Three operational taxonomic units of Eimeria are common in Nigerian chickens and may undermine effective molecular diagnosis of coccidiosis.

BACKGROUND: Chicken is fast becoming the world's most consumed meat. As a consequence poultry health is more important now than ever before, with pathogens of chickens recognised as serious threats to food security. One such threat are Eimeria species parasites, protozoa which can cause the disease coccidiosis. Eimeria can compromise economic poultry production and chicken welfare, and have serious consequences for poor livestock keepers. Seven Eimeria species that infect chickens are recognised with a global enzootic distribution. More recently three cryptic Operational Taxonomic Units (OTUx, y and z) have been described in populations of Eimeria recovered from chickens in Australia. Two of the three OTUs have also been detected in sub-Saharan Africa, but their occurrence, pathology and the risk they pose is largely unknown. RESULTS: Nigeria has witnessed a dramatic expansion in poultry production and is now the largest poultry producer in Africa. Here, faecal samples collected from nine of 12 commercial chicken farms sampled in Kaduna state, Nigeria, were found to contain eimerian oocysts. After amplification by in vivo propagation all three cryptic OTU genotypes were detected using polymerase chain reaction (PCR), including OTUy for the first time outside of Australia. Comparison with a widely used, established Eimeria species-specific PCR assay revealed failure to detect the OTU genotypes. CONCLUSIONS: All three of the Eimeria OTU genotypes appear to be common in north-western Nigeria. The failure of a leading species-specific molecular assay to detect these genotypes indicates a risk of false negative Eimeria diagnosis when using molecular tools and suggests that the spatial occurrence of each OTU may be far wider than has been recognised. The risk posed by these novel genotypes is unknown, but it is clear that a better understanding of Eimeria occurrence is required together with the validation of effective diagnostics.

Identification and characterization of sialidase-like activity in the developmental stages of Amblyomma variegatum.

Amblyomma variegatum F. are obligate hematophagous ectoparasites of livestock that serve as the vectors of Ehrlichia ruminantium (formerly known as Cowdria ruminantium), the causative agent of heartwater disease. In the light of the fact that they are blood-feeding, their salivary glands play prominent role in their acquisition of nutrients from the bloodmeal. Sialic acids are a major component of glycoprotein in mammalian blood fluid and cells. Sialome of hard ticks is still sparse. Here, for the first time, the possible expression of sialidase in A. variegatum was investigated. Our finding established the presence of type II sialidase-like activity in the three stages (larva, nymph, and adult) of the fed and unfed tick. There was no statistically significant difference in sialidase activity in the various stages of this ectoparasite (P > 0.05). The enzyme was purified by combination of salting out and ion exchange chromatography on DEAE--cellulose and hydroxylapatite columns. Characterization of the enzyme revealed that it is optimally active at 40 degrees C and pH 5.5, and is activated by bivalent cations Zn2+ or Fe2+. The enzyme has a Km of 0.023 mM and Vmax of 0.16 millimol/min with Fetuin as the substrate. To assess the susceptibility of some mammalian cells to the tick sialidase, we prepared erythrocyte ghost cells from different animals, which were incubated with the enzyme. Results revealed that the ruminant cells were better substrates. Our work and findings contribute to the preliminary characterization of the A. variegatum salivary proteome, and may pave way to the development of new acaricides.

Semen sialic acid surge and modulation of alpha-L-fucosidase activity: possible link to loss in reproductive capacity during trypanosomiasis.

The profiles of semen sialic acid and the enzyme alpha-L-fucosidase were studied in rams undergoing chronic infection by Trypanosoma congolense. Our data showed a significant surge in the level of sialic acid with parasitaemia. The pattern followed a polynomial function we had reported for erythrocyte sialic acid in mice undergoing acute infection by T. congolense. The activity of the enzyme alpha-fucosidase decreased progressively with approximately 60% decrease at the end of the 14 weeks of infection. Representative semen samples from the control and infected rams were subjected to kinetic characterization. While the uninfected semen sample showed two active pH peaks at 4.5-5.5 and at 6.8-7.2, respectively, there was an apparent shift to only a single pH optimum at 4.5-5.5 for the pathological semen. The fucosidases from both sources were optimally active at 35 degrees C albeit with contrasting activation energies (E(a)) with values 20.58 and 35 kJ/mol for the control and infected semen, respectively. Kinetic studies using methylumbelliferyl-beta-fucoside (4MU-Fuc) as substrate gave K(M) and V(max) values of 3.25 microM and 14.6 micromol. min(-1) mg(-1), respectively for the control semen. The values for the infected semen were 18.25 microM and 10.5 micromol. min(-1) mg(-1), respectively. The significance of these results is discussed as they relate to loss in reproductive capacity in trypanosomoses.

