Seroprevalence of anti-Toxoplasma gondii IgG antibody and risk factors among abattoir workers in Uyo, Southern Nigeria.

CONTEXT: The asymptomatic nature of toxoplasmosis with its severe consequences, especially in populations with high HIV infection rate, makes it a disease of public health importance. Being a zoonosis, Toxoplasma gondii infection may be high among people who work with animals. Thus, there is the need to determine the prevalence of the infection among abattoir workers in Uyo, where HIV prevalence rate is in double digits, which could lead to reactivation of toxoplasmosis in those with immunosuppression. AIM: To determine the prevalence and associated risk factors of T. gondii infection in abattoir workers in Uyo. SETTINGS AND DESIGN: A descriptive cross-sectional study that was conducted among persons who work with livestock and birds, in Uyo and its environs. METHODOLOGY: The study was conducted among 339 workers in abattoirs in Uyo and its environs. Data were collected using interviewer-administered questionnaire and detection of IgG antibodies to T. gondii using ELISA technique and HIV testing using the national algorithm. Data were analyzed using STATA statistical software version 20, and statistically significant level was set at P < 0.05. RESULTS: The seroprevalence of T. gondii IgG antibodies among the study participants was 55.8%. At a univariate level, many factors were associated with T. gondii seropositivity; they were age, sex, level of education, ethnicity, occupation in the abattoir, type of animal exposure, always washing hands before eating in the workplace, and taking of raw/unpasteurized milk. However, after multivariate analysis, participants exposed to poultry, butchers/raw meat sellers, and those who had worked for more than 5 years in abattoirs were at a greater risk of being T. gondii IgG seropositive; odds ratio (OR) 5.46 [confidence interval (CI) 1.88, 15.86]; OR 1.89 (CI 1.14, 3.14), and OR 1.25 (CI 1.25, 6.42), respectively. CONCLUSION: T. gondii IgG seroprevalence is high among abattoir workers in Uyo, and there is a need for Akwa Ibom State government to establish enlightenment and prevention programs for these occupationally exposed and other populations at risk of infection.

Expression of a mutated phospholipase A2 in transgenic Aedes fluviatilis mosquitoes impacts Plasmodium gallinaceum development.

The genetic manipulation of mosquito vectors is an alternative strategy in the fight against malaria. It was previously shown that bee venom phospholipase A2 (PLA2) inhibits ookinete invasion of the mosquito midgut although mosquito fitness was reduced. To maintain the PLA2 blocking ability without compromising mosquito biology, we mutated the protein-coding sequence to inactivate the enzyme while maintaining the protein's structure. DNA encoding the mutated PLA2 (mPLA2) was placed downstream of a mosquito midgut-specific promoter (Anopheles gambiae peritrophin protein 1 promoter, AgPer1) and this construct used to transform Aedes fluviatilis mosquitoes. Four different transgenic lines were obtained and characterized and all lines significantly inhibited Plasmodium gallinaceum oocyst development (up to 68% fewer oocysts). No fitness cost was observed when this mosquito species expressed the mPLA2.

Driving midgut-specific expression and secretion of a foreign protein in transgenic mosquitoes with AgAper1 regulatory elements.

The Anopheles gambiae adult peritrophic matrix protein 1 (AgAper1) regulatory elements were used to drive the expression of phospholipase A2 (PLA2), a protein known to disrupt malaria parasite development in mosquitoes. These AgAper1 regulatory elements were sufficient to promote the accumulation of PLA2 in midgut epithelial cells before a blood meal and its release into the lumen upon blood ingestion. Plasmodium berghei oocyst formation was reduced by approximately 80% (74-91% range) in transgenic mosquitoes. Blood-seeking behaviour and survival of AgAper1-PLA2 transgenic mosquitoes were comparable to sibling wild-type mosquitoes, while fertility was substantially lower. Ultrastructural studies suggest that decreased fitness is a consequence of internal damage to midgut epithelial cells.

Structure of the induced antibacterial protein from tasar silkworm, Antheraea mylitta. Implications to molecular evolution.

The crystal structure of an antibacterial protein of immune origin (TSWAB), purified from tasar silkworm (Antheraea mylitta) larvae after induction by Escherichia coli infection, has been determined. This is the first insect lysozyme structure and represents induced lysozymes of innate immunity. The core structure of TSWAB is similar to c-type lysozymes and alpha-lactalbumins. However, TSWAB shows significant differences with respect to the other two proteins in the exposed loop regions. The catalytic residues in TSWAB are conserved with respect to the chicken lysozyme, indicating a common mechanism of action. However, differences in the noncatalytic residues in the substrate binding groove imply subtle differences in the specificity and the level of activity. Thus, conformational differences between TSWAB and chicken lysozyme exist, whereas functional mechanisms appear to be similar. On the other hand, alpha-lactalbumins and c-type lysozymes exhibit drastically different functions with conserved molecular conformation. It is evident that a common molecular scaffold is exploited in the three enzymes for apparently different physiological roles. It can be inferred on the basis of the structure-function comparison of these three proteins having common phylogenetic origin that the conformational changes in a protein are minimal during rapid evolution as compared with those in the normal course of evolution.

Identification and characterisation of a silkworm ABC transporter gene homologous to Drosophila white.

