Vaccination of Poultry Against Influenza.

The complexity of influenza A virus (IAV) infections in avian hosts leads to equally complex scenarios for the vaccination of poultry. Vaccination against avian influenza strains can be used to prevent infections from sources with a single strain of IAV. It has been used as a part of outbreak control strategies as well as a way to maintain production for both low and high pathogenicity outbreaks. Unlike other viral pathogens of birds, avian influenza vaccination when used against highly pathogenic avian influenza virus, is tied to international trade and thus is not freely available for use without specific permission.

Vacunación de aves comerciales contra la influenza aviar. La complejidad de las infecciones por el virus de la influenza A en las aves hospedadoras conduce a escenarios igualmente complejos para la vacunación en la avicultura. La vacunación contra cepas de influenza aviar se puede utilizar para prevenir infecciones provenientes de fuentes con una sola cepa del virus de influenza. Se ha utilizado como parte de las estrategias de control de brotes, así como como una forma de mantener la producción tanto en brotes de baja como de alta patogenicidad. A diferencia de otros patógenos virales de las aves, la vacunación contra la influenza aviar, cuando se usa contra el virus de la influenza aviar altamente patógeno, está vinculada al comercio internacional y por lo tanto, no está disponible para su uso sin un permiso específico.

Enteric Viruses Associated with Mid-growth Turkey Enteritis.

Since August 2014, the University of Minnesota Veterinary Diagnostic Laboratory has received cases of turkey enteritis that are clinically different from previously described cases of poult enteritis syndrome and light turkey syndrome. The birds develop dark green and extremely foul-smelling diarrhea starting at 8-10 wk of age, which may last up to 15-16 wk of age. The affected turkey flocks show poor uniformity, and feed conversion and market weights are reduced. Multiple-age farms are affected more often than the single-age farms. Morbidity varies from flock to flock and in some cases reaches 100%. At necropsy, undigested feed with increased mucus is observed in the intestines along with prominent mucosal congestion and/or hemorrhage. Microscopically, lymphocytic infiltrates expand the villi in duodenum and jejunum to form lymphoid follicles, which are often accompanied by heterophils. Next generation sequencing (Illumina Miseq) on a pool of feces from affected birds identified genetic sequences of viruses belonging to Astroviridae, Reoviridae, Picornaviridae, Picobirnaviridae, and Adenoviridae. On testing pools of fecal samples from apparently healthy (16 pools) and affected birds (30 pools), there was a higher viral load in the feces of affected birds. Picobirnavirus was detected only in the affected birds; 20 of 30 pools (66.7%) were positive. These results indicate that a high viral load of turkey picobirnavirus alone, or in association with novel picornaviruses, may be a cause of this new type of turkey enteritis.

Environmental Sampling for Influenza A Viruses in Turkey Barns.

We have examined a variety of sampling strategies for detecting pathogens in turkey flocks undergoing infections with low pathogenicity avian influenza virus (LPAIV). We found that viral RNA was widely distributed in the barn environment of turkey flocks undergoing an active LPAIV infection and was in both water and drinker biofilm samples. Viral RNA was concentrated in drinker biofilm and sediment and was detectable using real-time reverse-transcription polymerase chain reaction (RRT-PCR) and by virus isolation. Drinker biofilm sample results correlated with concurrently collected oropharyngeal (OP) sample results from flocks on a farm with LPAI in which the two sampling strategies were directly compared. To evaluate the utility of biofilm sampling for the detection of highly pathogenic avian influenza virus (HPAIV), biofilm and OP swabs from mortality pools were collected daily from negative turkey flocks on an HPAI-positive premise. The biofilm swabs were positive 1-2 days prior to positives appearing in the OP sample pools. The drinker biofilm sampling strategy overcame the difficulty of finding a subclinical infectious bird in a population by collecting material from a large number of individuals and testing a sample in which a positive signal persists for several days to weeks. The sampling method is convenient for use in turkey barns and has been reliably used in both active and passive surveillance programs for LPAIV and HPAIV using RRT-PCR.

