Characterization of recombinant adeno-associated virus-2 as a vehicle for gene delivery and expression into vascular cells

Investigations into the incidence of Salmonella abortus ovis (S. abortus ovis) infections should not be neglected in diagnosis of ovine abortion cases. For detection of this pathogen agent, direct cultivation, as well as pre-enrichment combined with enrichment procedures in tetrathionate or Rappaport-Vassiliadis medium, should be performed with respect to the features of S. abortus ovis. The detection limit of S. abortus ovis using pre-enrichment and enrichment media could be determined using 6.5 x 10(3)-6.5 x 10(4) bacteria. For subsequent cultivation of S. abortus ovis Gassner, XLD or Rambach agar are suitable. In infected sheep showing no excretion of S. abortus ovis, the pathogen can be detected by serological studies using microagglutination or the ELISA test. The ELISA test proved to be more sensitive than the microagglutination test, detecting antibodies against S. abortus ovis in 17% of the 814 sheep tested. The microagglutination test revealed positive results in only 2% of the sheep tested.     

A filter immunobinding technique for the rapid detection and simultaneous identification of avian and bovine mycoplasmas

A filter immunobinding (FIB) method was developed for the detection and identification of mycoplasmas. Type strains of a total of 18 avian and bovine mycoplasma species propagated in broth media were diluted and immobilized on a nitrocellulose membrane as antigens for investigating the specificity with rabbit hyperimmune sera. Non-specific FIB reactions were easily eliminated by the procedure of absorbing rabbit hyperimmune sera in the broth. Absorbed rabbit hyperimmune sera exhibited clear species-specificity with mycoplasma antigens by the FIB. These specific reactions always agreed with the results of identification by tests of biochemical properties and growth inhibition for the isolates of M. bovirhinis, M. bovis, M. columbinum, M. columborale, M. gallisepticum and M. synoviae. Some bovine mycoplasma species, which were impossible to identify by growth inhibition test, because of their strong production of film and spots on the agar, specifically reacted with absorbed rabbit hyperimmune sera against M. bovis in the FIB. The detection limit of mycoplasmas by this method was about 10(4)-10(5) colony-forming units/ml, which is lower than that of colony determination on agar. The FIB seems to be a useful technique for rapid detection and simultaneous identification of mycoplasmas.     

Sweat conductometry in cellulose acetate foil--a simplified method for diagnosis of cystic fibrosis

A trial was designed to test the efficacy of vaccination of lambs against field challenge with T. ovis eggs using 3 different levels of pasture contamination. Three concentric paddocks (1.62 hectares each) were used and the inner paddock was heavily contaminated with T. ovis eggs by tethering 2 dogs, experimentally infected with T. ovis, at 5 different points in the paddock over a period of 1 month. Four groups of lambs were used. Sixty lambs, from a property known to be free of T. ovis for 2 years previously, were divided into 2 groups of 30 each; one group was vaccinated with T. ovis culture antigen (vaccinated group=V) while the other was vaccinated with culture medium without antigen (sham-vaccinated group=SV). Thirty mature sheep were obtained from a property with a recent record of T. ovis infection (previous natural exposure to infection group=PNE) and 15 lambs were artificially reared from birth under cestode-egg-free conditions (artificially reared groups= AR). Ten animals from groups V, SV, and PNE, and 5 AR lambs were grazed on each of the 3 concentric paddocks for 6 weeks, and then all animals were slaughtered and their total muscle mass (including heart, diaphragm, tongue and masseters) examined, by slicing, for T. ovis cysticerci. The mean total numbers of T. ovis cysticerci recovered were as follows: Inner paddock, PNE 45.5, AR 47.6, SV 107.8, V 6.5; Middle paddock, PNE 5.2, AR 9.0, SV 8.2, V 0.3; Outer paddock, PNE 2.9, AR 2.8, SV 2.3, V 0.2. There were 4 major conclusions from this study. Firstly, vaccination was more effective than previous natural exposure to infection in preventing establishment of new cysticerci. Secondly, although vaccination effectively reduced the numbers of cysticerci, those that became established were mostly viable at the time of slaughter. In contrast, PNE sheep had greater numbers of cysticerci but the majority were degenerating. Vaccination, therefore, did not seem to stimulate a complete immunological response. Thirdly, the presence of T. hydatigena in lambs did not prevent subsequent infection with T. ovis. Fourthly, T. ovis eggs were dispersed from the inner paddock to the outer paddock.     

