHV-4) è un gamma-herpesvirus ed il suo genoma è costituito da una molecola lineare di DNA a doppio filamento con una struttura genomica di tipo B, caratterizzata dalla presenza di un’unica lunga sequenza centrale (LUR) fiancheggiata da multiple sequenze poli-ripetute (prDNA). BoHV-4 è stato isolato sia da animali sani sia da animali con differenti patologie, tra cui malattie oculari e respiratorie, ma soprattutto da casi di metriti, endometriti, vaginiti o aborti. Generalmente, il ruolo svolto dal virus in questo tipo di patologie è associato alla compresenza di altri tipi di patogeni, che possono essere virus, come nel caso del Virus Della Diarrea Virale Bovina (BVDV), o più frequentemente batteri. Usualmente, l’iniziale difesa dell’endometrio bovino nei confronti dei microbi si fonda sul sistema immunitario innato e l’attivazione di specifici recettori cellulari determina la sintesi e la produzione di citochine e chemochine pro infiammatorie, che possono essere in grado di modulare la replicazione di BoHV-4. Il genoma di BoHV-4 possiede due principali trascritti per i geni Immediate Early (IE), trai quali ORF50/IE2 è il più importante ed il suo prodotto, Rta/ORF50, è fortemente conservato tra tutti gli Herpesvirus. Esso è responsabile della diretta trans-attivazione di numerosi geni virali e cellulari e può essere modulato da differenti stimoli extracellulari. Precedentemente è stato dimostrato come il TNF-, prodotto dalle cellule stromali e dai macrofagi all’interno dell’endometrio, in conseguenza ad infezione batterica, sia in grado di aumentare la replicazione di BoHV-4 attraverso l’attivazione del pathway di NFkB e direttamente agendo sul promotore di IE2. Per queste ragioni, è risultato di forte interesse investigare quali potessero essere, invece, i fattori limitanti la replicazione di BoHV-4. In questo lavoro è stata studiata la relazione tra cellule endometriali stromali bovine infettate con l’Herpesvirus Bovino 4 e l’interferon gamma (IFN-) ed è stata dimostrata la capacità di questa molecola di restringere la replicazione di BoHV-4 in maniera IDO1 indipendente ed IE2 dipendente. Inoltre, la presenza di alcuni elementi in grado di interagire con l’ IFN-γ, all’interno del promotore di IE2 di BoHV-4, ha confermato questa ipotesi. Basandoci su questi dati, abbiamo potuto supporre l’esistenza di uno stretto vincolo tra l’attivazione dell’asse dell’interferon gamma e la ridotta replicazione di BoHV-4, andando a porre le basi per una nuova efficiente cura e prevenzione per le patologie uterine. Poiché il meccanismo corretto attraverso il quale BoHV-4 infetta l’endometrio bovino non è ancora ben compreso, è stato interessante approfondire in maniera più accurata l’interazione presente tra il virus ed il substrato endometriale, analizzando le differenze esistenti tra cellule infettate e non, in termini di espressione genica. Basandoci su dati preliminari ottenuti attraverso analisi con RNA sequencing (RNAseq), abbiamo visto come numerosi geni risultino over-espressi in seguito ad infezione con BoHV-4 e come, tra questi, la Metalloproteasi 1 sia uno dei più interessanti, a causa delle sue possibili implicazioni nello sviluppo delle patologie dell’endometrio uterino bovino. Successive analisi, effettuate tramite westernblotting e real time PCR, sono state in grado di confermare tale dato, sottolineando l’efficacia di un nuovo approccio sperimentale, basato sul RNAseq, per lo studio dell’insorgenza delle patologie.

