Cell cycle synchronization and analysis of apoptosis‐related gene in skin fibroblasts from domestic cat (Felis silvestris catus) and kodkod (Leopardus guigna)

The kodkod population is in constant decrease and the somatic cell nuclear transfer (SCNT) might help to preserve the genetic pool of this species. The cell cycle synchronization of donor cells plays a crucial role in SCNT. The objective of this research was to evaluate two different methods for quiescence induction, serum starvation (SS) and contact inhibition (CI), both for 1, 3 and 5 days, on skin fibroblast from domestic cat and kodkod. Flow cytometry analysis revealed that in domestic cat, SS and CI, both at 3 and 5 days, increased the percentage of fibroblasts in G0/G1 compared to growing cells (GC) (p < .05). In kodkod, only SS for 3 and 5 days and CI for 1 and 3 days increased the percentage of fibroblasts in G0/G1 compared to GC (p < .05). Viability analysis by differential staining revealed that SS for 5 days decreased the proportion of live fibroblasts in domestic cat and kodkod (p < .05). Regarding gene expression analysis, in domestic cat fibroblasts, no differences were found in the BAX/BCL2 ratio in SS and CI (both at 1, 3 and 5 days) compared to GC. In kodkod fibroblasts, BAX/BCL2 ratio was increased in CI at 3 and 5 days compared to SS at 3 and 5 days (p < .05). In conclusion, in kodkod fibroblasts SS for 5 days and CI after 3 days might have a negative impact on cellular viability. According to these results, we suggest SS for 3 days for cell cycle synchronization in kodkod fibroblasts.     

Cellular and humoral immune response of foals to vaccination with Corynebacterium equi.

Transformation of peripheral blood lymphocytes from pony foals vaccinated and subsequently infected with Corynebacterium equi was studied. Three foals were vaccinated on two occasions using a formalinized C. equi vaccine with aluminum hydroxide as an adjuvant. Three nonvaccinated foals served as controls. Foals were challenged intratracheally with 9 x 10(9) C. equi six weeks after the initial vaccination.Foals survived this infection for one to two weeks. Significant lymphocyte transformation in response to C. equi antigens was detected in two vaccinated foals at the third week after initial vaccination and in all vaccinated animals at the fifth week. No statistically significant transformation was seen in nonvaccinated foals before infection. Vaccinated and nonvaccinated foals showed responsive lymphocytes following challenge. Vaccination offered no obvious protection against experimental challenge but this failure was probably due to an excessive infective dose of organisms. Low levels of humoral antibodies were detected in some challenged foals. The pathological changes in the lungs of infected animals were comparable with, but more fulminating than, changes observed in the natural disease.     

Pulmonary response to intratracheal challenge with Pasteurella haemolytica and Pasteurella multocida.

Calves were inoculated intratracheally with 5 X 10(7), 5 X 10(8), or 5 X 10(9) colony forming units of either 18-hour stationary phase cultures or 4-hour log phase cultures of Pasteurella haemolytica. The log phase culture at all concentrations produced more severe clinical signs, hematological changes and pulmonary lesions at postmortem examination than did the corresponding stationary phase culture. More severe effects were seen with the larger doses especially with the log phase culture. Fibrinous bronchopneumonia with focal or multifocal necrosis was consistently produced by both the stationary and log phase cultures. To determine if this lesion was peculiar to P. haemolytica or whether it could be produced generally by rapidly growing Gram negative organisms, a 4-hour log phase culture of Pasteurella multocida was prepared in an identical manner to that used for the culture of P. haemolytica and given to calves intratracheally at the high bacterial dose (5 X 10(9]. The P. haemolytica produced more severe clinical, hematological and morphological changes than did the P. multocida. The lesions observed with P. multocida differed morphologically from those of P. haemolytica; there was a suppurative exudative component and minimal to no necrosis with P. multocida. It appears that an important pathogenic principle is produced by the rapidly growing P. haemolytica that causes it to produce a more severe clinical disease and more necrotizing pulmonary lesions than P. multocida.     