Determination of L-cystine aminopeptidase (serum oxytocinase, EC 3.4.1.2) during normal pregnancy

The effect of a program to control Teania ovis cysticercosis was investigated on 32 adjacent farms at Mt Manypeaks in Western Australia. Farmers were advised not to feed dogs any raw sheep meat or offal and they were supplied with sufficient cestocide to treat all their dogs every 2 months. Other aspects such as the need for control of the movement of dogs and the correct disposal of offal from home killing of sheep were discussed and recommendations made. The success of the program was determined by recording the incidence of Cysticercus ovis in lambs born on the farms and killed at a local abattoir, and the incidence of T. ovis in dogs on the farms. Before the trial 6.9% of lambs were infected with C. ovis and 11 of the 32 farms had a T. ovis infected dog. The C. ovis incidence figures fell to 2.8%, 0.5%, 1.8% and 0.3% for the four years of the trial, and only 1 dog, a recently introduced puppy, was found to be infected with T. ovis. The results are discussed and reasons for the success of the program as a whole and apparent individual farm failures are discussed.     

Prevention of carbon tetrachloride-induced hepatotoxicity in the rat by H. rosasinensis anthocyanin extract administered in ethanol

A serologic survey was carried out in order to detect antibodies against Babesia ovis in a large population of Spanish ibex (Capra pyrenaica) from a hunting reserve in Catalonia, northeastern Spain. For this purpose, an indirect fluorescent antibody test (IFAT) was developed using a B. ovis isolate of ovine origin as antigen. Of the total 475 sera tested, 155 (32.6%) showed titres between 1:160 and 1:1280 and were considered positive. These results reveal that exposure of Spanish ibex to B. ovis is common in the studied area. No significant differences could be detected when comparing season or year of capture and age or sex of the animals in positive and negative samples. A high proportion of low titres was found in comparison to those reported by other researchers in sheep in Spain; this could be a consequence of the existence of some minor antigenic differences between B. ovis of domestic sheep and that found in Spanish ibex.     

Reduced egg counts in mixed infections with Oestrus ovis and Haemonchus contortus: influence of eosinophils?

In all, 2 groups of lambs were infected either with Oestrus ovis first-instar larvae or with 10,000 third-stage larvae (L3) of Haemonchus contortus. Another group of lambs was infected with both parasites. Fecal nematode egg counts, plasma pepsinogen concentrations, specific O. ovis enzyme-linked immunosorbent assay (ELISA) antibodies, and blood eosinophil counts were monitored and compared to with the values recorded for a control group of uninfected lambs. There was no significant difference between the burden of H. contortus found in mixed and single infections. However, the nematode egg production was significantly depressed in mixed infections. O. ovis affects the population of H. contortus at least by decreasing the parasite egg output. This effect may be mediated through the increase in eosinophil production stimulated by the presence of O. ovis.     

Helminth species of goats in Germany

The helminth fauna of the gastrointestinal tract of 25 and the respiratory organs and the livers of 6 German goats was qualitatively and quantitatively examined. One trematode species (Dicrocoelium dendriticum), 2 species of cestodes (Moniezia expansa and metacestodes of Taenia hydatigena) and 28 species of nematodes (24 in the gastrointestinal tract and 4 in the lungs) were recorded. Two goats were infested with Oestrus ovis larvae. The most prevalent species were Ostertagia circumcincta and Chabertia ovina (84% each), Ostertagia trifurcata and Oesophagostomum venulosum (76% each), Ostertagia pinnata and Trichostrongylus colubriformis (64% each), Trichuris ovis (60%) and Skrjabinema ovis and Trichuris globulosa (56% each). The highest mean worm counts were seen in goats infected with Skjabinema ovis (4003), Ostertagia circumcincta (2501), Trichostrongylus axei (1825), Trichostrongylus colubriformis (1578) and Nematodirus battus (1050). Totally, the goats did harbour more nematodes in the abomasum (3734) than in the small intestine (1707) or the large intestine (2343). The lungs were parasitized by Cystocaulus ocreatus, Muellerius capillaris, Neostrongylus linearis and Protostrongylus rufescens.     

Seroprevalence of Babesia ovis in mouflon sheep in Spain

A serological survey detected antibodies against Babesia ovis in mouflon sheep (Ovis musimon) from two different reserves located in Catalonia in northeastern Spain. An indirect fluorescent antibody test (IFAT) was developed using a B. ovis isolate of ovine origin as the antigen. Of 50 sera tested, six (12%) showed titres between 1:160 and 1:640 and were considered positive. These results indicate that exposure of mouflon to Babesia ovis is common in this region.     