Valutazione della risposta immunitaria cellulare e fenotipizzazione linfocitaria nel suino in corso di infezione naturale da virus della PRRS (“Porcine Reproductive and Respiratory Syndrome”) e dopo stimolazione di PBMC (“Peripheral Blood Mononuclear Cells”) in vitro con Peptide Killer (KP)

SUMMARY The Porcine Reproductive and Respiratory Syndrome (PRRS) virus is one of the most spread pathogens in swine herds all over the world and responsible for a reproductive and respiratory syndrome that causes severe heath and economical problems. This virus emerged in late 1980’s but although about 30 years have passed by, the knowledge about some essential facets related to the features of the virus (pathogenesis, immune response, and epidemiology) seems to be still incomplete. Taking into account that the development of modern vaccines is based on how innate and acquire immunity react, a more and more thorough knowledge on the immune system is needed, in terms of molecular modulation/regulation of the inflammatory and immune response upon PRRSV infection. The present doctoral thesis, which is divided into 3 different studies, is aimed to increase the knowledge about the interaction between the immune system and the PRRS virus upon natural infection. The objective of the first study entitled “Coordinated immune response of memory and cytotoxic T cells together with IFN-γ secreting cells after porcine reproductive and respiratory syndrome virus (PRRSV) natural infection in conventional pigs” was to evaluate the activation and modulation of the immune response in pigs naturally infected by PRRSV compared to an uninfected control group. The course of viremia was evaluated by PCR, the antibody titres by ELISA, the number of IFN-γ secreting cells (IFN- SC) by an ELISPOT assay and the immunophenotyping of some lymphocyte subsets (cytotoxic cells, memory T lymphocytes and cytotoxic T lymphocytes) by flow cytometry. The results showed that the activation of the cell-mediated immune response against PRRSV is delayed upon infection and that however the levels of IFN-γ SC and lymphocyte subsets subsequently increase over time. Furthermore, it was observed that the course of the different immune cell subsets is time-associated with the levels of PRRSV-specific IFN-γ SC and this can be interpreted based on the functional role that such lymphocyte subsets could have in the specific production/secretion of the immunostimulatory cytokine IFN-γ. In addition, these data support the hypothesis that the age of the animals upon the onset of infection or the diverse immunobiological features of the field isolate, as typically hypothesized during PRRSV infection, are critical conditions able to influence the qualitative and quantitative course of the cell-mediated immune response during PRRSV natural infection. The second study entitled “Immune response to PCV2 vaccination in PRRSV viremic piglets” was aimed to evaluate whether PRRSV could interfere with the activation of the immune response to PCV2 vaccination in pigs. In this trial, 200 pigs were divided into 2 groups: PCV2-vaccinated (at 4 weeks of age) and PCV2-unvaccinated (control group). Some piglets of both groups got infected by PRRSV, as determined by PRRSV viremia detection, so that 4 groups were defined as follows: PCV2 vaccinated - PRRSV viremic PCV2 vaccinated - PRRSV non viremic PCV2 unvaccinated - PRRSV viremic PCV2 unvaccinated - PRRSV non viremic The following parameters were evaluated in the 4 groups: number of PCV2-specific IFN-γ secreting cells, antibody titres by ELISA and IPMA. Based on the immunological data analysis, it can be deduced that: 1) The low levels of antibodies against PCV2 in the PCV2-vaccinated – PRRSV-viremic group at vaccination (4 weeks of age) could be related to a reduced colostrum intake influenced by PRRSV viremia. 2) Independently of the viremia status, serological data of the PCV2-vaccinated group by ELISA and IPMA does not show statistically different differences. Consequently, it can be be stated that, under the conditions of the study, PRRSV does not interfere with the antibody response induced by the PCV2 vaccine. 3) The cell-mediated immune response in terms of number of PCV2-specific IFN-γ secreting cells in the PCV2-vaccinated – PRRSV-viremic group seems to be compromised, as demonstrated by the reduction of the number of IFN-γ secreting cells after PCV2 vaccination, compared to the PCV2-vaccinated – PRRSV-non-viremic group. The data highlight and further support the inhibitory role of PRRSV on the development and activation of the immune response and highlight how a natural infection at early age can negatively influence the immune response to other pathogens/antigens. The third study entitled “Phenotypic modulation of porcine CD14+ monocytes, natural killer/natural killer T cells and CD8αβ+ T cell subsets by an antibody-derived killer