Agar gel immunodiffusion test for diagnosis of clinical paratuberculosis in cattle

During a 19-month period, the agar gel immunodiffusion (AGID) test was used as an aid in differential diagnosis of paratuberculosis in 51 cattle with a history of chronic weight loss and/or chronic diarrhea. Thirty-three cattle were AGID test-positive for paratuberculosis. Twenty-eight cattle (87.5%) yielded Mycobacterium paratuberculosis from bacterial culture of feces. Four cattle were confirmed as having paratuberculosis on necropsy. One had a negative fecal culture but was lost to follow-up. Thus, 32 of the 33 AGID test-positive cattle (96.9%) were confirmed as paratuberculous by culture or necropsy. Of the 18 cattle that were AGID test-negative, 17 (94%) also were culture-negative after 12 to 20 weeks' incubation of the culture tubes. Direct fecal smears from 29 confirmed paratuberculous cattle were examined. Twenty-three (79.3%) had smears positive for M paratuberculosis. Fifteen culture-negative cattle were examined by direct smear; the results were negative for all.     

Detection of antibodies to Mycoplasma gallisepticum vaccine ts-11 by an autologous pMGA enzyme-linked immunosorbent assay

Mycoplasma gallisepticum is a poultry pathogen that causes respiratory disease and loss of egg production worldwide. A live attenuated vaccine, ts-11, has been used for control of M. gallisepticum in several countries. The rapid serum agglutination test is usually used as an indicator of flock response to vaccination; however, in some flocks, the detected response may be weak or absent. With the use of specific monoclonal antibodies against M. gallisepticum strain S6 pMGA in immunoaffinity purification, the major membrane antigen of ts-11 was purified. An indirect enzyme-linked immunosorbent assay (ELISA) was developed with the purified antigen, and its potential for detection of antibodies induced after ts-11 vaccination was compared with an indirect ELISA with M. gallisepticum strain S6 pMGA. In the presence of high levels of ts-11-induced antibodies, both antigens detected similar numbers of positive sera. However, when lower levels of antibodies were present, ts-11 pMGA showed a higher sensitivity than S6 pMGA. Further examination of ts-11 pMGA with Mycoplasma synoviae-infected chicken sera revealed that ts-11 pMGA is specific for M. gallisepticum antibodies. With a panel of sera from ts-11-vaccinated or non-ts-11-vaccinated field chickens, the ts-11 pMGA ELISA was found to be more sensitive than the commercial rapid serum agglutination test in detecting antibodies to ts-11 vaccine. The results from this study suggest that the major membrane antigen of M. gallisepticum may have slightly different antigenic profiles in different strains, thereby necessitating the use of autologous antigens in serodiagnostic assays to increase sensitivity of the tests for mycoplasma antibodies. Thus, the low level of antibody response after ts-11 vaccination is, at least partially, due to the low ability of the current diagnostic antigens to bind ts-11 antibodies.     

Biological Control of the Larger Grain Borer, Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae)

Biological control was first considered shortly after the accidental introduction of Prostephanus truncatus in Africa in the early 1980's. This article reviews first the history of the biological control efforts with a focus on the results and recommendations of the initial international meetings and foreign exploration projects, followed by a discussion of the more important biocontrol agents, in particular the histerid Teretrius nigrescens. Since the release of T. nigrescens in Africa a number of techniques have been employed by different research groups to measure the effect of the natural enemy on P. truncatus, including pheromone trapping, field experiments, gut analysis grain store surveys, simulation modelling, and statistical and economic analysis the conclusions of these various studies have been largely equivocal, and the results of studies with recent data suggest that uncertainty is growing. Further resources and time are needed both for a proper evaluation and for the further development of control strategies compatible with biological control.     