Muestreo ambiental para el virus de influenza A en casetas de pavos. Se han examinado una variedad de estrategias de muestreo para detectar patógenos en parvadas de pavos que sufren infecciones con el virus de la influenza aviar de baja patogenicidad (con las siglas en inglés LPAIV). Se encontró que el ARN viral se distribuyó ampliamente en el ambiente de las casetas con parvadas de pavos con infección activa por el virus de la influenza aviar de baja patogenicidad y se determinó tanto en muestras de agua como en muestras de la biopelícula de bebederos. El ARN viral se concentró en la biopelícula y en el sedimento de bebederos y se detectó mediante transcripción reversa y reacción en cadena de la polimerasa en tiempo real (RRT-PCR) y mediante el aislamiento del virus. Los resultados de la muestra de la biopelícula del bebedero se correlacionaron con los resultados de la muestra orofaríngea (OP) colectada de forma simultánea de parvadas en una granja con influenza aviar de baja patogenicidad en las que se compararon directamente las dos estrategias de muestreo. Para evaluar la utilidad del muestreo de la biopelícula para la detección del virus de la influenza aviar altamente patógena (HPAIV), se recolectaron diariamente biopelículas e hisopos orofaríngeos de grupos de mortalidad de parvadas de pavos negativas en una granja positiva para la influenza aviar de alta patogenicidad. Los hisopos de biopelículas fueron positivos de uno a dos días antes de que aparecieran resultados positivos en las muestras orofaríngeas agrupadas. La estrategia de muestreo de la biopelícula del bebedero eliminó la dificultad de encontrar un ave infectada subclínicamente en una población al recolectar material de un gran número de individuos y analizar una muestra en la que persiste una señal positiva durante varios días o semanas. El método de muestreo es adecuado para su uso en casetas de pavos y se ha utilizado de manera confiable en los programas de vigilancia activa y pasiva para el virus de influenza aviar tanto de baja como de alta patogenicidad utilizando transcripción reversa y reacción en cadena de la polimerasa en tiempo real.

Transmissible viral proventriculitis identified in broiler breeder and layer hens.

Transmissible viral proventriculitis (TVP) is a recognized cause of production losses in broiler chickens, but previously it has not been reported in broiler breeder and commercial layer hens. In this study, TVP was identified in broiler breeder and commercial layer hens, 9-20 wk of age, based on histopathologic detection of characteristic microscopic lesions. Microscopic lesions in proventriculi of affected hens consisted of glandular epithelial necrosis, ductal epithelial hyperplasia, replacement of glandular epithelium with ductal epithelium, and diffuse interstitial lymphoid infiltration. Additionally, chicken proventricular necrosis virus (CPNV), a virus previously identified as the etiology of TVP in broiler chickens, was detected in proventriculi of TVP-affected hens using a reverse transcriptase-polymerase chain reaction procedure. The findings identify TVP as a potential cause of production losses in broiler breeder and commercial layer hens and provide additional evidence for etiologic involvement in TVP by CPNV.

Detection of chicken proventricular necrosis virus (R11/3 virus) in experimental and naturally occurring cases of transmissible viral proventriculitis with the use of a reverse transcriptase-PCR procedure.

