No effect of turkey herpesvirus infections on the outcome of avian pneumovirus infections in turkeys.

To determine whether turkey herpesvirus (HVT) impairs the aspecific and specific defense against an avian pneumovirus (APV) infection, specific-pathogen-free turkeys were inoculated at 7 days of age with HVT and 1, 5, or 7 wk later with APV. Clinical signs, APV replication, and development of antibodies against APV were evaluated. No differences were found between the birds that received both HVT and APV and those that received only APV.     

Change of porcine circovirus 2 target cells in pigs during development from fetal to early postnatal life

Change of porcine circovirus 2 (PCV2) target cells during development from fetal to postnatal life in pigs was examined. PCV2 inoculation was performed in fetuses in utero at either 57, 75 or 92 gestational days and in piglets at 1 day of age. Twenty-one days after virus inoculation, PCV2-infected cells in the heart, lungs, liver, spleen and inguinal lymph nodes were localized and immuno-phenotyped by double-immunofluorescence labeling using different cell markers and PCV2-antibodies. During fetal life, viral antigens were detected in cardiomyocytes, hepatocytes and macrophages and infected cell numbers decreased with increasing fetal age at inoculation. The heart contained the highest number of infected cells and cardiomyocytes were the main target cell. Postnatally, macrophages were the only target cell type in different organs and infected cell numbers were similar to those of fetuses inoculated at 92 days of gestation. One piglet showed exceptionally high number of infected cells in different organs with values 13-513-fold higher compared to littermates. In this piglet, the majority of infected cells in lymphoid tissues could not be typed. This study reveals that PCV2 target cells change from cardiomyocytes, hepatocytes and macrophages during fetal life to only macrophages postnatally.     

Antibody-induced internalization of viral glycoproteins in pseudorabies virus-infected monocytes and role of the cytoskeleton: a confocal study

Addition of pseudorabies virus (PrV)-specific polyclonal immunoglobulins to PrV-infected monocytes induces internalization of plasma membrane anchored viral glycoproteins. This process may interfere with antibody-dependent cell lysis and resembles the well-studied physiological endocytosis process. A confocal study was designed to investigate whether the major cellular components, involved in physiological endocytosis (clathrin, actin, dynein and microtubules), play a role in this virological internalization process. In order to visualize the interaction of endosomes, which contain the internalized viral glycoproteins, with clathrin, actin, dynein and microtubules, a double labeling of viral glycoproteins and different cellular proteins was performed. Porcine monocytes were inoculated with the PrV-strain 89V87 at a multiplicity of infection of 50 for 13h. After the addition of FITC-labeled porcine polyclonal PrV-specific antibodies, cells were fixed with para-formaldehyde at different time points and afterwards permeabilized. The different cellular components were visualized with monoclonal antibodies and a Texas Red-conjugate, with the exception of actin, which was stained with phalloidin-Texas Red. The cells were analyzed by confocal microscopy. A clear co-localization was observed between the viral glycoproteins and clathrin and dynein during the internalization process. The microtubules were in close contact with the internalized vesicles. For actin no co-localization could be observed. It can be stated that clathrin, dynein and microtubules, important components during physiological endocytosis, are also of importance during the antibody-induced internalization of viral glycoproteins.     

正交试验法优化普抗合剂制备工艺

以干浸膏得率和黄芩苷提取率为考核指标,采用正交试验法对普抗合剂水提取醇沉淀制备工艺进行考察.普抗合剂最佳制备工艺方案为加水量10倍,提取2次,每次1 h,55%的乙醇沉淀杂质 .该工艺科学合理,适合于大规模工业生产.     