Persistence of Anaplasma ovis infection and conservation of the msp-2 and msp-3 multigene families within the genus Anaplasma

Goats which have recovered from acute Anaplasma ovis infection remain seropositive, although infected erythrocytes cannot be detected by microscopic examination. Persistence of A. ovis 17 to 21 months following experimental infection was demonstrated by PCR detection of the msp-5 gene. Quantitative analysis of persistent rickettsemia over time showed that all levels were below the limit of microscopic detection and ranged from a low of 10(2) organisms/ml to peaks of 10(6) organisms/ml. Two patterns of persistent rickettsemia were observed: the first was characterized by cyclic fluctuations at 6- to 9-week intervals, similar to the pattern described for A. marginale-infected cattle, while in the second pattern, repetitive cycles did not occur and the rickettsemia levels were relatively constant. The msp-2 and msp-3 multigene families, which provide the genetic capacity for outer membrane protein antigenic variation during persistent A. marginale rickettsemia, were identified in the A. ovis genome by Southern blot analysis, and expression of an MSP-2 homologue was confirmed by using immunoblots.     

Characterization of Anaplasma isolates from eland (Taurotragus oryx). Pathogenicity in cattle and sheep and DNA profiles analysis

Two eland Anaplasma isolates, AnapE1, from Kenya, and AnapE2, from South Africa were characterised. Their characterization was based on their pathogenicity to intact and splenectomized cattle and sheep and also their DNA profiles. Their DNA profiles were analysed and compared to Anaplasma marginale, A. ovis and A. centrale after endonuclease restrictions and probing with Anaplasma DNA probes, AC5-12 and AC-1. The results of the pathogenicity trials showed AnapE1 to be similar to A. ovis and AnapE2 an isolate of A. marginale. On DNA profiles, AnapE1 was close to A. ovis, with differences that occur even in same Anaplasma species isolates from different locations. On the other hand, AnapE2, resembled one of the A. marginale isolates known to occur in South Africa. The DNA profiles correlated well with the pathogenicity results. It is concluded that elands are carriers of both A. marginale and A. ovis parasites and are therefore important reservoirs that need attention in epidemiology of anaplasmosis.     

Evaluation of the clinical impact of endoscopic ultrasonography in gastrointestinal disease

A total of 120 gastro-intestinal tracts and 960 faecal samples were examined to assess the prevalence and seasonal changes in the gastro-intestinal helminth parasites of Red Sokoto (maradi) goats slaughtered at Ibadan between May 1991 and April 1992. Egg types of strongyles, Strongyloides, Trichuris, Skrjabinema, Dicrocoelium and Moniezia were encountered in 93%, 83%, 44%, 0.9%, 2.3% and 31% of the faecal samples respectively. However, only strongyle, Strongyloides and Trichuris eggs occurred in large numbers and were more common during the rainy season than in the dry season. The parasites recorded and their prevalences were Haemonchus contortus (90.0%), H. ovis (5.0%), Strongyloides papillosus (80.8%), Trichostrongylus colubriformis (78.3%), T. axei (69.2%), Trichuris ovis (72.5%), T. globulosa (38.3%), Oesophagostomum columbianum (67.5%), Cooperia curticei (58.3%) Gaigeria pachyscelis (40.8%), Skrjabinema ovis (5.0%), Nematodirus battus (5.8%), Moniezia expansa (29.2%), M. benedeni (10.0%), Paramphistomum spp. (5.0%) and Cysticercus tenuicollis (33.3%). Haemonchus ovis is reported for the first time in Nigeria. Mixed infections were most prevalent. Young goats were more commonly infected and had higher worm counts than adult goats. Only Haemonchus, Trichostrongylus, Strongyloides and Cooperia spp. occurred in large numbers. Irrespective of the age of the goats, higher worm counts were generally encountered during the rainy season than in the dry season. The results are discussed in relation to the control of helminthiasis in grazing animals in Nigeria.     

Characterization of an immuno-dominant antigen in Brucella ovis and evaluation of its use in an enzyme-linked immunosorbent assay

