Does isometamidium chloride treatment protect tsetse flies from trypanosome infections during SIT campaigns?

African animal trypanosomosis is a major pathological constraint to cattle breeding across 10 million km2 of sub-Saharan West African countries infested by tsetse flies, their cyclic vectors. The release of sterile males (sterile insect technique [SIT]) is a potentially important control technique aimed at eliminating the vectors. Prior to release, tsetse are generally treated with isometamidium chloride, a trypanocide, to prevent them from transmitting parasites. The present study investigated the preventive action of isometamidium chloride (0.5 mg/L) on the subsequent susceptibility of tsetse released into the wild. A total of 1755 Glossina palpalis gambiensis Vanderplank and 744 Glossina tachinoides Westwood were released, of which 50 and 48, respectively, were recaptured 22-43 days after release. Their probosces were analysed by polymerase chain reaction to identify mature infections with three trypanosome species (Trypanosoma vivax, Trypanosoma brucei sensu lato and Trypanosoma congolense savannah type). Two mature infections with T. vivax and four with T. congolense were detected, indicating that the use of this treatment regimen in an SIT campaign would not totally prevent sterile males from transmitting trypanosomes.     

Selection of susceptible and refractory lines of Glossina morsitans centralis for Trypanosoma congolense infection and their susceptibility to different pathogenic Trypanosoma species

Abstract .In a single generation of selection, two lines of Glossina morsitans centralis were established that differed significantly in susceptibility to Trypanosoma congolense clone IL 1180. Reciprocal crosses demonstrated that susceptibility was a maternally inherited trait. Differences between the lines, to all phases of the trypanosome infection, were maintained for eight generations, whereas differences in susceptibility to midgut infections were maintained for twenty-eight generations. Thereafter, the lines did not differ in susceptibility to Trypanosoma congolense IL 1180. Susceptibility to infections with Trypanosoma congolense IL 1180 was only a weak predictor of susceptibility to T. congolense clones IL 13-E3 and K60/1, as well as clone T. brucei brucei STIB 247-L. However, the susceptible and refractory lines displayed these phenotypes when tested with Trypanosoma vivax, indicating that the factors that affect susceptibility to trypanosomes are expressed both within and outside the midgut.     

Trypanosoma congolense: host responses following tsetse transmitted infection of Kilifi isolates in goats

East African x Galla goats, when infected with Trypanosoma congolense isolates from the Kilifi area of Kenya by Glossina morsitans centralis, did not develop the characteristic chancre reaction at the bite sites, whereas bites of tsetse infected with the cloned T. congolense IL.1180 from Serengeti, Tanzania, resulted in chancres in the same goats. Histological changes could not be observed in skin biopsies collected 8 or 9 days after infection with Kilifi isolates. However, all goats became parasitemic about 10 days after challenge. It is concluded that the absence of chancre development is a characteristic feature of T. congolense parasites from Kilifi. The isoenzyme analysis of clones of two T. congolense Kilifi isolates and the T. congolense clone IL.1180 indicated that they belong to different zymodemes. Neutralizing antibodies to homologous metacyclic variable antigen types were detected in six out of seven (86%) of the sera from goats infected with a clone or stock of a T. congolense Kilifi isolate, 20 days after infection. Goats primed by tsetse transmitted infection with a stock or clone of T. congolense from Kilifi and treated with Berenil were, in three out of eight cases (37%), not immune to homologous challenge. It is suggested that the reduced immune response to metacyclic variable antigen types could be a result of the absence of cellular infiltration, i.e., chancre development in the skin at the tsetse bite site. It is concluded that the use of the chancre reaction as a marker for serodeme analysis of recently isolated stocks of T. congolense from Kilifi was not feasible.     

Trypanosoma congolense: expression of a heat shock protein 70 and initial evaluation as a diagnostic antigen for bovine trypanosomosis

A 69-kDa immunodominant protein of Trypanosoma congolense was identified as a member of the hsp70 family that is homologous to mammalian BiP. We report here the expression of the gene encoding the T. congolense BiP in a bacterial system. Dot blotting of the truncated recombinant proteins confirmed that BiP antigenicity is mainly located in the C-terminal third of the molecule. A recombinant fragment corresponding to this region was used as an antigen in an indirect ELISA and an initial evaluation of its diagnostic potential for bovine trypanosomosis was performed. The test showed limited sensitivity for detection of primary-infected cattle but was capable of accurately detecting secondary infections. As BiP and its derivatives may be produced at low cost under stable forms allowing standardization of the tests, they warrant further evaluation as antigens fro serodiagnosis of bovine trypanosomosis.     

