Effects of Confluent, Roscovitine Treatment and Serum Starvation on the Cell-cycle Synchronization of Bovine Foetal Fibroblasts

The present study was designed to examine the effects of cell-cycle synchronization protocols, such as confluent, roscovitine treatment and serum starvation, in bovine foetal fibroblasts on synchronization accuracy at G0/G1, viability, apoptosis, necrosis and ploidy for use as a nuclei donor. The cells in 5-10 passages were randomly allocated into three treated groups. Cells were cultured either in Dulbecco's modified Eagle's medium (DMEM) + 10% foetal bovine serum (FBS) until 90% confluent (group 1, confluent), in DMEM + 10% FBS + 30 microM roscovitine for 12 h (group 2, roscovitine), or in DMEM + 0.5% FBS for 5 days (group 3, serum starvation). Most of the cells (>80%) in all groups were arrested at the G0/G1 stage. Although the rates did not differ, cells in group 1 showed an increased cell population arrested at the G0/G1 phase. Significantly (p < 0.05) higher rates of apoptosis occurred in group 3 than in group 1 and 2 (10% vs 6% and 6%, respectively). No differences in chromosomal abnormality were observed among groups. However, by increasing the number of cell culture passages up to 15, significantly (p < 0.05) higher chromosomal abnormality was observed than in 5 and 10 passages (39% vs 28% and 23%, respectively) in group 1. The results clearly indicated that bovine foetal fibroblasts could be effectively synchronized at G0/G1 stages by all the three different treatments, confluent, roscovitine and serum starvation. However, cells in confluent showed reduced apoptosis and necrosis when they underwent 5-10 passages, exhibiting increased percentage of cells with stable chromosome diversity. Hence, cells in confluent merit further studies before they could be used as nuclear donors.     

Comparative pathogenicity of three genetically distinct types of Trypanosoma congolense in cattle: clinical observations and haematological changes

The pathology of African bovine trypanosomosis was compared in Zebu cattle subcutaneously inoculated with three clones of trypanosomes corresponding to the three genetically distinct types of Trypanosoma congolense; savannah-type, west African riverine/forest-type and kilifi-type. All inoculated animals became parasitaemic between 7 and 11 days post-infection (dpi). The savannah-type showed consistently higher levels of parasitaemia and lower packed red cell volume percentages and leukocyte counts than the other two types. The syndrome was also more severe in the savannah-type and led inexorably to death between 29 and 54 dpi while animals with the forest or the kilifi-types recovered from earlier symptoms and haematological alterations after 3 months of infection. By the end of the experiment, the animals self-cured from the forest-type infection and the kilifi-type passed under control. The results of the present study indicated clear difference in pathogenicity between the three types of T. congolense; the savannah-type was virulent while the forest-type was of low pathogenicity and the kilifi-type was non-pathogenic.     

Seroprevalence of T. gondii in sheep from Marrakech, Morocco

Toxoplasma gondii is a ubiquitous intracellular protozoan parasite transmitted by food. Concerning this parasite, there are few studies done in Morocco. In this study, 261 sera from sheep intended for consumption in Marrakech were subjected to the Toxoplasma ELISA based serology test for the detection of anti-T. gondii specific IgG confirming a past infection. Of the total tested 72 (27.6%) sera were positive for IgG. This result shows that the seroprevalence approaches the world average and is similar to what is found in other cities of Morocco. This has prompted us to investigate other animal species in the region in order to evaluate the degree of contamination by this parasite as well as the potential risk incurred on consumption of their meat.     

Monensin controlled-release intraruminal capsule for control of bloat in pastured dairy cows

Monensin, a polyether ionophore antibiotic, is potentially an important agent for bloat relief in dairy cows grazing temperate legume-based pasture. A series of studies was undertaken to determine the effect of monensin, when delivered continuously in the rumen of lactating dairy cows by means of controlled-release capsules (monensin CRC). Such devices release approximately 300 mg/head/day for 100 d. A short-term pilot study made at Ruakura, New Zealand, tested monensin CRC in cows selected for high susceptibility to bloat and grazing lucerne (Medicago sativa) or red clover (Trifolium pratense). Treatment significantly reduced the incidence of bloat, while milk yield and protein yield were increased. There was no effect on fat yield. Following the pilot study, 6 large-scale field experiments involving a total of 368 lactating dairy cows, were made in Australia and New Zealand to confirm the effectiveness of monensin CRC for bloat control and to measure the effect of such treatment on milk production and composition. A severe bloat problem occurred in 2 experiments, mild bloat occurred in 2 others, while no visual signs of bloat were observed in the remaining 2 experiments. Bloat was significantly (P less than 0.05) reduced by monensin CRC treatment when data was pooled over the 4 experiments in which bloat occurred. Daily milk yield was increased in all experiments from a mean of 17.7 in untreated groups to 18.8 kg/head/day (P less than 0.05) in monensin CRC-treated cows. Protein percentage was not affected by treatment, while there was a decrease from 4.29 to 4.10% fat, although total fat yield was not affected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Hormonal Supplementation on Nuclear Maturation and Cortical Granules Distribution of Canine Oocytes During Various Reproductive Stages