A panel of 45 Brucella ovis serologically positive sera were tested in immunoblots against B. ovis outer membrane proteins Omp31 and Omp25, purified by preparative SDS-gel electrophoresis. Forty-three sera reacted with Omp31, while only 11 reacted with Omp25, suggesting that Omp31 is identical to the previously reported immuno-dominant 29-kDa protein. Attempts to purify Omp31 on a larger scale by using procedures such as ion exchange-, reversed phase-, affinity- and gel filtration chromatography suggested that the outer membrane proteins were aggregated with rough lipopolysaccharide. Only denaturing SDS-gel filtration chromatography was able to separate proteins of about 29 kDa from rough lipopolysaccharide but did not separate Omp31 from Omp25 in B. ovis preparations. When used in an enzyme-linked immunosorbent assay, this 29-kDa protein preparation was less sensitive and less specific than the routinely used heat-extracted B. ovis antigen. A readily available recombinant E. coli, expressing the gene for Omp31 from Brucella melitensis 16 M, was used to extract and enrich recombinant Omp31 by a temperature-dependent Triton X-114-based technique. When this material was used in immunoblots with the 45 sera from B. ovis-infected sheep and with 10 monoclonal antibodies, raised against B. ovis Omp31, major differences in the antibody reactivity between the recombinant B. melitensis Omp31 and the B. ovis Omp31 were found. Such differences were unexpected because of the known structural and immunological relatedness of outer membrane proteins from various Brucella species. These results indicated that the antibody-response in B. ovis naturally-infected sheep against the immuno-dominant Omp31 was directed against epitopes which were only accessible when the protein was aggregated with rough lipopolysaccharides, or which were formed after aggregation but were not present in the recombinant protein.     

Efficacy of orally administered invermectin against larval stages of Oestrus ovis in sheep

The efficacy of ivermectin administered orally at a dosage rate of 0.2 mg/kg liveweight against naturally acquired larval infestations of Oestrus ovis in sheep was 100% in a field trial. Ten sheep were free from infestation by first, second and third instar larvae of O. ovis 12 days post treatment, whereas 10 control sheep harboured 37.7 larvae on average, most of them first stage.     

Clinical, clinico-pathological and serological studies of Babesia ovis in experimentally infected sheep

Clinical, clinico-pathological and serological studies were performed in sheep experimentally infected with Babesia ovis. Acute babesiosis occurred in all the lambs infested with adult Rhipicephalus bursa ticks and in one lamb infested with the larvae. The rate of parasitaemia and the degree of anaemia were not correlated. Decrease in the packed-cell volume ranged from 30 to 40%. Parasitized erythrocytes were not observed to block capillaries in the brain, which explained the absence of nervous symptoms in acute babesiosis. The kidneys were the most severely affected organs, exhibiting acute glomerulonephritis. The lesions observed were suggestive of vascular alteration and vascular stasis, leading to anoxia of the tissues. A disseminated intravascular coagulation (DIC) syndrome was recorded in sheep infected with babesiosis. A marked increase in the enzymes of the transaminase groups, mainly aspartate aminotransferase (AST), was observed. Enzymatic changes (increases in AST, alanine aminotransferase (ALT) and lactic dehydrogenase (LDH) and decreases in sorbitol dehydrogenase (SDH), alkaline phosphatase (ALP) and malic enzyme (MEZ)), decreases in total proteins and albumin, and increases in urea and creatinine might reflect the degree of severity of the damage to the liver and kidney tissues. Most of the lambs (85%) that were infested with larvae, and all lambs infested with adult R. bursa ticks, reacted serologically to B. ovis antigen. The serological reactions following infestation with the larvae occurred much later than those following infestation with the adult stage. The lambs which were infested with larvae showed mild clinical reactions when challenged by infected R. bursa adults, as compared with the reactions to the challenge in naive control animals. The serological findings, in addition to the fact that one splenectomized lamb reacted to larval infestation with acute ovine babesiosis, show that the preimaginal stages of R. bursa can transmit B. ovis, usually causing a sub-clinical disease. It is suggested that infections derived from preimaginal ticks in the winter can preimmunize sheep for the subsequent more severe infections derived from adult ticks in the summer. Furthermore, in the absence of a reliable vaccine against B. ovis, grazing flocks in the enzootic regions should be exposed to the preimaginal stages during their activity period (October-February) before exposure to the adult ticks in spring and summer (April-July).     

The cellular response of lambs to Brucella ovis before and after birth

The immunological response of lambs to Brucella ovis before and after birth was investigated. The establishment of indwelling cannulas in the efferent prescapular lymphatic ducts of foetal lambs allowed continual monitoring of the immune response of a single lymph node. Foetal lambs in the last trimester of pregnancy were shown to mount a strong cell-mediated immune response to B. ovis. Lymphocytes from the challenged lymph node stimulated with B. ovis in vitro usually first reacted significantly and had highest [3H]-thymidine incorporation between 4 and 6 days after primary and secondary challenge, whereas, lymphocytes from the unchallenged node did not exhibit significant [3H]-thymidine incorporation until some 24 h later. Lymphocytes from these lambs challenged as foetuses still exhibited significant [3H]-thymidine incorporation in response to B. ovis for 4 to 5 months after birth. The proportion of surface immunoglobulin-positive cells in efferent prescapular lymph of unchallenged lambs ranged from 0.5 to 2.0% but after B. ovis challenge this proportion ranged from 2.7 to 8.7% between 4 to 6 days after challenge. By 9 to 12 days after challenge, the proportion had declined to pre-challenge values.     

