Immunophenotype and functional properties of feline dendritic cells derived from blood and bone marrow

Dendritic cells (DCs) are a heterogeneous population of cells of fundamental importance in initiating innate as well as specific immune responses. The identity and function of DCs in the cat are unknown, although they are likely pivotal in the response to infection. In this study, feline DCs were derived by 3-10-day culture of adherent blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) in the presence of IL 4 and GM-CSF. BMMC consistently yielded a greater number of DCs than PBMC, and there were fewer macrophages than DC from both compartments. DCs expressed a distinct constellation of surface molecules, which included CD1a, CD1b, and CD1c, CD11b, CD14, and 2-3-fold higher levels of MHC class I and II molecules than co-cultured macrophages or fresh blood monocytes. DCs displayed typical cytoplasmic processes, limited non-specific esterase activity, and acquired antigen by phagocytosis, pinocytosis, and binding to specific receptors. Cytokine-exposed cells induced proliferation of allogeneic lymphocytes. Thus, the cells derived by these culture conditions had markers and functions analogous to immature myeloid DCs. Availability of feline DCs will enable investigation of their role in infectious disease and their potential therapeutic application.     

The Effect of Anthelmintic Capsules on the Egg Output and Larval Viability of Drug-resistant Parasites

Challenge with an equal mix of drug-resistant and drug-susceptible larvae of Teladorsagia circumcincta resulted in infections in groups of lambs (n = 6) either untreated or given controlled-release capsules, containing either albendazole or ivermectin. Lambs treated with albendazole capsules contained similar numbers of adult worms at necropsy to the other groups but had no detectable faecal egg count. Animals treated with ivermectin capsules had similar worm burdens and faecal egg counts to the control group but the worms had significantly higher numbers of eggs in utero. These results provide evidence for suppression of egg production by both anthelmintic treatments. The observation that albendazole caused a significant reduction in the developmental success of parasite eggs also has implications for the use of faecal egg count as an indicator for pasture contamination with resistant parasites. In two further groups of lambs, either untreated or given albendazole capsules, treatment caused a significant reduction in egg count and adult worm burden of Trichostrongylus colubriformis. No significant effects were observed on in utero egg counts or egg viability and the apparent effect on the number of eggs produced in faeces per adult female was not significant (p = 0.077). There was, therefore, no evidence that albendazole controlled-release capsules caused suppression of egg output in this species.     

Comparison of antibody values in sera of pigs vaccinated with a subunit or an attenuated vaccine against classical swine fever

Ten pigs, aged 85 days, were vaccinated with a subunit vaccine containing 32 g of classical swine fever virus glycoprotein E2 (gp E2) (group 1), and a further 10 pigs were vaccinated with a C strain vaccine (10^4±0.15 TCID₅₀/ml), produced by amplification in minipig kidney (MPK) cell culture (group 2). Nine non-vaccinated pigs served as a control group (group 3). Serum samples were collected before (day 0) and at 4, 10, 21 and 28 days after vaccination and were analysed by two commercially available enzyme immunoassays and by a neutralizing peroxidase-linked assay (NPLA). At the same times, peripheral blood was taken for determining the total leukocyte count and the body temperature was taken daily. Antibodies were not detected in serum samples collected before vaccination (day 0), and no side-effects that could be connected with vaccination were observed during the trial. Ten days after vaccination 6/10 pigs vaccinated with the subunit vaccine were seropositive. On days 21 and 28, the ratios of serologically positive to vaccinated pigs were 9/10 and 10/10, respectively. Four of the ten pigs that were vaccinated with the C strain vaccine were positive on day 21 and 9/10 on day 28. However, the results of the NPLA showed that only 4/10 pigs had an antibody titre >1:32 at the end of the trial in both the vaccinated groups, even though the subunit vaccine initiated an earlier and higher level of neutralizing antibodies than the vaccine produced from the C strain. Challenge was performed 28 days after vaccination on four randomly selected pigs from both vaccinated groups. The pigs survived the challenge without showing any clinical signs of classical swine fever (CSF), while two nonvaccinated control pigs died on the 10th and 12th days after infection.     

