A dose-escalation study of combretastatin A4-phosphate in healthy dogs.

Combretastatin A4-Phosphate (CA4P) is a vascular disrupting agent revealing promising results in cancer treatments for humans. The aim of this study was to investigate the safety and adverse events of CA4P in healthy dogs as a prerequisite to application of CA4P in dogs with cancer. Ten healthy dogs were included. The effects of escalating doses of CA4P on physical, haematological and biochemical parameters, systolic arterial blood pressure, electrocardiogram, echocardiographic variables and general wellbeing were characterised. Three different doses were tested: 50, 75 and 100 mg m-2 . At all 3 CA4P doses, nausea, abdominal discomfort as well as diarrhoea were observed for several hours following administration. Likewise, a low-grade neutropenia was observed in all dogs. Doses of 75 and 100 mg m-2 additionally induced vomiting and elevation of serum cardiac troponine I levels. At 100 mg m-2 , low-grade hypertension and high-grade neurotoxicity were also observed. In healthy dogs, doses up to 75 mg m-2 seem to be well tolerated. The severity of the neurotoxicity observed at 100 mg m-2 , although transient, does not invite to use this dose in canine oncology patients.

A Refined Guinea Pig Model of Foot-and-Mouth Disease Virus Infection for Assessing the Efficacy of Antiviral Compounds.

An antiviral containment strategy for foot-and-mouth disease (FMD) outbreaks could support or replace current contingency plans in case of an outbreak in Europe and could spare many healthy animals from being pre-emptively culled. Recently, substantial progress has been made towards the development of small molecule drugs that inhibit FMD virus (FMDV) replication in vitro. For the initial in vivo evaluation of antiviral lead molecules, a refined FMDV-infection model in guinea pigs (GP) is herewith described. This GP model was validated by demonstrating the antiviral effect of T-1105 (an influenza virus inhibitor with reported activity against FMDV). Sixteen animals were orally administered with T-1105 twice daily (400 mg/kg/day) for five consecutive days and inoculated intraplantarly with 100 GPID50 of the GP-adapted FMDV strain O1 Manisa 1 h after the first administration. The efficacy of T-1105 was compared with that of prophylactic vaccination with a highly potent double-oil emulsion-inactivated O1 Manisa vaccine. Ten animals received a single, full (2 ml) cattle vaccine dose and were inoculated 3 weeks later. Fourteen T-1105-treated and all vaccinated GP were completely protected from generalization of vesicular lesions. At 2 dpi, viral RNA was detected in serum of 9/16 T-1105-treated and of 6/10 vaccinated animals. At 4 dpi, viral RNA was detected in serum, organs and oral swabs of half of the T-1105-treated animals and only in the serum of 1/10 of the vaccinated animals. Mean viral RNA levels in serum and organs of T-1105-treated and vaccinated animals were reduced compared to untreated controls (P < 0.01). T-1105 conferred a substantial clinical and virological protection against infection with O1 Manisa, similar to the protection afforded by vaccination. These results validate the suitability of the enhanced GP model for the purpose of initial evaluation of inhibitors of FMDV replication and illustrate the potential of selective inhibitors of viral replication to control FMD outbreaks.

Haemorrhagic diathesis in neonatal calves: an emerging syndrome in Europe.

In 2008 and 2009 a large number of cases of haemorrhagic diathesis (HD) in neonatal calves were reported in different European countries. In Flanders, 84 cases of neonatal HD in 30 herds were reported in this period. The disease typically affects calves younger than 1 month old from different breed and gender. Prominent clinical signs are cutaneous bleeding, petechiae on all mucosae, melena and often high fever. Early in the disease, the mental state of the animals is uncompromised. The typical haematological finding is pancytopenia, with severe to complete thrombocytopenia being the cause of the increased susceptibility to bleeding. In seven of the affected herds blood samples of calves of the same age group as the clinical case were collected and on six of those farms at least one subclinical case could be identified. Necropsy findings were generalized petechiae, ecchymoses or haemorrhages and variable lymphadenopathy. Histopathology of haemorrhagic lesions revealed multifocal extravasation of red blood cells (haemorrhage) with preservation of tissue architecture and absence of other abnormalities. Total bone marrow aplasia and depletion of all lymphoid tissue was the most prominent finding on histology. Activated macrophages and haemophagocytosis were seen on bone marrow cytology from two live calves. Polymerase chain reaction for bovine viral diarrhoea virus, bluetongue and epizootic haemorrhagic disease virus was negative. Several attempts to isolate a viral agent were unsuccessful.