peptide (KP)” was aimed to determine whether and how the killer peptide (KP) could modulate the immune response in terms of activation of specific lymphocyte subsets. This is a preliminary approach also aimed to subsequently evaluate such KP with a potential antivural role or as adjuvant. In this work, pig peripheral blood mononuclear cells (PBMC) were stimulated with three KP concentrations (10, 20 and 40 g/ml) for three time points (24, 48 and 72 hours). TIME POINTS (hours) KP CONCENTRATIONS (g/ml) 24 0-10-20-40 48 0-10-20-40 72 0-10-20-40 By using flow cytometry, the qualitative and quantitative modulation of the following immune subsets was evaluated upon KP stimulation: monocytes, natural killer (NK) cells, natural killer T (NKT) cells, and CD4+ and CD8α/β+ T lymphocyte subsets. Based on the data, it can be deduced that: 1) KP promotes a dose-dependent activation of monocytes, particularly after 24 hours of stimulation, by inducing a monocyte phenotypic and maturation shift mainly involved in sustaining the innate/inflammatory response. 2) KP induces a strong dose-dependent modulation of NK and NKT cells, characterized by an intense increase of the NKT cell fraction compared to NK cells, both subsets involved in the antibody-dependent cell cytotoxicity (ADCC). The increase is observed especially after 24 hours of stimulation. 3) KP promotes a significant activation of the cytotoxic T lymphocyte subset (CTL). 4) KP can modulate both the T helper and T cytotoxic phenotype, by inducing T helper cells to acquire the CD8α thus becoming doube positive cells (CD4+CD8+) and by inducing CTL (CD4-CD8+high) to acquire the double positive phenotype (CD4+CD8α+high). Therefore, KP may induce several effects on different immune cell subsets. For this reason, further research is needed aimed at characterizing each “effect” of KP and thus identifying the best use of the decapeptide for vaccination practice, therapeutic purposes or as vaccine adjuvant. RIASSUNTO Il virus della PRRS (Porcine Reproductive Respiratory Syndrome) è uno dei più diffusi agenti patogeni negli allevamenti suini di tutto il mondo, responsabile di una sindrome riproduttiva e respiratoria causa di gravi danni ad impatto sanitario ed economico. Questo virus è emerso attorno alla fine degli anni ’80 ma nonostante siano passati circa una trentina di anni, le conoscenze su alcuni punti essenziali che riguardano le caratteristiche del virus (patogenesi, risposta immunitaria, epidemiologia) appaiono ancora spesso incomplete. Considerando che lo sviluppo dei vaccini moderni è basato sui principi dell’immunità innata e acquisita è essenziale una sempre più completa conoscenza del sistema immunitario inteso come modulazione/regolazione molecolare della risposta infiammatoria e immunitaria in corso di tale infezione. Questo lavoro di tesi, suddiviso in tre diversi studi, ha l’intento di contribuire all’aumento delle informazioni riguardo l’interazione del sistema immunitario, con il virus della PRRS in condizioni di infezione naturale. L’obbiettivo del primo studio, intitolato “Associazione di cellule memoria, cellule citotossiche e cellule secernenti IFN- nella risposta immunitaria in corso di infezione naturale da Virus della Sindrome Riproduttiva e Respiratoria del Suino (PRRSV)” è stato di valutare l’attivazione e la modulazione della risposta immunitaria in suini naturalmente infetti da PRRSV rispetto ad un gruppo controllo non infetto. I parametri valutati sono stati la viremia mediante PCR, il titolo anticorpale mediante ELISA, il numero di cellule secernenti IFN- (IFN- SC) mediante tecnica ELISPOT e la fenotipizzazione di alcune sottopopolazioni linfocitarie (Cellule citotossiche, linfociti T memoria e linfociti T citotossici) mediante citofluorimetria a flusso. Dai risultati ottenuti è stato possibile osservare che l’attivazione della risposta immunitaria cellulo-mediata verso PRRSV appare ritardata durante l’infezione e che l’andamento, in termini di IFN- SC e dei cambiamenti delle sottopopolazioni linfocitarie, mostra comunque degli incrementi seppur successivi nel tempo. E’ stato inoltre osservato che gli andamenti delle diverse sottopopolazioni immunitarie cellulari appaiono temporalmente associati ai livelli di IFN- SC PRRSV-specifiche e ciò potrebbe essere interpretato sulla base del ruolo funzionale che tali sottopopolazioni linfocitarie potrebbero avere nella produzione/secrezione specifica della citochina immunoattivatrice IFN-. Questi dati inoltre supportano l’ipotesi che l’età degli animali alla comparsa dell’infezione o, come tipicamente ipotizzato nell’infezione da PRRSV, le differenti caratteristiche immunobiologiche dell’isolato di campo, sia condizioni critiche nell’ influenzare l’andamento qualitativo e quantitativo della risposta cellulo-mediata durante l’infezione naturale da PRRSV. Il secondo studio, dal titolo “Valutazione della risposta