Effects of Retinol on the in vitro Development of Bos Indicus Embryos to Blastocysts in Two Different Culture Systems

The objective of this study was to evaluate the effect of retinol on the in vitro development of early embryos of cultured Bos indicus (Expt 1) to the blastocyst stage in medium simplex of optimization (KSOM) or sintetic fluid of oviduct (SOF) or co-cultured (Expt 2) with an oviduct cell monolayer (OCM) in KSOM or SOF. A total of 3149 cumulus-oocyte complexes obtained by aspirating follicles (2-5 mm diameter) from ovaries of slaughtered animals were selected for IVM and incubated in TCM 199 supplemented with 25 mM HEPES at 39 degrees C in air with 5% CO(2) and maximum humidity for 24 h. In vitro fertilization (IVF) was performed in modified defined medium (mDM) medium. Eighteen hours after IVF, cumulus cells were removed and presumptive zygotes were randomly allocated to the experimental groups. Zygotes cultured (Expt 1) in KSOM + retinol, KSOM, SOF + retinol and SOF were incubated in maximum humidity at 39 degrees C, 5% CO(2), 5% O(2) and 90% N(2). Zygotes co-cultured (Expt 2) in KSOM + retinol + OCM, KSOM + OCM, SOF + retinol + OCM and SOF + OCM were incubated at 39 degrees C, 5% CO(2). In both experiments media were partially changed 48 h after IVF and unfertilized ova were removed. Afterwards embryos were kept in culture or co-culture for further 9 days. In Expt 1, blastocyst rates (day 7) were 14.6% (KSOM + retinol), 15.8% (KSOM), 16.4% (SOF + retinol) and 15.9% (SOF). In Expt 2, the blastocyst rates (day 7) were 25.4% (KSOM + retinol + OCM) 14.2% (KSOM + OCM), 24.3% (SOF + retinol + OCM) and 15.9% (SOF + OCM). The same influence profile of retinol was observed in the formation of the expanded (day 9) and hatched (day 11) blastocysts. The results obtained in Expt 2 demonstrated that the addition of 0.28 microg/ml retinol to the embryo culture media used in this study had a significant (p < 0.05) positive effect on bovine early embryonic development, under the conditions tested, and can be used to enhance in vitro embryo production.     

Infectious bursal disease virus (IBDV) induces apoptosis in chicken B cells

The ability of infectious bursal disease virus (IBDV) serotypes 1 and 2, and the role of VP4 of both serotypes as well as the capacity of three IBDV intermediate serotype 1-specific vaccine strains to induce apoptosis in a chicken B-lymphocyte cell line, DT40, were investigated using the TUNEL technique. It was observed that IBDV serotype 1 infected the DT40 cell line and directly induced apoptosis. In contrast, the non-pathogenic serotype 2 neither infected nor induced apoptosis, but was able to reduce the serotype 1-induced apoptosis when the two viruses were present in combination. VP4 of both serotypes did not induce apoptosis. IBDV VP2 of serotype 2 induced apoptosis in the same proportion and intensity as VP2 of serotype 1. IBDV intermediate vaccines varied in their ability to induce apoptosis in the DT40 cell line, which was also decreased-delayed in presence of serotype 2 IBDV. We hypothesize that both serotypes compete for the same receptor in DT-40 cells, and suggest that IBDV-induced apoptosis is a multistep process involving virus replication, protein expression, and release of virions.     

Differentiation of three whitefly-transmitted geminiviruses from the Republic of Yemen

Three viruses collected in southern Yemen in 1990, infecting watermelon, tobacco and tomato were shown to be transmitted by the whiteflyBemisia tabaci and to have particle morphologies typical of geminiviruses. Colonies ofB. tabaci collected from different locations and from different hosts were used in virus transmission tests with the same host range of plants. Colonies established from both watermelon and cotton in the Yemen were identified as the squash silverleaf-inducing B biotype. The culture host of the colony did not influence virus acquisition and transmission efficiencies to and from other hosts. The tobacco and tomato geminiviruses had a similar host range, but differed in their severity in some hosts. Both these viruses differed from the watermelon geminivirus in host range and symptoms.Datura stramonium, an alternative host for all three viruses, could be co-infected by the watermelon and tobacco viruses.B. tabaci was able to acquire both viruses from the co-infectedD. stramonium and infect seedlings of either original host plant species with their respective viruses orD. stramonium with both. The viruses were identified as watermelon chlorotic stunt virus, tobacco leaf curl virus and tomato yellow leaf curl virus and were distinguished by cross hybridisation.