Amino acid changes in the capsid protein of a reassortant betanodavirus strain: Effect on viral replication in vitro and in vivo

Betanodavirus reassortant strains (RGNNV/SJNNV) isolated from Senegalese sole harbour an SJNNV capsid featuring several changes with respect to the SJNNV‐type strain, sharing three hallmark substitutions. Here, we have employed recombinant strains harbouring mutations in these positions (r20 and r20 + 247 + 270) and have demonstrated that the three substitutions affect different steps of the viral replication process. Adsorption ability and efficiency of viral attachment were only affected by substitutions in the C‐terminal side of the capsid. However, the concurrent mutation in the N‐terminal side seems to slightly decrease these properties, suggesting that this region could also be involved in viral binding. Differences in the intracellular and extracellular production of the mutant strains suggest that both the C‐terminal and N‐terminal regions of the capsid protein may be involved in the particle budding. Furthermore, viral replication in sole brain tissue of the mutant strains, and especially double‐ and triple‐mutant strains, is clearly delayed with respect to the wt strain. These data support previous findings indicating that the C‐terminal side plays a role in virulence because of a slower spread in the fish host brain and suggest that the concurrent participation of the N‐terminal side is also important for viral replication in vivo.     

Design and validation of a RT‐qPCR procedure for diagnosis and quantification of most types of infectious pancreatic necrosis virus using a single pair of degenerated primers

Infectious pancreatic necrosis virus (IPNV) is an important virus which affects the salmonid aquaculture industry worldwide; therefore, it is important to develop rapid and reliable methods of diagnosis to detect the disease at early stages. Nowadays, RT‐qPCR is replacing other methods because it provides additional information on the viral load, which is important to have a better understanding of the virus replication level and of the stage of the infection and its risk level. The main problem stems from the high diversity of this virus, which can compromise the reliability of the diagnosis. In this study, we have designed an RT‐qPCR procedure for diagnosis and quantification of IPNV based on a single pair of primers targeted to segment B. The procedure has been validated, in vitro and in vivo, testing two different types of standards against seven reference strains and 23 field isolates from different types. The procedure is reliable for the detection of any type, with a detection limit of 31 TCID₅₀ mL^?1, 50 pfu mL^?1 or 66 RNA copies mL^?1, depending on the standard. All the standard curves showed high reliability (R² > 0.95). The results support the high reliability of this new procedure for the diagnosis and quantification of IPNV.     

Disease of geese caused by a new herpesvirus

A goose flock farmed outdoors in south-eastern Queensland suffered an outbreak of peracute disease with high death rate (97%). Small button ulcers and large plaques overlying lymphocyte aggregates were present on the mucosa of the small intestine of affected birds. Small white foci of necrosis and focal haemorrhages were seen in the livers. Numerous intranuclear inclusion bodies were observed microscopically in hepatocytes and a herpesvirus which grew rapidly in chicken kidney cells was isolated from tissues. Duck virus enteritis (DVE) was suspected but DVE antiserums failed to neutralise the virus. Further serological studies with a limited range of known avian herpesviruses have failed to identify the virus. Experimental transmission resulted in high mortality in geese (100%), lower mortality in ducklings and nil mortality in chickens. Surveillance studies showed no evidence of infection in domestic and wild birds beyond the original farm and the infection appears not to have been established in the area. Wild ducks, which were frequent visitors to the farm dam, were considered the most likely source of the infection.     

Identification of Cytokeratins in Bovine Sperm Outer Dense Fibre Fractions

Outer dense fibres (ODF) are important substructures of mammalian sperm tails that are involved in the regulation of sperm motility. In this study, we investigated the identity of several sodium dodecyl sulphate (SDS)‐insoluble ODF proteins. Bovine ODF were purified by separating sperm heads and tails using ultrasound and Percoll^® density gradient centrifugation. Sperm flagella were treated with the detergent cetyltrimethylammonium bromide (CTAB). CTAB‐insoluble material, which reportedly represents the ODF fraction, was collected, and electron microscopy confirmed a highly purified ODF fraction. We found after solubilization of this fraction with SDS that high amounts of insoluble material were retained after centrifugation. SDS‐insoluble material was collected and quantitatively dissolved in 8 M urea. SDS‐gel electrophoresis in the presence of urea revealed polypeptides with apparent molecular masses of approximately 25, 43, and 50 kDa. Subsequent immunoblotting with anti‐cytokeratin antibodies detected two urea‐soluble, SDS‐insoluble proteins with apparent molecular masses of 45 and 66 kDa. The 45‐kDa protein was identified as cytokeratin 19. An antibody reacting with a palette of cytokeratins (CK 1–18 and CK 20), KL1, was the only antibody that reacted with the 66‐kDa polypeptide. We conclude that sperm ODF fractions contain at least one each of type I and type II intermediate filaments. As keratins and intermediate filaments are described as rope‐like structures, we suggest that these intermediate filaments play an important structural or tension‐bearing role in sperm flagella.     