Kolaviron shows anti-proliferative effect and down regulation of vascular endothelial growth factor-C and toll like receptor-2 in Wuchereria bancrofti infected blood lymphocytes.

The anti-proliferative effect and down regulation of vascular endothelial growth factor C and toll like receptor-2 by kolaviron on Wuchereria bancrofti infected peripheral blood lymphocytes were investigated. Blood were collected from consenting volunteers in Talata Mafara, Nigeria, between the hours of 10pm to 12am, and microscopically identified for microfilariae. W. bancrofti positive samples were cultured for 72h treated with Doxycycline (2µg/ml) and kolaviron (5µg/ml) in vitro. Mitotic index, expression of vascular endothelial growth factor-C (VEGF-c), toll like receptor-2 (TLR-2) were determined using standard procedures. Mitotic index was significantly (P<0.05) reduced in the kolaviron treated group compared to negative control. Kolaviron also significantly (P<0.05) down regulated the expression of VEGF-c and TLR-2 when compared with the untreated group. In both cases, the effects of kolaviron was not significantly different (P<0.05) to that of doxycycline. Furthermore, strong positive correlations between mitotic index, VEGF-c and TLR-2 expressions were observed. The study suggests that kolaviron rich portion of Garcinia kola exhibited anti-proliferative effect and down regulation of VEGF-c and TLR-2 in W. bancrofti infected blood. Thus, the results from this study might have unravelled the potency of kolaviron in the management of complications associated with lymphatic filariasis.

Global warming and the possible globalization of vector-borne diseases: a call for increased awareness and action.

Human activities such as burning of fossil fuels play a role in upsetting a previously more balanced and harmonious ecosystem. Climate change-a significant variation in the usual pattern of Earth's average weather conditions is a product of this ecosystem imbalance, and the rise in the Earth's average temperature (global warming) is a prominent evidence. There is a correlation between global warming and the ease of transmission of infectious diseases. Therefore, with global health in focus, we herein opine a stepping-up of research activities regarding global warming and infectious diseases globally.

Cis-vaccenic acid induces differentiation and up-regulates gamma globin synthesis in K562, JK1 and transgenic mice erythroid progenitor stem cells.

Gamma globin induction remains a promising pharmacological therapeutic treatment mode for sickle cell anemia and beta thalassemia, however Hydroxyurea remains the only FDA approved drug which works via this mechanism. In this regard, we assayed the γ-globin inducing capacity of Cis-vaccenic acid (CVA). CVA induced differentiation of K562, JK1 and transgenic mice primary bone marrow hematopoietic progenitor stem cells. CVA also significantly up-regulated γ-globin gene expression in JK-1 and transgenic mice bone marrow erythroid progenitor stem cells (TMbmEPSCs) but not K562 cells without altering cell viability. Increased γ-globin expression was accompanied by KLF1 suppression in CVA induced JK-1 cells. Erythropoietin induced differentiation of JK-1 cells 24h before CVA induction did not significantly alter CVA induced differentiation and γ-globin expression in JK-1 cells. Inhibition of JK-1 and Transgenic mice bone marrow erythroid progenitor stem cells Fatty acid elongase 5 (Elovl5) and Δ(9) desaturase suppressed the γ-globin inductive effects of CVA. CVA treatment failed to rescue γ-globin expression in Elovl5 and Δ(9)-desaturase inhibited cells 48 h post inhibition in JK-1 cells. The data suggests that CVA directly modulates differentiation of JK-1 and TMbmEPSCs, and indirectly modulates γ-globin gene expression in these cells. Our findings provide important clues for further evaluations of CVA as a potential fetal hemoglobin therapeutic inducer.

A bloodstream Trypanosoma congolense sialidase could be involved in anemia during experimental trypanosomiasis.