Different clinical,virological, serological and tissue tropism outcomes of two new and one old Belgian type 1 subtype 1 porcine reproductive and respiratory virus (PRRSV) isolates

In this study, the pathogenic behavior of PRRSV 13V091 and 13V117, isolated in 2013 from two different Belgian farms with enzootic respiratory problems shortly after weaning in the nursery, were compared with the Belgian strain 07V063 isolated in 2007. Full-length genome sequencing was performed to identify their origin. Twelve weeks-old pigs were inoculated intranasally (IN) with 13V091, 13V117 or 07V063 (9 pigs/group). At 10 days post inoculation (dpi), 4 animals from each group were euthanized and tissues were collected for pathology, virological and serological analysis. 13V091 infection resulted in the highest respiratory disease scores and longest period of fever. Gross lung lesions were more pronounced for 13V091 (13%), than for 13V117 (7%) and 07V063 (11%). The nasal shedding and viremia was also most extensive with 13V091. The 13V091 group showed the highest virus replication in conchae, tonsils and retropharyngeal lymph nodes. 13V117 infection resulted in the lowest virus replication in lymphoid tissues. 13V091 showed higher numbers of sialoadhesin⁻ infected cells/mm² in conchae, tonsils and spleen than 13V117 and 07V063. Neutralizing antibody response with 07V063 was stronger than with 13V091 and 13V117. It can be concluded that (i) 13V091 is a highly pathogenic type 1 subtype 1 PRRSV strain that replicates better than 07V063 and 13V117 and has a strong tropism for sialoadhesin⁻ cells and (ii) despite the close genetic relationship between 13V117 and 07V063, 13V117 has an increased nasal replication and shedding, but a decreased replication in lymphoid tissues compared to 07V063.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0166-3) contains supplementary material, which is available to authorized users.     

Determination of Feeder Cell‐Based Cellular Niches Supporting the Colonization and Maintenance of Spermatogonial Stem Cells from Prepubertal Domestic Cat Testes

Recently, isolation and in vitro culture of putative spermatogonial stem cells (SSCs) in the domestic cat have been conducted. However, the cellular niche conditions that facilitate the establishment and long‐term maintenance of feline SSCs (FSSCs) have not been described. Therefore, we investigated the type of feeder cells used to stimulate colony formation and growth of FSSCs among the various factors in the FSSC niche. Spermatogonial stem cells isolated from feline testes were cultured on mitotically inactivated testicular stromal cells (TSCs) derived from cats, dogs and mice, and mouse embryonic fibroblasts (MEFs). The formation and growth of colonies derived from SSCs cultured on each type of feeder cell were identified at passage 0, and the morphology, alkaline phosphatase (AP) activity and expression of SSC‐specific genes in surviving colonies were investigated at passage 4. Among these diverse feeder cells, TSCs from cat showed the greatest colony formation, growth and maintenance of FSSCs, and SSC colonies cultured by passage 4 showed a typical dome‐shaped morphology, AP activity and expression of SSC‐specific genes (NANOG, OCT4, SOX2 and CD9). Accordingly, these results demonstrate that feline TSCs could be used as feeder cells to support the establishment and maintenance of SSCs from domestic cats.     

The dispersal of Culicoides brevitarsis in eastern New South Wales and associations with the occurrences of arbovirus infections in cattle

Distributions of the vector Culicoides brevitarsis Kieffer (Diptera: Ceratopogonidae) (determined from light trap data) and 2 arboviruses (determined from seroconversions in sentinel cattle) were studied in eastern New South Wales in 1993–94. C brevitarsis was recorded progressively from endemic areas on the north coast, to Nowra on the south coast, and westward to Scone, in the Hunter Valley. C brevitarsis also survived through winter at Paterson, in the Hunter Valley. Its apparently focal reappearance in this marginal area had no obvious effect on the broad pattern of its progression or the dispersal of Akabane and bluetongue viruses. These viruses were first recorded from foci near Coffs Harbour, on the mid-north coast. Their first occurrences at different locations were associated with those of C brevitarsis, but not with each other. The viruses were found only within the recorded limits of the vector's distribution. Delays between the initial occurrence of C brevitarsis and first evidence of virus transmissions at locations ranged from 2 to 7 months. The delays decreased away from the points of focus and were negatively associated with the time of initial occurrence of the vector. Seroconversions to the viruses were related to the presence of C brevitarsis. However, the densities of C brevitarsis had no apparent effect on the initial numbers of cattle seroconverting to either virus. The results support the conclusion that the progressions of C brevitarsis and Akabane and bluetongue viruses were the result of gradual movements by the vector.     

