Identification of optimal regions for phylogenetic studies on VP1 gene of foot-and-mouth disease virus: analysis of types A and O Argentinean viruses

An analysis of the informative content of sequence stretches on the foot-and-mouth disease virus (FMDV) VPI gene was applied to two important viral serotypes: A and O. Several sequence regions were identified to allow the reconstruction of phylogenetic trees equivalent to those derived from the whole VPI gene. The optimal informative regions for sequence windows of 150 to 250 nt were predicted between positions 250 and 550 of the gene. The sequences spanning the 250 nt of the 3' end (positions 400 to 650), extensively used for FMDV phylogenetic analyses, showed a lower informative content. In spite of this, the use of sequences from this region allowed the derivation of phylogenetic trees for type A and type O FMDVs which showed topologies similar to those previously reported for the whole VP1 gene. When the sequences determined for viruses isolated in Argentina, between 1990 and 1993, were included in these analyses, the results obtained revealed features of the circulation of type A and type O viruses in the field, in the months that preceded the eradication of the disease in this country. Type A viruses were closely related to an Argentinean vaccine strain, and defined an independent cluster within this serotype. Among the type O viruses analysed, two groups were distinguished; one was closely related to the South American vaccine strains, while the other was grouped with viruses of the O3 subtype. In addition, a detailed phylogeny for type A FMDV is presented.     

The pharmacokinetics and metabolism of ivermectin in domestic animal species

The pharmacokinetic properties of drugs are closely related to their pharmacological efficacy. The kinetics of ivermectin are characterised, in general terms, by a slow absorption process, a broad distribution in the organism, low metabolism, and slow excretion. The kinetics vary according to the route of administration, formulation, animal species, body condition, age, and physiological status, all of which contribute to differences in drug efficacy. Characterisation of ivermectin kinetics can be used to predict and optimise the value of the parasiticide effects and to design programmes for parasite control. This article reviews the pharmacokinetics of ivermectin in several domestic animal species.     

Lack of a type-2 glycosyltransferase in the fish pathogen Flavobacterium psychrophilum determines pleiotropic changes and loss of virulence

Flavobacterium psychrophilum is an important fish pathogen, responsible for Cold Water Disease, with a significant economic impact on salmonid farms worldwide. In spite of this, little is known about the bacterial physiology and pathogenesis mechanisms, maybe because it is difficult to manipulate, being considered a fastidious microorganism. Mutants obtained using a Tn4351 transposon were screened in order to identify those with alteration in colony morphology, colony spreading and extracellular proteolytic activity, amongst other phenotypes. A F. psychrophilum mutant lacking gliding motility showed interruption of the FP1638 locus that encodes a putative type-2 glycosyltransferase (from here on referred to as fpgA gene, Flavobacterium psychrophilum glycosyltransferase). Additionally, the mutant also showed a decrease in the extracellular proteolytic activity as a consequence of down regulation in the fpgA mutant background of the fpp2-fpp1 operon promoter, responsible for the major extracellular proteolytic activity of the bacterium. The protein glycosylation profile of the parental strain showed the presence of a 22 kDa glycosylated protein which is lost in the mutant. Complementation with the fpgA gene led to the recovery of the wild-type phenotype. LD₅₀ experiments in the rainbow trout infection model show that the mutant was highly attenuated. The pleiotropic phenotype of the mutant demonstrated the importance of this glycosyltranferase in the physiology and virulence of the bacterium. Moreover, the fpgA mutant strain could be considered a good candidate for the design of an attenuated vaccine.     