A reverse-transcriptase-polymerase-chain-reaction (RT-PCR) procedure was evaluated for detection of chicken proventricular necrosis virus (CPNV) in transmissible viral proventriculitis (TVP) -affected chickens. The RT-PCR procedure was compared with indirect immunofluorescence (IFA) and virus isolation for detection of CPNV in experimentally infected chickens. Microscopic lesions characteristic of TVP were detected on days 5-35 postexposure (PE) in CPNV-infected chickens; CPNV was detected by RT-PCR on days 3-14 PE in freshly collected proventriculi, and on days 1-14 PE in formalin-fixed paraffin-embedded (FFPE) proventriculi. CPNV was detected in proventriculi of experimentally infected chickens by IFA on days 3-10 PE, and by virus isolation on days 1-14 PE. With IFA used as a reference, sensitivity of the RT-PCR procedure with freshly collected and FFPE proventriculi was 88% and 100%, respectively; specificity was 83% and 86%, respectively. Proventriculi (FFPE) obtained from suspect TVP cases (n=19) were evaluated for presence of CPNV by RT-PCR and microscopic lesions consistentwith TVP. CPNV was detected by RT-PCR in proventriculi from 8/11 TVP (+) cases (24/36 tissue sections). TVP (+) cases were defined by microscopic lesions characteristic of TVP; CPNV was not detected in proventriculi (0/8 cases, 0/32 tissue sections) in the absence of these lesions. The association between presence of TVP-characteristic microscopic lesions and presence of CPNV was highly significant (P = 0.0014). These findings indicate the utility of the RT-PCR procedure for detection of CPNV and provide additional evidence for an etiologic role for this virus in TVP.

Mycoplasma iowae associated with chondrodystrophy in commercial turkeys.

Opportunistic observations of and necropsies from selected commercial (meat) turkey flocks revealed skeletal lesions consistent with chondrodystrophy, characterized by leg and vertebral deformities, occurring at very low incidences in turkeys from two primary breeds and various multiplier breeder flocks. Mycoplasma organisms were cultured and identified as Mycoplasma iowae by immunofluorescence and polymerase chain reaction from some of the vertebral lesions but not from leg joints. This is the first detailed description of the gross and microscopic lesions of vertebral chondrodystrophy associated with M. iowae, which should now be considered in the differential diagnosis of turkeys with these lesions.

Species Comparison of the Role of p38 MAP Kinase in the Female Reproductive System.

The p38 mitogen-activated protein kinases (MAPKs) are members of discrete signal transduction pathways that have significant regulatory roles in a variety of biological processes, depending on the cell, tissue and organ type. p38 MAPKs are involved in inflammation, cell growth and differentiation and cell cycle. In the female reproductive system, p38 MAPKs are known to regulate various aspects of the reproductive process such as mammalian estrous and menstrual cycles as well as early pregnancy and parturition. p38 MAPKs have also been implicated in alterations and pathologies observed in the female reproductive system. Therefore, pharmacologic modulation of p38 MAPKs, and inter-connected signaling pathways (e.g., estrogen receptor signaling, c-fos, c-jun), may influence reproductive physiology and function. This article provides a critical, comparative review of available data on the roles of p38 MAPKs in the mammalian female reproductive system and in reproductive pathophysiology in humans and preclinical species. We first introduce fundamental differences and similarities of the mammalian female reproductive system that should be considered by toxicologists and toxicologic pathologists when assessing the effects of new pharmacologic agents on the female reproductive system. We then explore in detail the known roles for p38 MAPKs and related molecules in female reproduction. This foundation is then extended to pathological conditions in which p38 MAPKs are thought to play an integral role.

Expression of Ki-67 in the uterus during various stages of the estrous cycle in rats.

Rats have an average estrous cycle of 4-5 days. There are four phases (proestrus, estrus, metesterus, and diestrus) in the estrous cycle in rodents. Histologic staging of the rodent estrous cycle is challenging and requires expertise. Thus, utilizing additional parameters such as cellular proliferation of the various components of the uterine microanatomy may assist with this process. Having an alternative method by which a pathologist can correctly identify the stages of the rodent estrous cycle would be valuable to the assessment and interpretation of safety studies for new drug candidates. This study was performed to investigate the microanatomic location of the uterine proliferative activity by image analysis and immunohistochemistry using Ki-67, a well-established marker of proliferating cells. Each stage of the rodent estrous cycle exhibited a different pattern of cellular proliferation. During proestrus, the lowest degree of cellular proliferation occurred in the glandular epithelial cells and the highest occurred in the myometrial cells. In estrus, lower levels of cellular proliferation were seen in the luminal and glandular epithelial cells, while a higher rate of proliferation occurred in myometrial cells followed by the stromal cells. At the metestrus stage, the highest cellular proliferation occurred in stromal and myometrial cells, while lesser proliferation was observed in luminal and glandular epithelial cells. This work demonstrates that in the rodent uterus there are cyclic changes in cellular proliferation in specific microanatomic uterine locations which can aid in the staging of the estrous cycle.