The release of Sialic acid (SA) into the serum by Trypanosoma congolense infected BalbC mice was investigated. A progressive increase in the level of serum SA corresponding to anemia and parasitemia was observed. At maximum parasitemia, the level of total SA from the red blood cells (RBC) dropped by about 45%. Solved polynomials revealed an association between free serum SA and RBC-SA. Positive roots of quadratics were used to predict complete cleavage of RBC-SA on day 7.01 and maximum accumulation of free serum SA on day 6.6. A steady rise in the level of serum sialidase (SD) activity and a low packed cell volume (PCV) with an increase in parasitemia were observed. Mice infused with galactose, methyl-beta-gal, lactose, mannose, or L-arabinose and challenged by intraperitoneal inoculation with Trypanosoma congolense neither developed anemia nor secreted free SA above the control level even though there was detectable SD activity. Bloodstream Trypanosoma congolense parasites were isolated using DEAE cellulose from heparinized blood of experimentally infected BalbC mice. The parasites were lysed with 0.2% Triton-CF 54 to release membrane bound SD. The activity of the SD was proportional to the number of parasites. The enzyme was partially purified on Q-Sepharose and Fetuin agarose columns successively. The final active fraction from the latter column was used as the partially purified SD. The enzyme had an optimum pH of 6 and was maximally active at 37 degrees C with a requirement for the divalent ions Ca(2+) and Mg(2+). The enzyme was highly specific for NeuAc5alpha2,3 lac and Methylumbelliferyl-Neu5Ac (4-MU-Neu5Ac) with K(M) values of 0.34 and 0.025 mM, respectively. It was inhibited competitively by 2,3-didehydroneuraminic acid (Neu5Ac2en) and para-nitro-phenyloxamic acid (pNPO) with inhibition binding constants K(i) of 65 and 215 micro M, respectively. In deviation from the procyclic trypanosomal SD, it lacked trans-sialidase (TS) activity. The possible role of a secreted bloodstream Trypanosoma congolense SD and the development of anemia in the pathogensesis of trypanosomiasis are discussed.

Trypanosoma evansi sialidase: surface localization, properties and hydrolysis of ghost red blood cells and brain cells-implications in trypanosomiasis.

A membrane-bound sialidase was isolated from blood stream (BS) Trypanosoma evansi partially purified and characterized. The enzyme is a glycosyl phosphatidyl inositol (GPI) membrane anchored protein. It was solubilized from T. evansi cells recovered from infected camel blood by detergent treatment with Triton CF 54 and partially purified by a series of chromatography steps. The enzyme was optimally active at pH 5.5 and 37 degrees C. It had a KM and Vmax values of 4.8 x 10(-6) M and 3.75 x 10(-6) mol/min x mg protein with Neu5Acalpha2, 3lac as substrate respectively. The KM and Vmax values with fetuin (4-nitrophenyl-oxamic acid) as substrate were 2.9 x 10(-2) M and 4.2 x 10(-3) mol/min x mg protein in the same respect. Kinetic analysis with methly umbelliferyl sialate (MU-Neu5Ac) gave KM and Vmax values of 0.17 mM and 0.84 mmol/min x mg protein respectively. The T. evansi SD could hydrolyse internally linked sialic acid residues of the ganglioside GM2, but was inactive towards colomic acid, and NeuSAc2, 6. lac. When ghost red blood cell (RBC) was used as substrate, it desialylated the RBC in the following order of efficiency; mouse, rat, camel, goat, and dog. Similarly, cerebral cells isolated from BalbC mouse was desialylated by the T. evansi SD. Inhibition studies using 2-deoxy-2, 3 didehydro-N-acetyl neuraminic acid (NeuAc2, 3en) against MU-Neu5Ac revealed a competitive inhibition pattern with Ki of 5.8 microM. The enzyme was also inhibited non-competitively by parahydroxy oxamic acid (pHOA), and competitively by N-ethylmaleimide and N-bromosuccinate with Ki values of 25, 42, and 53 microM, respectively. It was activated by Mg2+ ion and inhibited by Cu2+ and Zn2+.

Infectivity of macrophages and the histopathology of cutaneous lesions, liver and spleen is attenuated by leaf extract of Vernonia amygdalina in Leishmania major infected BALB/c mice.

Preliminary investigation of the in vitro and in vivo efficacies of different extracts from the leaves of Vernonia amygdalina (VA), a plant widely used in Nigeria was evaluated in Balb/C mice infected with a laboratory strain of Leishmania major (L. major). The ability of the methanol, hexane and aqueous extracts of the plant to suppress the infection rate and its cytotoxicity on macrophages and L929 cells were determined in the in vitro study. The in vivo study evaluated time course of infection, lesion progression and the histopathology of cutaneous lesions, liver and spleen after inoculation with metacyclic promastigotes. Methanolic extract of VA containing high levels of flavanoids, was the most potent extract as it showed the highest suppression on infectivity and viability of intracellular amastigotes at a concentration lower than that which elicited cytotoxicity on macrophages. Treatment of infected mice with methanolic extract of VA showed delayed onset of disease with a significant reduction in lesion size and attenuation of the histopathological outcome characterised by intact epidermis and less tissue destruction in skin, spleen and liver. In conclusion, these results demonstrate that VA has potent antileishmanial properties which warrants further investigation into the immunological mechanism.