The effect of trypanosomosis on pregnancy in trypanotolerant Orma Boran cattle

A study was undertaken to investigate the influence of trypanosomosis on the outcome of pregnancy in trypanotolerant Orma Boran (Bos indicus) exposed to natural tsetse challenge in an area of Kenya infested predominantly with Glossina pallidipes. Of 73 pregnant Orma heifers, 58 (79.5%) produced live calves at term, 13 (17.8%) aborted and 2 (2.7%) died of trypanosomosis. Of the 71 surviving animals, 22 (31%) were infected with Trypanosoma vivax , 21 (29.6%) T. congolense, and 26 (36.6%) had mixed infections with T. vivax and T. congolense. These results suggest that in areas of high trypanosomosis risk reproductive function is affected even in trypanotolerant cattle, and that both T. vivax and T. congolense can be responsible for the abortions observed in the field. It is suggested that maintenance of pregnancy in the face of trypanosome challenge was dependent on individual variation among the Orma cattle, but as challenge increased beyond the limits of effectiveness of trypanotolerance, disruption of pregnancy occurred.     

Suppressive action of Samorin on the cyclical development of pathogenic trypanosomes in Glossina morsitans centralis

Male Glossina sexually sterilized by gamma-irradiation are as efficient vectors of trypanosomiasis as fertile males. An attempt was made, using isometamidium chloride (Samorin), to interfere with the cyclical development of trypanosomes in sterile males, destined for use in the sterile insect release (SIR) method of tsetse eradication. The infection rate with mature Trypanosoma congolense Broden was effectively reduced in sterile male Glossina morsitans centralis Machado, when the flies were fed on an infected goat 2 days after they were fed as tenerals on an in vitro bloodmeal containing 8 micrograms Samorin/ml blood. The infection rates with mature T.vivax Ziemann and T.brucei brucei Plimmer & Bradford were completely suppressed at this drug dose. Whensterile teneral males were fed on a bloodmeal containing 12 micrograms/ml Samorin and given the infected bloodmeal 10 days later, infections by mature T.vivax, T.congolense and T.b.brucei were completely suppressed. Hence in the management of a tsetse eradication programme utilizing the SIR method, it is recommended that the sterile teneral male tsetse should, prior to release, be given a bloodmeal containing Samorin at 12-15 micrograms/ml blood. This will effectively suppress future disease transmission.     

Enhancement of translation initiation by A/T-rich sequences downstream of the initiation codon in Escherichia coli

The region located downstream of the initiation codon constitutes part of the translation initiation signal, significantly affecting the level of protein expression in E. coli. In order to determine its influence on translation initiation, we inserted random 12-base sequences downstream of the initiation codon of the lacZ gene. A total of 119 random clones showing higher beta-galactosidase activities than the control lacZ gene were isolated and subsequently sequenced. Analysis of these clones revealed that their insertion sequences are strikingly rich in A and T, but poor in G, with no consensus sequences among them. Toeprinting experiments and polysome profile analysis confirmed that the A/T-rich sequences enhance translation at the level of initiation. Collectively, the present data demonstrate that A/T richness of the region following the initiation codon plays a significant role in E. coli gene expression.     

A modified AFLP for Trypanosoma congolense isolate characterisation

The amplified fragment length polymorphism (AFLP) technique is a reliable and powerful DNA fingerprint tool for genetic characterisation and analysis. In this paper, we described a modified AFLP with high resolution for Trypanosoma congolense using one enzyme and agarose or Elchrom gel electrophoresis. Eleven allopatric and fourteen sympatric isolates of T. congolense savannah were used to assess the resolution of the method and its ability to characterise T. congolense isolates. Two enzymes (Eco RI or Bgl II) and corresponding non-selective and selective primers were used to identify the most appropriate combination. Patterns generated by Bgl II enzyme and a single selective primer A, C, G or T produced clear profiles. Each of the four selective primers produced different profiles for all the 25 T. congolense isolates. Due to the reduction in the number of bands, profiles could be analysed using agarose or Elchrom gels. Although comparison of a great number of samples could benefit from software help, this technique did not require flurochrome detection methods. The results of the present study demonstrated that this modified AFLP makes the characterisation of T. congolense easier while maintaining high resolution.     

