Diversity of Sand Flies (Diptera: Psychodidae: Phlebotominae) in Two Different Eco-Climatic and Endemic Zones of Cutaneous Leishmaniasis in Mali, West Africa.

Being the only established vectors of the protozoan parasites of the genus Leishmania, sand flies have become very important in all countries where leishmaniasis exists. To better understand the sand fly fauna, a taxonomic inventory study was carried out between January and March 2012 in Soudan savannah (Boundioba, Sikasso) and Sahelian (Tieneguebougou, Koulikoro) areas of Mali. CDC light traps were used to collect the sand flies. Collected sand flies specimens were cleaned with lacto-phenol and examined under a light microscope for species identification. In total, 14 species belonging to the genera Phlebotomus and Sergentomyia were identified. The genus Sergentomyia constituted 98.05% of collected sand flies versus 1.95% for the genus Phlebotomus. The most abundant species were Sergentomyia dubia Parrot, Mornet, & Cadenat, Sergentomyia shwetzi, Sergentomyia clydei Sinton, and Sergentomyia antennata Newstead. In Boundioba, the genus Phlebotomus was represented by two species (Phlebotomus duboscqi Neveu-Lemaire and Phlebotomus rodhaini Parrot), whereas only one species, Ph. duboscqi, was captured in Tieneguebougou. For the first time, three new species, Sergentomyia madagascariensis, Sergentomyia congolensis, and Sergentomyia dureni, were identified in Mali. More investigations are needed for a better entomological assessment of the transmission of cutaneous leishmaniasis in the different eco-climatic zones of Mali.

[Study of phlebotomines sand fly wildlife suburban location of Bamako (Mali) presence of Phlebotomus (Phlebotomus) duboscqi]. / Étude de la faune phlébotomienne des zones périurbaines de Bamako (Mali), présence de Phlebotomus (Phlebotomus) duboscqi.

During three months of sampling, one thousand nine hundred and thirty five sand flies belonging to thirteen species of Phlebotomine sandflies were collected in suburban location of Bamako. Phlebotomus duboscqi, which is the common vector of cutaneous leishmaniasis in Mali, was found for the first time in Bamako mostly within human houses, which can confirm the possibility of a local transmission of Leishmania major. Sergentomyia freetownensis was found for the first time in Mali, which raises to 15 the number of sand flies species identified in Mali.

Transportation of goats: effects on physiological stress responses and live weight loss.

The management of food animals prior to slaughter influences both profitability and animal well-being. This experiment was conducted as a split-unit design to determine live weight shrink and stress responses in goats due to differences in stocking density during transportation and holding. A total of 150 Spanish does were transported on two different days (replicate) and held overnight (18 h) without feed in low- (LD) or high-density (HD) groups. On each day, 75 does were transported 2.5 h with floor spaces of .18 m2 and .37 m2/animal in LD (25 does) and HD (50 does) groups, respectively. The average temperatures in the trailer during transportation were 34.6 and 35 degrees C, respectively, on d 1 and 2. All animals were blood-sampled before loading (PRELOAD) and four does from each treatment were sampled immediately after loading (POSTLOAD). Animals were blood-sampled in holding pens either at 0, 1, 2, 3, 4, or 18 h after transportation (time) to assess the time course (n = 8 does per time per replicate) of stress responses. Individual animals were weighed just before loading onto a trailer and after overnight holding to assess shrinkage. Treatment or treatment x time did not have a significant effect on any of the dependent variables studied. There were significant effects of time (P < .01) on plasma cortisol, glucose, and urea nitrogen (PUN) concentrations. Time also had significant effects (P < .01) on plasma creatine kinase (CK) activity, differential leukocyte counts (neutrophils, lymphocytes, monocytes, and eosinophils), and ratio of neutrophils to lymphocytes (N:L). However, plasma leptin concentrations were not influenced by time. Cortisol concentrations increased at POSTLOAD sampling, peaked at 0 h, and decreased thereafter before spiking again at 18 h of holding. The PUN was higher at 18 h than at other time periods studied. Plasma glucose concentrations increased and remained at higher levels at 0, 1, and 2 h and began decreasing at 3 h, reaching PRELOAD levels at 18 h. Plasma CK kinase activity peaked at approximately 2 h after transportation. The N:L ratio was higher at all time periods after transportation than prior to starting the journey, indicating a prolonged effect of transportation stress on the immune system. The mean (+/- SE) shrinkage losses were 10.2 +/- .68 and 9.8 +/- .68 in HD and LD treatment groups, respectively. The results indicate that the stress responses of goats due to transportation begin decreasing within 3 h after transportation. However, prolonged holding periods without feed may increase stress responses and bring about metabolic changes.