immunitaria nei confronti di una vaccinazione contro PCV2 in suini riscontrati PRRSV viremici e non viremici alla vaccinazione” ha avuto lo scopo di valutare se il virus della PRRS potesse andare ad interferire sull’attivazione della risposta immunitaria indotta da vaccinazione contro PCV2 nel suino. In questo lavoro sono stati arruolati 200 animali divisi in due gruppi, PCV2 Vaccinato (a 4 settimane di età) e PCV2 Non Vaccinato (controllo negativo). Alcuni suinetti di entrambi i gruppi, si sono naturalmente infettati con PRRSV, come determinato con l’analisi della viremia da PRRSV, per cui è stato possibile creare quattro sottogruppi, rispettivamente: PCV2 vaccinato - PRRSV viremico PCV2 vaccinato - PRRSV non viremico PCV2 non vaccinato - PRRSV viremico PCV2 non vaccinato - PRRSV non viremico Su questi quattro sottogruppi sono stati valutati i seguenti parametri: numero di cellule secernenti IFN- PCV2 specifiche, ed i titoli anticorpali mediante tecniche ELISA ed IPMA. Dall’analisi dei dati immunologici derivati dalle suddette tecniche è stato possibile dedurre che:  I bassi valori anticorpali nei confronti di PCV2 del gruppo Vaccinato PCV2-PRRSV viremico già al periodo della vaccinazione (4 settimane di età) potrebbero essere messi in relazione ad una ridotta assunzione di colostro legata allo stato di viremia da PRRSV  Indipendentemente dallo stato viremico, i dati sierologici del gruppo vaccinato PCV2 provenienti sia da ELISA sia da IPMA non mostrano differenze statisticamente significative. Di conseguenza è possibile affermare che in questo caso PRRSV non interferisce con la risposta anticorpale promossa dal vaccino PCV2.  La risposta immunitaria cellulo-mediata, intesa come numero di cellule secernenti IFN- PCV2 specifiche nel gruppo PCV2 vaccinato PRRS viremico sembra essere compromessa, come viene infatti dimostrato dalla diminuzione del numero di cellule secernenti IFN- dopo la vaccinazione contro PCV2, comparata con il gruppo PCV2 vaccinato- non viremico. I dati evidenziano ed ulteriormente sostengono il ruolo inibitorio del virus della PRRSV sullo sviluppo ed attivazione della risposta immunitaria e come un infezione naturale ad età precoci possa influenzare negativamente la risposta immunitaria ad altri patogeni/antigeni. Il terzo studio, intitolato “Modulazione fenotipica di: monociti CD14+, cellule natural killer (NK), T natural killer (NKT) e sottopopolazioni linfocitarie T CD4+ e CD8+ durante stimolazione con killer peptide (KP) nella specie suina” ha avuto come scopo quello di stabilire se e come il Peptide Killer (KP) potesse modulare la risposta immunitaria in termini di attivazione di specifiche sottopopolazioni linfocitarie. Si tratta di un approccio preliminare anche ai fini di successivamente valutare tale KP in un potenziale ruolo antivirale o come adiuvante. In questo lavoro, periferal blood mononuclear cells (PBMC) suine sono state stimolate con KP a tre diverse concentrazioni (10, 20 e 40 g/ml) per tre diversi tempi (24, 48 e 72 ore). TEMPI DI STIMOLAZIONE (ore) CONCENTRAZIONE DI KP (g/ml) 24 0-10-20-40 48 0-10-20-40 72 0-10-20-40 Mediante la citometria a flusso è stato dunque possibile analizzare il comportamento qualitativo e quantitativo di alcune sottopopolazioni linfocitarie sotto lo stimolo del KP, tra cui: monociti, cellule Natural Killer (NK), cellule T Natural Killer (NKT) e linfociti T CD4 e CD8+. Dai dati ottenuti è stato possibile dedurre che: 1) KP promuove un’attivazione dei monociti dose-dipendente in particolare dopo 24 ore di stimolazione, inducendo uno “shift” fenotipico e di maturazione monocitaria maggiormente coinvolto nel sostegno della risposta innata/infiammatoria. 2) KP induce una forte modulazione dose-dipendente di cellule NK e NKT con un forte aumento della frazione delle cellule NKT rispetto alle NK, sottopopolazioni entrambe coinvolte nella citotossicità cellulare mediata da anticorpi (ADCC). L’aumento è riscontrabile soprattutto dopo 24 ore di stimolazione. 3) KP promuove una significativa attivazione della sottopopolazione del linfociti T citotossici (CTL). 4) Per quanto riguarda la marcatura CD4+/CD8+ è stato dimostrato che KP ha la capacità di modulare sia il fenotipo T helper che T citotossico, inducendo le cellule T helper ad acquisire CD8 diventando quindi doppio positive (CD4+CD8+) ed inducendo il fenotipo CTL (CD4-CD8+high) ad acquisire il fenotipo doppio positivo (CD4+CD8α+high). Molti dunque potrebbero essere gli effetti che il decapeptide KP potrebbe esercitare sulle diverse sottopopolazioni del sistema immunitario, per questo motivo va evidenziata la necessità di impostare e attuare nuove ricerche che portino alla caratterizzazione di ciascuna “abilità” di KP e che conducano successivamente alla scoperta del migliore utilizzo che si possa fare del decapeptide sia dal punto di vista vaccinale, terapeutico oppure sotto forma di adiuvante vaccinale.