A response to BIO 2010: Transforming undergraduate education for future research biologists, from the perspective of the biochemistry and molecular biology major program at Kenyon College.

The National Research Council completed a major study of undergraduate biology education, BIO 2010-Transforming Undergraduate Education For Future Research Biologists (BIO 2010), funded by the Howard Hughes Medical Institute and the National Institutes of Health. The BIO 2010 report recommends that biology pedagogy should use an interdisciplinary approach incorporating a strong basis in mathematics and physical sciences. Many of the aims of BIO 2010 can be met by an interdisciplinary major program such as that of Biochemistry and Molecular Biology at Kenyon College. The Kenyon program effectively encourages students to connect biology with chemistry and mathematics and to develop a sound basis for research in the biological sciences. A continuing challenge is to balance the needs for depth of physical and mathematical understanding and breadth of diversity in biology.

Dexrazoxane (ICRF-187) protects cardiac myocytes against doxorubicin by preventing damage to mitochondria.

The clinically approved antioxidant cardioprotective agent dexrazoxane (ICRF-187) was examined for its ability to protect neonatal rat cardiac myocytes from doxorubicin-induced damage. Doxorubicin is thought to induce oxidative stress on the heart muscle, both through reductive activation to its semiquinone form, and by the production of hydroxyl radicals mediated by its complex with iron. Hydrolyzed dexrazoxane metabolites prevent site-specific iron-based oxygen radical damage by displacing iron from doxorubicin and chelating free and loosely bound iron. The mitochondrial stain MitoTracker Green FM and doxorubicin were shown by epifluorescence microscopy to accumulate in the myocyte mitochondria. An epifluorescence microscopic image analysis method to measure mitochondrial damage was developed using the mitochondrial membrane potential sensing ratiometric dye JC-1. This method was used to show that dexrazoxane protected against doxorubicin-induced depolarization of the myocyte mitochondrial membrane. Dexrazoxane also attenuated doxorubicin-induced oxidation of intracellular dichlorofluorescin. Annexin V-FITC/propidium iodide staining of myocytes was used to demonstrate that, depending on the concentration, doxorubicin caused both apoptotic and necrotic damage. These results suggest that doxorubicin may be cardiotoxic by damaging the mitochondria and dexrazoxane may be protective by preventing iron-based oxidative damage.

Prevention of doxorubicin-induced damage to rat heart myocytes by arginine analog nitric oxide synthase inhibitors and their enantiomers.

The clinical use of the widely used anticancer drug doxorubicin is limited by a dose-dependent cardiotoxicity. Doxorubicin can be reduced to its semiquinone free radical form by nitric oxide synthases (NOS). The release of lactate dehydrogenase (LDH) from doxorubicin-treated neonatal cardiac rat myocytes was used as a model of doxorubicin-induced cardiotoxicity. The NOS inhibitors N(G)-nitro-L-arginine methyl ester (L-NAME) and N(G)-monomethyl-L-arginine (L-NMMA) protected myocytes from doxorubicin as did their non-inhibitory enantiomers D-NAME and D-NMMA. Thus, these agents did not protect by inhibiting NOS. L-NAME, which does not act at the reductase domain of NOS, also had no effect on the production of the doxorubicin semiquinone by myocytes. Nitric oxide (NO) EPR spin trapping experiments showed that L-NAME reacted with various biological reducing agents to produce NO. Ascorbic acid was highly effective in reacting with L-NAME to produce NO, while glutathione, NADPH, and NADH were much less effective. Thus, these guanadino-substituted analogs of L-arginine likely protected through their ability to slowly produce NO by reaction with intracellular ascorbic acid. Thus, some caution must be exercised in their use. NO may exert its protective effects either by directly acting as an antioxidant or through some other NO-dependent pathway.