The generation and persistence of genetic variation in foot-and-mouth disease virus

Genetic variation in foot-and-mouth disease virus (FMDV) is of interest for at least two reasons. First, changes to the genes encoding capsid proteins results in antigenic variation, and affects vaccine efficiency and effectiveness of vaccination programs; second, genetic changes can lead to important insights into the transport of virus between countries, regions, herds, and even possibly individuals. Current estimates of RNA virus mutation rates suggest that an average of about one base mis-incorporation is likely to occur each time a single FMDV genome replicates. This should result in the introduction of every possible 1-step mutation from the progenitor genotype into the viraemia of a single infected animal many times a day. In the absence of purifying selection, a single infected animal should therefore generate a genetically very diverse population of virus.Viral-capsid sequences obtained from infected animals sampled over long-term FMDV epidemics suggest that these genetic changes accrue in a remarkably linear 'clock-like' fashion and at rates of around 1% change per year. While such a rate is generally regarded as quite high, it is actually somewhat lower than one might expect based on the rate at which viral diversity could be generated within a single animal. The difference might be explained in a variety of possible ways: (1) the mutation rate has been overestimated; (2) purifying selection is stronger than predicted; (3) only a restricted subset of excreted virus is actually infectious; (4) infected animals only excrete virus from a small partitioned subset of amplified virus, and that most of the generated viral diversity is unable to exit the animal; or (5) only a small fraction of all infected animals participate in the actual disease-transmission process.     

Ultrasonography and surgery of canine biliary diseases

Findings of hepatic and gallbladder ultrasonography were analyzed in 12 dogs with gallbladder and/or extrahepatic biliary tract obstruction and compared with the results of exploratory laparotomy. Hepatic ultrasonography demonstrated normal liver in 2 dogs and hepatic abnormalities in 10 animals. The following ultrasonographic diagnoses were established compared to surgical findings: gallbladder obstruction caused by bile sludge (correct/incorrect: 1/2, surgical diagnosis: choleliths in one case), gallbladder obstruction caused by neoplasm (0/1, surgical diagnosis: mucocele), gallbladder and extrahepatic biliary tract obstruction due to choleliths (3/3), extrahepatic biliary tract obstruction caused by pancreatic mass (1/1) and small intestinal volvulus (1/1). Bile peritonitis caused by gallbladder rupture (4/4) was correctly diagnosed by ultrasound, aided with ultrasonographically-guided abdominocentesis and peritoneal fluid analysis. Rupture of the gallbladder should be suspected in the presence of a small, echogenic gallbladder or in the absence of the organ together with free abdominal fluid during ultrasonography. Laparotomy was correctly indicated by ultrasonography in all cases. However, the direct cause of obstruction could not be determined in 2 of the 12 dogs by ultrasonography alone.     

Molecular and immunological characterisation of Theileria parva stocks which are components of the 'Muguga cocktail' used for vaccination against East Coast fever in cattle

The 'Muguga cocktail' which is composed of three Theileria parva stocks Muguga, Kiambu 5 and Serengeti-transformed has been used extensively for live vaccination against East Coast fever in cattle in eastern, central and southern Africa. Herein we describe the molecular characterisation of the T. parva vaccine stocks using three techniques, an indirect fluorescent antibody test with a panel of anti-schizont monoclonal antibodies (MAb), Southern blotting with four T. parva repetitive DNA probes and polymerase chain reaction (PCR)-based assays detecting polymorphism within four single copy loci encoding antigen genes. The Muguga and Serengeti-transformed stocks exhibited no obvious differences in their reactivity with the panel of MAbs, whereas Kiambu 5 differed with several MAbs. Kiambu 5 DNA was very distinct from the Muguga and Serengeti-transformed isolates in the hybridisation pattern with all four nucleic acid probes, whereas Muguga and Serengeti-transformed isolates exhibited minor differences and could not be discriminated with one of the probes. PCR amplification in combination with restriction fragment length polymorphism analysis indicated that Kiambu 5 was also markedly divergent from the Muguga and Serengeti-transformed stocks within two of the four antigen coding genes. The T. parva Serengeti-transformed stock did not contain a 130 base pair insert within the p67 sporozoite antigen gene, which has been observed previously in most T. parva parasites isolated from buffalo, and could not be discriminated from T. parva Muguga at any of the four single copy loci. Collectively the data indicate that two of the cocktail components T. parva Serengeti-transformed and Muguga are genetically closely related, while the third component Kiambu 5 is quite distinct. Based on the findings, there may be a need to include only one of the T. parva Muguga and Serengeti-transformed components in the immunising cocktail. The study demonstrates the value of molecular characterisation data for monitoring of live vaccines.     