Experimental vascular graft for liver transplantation

Hepatic artery thrombosis is a major cause of graft failure in liver transplantation. Use of donor interponates are common, but results are controversial because of necrosis or thrombosis after rejection. Reperfusion injury, hypoxia and free radical production determinate the survival. The aim of the study was to create an 'ideal' arterial interponate. Autologous, tubular graft lined with mesothelial cells, prepared from the posterior rectus fascia sheath, was used for iliac artery replacement in eight mongrel dogs for six months under immunosuppression. Patency rate was followed by Doppler ultrasound. Eight grafts remained patent and another two are patent after one year. The patency rate was good (median Doppler flow: 370 cm/sec) and there was no necrosis, thrombosis or aneurysmatic formation. The grafts showed viable morphology with neoangiogenesis, appearance of elastin, smooth muscle and endothelial cells. Electron microscopy showed intact mitochondrial structures without signs of hypoxia. Tissue oxygenation was good in all cases with normal (< 30 ng/ml) myeloperoxidase production. In conclusion, this autologous graft presents good long-term patency rate. Viability, arterialisation and low thrombogenicity are prognostic factors indicating usability of the graft in the clinical practice without the risk of rejection. Further investigations such as cell cultures and standardisation are necessary.     

Operation technique and healing process of telescopic ileocolostomy in dogs

The healing process of telescopic anastomoses was found in an animal experiment with 12 mongrel dogs. After the division of vessels an ileal segment of different length was invaginated into the lumen of the colon using single-layer interrupted sutures. The following four groups were used: Group A (n = 3): end-to-side ileocolostomy, single-layer interrupted suture (invagination length: 0 mm), survival time: 21 days. Group B (n = 3): invagination length: 20 mm, survival time: 7 days. Group C (n = 3): invagination length: 10 mm, survival time: 21 days. Group D (n = 3): invagination length: 20 mm, survival time: 21 days. At the end of the above survival times the anastomosis area was removed. The bursting pressure was measured and morphological as well as histological examinations were performed. In each case the 0-day look-alikes of anastomoses were performed using the remnant bowels, and bursting pressure measurements were done on these models as well. Anastomosis leakage did not occur. The serosal layer of the intracolonic part of the ileum disappeared during the healing process. The free surface of the intracolonic ileal segment became covered by the sliding mucosa of the colon and the prolapsing mucosa of the ileum. The following could be concluded after the experiments: The inner pressure tolerance of a telescopic ileocolostomy promptly after preparation is better than in case of another single-layer anastomosis. This fact results in increased safety against leakage on the first postoperative days. The inner pressure tolerance of the telescopic ileocolostomy increases during the healing process and it does not depend on the length of the invaginated part (0 day-20 mm: 56 mmHg +/- 6, Group A: 252 +/- 39, Group B: 154 +/- 19, Group C: 249 +/- 20, Group D: 298 +/- 2). There is no difference in pressure tolerance between the telescopic and the end-to-side single-layer interrupted anastomoses after the healing process. The invaginated section within the lumen of the large intestine does not suffer ischaemic or any other kind of damage. This inexpensive and simple anastomosis technique could be useful in the veterinary surgical practice as well.     

Characterisation of Hungarian porcine circovirus 2 genomes associated with PMWS and PDNS cases

The authors report the data of the first survey on the incidence of post-weaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS) in Hungary. A PCR method specific for the detection of porcine circovirus 2 (PCV-2) was developed, which proved to be suitable for diagnostic purposes. PCR screening of organ samples from pigs suspected to be affected with PMWS or PDNS revealed the presence of PCV-2 in 80% of the cases. Six PCV-2 genomes from Hungarian isolates were completely sequenced. Phylogenetic comparison with all the available PCV-2 sequences showed that porcine circoviruses circulating in Hungary are more variable than in several other European countries. Two Hungarian strains clustered together with the Spanish strains forming a distinct group; two others fell in a common group with the French, UK, and Dutch strains, whereas another two strains showed the closest relationship to two of the three known German PCV-2 sequences.     