Theileria orientalis: a review

Theileria orientalis (also known historically as T. sergenti and T. buffeli) is responsible for benign or non-transforming theileriosis, and exerts its major effect through erythrocyte destruction. The life cycle of T. orientalis is essentially similar to that of other Theileria species, except that the schizonts do not induce transformation and fatal lymphoproliferation. The pathogenesis of anaemia as a result of infection is not clearly established and may be multifaceted. Clinical signs of weakness, reluctance to walk and abortion are early but non-specific indications of disease, particularly if accompanied by a history of cattle being moved. Physical examination may reveal pallor (pale eyes, vaginal mucosa), pyrexia, and elevated heart and respiratory rates. T. orientalis is an economically important parasite of cattle in New Zealand, Australia and Japan, especially where naïve animals are introduced into an endemic area or in animals under stress. Increased awareness of the risks posed by the parasite is required to enable management practices to be implemented to minimise its impact.     

Blood supply of the ovine adrenal gland and its relevance in adrenal autotransplantation

SUMMARY The arterial supply and venous drainage of 62 left and 5 right ovine adrenal glands is described, and the contribution of individual arteries to successful adrenal gland autotransplantation was evaluated. Arterial flow was measured by direct collection from the draining adrenal vein. Assessment of function of the transplanted adrenal gland was made from survival of the sheep and by the cortisol response to infusion of ACTH and the aldosterone secretory response to infusion of angiotensin II or potassium. For the left adrenal, the principal arterial supply was from the renal artery in 21 (34%), a lumbar artery in 32 (52%), and the anterior mesenteric artery in 3. The total blood flow was 5.0 ± SEM 0.4 mL/min, the flow from the renal branch 2.3 ± 0.3 mL/min, and the principal lumbar branch 2.6 ± 0.3 mL/min. Venous drainage from the left adrenal was via a major adrenal vein to the left renal vein, but additional tributaries to the renal vein were present in 26% . The arterial supply to the adrenal is regional and omission of a branch at transplantation could result in infarction of portion of the gland. By defining arterial supply and measuring blood flow, selection of the appropriate artery or multiple arteries can achieve an adrenal gland autotransplant survival of 90% .     

Effects of Confluent, Roscovitine Treatment and Serum Starvation on the Cell-cycle Synchronization of Bovine Foetal Fibroblasts

The present study was designed to examine the effects of cell-cycle synchronization protocols, such as confluent, roscovitine treatment and serum starvation, in bovine foetal fibroblasts on synchronization accuracy at G0/G1, viability, apoptosis, necrosis and ploidy for use as a nuclei donor. The cells in 5-10 passages were randomly allocated into three treated groups. Cells were cultured either in Dulbecco's modified Eagle's medium (DMEM) + 10% foetal bovine serum (FBS) until 90% confluent (group 1, confluent), in DMEM + 10% FBS + 30 microM roscovitine for 12 h (group 2, roscovitine), or in DMEM + 0.5% FBS for 5 days (group 3, serum starvation). Most of the cells (>80%) in all groups were arrested at the G0/G1 stage. Although the rates did not differ, cells in group 1 showed an increased cell population arrested at the G0/G1 phase. Significantly (p < 0.05) higher rates of apoptosis occurred in group 3 than in group 1 and 2 (10% vs 6% and 6%, respectively). No differences in chromosomal abnormality were observed among groups. However, by increasing the number of cell culture passages up to 15, significantly (p < 0.05) higher chromosomal abnormality was observed than in 5 and 10 passages (39% vs 28% and 23%, respectively) in group 1. The results clearly indicated that bovine foetal fibroblasts could be effectively synchronized at G0/G1 stages by all the three different treatments, confluent, roscovitine and serum starvation. However, cells in confluent showed reduced apoptosis and necrosis when they underwent 5-10 passages, exhibiting increased percentage of cells with stable chromosome diversity. Hence, cells in confluent merit further studies before they could be used as nuclear donors.