Immunohistochemical detection of apolipoprotein B-100 and immunoglobulins (IgA, IgM, IgG) in the splenic arteries of aging dogs

Accumulation of lipids and hyalinosis in the splenic arteries of aged dogs are frequently detected by routine histopathologic examinations. The purpose of this study was to pinpoint the localization of canine apolipoprotein B-100 (CApoB-100) and immunoglobulins (IgA, IgM, IgG) in the splenic arteries of aging dogs (n = 80) through the use of immunohistochemical techniques. CApoB-100 deposits were found in the subendothelial space, extracellular matrix, and atheromatous lesions in the tunica media of the arteries in dogs > or = 6 years of age. Foamy cytoplasm of the infiltrated macrophages was also CApoB-100 immunopositive. In dogs > or = 10 years of age, almost all central arteries were CApoB-100 immunopositive. Hyaline deposits within the wall were characterized by immunopositivity against canine IgA, IgM, IgG, and albumin. Lipid accumulation in splenic arteries may be an age-related lesion and a precursor of the atheromatous plaques associated with splenic hemorrhage and infarcts later in life. In addition, deposition of immunoglobulins, probably mediated by immune complexes, may play an important role in the development of canine vascular diseases similar to human disease.     

Refinement of an orthotopic lung cancer model in the nude rat

Over 85% of people with lung cancer eventually succumb to this disease, largely because current chemotherapies are ineffective. The testing and validation of promising new approaches generally rely on achieving responses with cell lines in vitro or in tumor xenografts in nude mice. However, quite often the results seen with these models are not recapitulated in the clinic, thus necessitating the need for better animal models of lung cancer for preclinical testing of new therapies. One promising model is that of orthotopic lung cancer, where xenografts of human lung cancer are established in lungs of immunodeficient rodents. The problems associated with this model include poor rates of engraftment, limited tumor multiplicity, and a heightened risk for surgical trauma. The purpose of our study was to develop an efficient approach to engraftment of orthotopic tumors throughout the lungs of the Rowett nude rat. Initially, we augmented immunosuppression in the rats with whole-body X-irradiation and then used orotracheal cannulas to intratracheally instill human cancer cells from the Calu-6 cell line. This protocol produced a low rate of engraftment and low tumor multiplicity. The hypothesis that slight disruption of the pulmonary epithelium or the surfactant layer would allow better tumor engraftment was tested by coadministration of either pancreatic elastase or ethylenediaminetetraacetic acid (EDTA) along with the cell instillations. Lung tumor engraftment was evaluated 8 weeks after instillation. The inclusion of elastase or EDTA with the Calu-6 cells resulted in an 80-100% engraftment rate, respectively. Coadministration of EDTA resulted in significantly larger and greater numbers of tumors/lung than those in elastase-treated animals. Temporal studies demonstrated that small nodules were scattered throughout the lung parenchyma 5 weeks after instilling Calu-6 cells and EDTA. These nodules grew to coalesce and form large masses that effaced >75% of the parenchyma at 9 weeks postinstillation. The refinements made through our studies have led to the development of an orthotopic lung cancer model that should facilitate the evaluation of novel therapies designed to treat or impede lung cancer development.     

Pulmonary expression of tumor necrosis factor alpha, interleukin-1 beta, and interleukin-8 in the acute phase of bovine pneumonic pasteurellosis

Inflammatory cytokines are suspected to contribute to the pathogenesis of bovine pneumonic pasteurellosis (BPP) through neutrophil recruitment, leukocyte activation, and the induction of a broad array of soluble inflammatory mediators. An in vivo experimental model of BPP was used to characterize the pulmonary expression kinetics of tumor necrosis factor alpha (TNFalpha), interleukin-1 beta (IL-1beta), and interleukin-8 (IL-8) genes and proteins during the acute phase of disease development. Cytokine expression in bronchoalveolar lavage (BAL) fluid, BAL cells, and pneumonic lung parenchyma was quantitated by northern blot analysis, enzyme-linked immunosorbent assay (ELISA), and in situ hybridization at 2, 4, 8, 16, and 24 hours after endobronchial inoculation of Pasteurella (Mannheimia) haemolytica. Expression of TNFalpha, IL-1beta, and IL-8 was significantly increased in the airways and lung lesions of infected calves as compared with mock-infected controls. Although kinetic patterns varied, peak levels of cytokine mRNA occured within 8 hours postinfection (PI), and peak cytokine concentrations occurred within 16 hours PI. In all samples, IL-8 was expressed to the greatest extent and TNFalpha was least expressed. Expression of TNFalpha was restricted to alveolar macrophages. Alveolar and interstitial macrophages produced IL-1beta and IL-8 in the first 4 hours; bronchial and bronchiolar epithelial cells were also significant sources of IL-8 during this period. By 8 hours PI, neutrophils were the dominant source of both IL-1beta and IL-8. These findings demonstrate a spatial and temporal association between pulmonary expression of inflammatory cytokines and acute lung pathology, supporting the hypothesis that cytokines contribute to inflammatory lung injury in BPP.     