Determination of the immunotoxic potential of heavy metals on the functional activity of bottlenose dolphin leukocytes in vitro

Heavy metals may affect the immune system of cetaceans. But no information exists on their effects on the bottlenose dolphin (Tursiops truncatus) immune system, although this species is a coastal top predator which can bioaccumulate high concentrations of them. This work studies the effects of Hg (1, 5 and 10mg/L), Al (2,5, 25 and 50mg/L), Cd (1, 10, 20 and 40mg/L), Pb (1, 10, 20 and 50mg/L) and Cr (1 and 10mg/L), on the function of phagocytes and lymphocytes isolated from the peripheral blood of bottlenose dolphins under in vitro conditions. Cell viability, apoptosis, lymphocyte proliferation and phagocytosis were evaluated. Viability and lymphoproliferation were measured with Alamar Blue assay, and apoptosis and phagocytosis were evaluated with flow cytometry. Apoptosis was detected as mechanism of cell death after cadmium and mercury exposure. A significant reduction in the lymphoproliferative response was registered by exposure to 1mg/L of mercury, 10mg/L of cadmium and 50mg/L of lead. Decreased phagocytosis was also observed at 5mg/L of mercury, 50mg/L of aluminium and 10mg/L of cadmium. Chromium did not present any effects on any immune assay at the concentrations tested. The concentrations of heavy metals that were found to affect the functional activity of bottlenose dolphin leukocytes are within the environmental ranges reported in the tissues of bottlenose dolphins. These results support the hypothesis that exposure to these contaminants, particularly mercury and cadmium could lead to a reduction in host resistance to disease in these animals.     

Pseudorabies virus infection in mink: a host-specific pathogenesis

Pseudorabies virus (PRV) is an alphaherpesvirus that causes a neurological disease in many wild and domestic animals. The neuropathology elicited by PRV is quite consistent regardless of the host with the only exception of mink, in which it is characterized by a vasculopathy rather than by an encephalitis. In this study, we aimed to investigate the underlying pathogenic mechanism(s) of PRV infection in mink by using immunohistochemistry and laser capture microdissection (LCM) on material from naturally and experimentally infected animals. The inflammatory reaction induced by PRV was minimal or absent not only in the nervous system, where we identified a low number of macrophages and a few T lymphocytes, but also in the primary replication site, the oropharyngeal mucosa; however, the number of PRV-infected cells detected by immunohistochemistry was extremely high both in the peripheral mucosa and in the nervous tissue. On the other hand, the vascular pathology included parenchymal hemorrhages of various degrees and, in specific cortical areas of the brain, fibrinoid degeneration of the capillary walls. Detection of viral antigens by immunohistochemistry revealed infection of endothelial cells of capillaries situated both in the oropharyngeal mucosa and in the brain stem; the presence of PRV DNA in vessels was further demonstrated by PCR performed on LCM samples of brain capillaries. These results can be interpreted as supporting the idea that the different pathology of the disease in mink may be the consequence of an increased endotheliotropism of PRV in this species. Infection of the vessel wall may then lead to vascular pathology and impairment in endothelial cell function, resulting in a weak immune response to infection.     

Salmonella enterica serovar Choleraesuis derivatives harbouring deletions in rpoS and phoP regulatory genes are attenuated in pigs, and survive and multiply in porcine intestinal macrophages and fibroblasts, respectively

Live attenuated Salmonella enterica strains have been extensively studied as potential vectors for the oral delivery of heterologous antigens. Due to its ability to target immune cells, its specific mechanism for crossing the intestinal barrier, and its swine-restricted tropism, S. enterica subspecies enterica serovar Choleraesuis (S. Choleraesuis) has attracted a great deal of interest for the production of bacterial-based oral carriers specifically adapted to swine. In this study, two mutants of S. Choleraesuis were constructed and their attenuation and intracellular fate analysed with the purpose of engineering new attenuated live strains with improved properties as oral vaccine carriers. Those strains harboured a specific deletion either within the phoP or rpoS genes, which encode virulence-related regulators in S. Typhimurium. In comparison to the wild-type parental S. Choleraesuis, the mutant strains, especially DeltaphoP, were extremely low in virulence in the murine model and in the natural host, the pig. Moreover, when compared with a commercial live vaccine strain, SC-54, the two mutants showed a higher level of attenuation in mice and DeltaphoP also in pigs. In addition, DeltarpoS and DeltaphoP presented a proliferation and survival phenotype within swine intestinal primary fibroblast and macrophage cell cultures, respectively. Collectively, the present results indicate that the DeltarpoS and DeltaphoP strains of S. Choleraesuis gather adequate features to be potential candidates for vaccine vectors for the specific delivery of heterologous antigens adapted to pigs.     