Molecular characterization of a diagnostic DNA marker for domesticated tetraploid wheat provides evidence for gene flow from wild tetraploid wheat to hexaploid wheat

All forms of domesticated tetraploid wheat (Triticum turgidum, genomes AABB) are nearly monomorphic for restriction fragment length polymorphism (RFLP) haplotype a at the Xpsr920 locus on chromosome 4A (Xpsr920-A1a), and wild tetraploid wheat is monomorphic for haplotype b. The Xpsr920-A1a/b dimorphism provides a molecular marker for domesticated and wild tetraploid wheat, respectively. Hexaploid wheat (Triticum aestivum, genomes AABBDD) is polymorphic for the 2 haplotypes. Bacterial artificial chromosome (BAC) clones hybridizing with PSR920 were isolated from Triticum urartu (genomes AA), Triticum monococcum (genomes AmAm), and T. turgidum ssp. durum (genomes AABB) and sequenced. PSR920 is a fragment of a putative ATP binding cassette (ABC) transporter gene (designated ABCT-1). The wheat ABCT-1 gene is more similar to the T. urartu gene than to the T. monococcum gene and diverged from the T. urartu gene about 0.7 MYA. The comparison of the sequence of the wheat A genome BAC clone with that of the T. urartu BAC clone provides the first insight into the microsynteny of the wheat A genome with that of T. urartu. Within 103 kb of orthologous intergenic space, 37 kb of new DNA has been inserted and 36 kb deleted leaving 49.7% of the region syntenic between the clones. The nucleotide substitution rate in the syntenic intergenic space has been 1.6 x 10(-8) nt(-1) year(-1), which is, respectively, 4 and 3 times as great as nucleotide substitution rates in the introns and the third codon positions of the juxtaposed gene. The RFLP is caused by a miniature inverted transposable element (MITE) insertion into intron 18 of the ABCT-A1 gene. Polymerase chain reaction primers were developed for the amplification of the MITE insertion site and its sequencing. The T. aestivum ABCT-A1a haplotype is identical to the haplotype of domesticated tetraploid wheat, and the ABCT-A1b haplotype is identical to that of wild tetraploid wheat. This finding shows for the first time that wild tetraploid wheat participated in the evolution of hexaploid wheat. A cline of the 2 haplotype frequencies exists across Euro-Asia in T. aestivum. It is suggested that T. aestivum in eastern Asia conserved the gene pool of the original T. aestivum more than wheat elsewhere.     

Amplified fragment length polymorphism and metabolomic profiles of hairy roots of Psoralea corylifolia L

A reproducible protocol for establishment of hairy root cultures of Psoralea corylifolia L. was developed using Agrobacterium rhizogenes strain ATCC 15834. The hairy root clones exhibited typical sigmoid growth curves. Genomic and metabolomic profiles of hairy root clones along with that of untransformed control were analysed. Hairy root clones, Ps I and Ps II, showed significant differences in their amplified fragment length polymorphism (AFLP) profiles as compared to that of control, besides exhibiting Ri T-DNA-specific bands. These results amply indicate the stable integration of Ri T-DNA into the genomes of these clones. Further, the variations observed between clones in the AFLP profiles suggest the variable lengths and independent nature of Ri T-DNA integrations into their genomes. An isoflavonoid, formononetin, and its glycoside were present only in the hairy root clones while they were absent in the untransformed control. Variations observed in the metabolite profiles of these clones may be attributed to the random T-DNA integrations and associated changes caused by them in the recipient genomes. GC/MS analyses revealed the production of three and six clone-specific compounds in Ps I and Ps II, respectively, suggesting that the clones are dissimilar in their secondary metabolism. HPLC/UV-MS analyses disclosed substantial increases in the total isoflavonoids produced in Ps I (184%) and Ps II (94%) compared to untransformed control.     

Rescue of silent integrated polyoma genomes suggests homologous recombination between resident and transfected DNA fragments

Two defective polyoma virus genomes, deleted in the nucleotide sequences coding the N-termini of the tumor antigens, were introduced into Fisher 3T3 rat cells by DNA-mediated gene transfer (transfection). The resulting integrated genomes were incapable of conferring a transformed phenotype to the cells. However, after transfection of these lines with small polyoma fragments overlapping the deleted sequences, transformed clones were isolated. These clones were analyzed by Southern genomic blot hybridization and by isolation in E. coli of plasmids containing viral sequences excised following fusion with mouse polyoma growth-permissive cells. In all cases at least one intact copy of the early region of the polyoma genome was found. Furthermore, restriction sites adjacent to the initial inactive insertion remained unchanged in many of the transformed lines. These results show that functional restoration of the defective polyoma early region involves homologous recombination between the deleted viral genomes integrated in the cellular DNA and the transfecting viral fragments.     

Free and integrated forms of hepatitis B virus DNA in human hepatocellular carcinoma cells (PLC/342) propagated in nude mice.