Synchronization of cell division in eight-cell bovine embryos produced in vitro: effects of 6-dimethylaminopurine.

The methods used to achieve blastomere cell cycle synchronization in embryos used as nuclear donors during embryo reconstruction have been largely unsuccessful. The aim of this study was to determine the reliability of 6-dimethylaminopurine (6-DMAP), an inhibitor of maturation promoting factor, to half and to synchronize blastomere division in cleavage stage bovine embryos. A second goal was to assess its reversibility and toxicity in vitro. Eight-cell stage embryos obtained at 58 h after insemination were treated with several concentrations of 6-DMAP for 12 h. Treated embryos were assessed for cleavage arrest, chromatin morphology, DNA synthesis, histone H1 and scored for blastocyst formation and for hatching rate. They were subsequently fixed and the number of nuclei counted. Complete arrest of cell division was observed at concentrations of 3 mmol 6-DMAP l-1 and above. At these concentrations, interphase nuclei in arrest were noticeably larger compared with interphase nuclei of eight-cell control embryos. Removal from 6-DMAP led to release from cleavage arrest and was followed by synchronized mitosis, histone H1 kinase deactivation and re-entry into interphase within 4-5 h. Twenty-nine per cent of interphase nuclei were synthesizing DNA at the end of the 12 h treatment as indicated by BrdU analysis. At 2 h after removal from 6-DMAP, an abrupt decrease to 9% BrdU-positive nuclei was observed followed by an increase to 39% by 6 h and a decrease to 28% at 10 h. The ability of treated embryos to reach the blastocyst stage in vitro and the number of cells per blastocyst were reduced. These results indicate that 6-DMAP can reversibly arrest and synchronize cleavage to the fifth cell cycle in eight-cell bovine embryos. Although a decrease was observed in the proportion of blastocysts obtained after treatment, it is concluded that 6-DMAP is a useful tool for synchronization studies requiring donor nuclei at metaphase before fusion to recipient oocyte.

Synchronization of cell division in eight-cell bovine embryos produced in vitro: effects of aphidicolin.

To date, methods for synchronizing the cell division of ungulate embryos without reducing their developmental potential have not been reliable or simple. The overall objective of this study was to determine the reliability of aphidicolin, a powerful inhibitor of eukaryotic DNA synthesis, to arrest and synchronize blastomere division in cleavage-stage bovine embryos and to assess its reversibility and toxicity in vitro. Eight-cell stage embryos obtained at 58 h post insemination were treated with several concentrations of aphidicolin for 12 h. Treated embryos were assessed for cleavage arrest, chromatin morphology and DNA synthesis; scored for blastocyst formation and hatching rate; and fixed for determination of the number of nuclei. Complete arrest of cell division was observed at aphidicolin concentrations of 1.4 microM and above. At these concentrations, no morphological alteration to interphase chromatin was observed in treated embryos compared with the controls. Removal of aphidicolin led to at least a 4-h delay before resumption of DNA synthesis and cleavage. The ability of treated embryos to reach the blastocyst stage in vitro, the hatching rate and the number of cells per blastocyst were significantly reduced compared with the control group. Since the ability of treated embryos to develop to the blastocyst stage was significantly reduced even at the minimal effective dosage, it is concluded that aphidicolin is unlikely to provide suitable cell cycle synchronization without damage to the embryos.

Synchronization of cell division in eight-cell bovine embryos produced in vitro: effects of nocodazole.