A monoclonal antibody against a canine CD45 homologue: analysis of tissue distribution, biochemical properties and in vitro immunological activity

This report describes the characterisation of a monoclonal antibody (mAb), AB6, which recognises specifically a cluster of canine leukocyte surface molecules. The immunogen used for obtaining the AB6 mAb was a lysate of canine peripheral blood mononuclear cells (PBMC). This novel mAb belongs to the IgG2a isotype, and reacted in Western blot with four different canine leukocyte glycoproteins with apparent molecular weights of 180, 190, 205 and 220 kDa. The AB6 mAb recognised the majority of canine peripheral blood leukocytes as determined by flow cytometry (97%). It also exhibited a broad reactivity pattern against lymphoid and myeloid cells, inhibited the proliferation of mitogen-stimulated canine PBMC and did not recognise human PBMC and murine splenocytes. The biochemical properties, cell and tissue specificity, and in vitro biological activity of the AB6 mAb indicate that it recognises a canine CD45 homologue. The mAb could become a valuable diagnostic and research tool for the evaluation of immune functions in dogs.     

Sequence analysis of the msp4 gene of Anaplasma ovis strains

Anaplasma ovis (Rickettsiales: Anaplasmataceae) is a tick-borne pathogen of sheep, goats and wild ruminants. The genetic diversity of A. ovis strains has not been well characterized due to the lack of sequence information. In this study, we evaluated bighorn sheep (Ovis canadensis) and mule deer (Odocoileus hemionus) from Montana for infection with A. ovis by serology and sequence analysis of the msp4 gene. Antibodies to Anaplasma spp. were detected in 37% and 39% of bighorn sheep and mule deer analyzed, respectively. Four new msp4 genotypes were identified. The A. ovismsp4 sequences identified herein were analyzed together with sequences reported previously for the characterization of the genetic diversity of A. ovis strains in comparison with other Anaplasma spp. The results of these studies demonstrated that although A. ovismsp4 genotypes may vary among geographic regions and between sheep and deer hosts, the variation observed was less than the variation observed between A. marginale and A. phagocytophilum strains. The results reported herein further confirm that A. ovis infection occurs in natural wild ruminant populations in Western United States and that bighorn sheep and mule deer may serve as wildlife reservoirs of A. ovis.     

Evaluation of an antigen capture ELISA for the early diagnosis of Hypoderma lineatum in cattle under field conditions

An antigen capture or sandwich ELISA (sELISA) was evaluated for the diagnosis of Hypoderma lineatum in cattle under field conditions in northwestern Spain. The kinetics of circulating hypodermin C (HyC) and specific antibodies during the course of natural infestation were determined in a group of 10 Frisian calves. In addition, oesophagi and blood samples were taken from 105 cows at a slaughterhouse in order to compare three methods for the diagnosis of H. lineatum: sandwich ELISA for the detection of the antigen HyC (sELISA), indirect ELISA for the detection of antibodies anti-HyC (iELISA) and the detection of first instars (L1) in the oesophagus. In naturally infested cattle, HyC was present in circulation at low levels during the early and late phases of the infestation. However, in the middle phase, coinciding with the presence of L1 in the oesophagus, two peaks of increased HyC concentration were observed. Specific antibodies increased progressively until the first appearance of larvae in warbles on the back. There was no correlation between antigen or antibody levels and the number of grubs in the back. Prevalence of first instars in the oesophagi of slaughtered cows was 21.9% (23/105). The percentage of cattle that were positive for circulating antigen was slightly higher (24.8%), suggesting the recent destruction of migrating larvae in some animals. However, there was no correlation between the number of L1 and HyC levels. With the iELISA, 79% of the animals were positive to Hypoderma, which means that a high percentage of those animals have been exposed to the parasite but they had no apparent current infestation. The sELISA is a good tool to follow larval development within the host; however, the episodic elevation of HyC levels limits the usefulness of this test for the early diagnosis of Hypoderma under field conditions.