Spontaneous antibiotic resistance mutation associated pleiotropic changes in Escherichia coli O157:H7

Besides the well-known O157:H7 clone causing enterohaemorrhagic colitis and haemolytic uraemic syndrome in Europe, Japan and North America, the number of Escherichia coli isolates with non-motile (NM) phenotype has considerably increased. We supposed that spontaneous antibiotic resistance mutation could cause this phenotypic change. To model our hypothesis we isolated rifampicin--(Rif) and ampicillin--(Amp) resistant mutants from E. coli O157:H7 prototype strains 7785 and EDL933. Among Rifr mutants we could isolate strains with no or reduced motility, while the Ampr mutants became hypermotile. The biochemical profile of the mutants had not changed but phage sensitivity and generation time of the mutants were altered. Among the representative strains we did not find polymorphism with Southern blot analysis and no polymorphism was found in the fliC gene of the mutants. The described characteristics have proven to be stable. In a mice virulence assay by intravenous infections the virulence of the derivatives was also found to be changed. In summary, we found that the antibiotic-resistant phenotype in E. coli O157:H7 was coexpressed with several other phenotypic changes including motility and virulence. It can be assumed that expression of the involved phenotypes may be under the influence of a common regulatory cascade. Further work is needed to identify the components and mechanism of this regulatory system.     

Analysis of gene cassettes of streptomycin-spectinomycin resistance of Hungarian Salmonella enterica serotype Typhimurium strains

By PCR using the ant(3")-Ia primer pair the aadA gene was detected in 34 streptomycin- and spectinomycin-resistant Salmonella enterica serotype Typhimurium strains. Out of them 12 belonged to DT104 and 22 to non-DT104 phage type. Using different primer combinations it was demonstrated that this gene was integron-associated in all cases: in the DT104 strains it was generally contained by a 1 kb integron while in the majority of the non-DT104 strains by a 2.05 kb (less often by a 1.9 or 1 kb) integron. In the case of integrons carrying multiple cassettes the cassette containing the aadA gene was located closer to the 3' end of the integron. The aadA genes of DT104 and non-DT104 strains were different: in the former group the aadA2 gene, while in the latter group (constituted by strains of five different phages types as well as unclassifiable and untypable strains) the aadA1 gene could be identified. The RH50/RH51 primer pair described by Collis and Hall (1992) proved to be suitable for rapid discrimination between the aadA1 and aadA2 genes on the basis that the RH51 primer bound exclusively to the aadA2 gene.     

Histopathologic patterns of nephropathy in naturally acquired canine visceral leishmaniasis

Although the nephropathy of visceral leishmaniasis (VL) is known both in humans and dogs, histopathologic alterations have not been thoroughly studied. We examined renal alterations in 55 dogs with naturally acquired VL compared with five noninfected dogs from an endemic area in northeastern Brazil. Glomerulonephritis was found in 55 dogs, interstitial alterations in 53 dogs, and tubular changes in 43 dogs with VL. The glomerular alterations found were minor glomerular abnormalities (n = 8, 14.5%), focal segmental glomerulosclerosis (n = 10, 18.2%), mesangial proliferative glomerulonephritis (n = 17, 32.7%), membranoproliferative glomerulonephritis, (n = 18, 30.9%), crescentic glomerulonephritis (n = 1, 1.8%), and chronic glomerulonephritis (n = 1, 1.8%). Morphometric and ultrastructural studies complemented the analysis. The five control animals exhibited no glomerular alterations. The glomerular lesions were related to functional alterations. Considering that the alterations of canine and human nephropathy in VL are very similar, the data obtained in this study constitute an important contribution to the understanding of canine and human VL nephropathy.     

Pancreatic injury in equine acute abdomen evaluated by plasma trypsin activity and histopathology of pancreatic tissue

In cases of equine acute abdominal disease, where pancreatic damage is suspected, pancreatic damage can be assessed by measuring increased trypsin activity in the plasma of horses suffering intestinal obstruction and severe shock. The pancreas is particularly vulnerable to splanchnic hypoperfusion because it is a highly active tissue. In this study, 10 horses undergoing abdominal surgery for intestinal obstruction were assayed for trypsin activity on admission and, because of extensive intestinal lesions that were not amenable to surgery, euthanasia was selected; the pancreas was removed before euthanasia. Trypsin activity in the plasma of these horses was significantly higher than in healthy horses (196 ng/ml +/- 128.2 versus 28.5 ng/ml +/- 19.2; P = 0.0026). Light and transmission electron microscopy revealed slight to severe lesions of vacuolar degeneration, a few zymogen granules, dilation of the endoplasmic reticulum, and swelling of mitochondria in the exocrine pancreas. The activation of an inflammatory cascade occurring during strangulating intestinal obstruction could increase pancreatic anoxic lesions caused by severe shock and hypoperfusion in the horse. Further studies will show the significance of pancreatic lesions and the ensuing damage in equine acute intestinal obstruction and shock.