Epidemiological observations in a Corriedale flock affected by Brucella ovis

Brucellosis in sheep, caused by Brucella ovis, is primarily a chronic infectious disease of rams with epididymitis as its most characteristic lesion. Six hundred rams from an infected farm were clinically and serologically examined once a year, over a 3-year period. An increase from 2.1% to 6.3% in the prevalence of animals serologically positive to B. ovis occurred over the 3 years. However, the prevalence of rams with lesions in the reproductive tract declined from 14.2% to 6.5% in the third year following one year of strict culling of clinically affected and rams that were serologically positive for B. ovis. Clinical lesions found in the 179 affected rams fell into two main categories: rams with epididymitis and rams with affected lymph nodes. These results suggest that the prevalence of the disease relates mainly to the sexual activity of the animal and not to age in itself. A single cull based on the results of clinical examination and serological test results was unable to decrease the prevalence of B. ovis in an extensive Corriedale sheep flock.     

Effect of antibiotics contained in two Brucella selective media on growth of Brucella abortus, B. melitensis, and B. ovis

The MIC and the highest concentration enabling bacterial growth (CEG) of the antibiotics contained in two selective media were determined for Brucella abortus, B. melitensis, and B. ovis. The nalidixic acid and bacitracin contained in Farrell's selective medium were responsible for the inhibitory effects observed.     

Influence of endotoxin on contractility in the rat gastric fundus

Cefoperazone amphotericin teicoplanin (CAT) agar was developed from cefoperazone deoxycholate (mCCD) agar by modification of the selective antibiotics in order to permit growth of strains of Campylobacter upsaliensis. In this study, 35 strains of Campylobacter and Arcobacter were tested for their ability to grow on CAT and mCCD media using the ecometric method. Six of these strains were also tested using the modified Miles-Misra method. Overall, nineteen strains out of the 35 tested grew better on CAT than on mCCD agar, although for eight strains, the difference was slight. These differences could not be attributed solely to poorer growth of C. upsaliensis on mCCD agar. No strain grew better on mCCD than CAT agar. Eight of the 35 strains tested did not grow on mCCD agar at all, however, only one strain failed to grow on CAT medium. The two methods of testing gave similar results, although the Miles-Misra method was found to be more sensitive and less prone to subjective interpretation. All four CNUPC (catalase negative, urease positive campylobacter-like) strains, one strain of C. sputorum biovar, fecalis, one of two Arcobacter cryaerophilus strains (incubated at 30 degrees C, aerobically) could be detected only using CAT agar. In addition, for some strains of A. butzleri, C. upsaliensis and C. hyointestinalis, CAT medium gave better growth scores than mCCD agar. The level of cefoperazone in mCCDA is inhibitory to some campylobacter strains, but suboptimal growth of Arcobacter strains is more probably due to synergistic interaction between deoxycholate and cefoperazone. CAT agar supports the growth of a wider variety of Campylobacter and Arcobacter species than mCCD agar.     

Comparative analysis of antigens from different Brucella species using immunoblotting with antisera from immunized rabbits

Brucella antigens recognized by IgG antibodies in cell lysates from various Brucella species differing by the origin, biological, and virulent properties (including the reference, vaccine, and newly isolated strains) were compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins in SDS-cell lysates were separated by 12% SDS-PAGE and protein gels were stained with Coomassie brilliant blue R-250 and Silver reagent. SDS-PAGE showed differences in the protein profiles of 15 strains of different species. Immunoblotting revealed that rabbit S-antisera contained IgG reacting with S-LPS and identical proteins of 90 to 16 kDa belonging to B, melitensis, B. suis, B. abortus, and B. neotomae strains. B. canis strains had 4 antigens reacting with these antisera, whereas B. ovis had none. No agglutinating antibody were detected by the standard tube agglutination test with smooth Brucella strains in rabbit R-antisera. By contrast, immunoblotting analysis with these sera demonstrated common 90-16 kDa antigens in the strains of B. melitensis, B. suis, B. abortus, B. neotomae, and B. canis. B. ovis possessed none of these antigens. These results confirm that all Brucella species except B. ovis possess common protein antigens reacting with IgG.