A survey of more than 11 years of neurologic diseases of ruminants with special reference to transmissible spongiform encephalopathies (TSEs) in Greece

The first cases of scrapie were detected in Greece in a flock of sheep in October 1986. All the animals of the affected flock and all sheep in two flocks that were in contact were killed and buried. A systematic investigation of all available cases with signs indicating a neurological disease started in sheep and goats in late 1986, as well as in cattle in 1989. The investigation was based on clinical examination, necropsy or macroscopical examination of the brain and viscera, and histological examination of the brain in all animals except those with coenurosis. Histological examinations of specimens from the spinal cord and other tissues, and if considered necessary bacteriological, toxicological and serological examinations were also carried out. In October 1997, scrapie was diagnosed in sheep of a second flock (a mixed flock of sheep and goats), grazing in a pasture close to the place where scrapie was initially detected. All animals of the second flock were also killed and buried. Diagnosis in the first flock was based on clinical signs and histological lesions, and in the second immunoblotting was also used. Distinctive lesions of scrapie were found in the brain and/or the spinal cord of eight sheep with clinical signs from the two flocks. The lesions were revealed in the brain stem and/or in the cervical spinal cord, and tended to be symmetrical. In one sheep, severe lesions in the cortex of cerebral hemispheres and of the cerebellum were also found. In the brain of two sheep from the second flock the pathological isoform of PrP protein was detected. Despite the eradication scheme applied, scrapie in sheep reappeared after 11 years in a place close to where it occurred initially. This may indicate that the effectiveness of the eradication scheme implemented was not adequate and additional approaches may be needed.     

The detection of proviral DNA by semi-nested polymerase chain reaction and phylogenetic analysis of Czech Maedi-Visna isolates based on gag gene sequences

A semi-nested polymerase chain reaction (snPCR) for detecting proviral DNA of ovine lentivirus (OvLV) in peripheral blood mononuclear cells was developed. Primers for snPCR were situated within the gag gene of the Maedi-Visna virus (MVV) genome. A comparison between the snPCR and serological tests (agar gel immunodiffusion test, immunoblot) were performed using 98 ovine blood samples. Thirty (30.6%) of the 98 sheep examined had antibodies specific for the MVV. PCR showed 21 of them to be positive and nine seropositive animals to be PCR negative. Six of the 68 serologically negative sheep were found to be PCR positive, probably due to delayed seroconversion. The PCR amplification products of these six sheep were sequenced and subjected to phylogenetic analysis. The resulting phylogenetic tree of partial gag gene sequences confirmed that the ovine lentivirus genotype in the Czech Republic is more closely related to the prototype MVV isolates than to the caprine arthritis encephalitis viruses.     

Construction of an internal standard used in RT nested PCR for Borna Disease Virus RNA detection in biological samples

The highly neurotropic Borna Disease Virus (BDV), which belongs to the Mononegavirales order--Bornaviridae family--is generally detected using the RT-nested-PCR. If false positive results (often caused by laboratory contaminations) can be avoided, some false negative results which are mostly due to inhibitory effects of some reaction components and/or to sample preparation errors, can occur. Thus, in order to control the RT-PCR sample, an RNA internal standard molecule named "mimic" was constructed with the same primer recognition sites as the viral nucleic acids, flanking a heterologous DNA fragment of distinct molecular weight. Because of their different sizes, the mimic and viral PCR products can be easily discriminated by agarose gel electrophoresis. The co-amplification of both BDV and mimic RNA was performed on infected cells and on biological tissues such as the brain and blood, commonly known to contain PCR inhibitor components. After mimic sensitivity studies were achieved (2.5 fg of "p40 RNA mimic" and 0.25 fg of "p24 RNA mimic"), the competitive amplification reaction between both BDV and mimic RNA was performed on these tissues. The results confirmed that nervous tissue has an inhibitory effect on RT-PCR, which supports the necessity of BDV detection by a higher sensitive method such as RT nested PCR. Moreover, these results confirmed the interest of an internal standard for BDV RNA detection in biological samples.     

Equine conjunctival pseudotumors

Five horses presented with unilateral pink, smooth, nonulcerated conjunctival masses with histologic features characteristic of inflammatory pseudotumors, i.e. proliferative inflammatory lesions clinically resembling true neoplasia. Although causes for the inflammatory lesions were not determined, based on the presence histologically of mononuclear (predominantly lymphocytic) inflammatory cell infiltrates and the absence of infectious agents, parasites or foreign bodies, an immune-mediated pathogenesis was suspected. Affected horses ranged from 5 to 8 years of age with no apparent breed or sex predilection. Conjunctival lesions were nodular in two cases and relatively flat and more diffuse in three cases. Third eyelid lesions were present in three cases and two affected eyes had corneal involvement. Based on findings from these five cases, the prognosis for equine conjunctival pseudotumors appears to be good when lesions are treated by partial or complete surgical excision, local administration of anti-inflammatory agents, or a combination of surgery and anti-inflammatory therapy.     

CO2 stunning of broilers and turkey hens

Stunning of poultry is still not solved satisfactorily. This concerns the requirements of animal welfare, meat quality and working conditions in the lairage, stunning and debleeding area. In an investigation of combined CO2-/O2-stunning in a new gas stunning system stress reactions of the animals during the induction phase and stunning effectivity were recorded in 7,000 chicken and 3.825 turkeys. During the induction phase (here: chicken 41 sek./turkeys 25-65 sec.) the animals first staid calm and then showed beak-opening as a consequence of the breathing stimulating effect of CO2. As a further sign of the aversiveness against CO2 the animals showed head shaking and wing flapping. The used settings of gas concentrations and stunning time in the system investigated lead to a very deep stunning resp. Killing of the animals. Therefore the debleeding cut could be performed late (chicken 44-55 sec., turkeys 54-90 sec.) after leaving the system without animals regaining consciousness. The stunning of turkeys with a mixture of CO2 and oxygen is an improvement according to animal welfare requirements because unnecessary pain and suffering, happening very often with electrical stunning, can be avoided. As far as animal welfare in chicken stunning is concerned it must be evaluated if gas stunning means an improvement because stress during the till now relatively long induction phase must be put into relation with comparably lower stress caused by hanging upside down and a fast and safe working electrical stunning unit.     