Highly sensitive detection of swine vesicular disease virus based on a single tube RT-PCR system and DIG-ELISA detection.

A highly sensitive detection of swine vesicular disease virus (SVDV) based on a single tube RT-PCR system and digoxigenin (DIG)-PCR-ELISA detection was developed. Using a one tube RT-PCR system, optimisation of the PCR conditions and optimisation of the microwell hybridisation and colourimetric detection of the amplicons resulted in a method that could detect viral RNA in infected tissue culture fluid with a titre as low as 0.1 TCID50/100 microl. The same sensitivity was obtained with SVDV-spiked faeces, if the samples were pre-treated with 1,1,2-trichlorotrifluoroethane/chloroform and subsequently concentrated using an ultrafiltration system and RNA extracted with the Purescript kit. The specificity of the test was validated on 27 SVDV strains belonging to four different groups. No cross-reactivity with genetically and symptomatically related viruses was detected using RNA of foot-and-mouth disease virus (FMDV), porcine enterovirus (PEV), vesicular stomatitis virus (VSV), Coxsackie B5 virus (CV-B5) and encephalomyocarditis virus (EMCV). The test was validated successfully on clinical samples, being slightly more sensitive and much faster than virus isolation on cell cultures. Moreover the possibility of automating the procedure will allow the processing of large numbers of clinical samples.

Differentiation of the seven serotypes of foot-and-mouth disease virus by reverse transcriptase polymerase chain reaction.

A strategy for reverse transcriptase polymerase chain reaction (RT-PCR) using multiple primers was developed to detect and to differentiate the seven serotypes of foot-and-mouth disease virus (FMDV) simultaneously, quickly and accurately. The development of the test was carried out on virus isolates grown in tissue culture prior to cDNA synthesis and PCR using various sets of primers. Primers P33 and P32 were used for the consensus PCR to detect FMDV regardless of the serotype. Positive cDNA was assayed in two multi-primer PCR mixes containing type-specific primers capable of distinguishing between the seven serotypes. The serotype-specific primers were selected to correspond to regions of the genome coding for parts of the VP1 polypeptide that is responsible for the antigenic diversity of the virus group. Multi-primer mix P33-P(A-C-O-ASIA 1) gave products of 732, 596, 402, 292 bp for the A,C,O and ASIA 1 serotypes, respectively, and no target products for South African Territories serotypes (SAT 1, SAT 2 and SAT 3). The multi-primer mix P33-P(A-C-O-ASIA 1) was also capable of detecting a mixture of two different serotypes. Multi-primer mix P1-P(SAT 1-3-2) gave products of 246, 201 and 75 bp for the SAT 1, SAT 3 and SAT 2 serotypes, respectively, and no specific products for serotypes A, C, O and ASIA 1. This is the first PCR assay to be described that differentiates between the SAT serotypes of FMDV. The method has been applied to 25 cell-culture-derived isolates of the SAT serotypes of FMDV and the results were totally compatible with the standard techniques for FMDV detection and serotyping.

The serological response to foot-and-mouth disease vaccination is not affected by simultaneous infectious bovine rhinotracheitis/adenovirus/parainfluenza 3 vaccination.

Young calves were simultaneously vaccinated by subcutaneous route against foot-and-mouth disease (FMD) and against infectious bovine rhinotracheitis/adenovirus/parainfluenza-3 (IBR/Adeno/PI-3) by intranasal route. The serological response against the 3 FMD virus types of the FMD vaccine was clearly positive. There was no significant difference between results of simultaneous FMD and IBR/Adeno/PI-3 vaccination and FMD vaccination only.

Experimental transmission of enzootic bovine leukosis to cattle, sheep and goats: infectious doses of blood and incubation period of the disease.

A group of 49 BLV-free recipient animals (24 cattle, 15 sheep and 10 goats) were inoculated intradermally with serial dilutions of blood collected on two BLV-positive donor cows. One donor had a high lymphocytosis and high antibody titers to gpBLV antigens; these two parameters were low for the second donor. The number of lymphocytes which induced BLV infection in recipient animals varied widely with the donor. The high infectivity of a donor seemed to be correlated with high lymphocytosis and high antibody titers to gpBLV antigens. Identification and removal of infectious animals would reduce or stop the spread of the infection in a herd, and could be used in the strategy to eradicate the disease. A given inoculum can be infectious in sheep and, at the same time, harmless in cattle. The incubation periods, apparently shorter in sheep, were generally in the range from 2 to 5 weeks for the three species, and exceptionally above.