Primary hepatocellular carcinoma cells (PLC/342) propagated in nude mice produce hepatitis B surface antigen of subtype adr, as well as core particles containing viral DNA and DNA polymerase. Free and integrated forms of hepatitis B virus (HBV) DNA in the tumor were isolated by molecular cloning, and their nucleotide sequences were determined. Both of the two representative clones of free HBV DNA had the same genomic length (3,158 base pairs) and had two stop codons as well as two deletions in the envelope gene. None of the seven distinct clones of integrated HBV DNA possessed the entire viral genome. The integrated clone sequences had deletions and rearrangements, and only two clones possessed the envelope gene including the promoter and enhancer sequences. The C gene, which codes for core protein, was preserved in the two free clones and one of the integrated clones. The P gene, which codes for DNA polymerase, had deletions at two positions of 21 and 36 base pairs in both free clones, but was carried in toto by one of the integrated clones. The nucleotide sequences of the S genes of two free and four integrated clones, as well as their two inverted repeats, were compared. All of the eight sequences of the S gene possessed two nucleotide substitutions in common that were not displayed by any of the reported HBV genomes. The sequences differed from one another by only 1.2%. They differed, however, from 11 reported HBV genomes of subtype adr by 2.4%, from an ayr genome by 1.9%, from 2 adw genomes by 6.9%, and from 2 ayw genomes by 5.9%. These results indicate that all free and integrated HBV DNA species in the PLC/342 tumor cell evolved from a common progenitor. The free HBV DNA underwent nucleotide substitutions during several integration events, resulting in integrated HBV DNA copies that were similar in sequence but distinct from the reported HBV genomes.     

The isolation of the beta A-, beta C-, and gamma-globin genes and a presumptive embryonic globin gene from a goat DNA recombinant library

As an approach to understand how the expression of globin genes are regulated during development, clones containing globin DNA sequences were selected from a recombinant library of goat genomic DNA. The type of globin gene present in each of the recombinants was determined by cross-hybridization to the DNA of mouse alpha- and beta-globin cDNA-containing plasmids. Of 11 clones isolated, eight hybridized specifically to the DNA of the mouse beta-globin plasmid, while one clone hybridized only to the DNA of the alpha globin plasmid. The location of each globin sequence within its DNA insert was determined by a combination of restriction enzyme mapping and Southern transfer-hybridizations. Selected fragments were sequenced; comparisons of the amino acids coded for by these regions with those of the goat globins identified clones carrying beta A-, beta C-, and gamma-globin genes. Another recombinant coded for amino acid sequences resembling, but not identical with, the known goat globins, and was identified tentatively as containing an embryonic or epsilon-gene. Detailed analysis of the clone containing the beta C gene and an overlapping clone revealed that three other beta-like sequences are located 6, 12, and 21 kilobases on the 5'-side of the beta C gene. The globin sequence of the locus nearest to the beta C gene has an altered translation termination codon and, if transcribed and translated, would give a globin chain seven amino acids longer than the normal goat beta C-globin. In addition, the sequence following this termination codon is very AT-rich, unlike that of other globin genes. The recombinants described contain extensive regions of DNA surrounding the globin genes, making them useful for identifying regulatory sequences as well as determining the sequence organization of the goat globin genes.     

Mung bean nuclease cleaves preferentially at the boundaries of variant surface glycoprotein gene transpositions in trypanosome DNA

Telomere-linked genes coding for the variant surface glycoproteins (VSGs) of African trypanosomes have been difficult to clone because their flanking regions frequently lack restriction sites. Therefore, we constructed a genomic DNA library of fragments generated by digestion of purified trypanosome DNA with mung bean nuclease, an enzyme that cleaves before and after genes in Plasmodium falciparum DNA (McCutchan, T. F., Hansen, J. L., Dame, J. B., and Mullins, J. A. (1984) Science 225, 625-628). Southern hybridizations with several gene probes showed that under the appropriate conditions mung bean nuclease produces discrete trypanosome DNA fragments that are as clearly resolved on an agarose gel as restriction fragments. The majority of VSG genes are on fragments of about 1.7 kilobase pairs. To examine the sites of mung bean nuclease cleavage, the insert boundary sequences of eight recombinant clones in the library containing VSG genes were determined. In general, mung bean nuclease cleaved 300-800 base pairs in front of the VSG start codon and within 50 base pairs on either side of the termination codon. These regions also form the boundaries of VSG gene conversion events indicating that the enzyme recognizes, in part, a conformational structure rather than a specific sequence. The analyzed clones included both telomere-linked and interior basic copy VSG genes indicating that the library potentially contains all of the telomere-linked VSG genes in the genome.     

