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.     

Metabolic changes induced by regular submaximal aerobic exercise in meat-type rabbits

Pannon White growing rabbits (a group of 8) were exposed to treadmill exercise (3-9 m/s, 1.2-1.6 km/day) twice a day for 4 weeks, while additional 8 animals, kept inactive, were assigned as the control group. Weekly, 12 hours after exercise, venous blood was taken for serum metabolite and enzyme activity measurements. Total serum protein, albumin and creatinine levels significantly increased during the second half of the training, as compared to the control group. Triacylglycerol levels in the exercised group as compared to controls, however, were higher only after the first and the fourth weeks of the experiment. Resting non-esterified fatty acid (NEFA) concentration of the trained rabbits was lower at the end of the trial. On the other hand, there were no significant differences, as compared to the respective controls, in serum urea, total and HDL cholesterol levels. At the end of the exercise alkaline phosphatase activity was higher and total lactate dehydrogenase activity was lower in the trained rabbits. Serum alanine aminotransferase, aspartate aminotransferase and gamma-glutamyl transpeptidase activities were not changed, while creatine kinase activity was slightly lower in the trained group. The serum cortisol concentration was not different in the trained and control rabbits.     

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.     

Co-feeding studies of ticks infected with Anaplasma marginale

Ticks often cluster at preferred feeding sites on hosts, and the co-feeding of ticks at the same site has been shown to increase feeding success and the transmission of some pathogens. While the major route of infection of ticks with pathogens is via the bloodmeal during feeding on a parasitemic host, non-systemic transmission of viruses and spirochetes has been shown to occur from infected to uninfected ticks at common feeding sites on uninfected hosts. In this research, two separate studies were done using the tick-borne rickettsial pathogen of cattle, Anaplasma marginale. In one study we tested whether A. marginale could be transmitted non-systemically from infected to uninfected Dermacentor variabilis males while co-feeding on rabbits. Infection of ticks was determined by allowing them to transmission feed on susceptible cattle and by DNA probe and microscopy studies on salivary glands. In the second study, we tested whether the co-feeding of male and female ticks on parasitemic cattle would increase the acquisition and development of A. marginale in males. A. marginale infections in salivary glands were determined by quantitative PCR after the ticks were allowed to transmission feed on susceptible cattle. Non-systemic transmission of A. marginale did not occur from infected and uninfected ticks that fed at the same site on rabbits and, therefore, does not appear to be a means of A. marginale transmission. A. marginale infections in male ticks were not increased while co-feeding with females. Thus, co-feeding of adult Dermacentor spp. does not appear to influence the dynamics of A. marginale transmission.     

Gene discovery in Eimeria tenella by immunoscreening cDNA expression libraries of sporozoites and schizonts with chicken intestinal antibodies

Specific antibodies were produced ex vivo from intestinal culture of Eimeria tenella infected chickens. The specificity of these intestinal antibodies was tested against different parasite stages. These antibodies were used to immunoscreen first generation schizont and sporozoite cDNA libraries permitting the identification of new E. tenella antigens. We obtained a total of 119 cDNA clones which were subjected to sequence analysis. The sequences coding for the proteins inducing local immune responses were compared with nucleotide or protein databases and with expressed sequence tags (ESTs) databases. We identified new Eimeria genes coding for heat shock proteins, a ribosomal protein, a pyruvate kinase and a pyridoxine kinase. Specific features of other sequences are discussed.     

Characterization of Cryptosporidium spp.--recent developments and future needs

Cryptosporidia, widely distributed protozoan parasites of vertebrates have recently attracted increasing interest due to several serious waterborne outbreaks, the life-threatening nature of infection in immunocompromised patients, and the realization of economic losses caused by these pathogens in livestock. Genetic polymorphism within Cryptosporidium spp. is being detected at a continuously growing rate, owing to the widespread use of modern molecular techniques. The aim of this paper is to review the current status of taxonomy, genotyping, molecular phylogeny, and characterization of cryptosporidia, and to highlight the need for polyphasic typing, i.e. an integrated approach comprising standardized morphologic, biologic, and molecular methods for describing Cryptosporidium species and isolates, and for establishing "virtual" reference strains.     