Clinical Effects of Experimental Dual Infections with Porcine Reproductive and Respiratory Syndrome Virus Followed by Swine Influenza Virus in Conventional and Colostrum‐deprived Pigs

Previous studies demonstrated that experimental dual infections of pigs with porcine reproductive and respiratory syndrome virus (PRRSV) followed by H1N1 influenza virus cause more severe disease and growth retardation than the respective single virus infections. Here three experiments were undertaken to better define the clinical impact of combined PRRSV‐H1N1 infections in conventional and caesarean‐derived colostrum‐deprived (CDCD) pigs. Groups of pigs were inoculated by aerosol with PRRSV followed by H1N1 at 3‐, 7‐ or 14‐day intervals. During the post‐H1N1 period, mean body temperatures, respiratory signs and mean weight gains in the PRRSV‐H1N1 inoculated groups were recorded and compared with those in uninoculated controls (experiments 1 and 2) or in singly virus‐inoculated pigs (experiment 3). In a first experiment with conventional pigs, the PRRSV‐3d‐H1N1 and PRRSV‐7d‐H1N1 infections induced mean body temperatures >40.5°C during 8 days (peaks 41.1 and 41.6°C, respectively) and mean growth reductions of 3.4 and 4.8 kg, respectively, during the 2 weeks after H1N1, along with marked depression and respiratory disease. The PRRSV‐14d‐H1N1 infection, on the contrary, was largely subclinical. In a second experiment with conventional pigs, PRRSV‐3d‐H1N1 and PRRSV‐7d‐H1N1 infections were clinically milder, with smaller increases in mean body temperatures (peak 40.5°C in both groups) and growth reductions (1.4 and 1.6 kg, respectively). In both groups, only one pig showed prominent general and respiratory signs. In a final experiment with CDCD pigs, PRRSV‐7d‐H1N1 infection had minimal effects on mean clinical performances and growth and, except for one pig that was severely affected, differences with the single virus inoculations were negligible. Thus, both the time interval between infections and the sanitary status of pigs can affect the clinical outcome of dual PRRSV‐H1N1 infections. However, factors so far unknown seem to cause large variations in the clinical response between individual pigs.     

Key role of Chlamydophila psittaci on Belgian turkey farms in association with other respiratory pathogens

Two hundred turkey sera from eight Belgian and two French farms were tested for the presence of antibodies against avian pneumovirus (APV), Ornithobacterium rhinotracheale (ORT), Mycoplasma gallisepticum, Mycoplasma meleagridis and Chlamydophila psittaci. At slaughter, C. psittaci, APV and ORT antibodies were detected in 94, 34 and 6.5% of the turkeys, respectively. No antibodies against M. gallisepticum or M. meleagridis were present. Additionally, turkeys on three Belgian farms were examined from production onset until slaughter using both serology and antigen or gene detection. All farms experienced two C. psittaci infection waves, at 3-6 and 8-12 weeks of age. Each first infection wave was closely followed by an ORT infection starting at the age of 6-8 weeks, which was still detectable when the second C. psittaci infection waves started. Animals on farm A were not vaccinated against APV leading to an APV subtype B outbreak accompanying the first C. psittaci infection wave. Despite subtype A APV vaccination on farms B and C, the second C. psittaci infection waves were accompanied (farm B) or followed (farm C) by a subtype B APV infection. On all farms respiratory signs always appeared together with a proven C. psittaci, APV and/or ORT infection. This study suggests an association between C. psittaci, APV and ORT, and indicates the multi-factorial aetiology of respiratory infections in commercial turkeys. All three pathogens should be considered when developing prevention strategies for respiratory disease.