AFLP fingerprinting shows that a single Prymnesium parvum harmful algal bloom consists of multiple clones

Due to slow rates of molecular evolution, DNA sequences used to identify and build phylogenies of algal species involved in harmful algal blooms (HABs) are generally invariant at the intraspecific level. This means that it is unknown whether HAB events result from the growth of a single clone, a few dominant clones, or multiple clones. This is true despite the fact that several physiological and demographic traits, as well as toxicity, are known to vary across clones. We generated AFLP fingerprints from a set of 6 clonal isolates, taken from a bloom of Prymnesium parvum at a striped bass mariculture facility. This new haptophyte bloom was recently implicated in fish kills at several sites in the United States. The AFLP fragments were highly reproducible and showed that all isolates were distinguishable due to abundant AFLPs unique to single isolates. These results demonstrate that blooms can be genetically diverse outbreaks and indicate that AFLP can be a powerful molecular tool for characterizing and monitoring this diversity.     

Primary structure of a proline-rich zein and its cDNA

Eighty-five cDNA clones for γ-zein (proline-rich zein) from a cDNA expression library were isolated using specific antibody and cDNA probes. Nucleotide sequences of seven independent clones were determined and found to be identical in regions where they overlapped. The primary structure of the mature protein, determined from the sequence of one near full-length clone, consists of 204 amino acids. It has a molecular weight of 21,824 daltons, about 5 kilodaltons less than that estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal one-half of the sequence contained eight essentially identical tandem repeats of the hexapeptide Pro-Pro-Pro-Val-His-Leu and two of the octapeptide Gln-Pro-His-Pro-Cys-Pro-Cys-Gln. The codon specifying the third proline in the hexapeptide repeating units is identical (CCG) in all of the eight repeats. The coding region has a very high G-C content (69.8%). The multiple charge components of γ-zein detected by isoelectric focusing do not seem to be encoded by members of a multigene family. Moreover, it was found that the codon preference in γ-zein is, in fact, the base preference in the wobble position. A codon usage value was devised to express this phenomenon.     

Effects of Trypanosoma vivax and Trypanosoma congolense infections on the reaction time and semen characteristics of Zebu (Bunaji) x Friesian crossbred bulls

The effect of trypanosomosis on reaction time and semen characteristics of 12 Zebu (Bunaji) x Friesian crossbred bulls aged between 3 and 5 years was studied for a duration of 12 weeks. Four of the bulls were infected with Trypanosoma vivax, another four with Trypanosoma congolense and the remaining four bulls served as controls. Rectal temperatures and haematological parameters were monitored twice weekly. The pre-infection mean value of the rectal temperature was 38.3 degrees C, and this rose to a mean of between 40.5 and 41.1 degrees C in the infected animals. Concurrently, the infected animals exhibited signs of anaemia shown by pale mucous membranes and decreased packed cell volume (PCV), weight loss, lethargy, weakness and dullness.The reaction time (ejaculation time) of semen collection significantly increased from a pre-infection mean value of 20.46-25.14 s to a mean of 290.33-301.15 s within 12 weeks post-infection. Semen characteristics deteriorated progressively within the same period in the infected bulls. There were highly significant and drastic decreases in sperm concentration and volume of semen and increases in sperm morphological defects. By the third week, all the infected bulls were unfit for breeding because of very poor semen characteristics. Deterioration, also characterized by oligospermia at 6 weeks post-infection in all bulls which later culminated in azoospermia in two bulls infected with T. vivax and two bulls infected with T. congolense continued to the end of the investigation. The present results indicate that trypanosomosis due to T. vivax and T. congolense infections is very pathogenic and devastating in its effect on the reaction time (ejaculation time) and semen characteristics which resulted in very poor semen quality. The practical implication is infertility and sterility in Zebu x Friesian crossbred bulls in trypanosome endemic areas.     

Sample sequencing of a Salmonella typhimurium LT2 lambda library: comparison to the Escherichia coli K12 genome

As part of the ongoing sequencing of the complete Salmonella typhimurium LT2 genome, a partly ordered set of 416 lambda clones has been developed, representing over 90% of the genome. The average insert size is 17 kb. Sequences were obtained from both ends of each clone in this set. A total of over 600 kb of sequence has been deposited in the genome survey sequence section of GenBank. This resource of clones is available from the Salmonella Genome Stock Center. A preliminary comparison with the Escherichia coli K12 genome indicates that there are likely to be many hundred insertion deletion events, encompassing more than one gene, that distinguish these genomes. Fully 30% of the S. typhimurium sequences have no close homologs in the GenBank database.