Experimental infection of Black Belly and INRA 401 straight and crossbred sheep with trichostrongyle nematode parasites

Compared to INRA 401 lambs reared in France, Black Belly (BB) lambs reared in Guadeloupe (F.W.I.) were highly resistant to both primary and secondary experimental infection with Haemonchus contortus. To investigate this huge inter-breed difference, a nucleus flock of BB was constituted, and experiments were conducted to: (i) confirm this difference in lambs born in France, (ii) check whether it was similar for Trichostrongylus colubriformis and Teladorsagia circumcincta, and (iii) find out whether this difference was age-related. Forty BB lambs, 84 F1 lambs (BB siresxINRA 401 ewes) and 88 INRA 401 lambs born in two cohorts were used in an experimental design involving three host breeds, both genders and two age-groups (3.5- and 7-month-old when first infected). The limited availability of BB lambs made the study incomplete. Infection consisted of the administration of two doses of 10,000 infective larvae of one of the nematode species, separated by an anthelmintic treatment and an interval of 1 week before the second dose was administered. Fecal egg counts (FECs) were done on Days 28 and 35 after each infection; ewe lambs of the INRA 401 and F1 breeds were necropsied, the worm burden was established, the length of the female worms measured and the eggs in utero counted. For H. contortus and T. colubriformis, the FEC was lower in the BB than in the INRA 401 lambs, and the FEC found for the F1 lambs was intermediate. For T. circumcincta, only data for the F1 and INRA 401 lambs were available, and the FECs for these breeds were the same. In all three breeds, the FEC determined after the second dose was significantly lower than that found after the first dose in the ewe lambs, but not in the ram lambs. Infecting the lambs when they were 7- instead of 3.5-month-old significantly lowered egg excretion of both parasites in the F1 (P<0.0001), but not the INRA 401 lambs. Worm numbers in the F1 and in the INRA 401 ewe lambs confirmed the FEC data, many F1 lambs being free of T. colubriformis. The H. contortus female worms were shorter and had fewer eggs in utero in the F1 than in the INRA 401 lambs. A higher proportion of T. circumcincta was at the fourth larval stage in the F1 lambs. In conclusion, the BB breed is much more resistant than INRA 401 to H. contortus, with the F1 lambs being more like the BBs after the second dose. This difference was also found for T. colubriformis and, to a lesser extent, for T. circumcincta.     

Effect of freezing goat milk samples on recovery of intramammary bacterial pathogens

With the aim of evaluating the effect of freezing goat milk samples on recovery of intramammary pathogens, 1200 milk samples from udder halves with subclinical intramammary infection were studied. Samples (20 ml) were frozen at -20 and at -80 degrees C. Thawing was carried out at room temperature at 7, 14, 21, 28, 58, 118, 178, 236 and 730 days after collection and bacteriological analyses were carried out to determine the number of colony forming units/ml (CFU/ml). Mixed model statistical analysis showed that bacterial group, temperature of storage, interaction of bacterial group and temperature of storage and the interaction of bacterial group, time and temperature of storage were statistically significant effects. For coagulase negative staphylococci (CNS), least squares means of log CFU/ml recovered at -20 and -80 degrees C were not different. Nevertheless, for Gram negative bacilli (GNB) a significant decrease was detected in samples frozen at -20 vs. -80 degrees C. At both temperatures and at different times of storage, significant increases were detected between log CFU/ml of CNS and values on day zero. At -20 degrees C, a significant decrease in GNB recovery was detected between freezing days zero and 730. This difference was not detected when goat milk samples infected by GNB were frozen at -80 degrees C. The results show that frozen milk samples can be useful in goat subclinical mastitis control programs.     

Predominance of the papGII allele with high sequence homology to that of human isolates among avian pathogenic Escherichia coli (APEC)

Avian pathogenic Escherichia coli (APEC) are often found in poultry and are responsible for a set of diseases, commonly referred to as avian colibacillosis. One of the important virulence factors is adhesion to different epithelial surfaces, which is mediated by pili. P pili are thought to play a role by means of their PapG adhesin, which occurs in three molecular variants: PapGI, PapGII and PapGIII. This study is the first to determine and analyse the distribution of the different papG alleles in APEC. Our results show a significant predominance of the papGII allele above all other alleles or allele combinations. No statistically significant associations could be found between papG allele distribution and the type of bird, organ of isolation and O serogroup. Finally, the papGII and papGIII sequences showed high homology with mammalian (including human) source papG sequences.