The aim of this study was to evaluate the effects of hCG, progesterone and oestradiol supplementation on nuclear and cytoplasmic maturation of canine oocytes cultured for 24, 48, 72 and 96 h. Oocytes obtained from 18 healthy bitches were divided into three groups according to their reproductive status (follicular, luteal and anoestrus stages) and cultured in TCM 199 + 25 UI/ml of hCG + 1 μg/ml of progesterone + 1 μg/ml of 17‐β oestradiol or without hormonal supplementation (control) for different periods. Then, they were stained with FITC‐LCA‐Hoescht for chromatin configuration and cortical granules distribution and evaluated under an epifluorescence microscope. Culture time and the influence of different stages of the oestrous cycle were also evaluated. The present study demonstrated that there was no significant difference among the reproductive stages. With regards to culture medium, only oocytes from the supplemented medium were able to complete meiosis; however, significant difference was only noticed in the percentage of MI stage oocytes (p < 0.05) in the follicular and luteal group at 72 h of culture. Most oocytes in germinal vesicle, germinal vesicle breakdown and metaphase I stage had cortical granules distributed throughout the cytoplasm (immature pattern), irrespective of the culture period (p < 0.05). Cortical granules distributed immediately beneath the plasma membrane (mature) was only observed in metaphase II stage oocytes, but not all of them presented matured cytoplasm. Our results reveal that cortical granules distribution in canine oocytes matured in vitro did not progressed in correspondence with nuclear stage changes and are in accordance with those from other species.     

Serotypes of Erysipelothrix rhusiopathiae in Australian pigs, small ruminants, poultry, and captive wild birds and animals

Serotypes of 93 Australian isolates of Erysipelothrix rhusiopathiae from diseased domestic animals and poultry and a variety of captive wild birds and animals were determined by double diffusion gel precipitation. Two isolates, from the faeces of a swallow were also examined. Serotypes 1a, 1b and 2 were isolated from pigs and serotypes 1a, 1b, 2, 5, 15 and 21 from sheep or goats. Erysipelas in poultry was attributed to serotypes 1b, 5, 15 and 16. In captive wild birds serotypes 1b, 5, 6, 8, 14, 21 and an isolate reactive with antiserum to strain Seehecht were associated with septicaemic deaths. Single isolates from tissues of a bilby (Macrotis lagotis), black rat (Rattus rattus), brown snake (Pseudechis australis) and a bandicoot (Isoodon macrouris) were classified as serotypes 4, 4, 7, and 10 respectively. Six isolates were not able to be typed. Serotype 1b was the most widely distributed and most common (28%), being associated with disease in pigs, sheep, poultry and wild birds. Serotypes 1a or 2 were found in a more restricted range of animals, being commonly associated with erysipelas in pigs, less commonly in sheep and infrequently in other species. From diseased pigs, 26 of 33 isolates (79%) were serotypes 1a and 1b.     

Comparison of inoculation regimes for the experimental production of swine erysipelas arthritis: II Serological findings in a gel diffusion precipitin test and enzyme-linked immunosorbent assay

The serological response of pigs to Erysipelothrix rhusiopathiae inoculation was monitored by a gel diffusion precipitin test (GDPT) using a crude, serotype-specific, autoclaved antigen and an enzyme-linked immunosorbent assay (ELISA) using a heat-extracted, alcohol precipitated and molecular seived antigen previously shown to react with serum from pigs infected with serotypes 1 or 2. All pigs receiving 3 or 5 weekly intravenous inoculations of either a highly virulent (VRS 229) or a lowly virulent isolate (VRS 252) produced GDPT-reactive antibody within 3 weeks, but only 44% were still reactive at 8 to 9.5 weeks. The ELISA response was significantly higher in pigs inoculated with the highly virulent strain, and was similar in pigs receiving 3 or 5 doses of either strain. In a dose-response trial, after 3 doses of VRS 229, GDPT reactivity occurred earlier and was stronger in pigs given higher doses of E. rhusiopathiae, but the response peaked 3 to 5 weeks after the start of challenge and was short lived. GDPT reactivity correlated with dose, but not with the severity of arthritis. The ELISA demonstrated specific IgG antibody was present by 2 weeks, and persisted to at least 11 weeks. The ELISA reactivity was significantly higher in pigs with arthritis than in pigs that received low doses and were not arthritic. Within groups of pigs with arthritis a significant, dose dependent, linear ELISA response developed but did not correlate with the presence or degree of arthritis at slaughter. Non-arthritic pigs had similar low ELISA responses to uninoculated controls.     

Prion protein gene polymorphism in four West African sheep populations

A total of 162 individuals, belonging to three Burkinabé and one Niger sheep populations, were analysed for prion protein (PrP) gene polymorphism at codons 136, 154 and 171. The ARQ allele was the most frequent in both the Burkinabé (86.7%) and the Niger (67.5%) sheep populations. The highly sensitive allele VRQ was not found in the sampled individuals. The highly resistant ARR allele was in very low frequency in the Burkina-Sahel (4.4%) and Mossi (3.2%) populations and was not present in the Djallonké and Touareg populations. Only 4 out of 15 possible PrP genotypes were identified in the sampled individuals. No favourable ARR/ARR genotypes were found in either of the breeds. Sequencing a subgroup of the samples allowed the identification of other five polymorphisms on the PrP gene sequence at codons 116, 138, 151, 237 and 240. The very low frequency of the ARR allele in the West African sheep should dissuade the implementation of a preventive selection programme aimed to increase resistance to scrapie, to avoid an extreme erosion of the genetic stock.