Detecção de Enterococcus resistentes a vancomicina em criações comerciais de ovinos e caprinos das regiões centro-leste e nordeste do Estado de São Paulo

As exigências das condições higiênico-sanitárias na produção de animais de interesse zootécnico vêm aumentando progressivamente dada à necessidade de aliar-se produtividade a produtos de alta qualidade para atender a mercados consumidores cada vez mais exigentes. Nesse sentido, a utilização de antimicrobianos, tanto na profilaxia como na terapêutica, permanece como estratégia de controle para vários microrganismos patogênicos, de importância não apenas para a produção animal como também para a saúde humana, ainda que restrições ao uso indiscriminado desses produtos têm se intensificado. Não obstante, o uso excessivo desses produtos está associado à seleção de microrganismos resistentes nas áreas de produção. Por outro lado, investigações sobre circulação de cepas resistentes em rebanhos animais, até então restritas a populações humanas, ainda permanecem limitadas no Brasil. Bactérias do gênero Enterococcus, integrantes usuais da microbiota gastrointestinal animal e humana, são indicadoras ambientais de contaminação fecal e tem-se tornado objeto de preocupação em saúde pública e veterinária dada a ocorrência de cepas resistentes à vancomicina (VRE). O presente trabalho teve como objetivo isolar, quantificar e caracterizar VRE presentes em amostras fecais de ovinos oriundos de pequenas propriedades das regiões centro-leste e nordeste do estado de São Paulo. Para tanto, 132 amostras fecais foram coletadas diretamente do reto dos animais ou do piso das instalações. As amostras foram semeadas em ágar m-Enterococcus e subcultivadas em Ágar Bile Esculina acrescido de 6 µg/mL de vancomicina (ABEV), para confirmação de Enterococcus spp e detecção de cepas resistentes. Procedeu-se igualmente a observação da morfologia, características tintoriais, bioquímicas e moleculares. O número máximo de Enterococcus spp. encontrado foi de 2,6 × 105 e 1,70 × 105 UFC/g de fezes do ambiente e dos animais, respectivamente. Na caracterização bioquímica espécies mais prevalentes foram: Enterococcus faecalis e Vagococcus fluvialis. No ABEV, houve crescimento de colônias VRE em 33 das 84 amostras de ovinos-caprinos e em 21 das 48 amostras ambientais, representando, respectivamente 46,7% e 29,3% das amostras analisadas. A análise por multiplex PCR das 54 cepas VRE obtidas indicaram que 23 (43%), 22 (41%), 2 (3,5%) e 2 (3,5%) foram positivas, respectivamente, para os genes vanC2/C3, vanC1, vanA e vanB, sendo que para 5,3% dos isolados nenhum produto foi amplificado, sugerindo a possível ocorrência de genes dos demais grupos van conhecidos entre os isolados. Os resultados obtidos indicam, de forma inédita no país, a circulação de VRE em propriedades produtoras de ovinos e caprinos, sem ocorrência de manifestações clínicas aparentes nos animais, porém com possíveis riscos à saúde dos produtores e profissionais envolvidos, bem como a eventuais consumidores.

Potential Adverse Effects of the Alaska Natural Gas Pipeline on Arctic Caribou and Proposed Mitigation

The Alaska Natural Gas Pipeline (ANGP) is proposed for construction on the North Slope in 2016. It will be aligned through Arctic caribou habitat and evidence shows that caribou are negatively affected by human development. This Capstone identifies potential adverse affects of ANGP on Arctic caribou using interviews from expert caribou biologists and the 1977 Trans-Alaska Pipeline System (TAPS) as a model. Based on a synthesis of the interviews and TAPS analysis, this capstone proposes and examines a set of seventeen conservation measures to be implemented during construction and operation of ANGP to minimize adverse impacts on caribou herds. These conservation measures can be used as a baseline for future developments on the North Slope to promote caribou herd management.