Erythrocyte surface sialic acid levels of clinically healthy mongrel and exotic (alsatian and terrier) breeds of dogs.

The erythrocyte surface sialic acid concentration of clinically healthy mongrel and exotic (Alsatian i.e. German shepherd and Terrier) breeds of dogs was analyzed in order to determine their role in the genetic resistance of these breeds of dogs to diseases that cause anaemia. The mean erythrocyte surface sialic acid (ESA) concentration was 57.08 +/- 1.67, 34.50 +/- 2.30 and 20.20 +/- 3.54 mg/dl for Mongrel, Alsatian (German shepherd) and Terrier breeds of dogs, respectively, on acid hydrolysis. The mean values of ESA obtained following enzymic hydrolysis of haemoglobin-free erythrocyte membranes using Clostridium chauvoei (Jakari strain) sialidase were 49.08 +/- 0.41, 30.97 +/- 1.82 and 18.64 +/- 0.75 mg/dl for Mongrel, Alsatian (German shepherd) and Terrier dogs respectively. When Trypanosoma vivax sialidase was used the ESA values obtained were 50.81 +/- 0.37, 41.70 +/- 0.94 and 19.65 + 0.65 mg/dl for Mongrel, Alsatian (German shepherd) and Terrier breeds of dogs respectively. This represents a statistically significant difference (P < 0.001) between the mean ESA concentration of all the breeds of dogs investigated in this study. The higher mean ESA concentration in Mongrel dogs, compared to the exotic breeds may be responsible for their resistance to disease conditions, whose aetiologic agents produce neuraminidase and also cause anaemia.

Effective measures for controlling trypanosomiasis.

African trypanosomiasis, otherwise known as sleeping sickness in humans and 'Nagana' in cattle, is a disease that is resurgent in Africa. Research on the disease suggests that the development of a vaccine is still far away; even existing drugs are becoming ineffective on account of the emergence of drug-resistant trypanosomes. All this contributes to heavy economic losses and a sociopolitical crisis in the continent, thus underscoring the pressure to intensify research for inexpensive, less toxic and affordable trypanocides. This review discusses the current treatment of trypanosomiasis and the progress made towards the effective control of trypanosomiasis.

Correlation of gastric mucosal damage with sialic acid profile in rats: effect of hydrochloric acid, pepsin and hypertonic saline.

Sialic acids occupy terminal positions on gastric mucus glycoprotein where they contribute to the high viscosity of mucin. Desialylation of mucus may lead to degradation of the mucus and eventually to the breakdown of the gastric mucus barrier. The effect of a variety of damaging agents (0.1 M HCl, 2 mg ml(-1) pepsin and 2 M NaCl) on sialic acid profile was determined in pylorus-ligated rats. The relationship between sialic acid, galactose, pyruvate and the extent of gastric mucosal damage were studied. Instillation of pepsin significantly increased total sialic acid, galactose and macroscopic mucosal lesions in the stomach. Instillation of 0.1 M HCl reduced the total sialic acid but this decrease was not significant. Acidity led to a significant increase in the amount of free sialic acid in the gastric instillates and the macroscopic lesions induced by acid was not significantly different from the control animals (0.15 M NaCl). 2 M NaCl induced the macroscopic lesions in the stomach and also free sialic acid in the instillates. Pepsin potentiates the action of 2 M NaCl. In all the agents examined with the exception of acid, it was observed that an increase in free sialic acid and galactose was accompanied by gastric mucosal erosion and elevation of pyruvate concentration. It is concluded that gastric acidity alone is not inherently damaging and that resistance of gastric mucosa to destructive agents may be dependent on the integrity of the sialic acids.

Characterization of sialidase from Entamoaeba hystolitica and possible pathogenic role in amebiasis.