Current methods of arresting and synchronizing cell division have not been very successful and have had few applications in embryo studies. Our objective was to determine the reliability of a metaphase arrest agent, nocodazole, for halting and synchronizing blastomere division in cleavage-stage bovine embryos, and to verify its reversibility and toxicity in vitro. Eight-cell-stage embryos obtained at 58 hr postinsemination were treated with varying concentrations of nocodazole for 12 hr. Treated embryos were assessed for cleavage arrest, chromatin morphology, DNA synthesis, and histone H1 and myelin basic protein (MBP) kinase activity, and were scored for blastocyst formation and hatching rate. They were subsequently fixed to count the number of nuclei. Complete arrest of cell division was observed at concentrations of 0.4 micrograms ml-1 and above. Removal from nocodazole treatment led to immediate release from cleavage arrest, and was followed by synchronized mitosis, histone H1 kinase deactivation, and reentry into interphase within 3-5 hr. DNA synthesis was reinitiated at 6 hr after release. Although cell numbers and hatching rate decreased, the proportion of embryos reaching blastocyst stage was not significantly affected in nocodazole-treated embryos. It is concluded that nocodazole is a suitable choice for the cell-cycle synchronization of donor embryos for use in studies on the interactions between nucleus and cytoplasm during early embryogenesis.

Sequence comparison of mitochondrial tRNA genes and origin of light strand replication in Bos taurus and Nellore (Bos indicus) breeds.

Although the complete bovine mitochondrial DNA molecule has been previously sequenced and sequence comparisons of the mitochondrial displacement loop have been performed, detailed sequence information is limited on coding regions of mitochondrial DNA within and among breeds of Bos taurus and Bos indicus. This study analysed polymorphism of the mitochondrial DNA transfer RNA genes for tryptophan, alanine, asparagine, cysteine, tyrosine and the origin of light strand replication among Ayrshire, Canadian, Belgium Blue, Brown Swiss, Hereford, Jersey, Limousine, Piedmontaise, Red Angus, Simmental (Bos taurus) and a Nellore (Bos indicus). Nucleotide sequence analysis of a 420-bp fragment of mitochondrial DNA comprising the five transfer RNA genes showed 100% homology among single individuals of the Bos taurus breeds. The Nellore breed showed guanine to adenine substitutions in the DHU arm of asparagine tRNA and in the origin of light-strand replication. This equates to a 0.5% sequence difference between the Nellore and Bos taurus breeds and may reflect an independent evolutionary origin of the species.

Effects of cell-cycle-arrest agents on cleavage and development of mouse embryos.

In mammals, there are no reliable methods for synchronizing cell division of early embryos without reducing their ability to develop into blastocysts and fetuses. The present study was undertaken to examine the in vitro inhibition of cell division of four-cell mouse embryos by cell cycle arrest agents. The reversibility of the agents was also tested by examining the developmental ability of treated embryos. Four-cell mouse embryos obtained at 54 hr post-human chorionic gonadotrophin (post-hCG) were cultured for 4, 8, 12, or 16 hr in media supplemented with either nocodazole, an inhibitor of tubulin polymerization, 6-dimethylaminopurine (6-DMAP), an inhibitor of maturation promoting factor (MPF) activation, or aphidicolin, a specific inhibitor of DNA polymerase alpha. Reversibility and toxicity of these agents were both dose and time dependent. For all three agents, prolonging cleavage arrest for 8 or 16 hr (at the effective concentrations) caused embryo lethality. Although nocodazole treatment was least cytotoxic, 6-DMAP and aphidicolin concentrations which induce cleavage arrest were detrimental to development beyond the blastocyst stage. The results of this study show that the development of embryos treated with these three cell-cycle-arrest agents is dose and incubation time dependent. Toxic effects beyond the blastocyst stage could only be minimized for nocodazole by reducing the exposure time of treatment and concentration of the mitotic inhibitor. However, these results render doubt on the usefulness of 6-DMAP and aphidicolin for synchronization studies leading to embryo transfer procedures.