International harmonisation of anthelmintic efficacy guidelines

The "International Co-operation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH)" is an international programme of co-operation between regulatory authorities and the animal health industries of the European Union, Japan, and the United States of America which aims to harmonise the technical requirements for the registration of veterinary medicinal products. Australia and New Zealand participate as active observers. The objective of the present paper is to present the guidelines established by the working group on Anthelmintic Efficacy Guidelines: (1) efficacy of anthelmintics: general requirements (VICH GL7); (2) efficacy of anthelmintics: specific recommendations for bovines (VICH GL12); (3) efficacy of anthelmintics: specific recommendations for ovines (VICH GL13); (4) efficacy of anthelmintics: specific recommendations for caprines (VICH GL14). These guidelines do not consist of rigid stipulations, but make clear recommendations on the minimal standards needed. To the veterinary profession, livestock producers and animal owners, harmonisation should mean quicker access to safer and more effective veterinary anthelmintics. In general, products should be relatively more affordable because of the reduction in registration costs and efficient use of resources by the regulatory authorities.     

Canine model and genomic structural organization of glycogen storage disease type Ia (GSD Ia)

A canine model of glycogen storage disease Ia (GSD Ia), similar clinically, biochemically, and pathologically to the human disease, was established by crossbreeding Maltese and Beagle dogs carrying a mutated, defective glucose-6-phosphatase (G-6-Pase) gene. Ten puppies were born in three litters from these crossbreedings. Six were homozygous for the previously described M121I GSD Ia mutation. Of these six affecteds, two were stillborn, and one died at 2, 32, and 60 days of life, respectively (puppies A, B, C, D, E), while one is alive at age 15 months (puppy F). Affected puppies exhibited tremors, weakness, and neurologic signs when hypoglycemic. They had postnatal growth retardation and progressive hepatomegaly. Biochemical abnormalities included fasting hypoglycemia, hyperlactacidemia, hypercholesterolemia, hypertriglyceridemia, and hyperuricemia. Microscopic examination of tissues from affected puppies showed diffuse, marked hepatocellular vacuolation, with distended clear hepatocytes and central to marginally located rounded nuclei. In the kidneys of puppies D and E, there was segmental glomerular sclerosis and vacuolation of proximal convoluted tubular epithelium. Biochemical analysis revealed increased liver glycogen content and isolated markedly reduced G-6-Pase enzyme activity in liver and kidney. The canine G-6-Pase gene was characterized by screening a canine genomic library. It spans approximately 11.8 kb and consists of five exons with >90% amino acid sequence homology to the derived human sequence. The first 1.5 kb of the 5' region was sequenced and contains several putative response element motifs homologous to the human 5' region. Establishment of this canine colony of GSD Ia that closely resembles human disease and isolation of the canine genomic gene provides an excellent model for studying pathophysiology and long-term complications and an opportunity to develop novel therapeutic approaches such as drug and gene therapy.     

Immunohistochemical demonstration of Leu-7 (HNK-1), Neurone-specific Enolase (NSE) and Protein-Gene Peptide (PGP) 9.5 in the developing camel (Camelus dromedarius) heart

The development of the heart-conducting system has been controversially discussed. The common opinion that these specialized myocytes originate from mesodermal precursors has been challenged when nerve-specific antigens (Leu-7, NF, GIN2) were demonstrated in embryonic hearts of various species, suggesting a neural crest contribution to the embryonic conducting tissue. Anti-Leu-7 (HNK-1) antibodies were reported to reliably mark the conducting system in developing rat, chicken and human hearts. The present investigation was carried out on the hearts of 15 camel fetuses at 35, 45, 60, 75 and 100 cm crown-rump length (three specimens for each stage), in addition to three adult hearts. We investigated the antigenicity of cardiac structures for Leu-7, NSE (Neurone specific Enolase) and PGP (Protein Gene Peptide) 9.5. In all specimens investigated, both NSE and PGP 9.5 were expressed by cardiac nerves and conducting system components. The sinuatrial and atrioventricular nodes, the atrioventricular bundle as well as subendocardial and intramyocardial Purkinje fibers were stained. In contrast, the developing conducting system did not react with anti-Leu-7 antibody, although Leu-7 antigenicity was strongly expressed by the developing cardiac nerves. In adult camel hearts, the same pattern of immunoreactivity for the markers studied was still retained. Our results show that the expression of marker proteins for the developing conducting system is species-specific. Therefore, these markers are of little significance in discussions on the possible neurogenic nature of the heart conducting tissue.