Sialidase from Entamoeba histolytica was purified to apparent electrophoretic homogeneity by chromatography on hydroxyapatite, reactive brown agarose, fetuin/agarose and by fast performance liquid chromatography on a MonoQ column. The enzyme had a molecular mass of 65 kDa, as determined by SDS-PAGE. It had an optimum pH of 5.5 and was maximally active at 37 degrees C. It required Ca(2+) and Mg(2+) for activation but was strongly inhibited by Cu(2+), Fe(2+) and Zn(2+). The E. histolytica sialidase exhibited high specificity for Neu5Acalpha2,3lac and methylumbelliferyl-Neu5Ac (4-MU-Neu5Ac) with K(m) values of 0.144 mM and 0.059 mM, respectively. The enzyme was not active against colomic acid and the gangliosides GM(1) and GD(1). It was activated in the presence of lactose and galactose, but was unaffected by glucose, sucrose and mannose. The enzyme was inhibited competitively by 2,3,didehydroneuraminic acid and para-nitro-phenyloxamic acid with inhibition binding constants ( K(i)) of 30 micro M and 185 micro M, respectively. The motility of intact E. hystolytica cells was enhanced about 6-fold in the presence of 0.05-0.1 mM Neu5Acalpha2,3lac, 4-MU-Neu5Ac and fetuin. However, the motility of the parasite was highly diminished when incubated with Neu5Acalpha2en and sialic acid-containing compounds. Lysed E. histolytica trophozoites were found to lack neuraminic acid.

A 45-kDa midgut glycoprotein from Anopheles albimanus mosquito mediates the killing of trypanosomes.

Trypanosomes do not inhabit or grow in anopheles mosquitoes, the vector for the transmission of Plasmodium parasites the causative agent for malaria. The possession of lytic factors by the anopheline mosquito was thus considered. Head and midgut sections prepared in phosphate buffered saline were tested for trypanocidal action against T. congolense. While the head section was inactive towards the trypanosomes, the midgut extract at 0.2 mg ml(-1) diminished the motility of the parasites within 2 min of incubation; killing 50% of the population after 5 min. At 0.5 mg ml(-1) of the extract, about 90% of the parasites were killed within 2 min of incubation. The midgut fraction was subjected to a purification protocol involving successive chromatography on: octyl-sepharose, reactive brown agarose and fetuin-agarose columns. A final trypanocidal active fraction (gp45), which moved homogeneously during electrophoresis as a 45-kDa protein, was recovered from the fetuin-agarose column. The protein reacted positively with thiobarbituric acid, which suggests it is a sialoglycoprotein. Desialylation of the glycoprotein nullified its trypanocidal activity on T. congolense. Similarly, when the saccharides, lactose, methyl-beta-galactoside, lactulose, methyl-umbelliferyl-beta-galactoside (MU-Gal), were included in the culture medium, they inhibited the gp45 trypanocidal activity. Asialo-fetuin and asialo-RBC also inhibited the gp45-induced killing of T. congolense cells. The potential use of anopheline 45 kDa protein in the production of transgenic tsetse flies (Glossina spp.) in the control of trypanosomiasis is discussed.

Aryl sulfatase from Naja nigricolis venom: characterization and possible contribution in the pathology of snake poisoning.

The venom of Naja nigricolis was found to contain a high level of the enzyme aryl sulfatase. The enzyme was isolated from the venom of N. nigriclois and purified to electrophoretic homogeneity by gel chromatography on Sephadex G-100, DEAE-cellulose, and phenyl-sepharose columns. The enzyme was optimally active at pH 5 and 40 degrees C. Arrhenius plot for the determination of the activation energy (E(a)) gave the value 25 kJ/mol with a half-life (t(1/2)) of 5 min at 50 degrees C. It was highly activated by Fe(2+) and Ca(2+) and inhibited by Co(2+) and Mn(2+). The enzyme catalyzed the hydrolysis of the fluorescent compound methylumbelliferyl-sulfate (MU-SO(4)). Double reciprocal plots of initial velocity data, using MU-SO(4) as substrate, gave a K(M) value of 110 microM and V(max) of 225 micromol min(-1) x mg(-1). N. nigricolis Aryl sulphatase also hydrolyzed chondroitin-4-sulphate. It was inhibited competitively by N-acetyl glucosamine sulfate (GlcNAc-SO(4)), glucose-6-sulfate (Glc-6-SO(4)), and glucose 1-sulfate (Glc-1-SO(4)). Extrapolated inhibition binding constants (K(i)) gave the values of 3, 25, and 315 microM for GlcNAc-SO(4), Glc-6-SO(4), and Glc-1-SO(4) respectively.