Infection of a canine macrophage cell line with leishmania infantum: determination of nitric oxide production and anti-leishmanial activity

We have previously shown that resistance to Leishmania infantum in dogs is associated with a Th1 type of immune response. In this study, we use a canine macrophage cell line (030-D) that can readily be infected with this protozoan parasite. Our aim is to further characterize the effector mechanisms involved in killing of Leishmania parasite in dogs. We observed that activation of 030-D cells by incubation with a supernatant derived from a Leishmania-specific T cell line containing IFN-gamma, TNF-alpha and interleukin-2 (IL-2) resulted in enhanced nitric oxide (NO) production by these cells. In addition, we observed enhanced anti-leishmanial activity of infected 030-cells after activation. Both, NO production and anti-leishmanial activity were abrogated by addition of L-N(G)-nitroargininemethyl ester (L-NAME), an analogue of L-arginine. Thus, NO play an important role in the anti-leishmanial activity of these canine macrophages. We propose the infection of the 030-D cell line as a good in vitro model to further investigate parasite-host cell interactions in dogs, a natural host of Leishmania parasites.     

Lateral musculature in the whitemouth croaker (Micropogonias furnieri): its characterization with respect to different gonadal conditions

Histochemical and ultrastructural studies were performed on the lateral musculature from individual female whitemouth croaker, Micropogonias furnieri, at the anterior, medium and posterior regions. Based upon histochemical myosin-ATPase (m-ATPase) determination, diverse types of red, pink and white fibres were discerned. Red muscle had abundant mitochondria and stained intensely for aerobic enzymes, white muscle scarcely stained for the same enzymes and pink muscle responded in an intermediate manner. Both white and pink muscle had few mitochondria. The relative proportion of red muscle increased towards the caudal region; pink muscle diminished towards this region and white muscle modified its proportion only in the anterior region. m-ATPase activity showed differences in relation to the gonadal condition along the body, particularly in the white fibres at the anterior and medium regions.     

Effects of porcine reproductive and respiratory syndrome virus (isolate tw91) on porcine alveolar macrophages in vitro

To verify the role of porcine reproductive and respiratory syndrome virus (PRRSV) infection on pulmonary defense mechanisms, alterations in the viability, morphology, and various functions of porcine alveolar macrophages (AMs) were evaluated in vitro for 2-72 h after exposure to a Taiwan isolate, tw91, at a multiplicity of infection (MOI) of 0.1. A low but constant rate of infection, around 5%, was seen in AMs from the PRRSV-infected group throughout the study. When compared with a mock-infected group, AMs from the PRRSV-infected group had a significantly lower viability at 18-72 h post-infection (HPI) as determined by trypan blue dye exclusion. Also during this time period, the cells showed morphological changes, including rounding, bleb formation, and rupture. The phagocytic and microbicidal capacity of AMs against Candida albicans was significantly inhibited after 6 HPI. Although the total amount of superoxide anion (O2-) and hydrogen peroxide (H2O2) produced by the AMs was reduced after 18 and 12 HPI, respectively, the amount of production was enhanced in both reactive oxygen species on a per viable cell basis after 12 HPI. In contrast, the level of bioactive tumor necrosis factor alpha (TNF-alpha) secretion, either total or on a per viable cell basis, was markedly reduced soon after PRRSV infection, up to 36 HPI, followed by a rebound thereafter. Prostaglandin E2 (PGE2) production was enhanced, both in total and on a per viable cell basis, in the first 6 h of infection, especially at 2 HPI. However, it became lower than that of the control after 36 HPI. The results indicated that PRRSV infection could cause, directly and/or indirectly, not only death of AMs but also adverse alterations in their morphology and function, although some of the effects seemed to be reversible. Because AMs are crucial to the host against airborne pathogens, PRRSV infection may potentially predispose pigs to secondary pulmonary infections.     

Syringohydromyelia in Cavalier King Charles spaniels

Syringohydromyelia secondary to foramen magnum overcrowding is described in seven Cavalier King Charles spaniels. Clinical signs were consistent with a central spinal cord lesion. The most common signs were persistent scratching at the shoulder region with apparent neck, thoracic limb, or ear pain and thoracic limb lower motor neuron deficits. The diagnosis was made by magnetic resonance imaging. The syringohydromyelia is postulated to be a consequence of an occipital bone malformation resulting in a small caudal fossa and cerebellar herniation. Clinical signs improved but did not completely resolve when the dogs received treatment with corticosteroids or nonsteroidal anti-inflammatory drugs.     

Dirofilaria repens in a cat with acute liver failure

Acute liver failure was diagnosed in a 12-year-old cat. Fine needle aspirate cytology revealed high numbers of unsheathed microfilariae and a hepatocellular reaction with no evidence of bacterial infection. The microfilariae were identified as those of Dirofilaria repens by acid phosphatase staining. The high number of microfilariae seen in both the blood and the liver aspirate samples as well as the favourable response to ivermectin amongst other drugs administered, is suggestive that D. repens was the cause of the liver insult. A positive result obtained with an antigen-capture ELISA (Dirochek) for Dirofilaria immitis antigen was interpreted as false. This is the 1st report of Dirofilaria repens for South Africa.