In the silkworm, Bombyx mori, many eye- and egg-colour mutations affecting the synthesis and accumulation of ommochrome pigments have been described. In order to understand the pigment precursor transporters involved, ABC transporter genes homologous to the Drosophila white gene were isolated from the silkworm. Reverse transcriptase-polymerase chain reactions (RT-PCR) using embryonic mRNA amplified three cDNA fragments, named Bmwh1, Bmwh2 and Bmwh3 that showed homology to the white gene. Since Bmwh3 shows the highest degree of sequence identity and a similar expression pattern to the Drosophila homologue, we characterised this gene further. A 2667-bp Bmwh3 cDNA isolated from an embryonic library has one ORF encoding a polypeptide of 687 amino acids. The predicted protein has one ATP-binding domain, six transmembrane-spanning segments and high similarity to the Drosophila WHITE protein. Southern analysis indicates that Bmwh3 is a single-copy gene. Polyadenylated Bmwh3 transcripts about 2.7 kb long were detected in eggs, Malpighian tubules and pupal heads, but not in testes, posterior silk glands or fat body cells. The level of Bmwh3 mRNA was reduced in w3 and w3ol mutants but normal in other egg- and eye-colour mutants, suggesting that Bmwh3 correspond to the w3 locus. Genetic analysis was used to map the cloned gene to chromosome 10.

Microsatellites in the silkworm, Bombyx mori: abundance, polymorphism, and strain characterization.

We have isolated and characterized microsatellites (simple sequence repeat (SSR) loci) from the silkworm genome. The screening of a partial genomic library by the conventional hybridization method led to the isolation of 28 microsatellites harbouring clones. The abundance of (CA)n repeats in the silkworm genome was akin to those reported in the other organisms such as honey bee, pig, and human, but the (CT)n repeat motif is less common compared to bumble bee and honey bee genomes. Detailed analysis of 13 diverse silkworm strains with a representative of 15 microsatellite loci revealed a number of alleles ranging from 3 to 17 with heterozygosity values of 0.66-0.90. Along with strain-specific microsatellite markers, diapause and non-diapause strain-specific alleles were also identified. The repeat length did not show any relationship with the degree of polymorphism in the present study. The co-dominant inheritance of microsatellite markers was demonstrated in F1 offspring. A list of primer sequences that tag each locus is provided. The availability of microsatellite markers can be expected to enhance the power and resolution of genome analysis in silkworm.

Genetic characterization of the silkworm Bombyx mori by simple sequence repeat (SSR)-anchored PCR.

Thirteen diverse strains of the silkworm Bombyx mori were analysed using the simple sequence repeat anchored polymerase chain reaction (SSR-anchored PCR) or Inter-SSR-PCR (ISSR-PCR). A set of four 5'-anchored and two 3'-anchored repeat primers amplified a total of 239 bands out of which 184 (77%) were polymorphic. The 5'-anchored primers revealed more distinct polymorphic markers than the 3'-anchored primers and the ISSR-PCR method showed greater variability than RAPDs. The strain-specific pattern was shown to be inherited and segregated in a Mendelian fashion. A dendrogram constructed using the UPGMA method revealed two distinct groups, one comprising nondiapausing and one comprising diapausing strains. These results suggest that the ISSR-PCR method is potentially useful for genetic fingerprinting of silkworm genotypes and as a mapping tool in the silkworm.

Purification and partial characterization of an induced antibacterial protein in the silkworm, Bombyx mori.

Injection of live Escherichia coli into larvae of the silkworm, Bombyx mori, induces antibacterial activity in the hemolymph. The major induced antibacterial activity was purified in two steps by CM-Sephadex C-50 and Sephadex G-100 column chromatography. After trypsin treatment, the purified antibacterial protein lost its activity and the antibacterial activity was found to be partially heat labile. The purified protein was a single polypeptide chain of molecular weight 16 kDa. The 20 N-terminal amino acid sequence of the protein was determined and this sequence showed homology with the N-terminal amino acid sequence of lysozymes reported in other species. The purified protein was found to have comparable antibacterial activity against both E. coli and Micrococcus luteus. The purification of antibacterial protein and the antibacterial properties of the purified protein are discussed.

Expression of a Bombyx cytoplasmic actin gene in cultured Drosophila cells: influence of 20-hydroxyecdysone and interference with expression of endogenous cytoplasmic actin genes.

The expression of the Bombyx cytoplasmic actin A3 gene and its response to 20-hydroxyecdysone are studied after transfection in hormone responsive Drosophila cells and are compared to the expression of homologous resident genes. The host cells accumulate correct transcripts of the Bombyx gene in a gene dosage dependent way. The relative amount of endogenous cytoplasmic actin mRNAs is decreased in transfected cells, whether the transgene is integrated into the genome or not. When 20-hydroxyecdysone is added to the culture medium, the accumulation of the foreign mRNA is decreased whereas those of endogenous cytoplasmic actin transcripts are increased. These results are discussed in terms of competition for transcription and regulatory factors.

A cytoplasmic actin gene from the silkworm Bombyx mori is expressed in tissues of endodermal origin and previtellogenic germ cells of transgenic Drosophila.

A cytoplasmic actin gene from Bombyx mori introduced into Drosophila melanogaster by P-element mediated transformation, is efficiently transcribed in larvae, pupae and adults of the host. The exogenous mRNA has the same size as the one observed in the Bombyx cells and the intron located within the coding region is properly excised, indicating a correct recognition of the exogenous sequences by the Drosophila transcriptional and splicing machineries. The expression of the Bombyx gene in Drosophila tissues was determined by transforming flies with a hybrid gene in which a large part of the Bombyx actin coding sequences was replaced by those of the bacterial lac Z gene. This chimaeric gene is specifically and highly expressed, from the embryo to the adult of the transgenic lines, in tissues of endodermal origin, the midgut and its derivatives, i.e. gastric caeca, the outer layer of the proventriculus, and in the Malpighian tubules. This gene is also expressed, at a lower level, in germ cells but restricted to the sixteen cell cysts during previtellogenesis. The expression of the Bombyx gene during development of transgenic flies was compared to that of the two Drosophila endogenous cytoplasmic actin genes and the results are discussed.