Spatial clustering of pathology submissions during the initial introduction and spread of avian influenza H5N1 in poultry in Nigeria in 2006-2007.

Highly pathogenic avian influenza (HPAI) virus H5N1 spread throughout Nigeria between 2006 and 2007. Bird samples collected across the country were submitted through the free-of-charge (FOC) program to the National Veterinary Research Institute, Vom (NVRI-Vom) laboratory. The present article describes the spatial distributions and evaluated clustering of the FOC submissions from poultry farms at the global, local, and focal levels between 2006 and 2007 epidemic in Nigeria. Spatial statistics evaluating clustering of the FOC submissions were implemented using the Moran's I test, the purely spatial cluster analysis with the SaTScan Poisson model, and the Bithell's linear score test. A significant global clustering of the FOC submissions was observed. Significant local clusters of submissions were observed in the North-East, North-Central, and South-West zones. There was significant decline in FOC submissions with increasing distance from NVRI-Vom. These results indicated that the geographic area of influence of the FOC submission program in Nigeria was limited to regions closer to the diagnostic laboratory. This work provides a detailed insight into the surveillance activities during the HPAI outbreaks in Nigeria, and should assist policy-makers and field veterinarians to improve the effectiveness of national eradication plans in the face of any outbreak of animal diseases.

Spatio-temporal epidemiology of highly pathogenic avian influenza (H5N1) outbreaks in Nigeria, 2006-2008.

From 2006 to 2008, outbreaks of highly pathogenic avian influenza A (HPAI) virus of the H5N1 subtype occurred among poultry in Nigeria. We described the spatio-temporal patterns of the HPAI H5N1 outbreaks in Nigeria. Data of suspected and laboratory confirmed outbreaks maintained at the National Veterinary Research Institute Vom was analyzed using descriptive and exploratory analyses, GIS mapping, global and local spatial statistical analyses using the Cuzick-Edwards' (C-E) test and SaTScan Space-Time Scan Statistic. A total of 1654 suspected outbreaks were reported from 32 of the 36 States and the Federal Capital Territory (FCT), 299 were confirmed HPAI H5N1 positive from 27 states and FCT. The outbreaks occurred as three distinct epidemic waves with peak periods of January-March mainly in the North-West, North-Central and North-East regions during 2006 and 2007 and July-September in the South-West and South-South regions in 2007. Three spatio-temporal clusters were identified extending across States and international borders, consistent with disease transmission occurring through local and long-distance spread. This calls for enhanced strategies by the states and regional authorities to improve surveillance, prevention and control measures at the states, national and international levels.

Babesia canis and Babesia rossi co-infection in an untraveled Nigerian dog.

A sexually intact 6-month-old female Alsatian dog was presented to the Veterinary Clinic of the National Veterinary Research Institute, Vom, Plateau State, Nigeria, for the following complaints: anorexia, hemoglobinuria, fever, tick infestation and general malaise. Microscopy revealed piroplasms with a wide range of sizes (1-5 µm in length) in red blood cells, raising a suspicion of a co-infection with two or more Babesia species. Specific PCR assays for canine Babesia spp. and DNA sequencing revealed the presence of Babesia canis and Babesia rossi co-infection. This study constitutes the first report of co-infection with B. canis and B. rossi in the West African sub-region and the first report of autochthonous B. canis on the African continent. Practitioners should be aware of potential changes in the species/sub-species of Babesia causing canine babesiosis in this region.

Evolutionary dynamics of multiple sublineages of H5N1 influenza viruses in Nigeria from 2006 to 2008.

Highly pathogenic A/H5N1 avian influenza (HPAI H5N1) viruses have seriously affected the Nigerian poultry industry since early 2006. Previous studies have identified multiple introductions of the virus into Nigeria and several reassortment events between cocirculating lineages. To determine the spatial, evolutionary, and population dynamics of the multiple H5N1 lineages cocirculating in Nigeria, we conducted a phylogenetic analysis of whole-genome sequences from 106 HPAI H5N1 viruses isolated between 2006 and 2008 and representing all 25 Nigerian states and the Federal Capital Territory (FCT) reporting outbreaks. We identified a major new subclade in Nigeria that is phylogenetically distinguishable from all previously identified sublineages, as well as two novel reassortment events. A detailed analysis of viral phylogeography identified two major source populations for the HPAI H5N1 virus in Nigeria, one in a major commercial poultry area (southwest region) and one in northern Nigeria, where contact between wild birds and backyard poultry is frequent. These findings suggested that migratory birds from Eastern Europe or Russia may serve an important role in the introduction of HPAI H5N1 viruses into Nigeria, although virus spread through the movement of poultry and poultry products cannot be excluded. Our study provides new insight into the genesis and evolution of H5N1 influenza viruses in Nigeria and has important implications for targeting surveillance efforts to rapidly identify the spread of the virus into and within Nigeria.

Highly pathogenic avian influenza virus subtype H5N1 in Africa: a comprehensive phylogenetic analysis and molecular characterization of isolates.

Highly pathogenic avian influenza virus A/H5N1 was first officially reported in Africa in early 2006. Since the first outbreak in Nigeria, this virus spread rapidly to other African countries. From its emergence to early 2008, 11 African countries experienced A/H5N1 outbreaks in poultry and human cases were also reported in three of these countries. At present, little is known of the epidemiology and molecular evolution of A/H5N1 viruses in Africa. We have generated 494 full gene sequences from 67 African isolates and applied molecular analysis tools to a total of 1,152 A/H5N1 sequences obtained from viruses isolated in Africa, Europe and the Middle East between 2006 and early 2008. Detailed phylogenetic analyses of the 8 gene viral segments confirmed that 3 distinct sublineages were introduced, which have persisted and spread across the continent over this 2-year period. Additionally, our molecular epidemiological studies highlighted the association between genetic clustering and area of origin in a majority of cases. Molecular signatures unique to strains isolated in selected areas also gave us a clearer picture of the spread of A/H5N1 viruses across the continent. Mutations described as typical of human influenza viruses in the genes coding for internal proteins or associated with host adaptation and increased resistance to antiviral drugs have also been detected in the genes coding for transmembrane proteins. These findings raise concern for the possible human health risk presented by viruses with these genetic properties and highlight the need for increased efforts to monitor the evolution of A/H5N1 viruses across the African continent. They further stress how imperative it is to implement sustainable control strategies to improve animal and public health at a global level.

Reassortant avian influenza virus (H5N1) in poultry, Nigeria, 2007.

Genetic characterization of a selection of influenza virus (H5N1) samples, circulating in 8 Nigerian states over a 39-day period in early 2007, indicates that a new reassortant strain is present in 7 of the 8 states. Our study reports an entirely different influenza virus (H5N1) reassortant becoming predominant and widespread in poultry.

The comparative pathogenicity of strains of eight serovars and untypable strains of Mannheimia haemolytica in experimental pneumonia of sheep.

The experimental induction of pneumonic pasteurellosis in groups of conventionally reared lambs by 8 serovars (A1, A2, A6, A7, A8, A9, T10, and A11) and untypable (UT) strains of Mannheimia haemolytica (Mh) were examined and compared. The groups of lambs were inoculated intratracheally with 1.4 x 10(8) +/- 0.6 x 10(8) (mean +/- SD) colony-forming units of the Mh serovars or UT isolates in the 6-hour log phase of growth. The variables measured as indicators of disease severity were clinical score, percentage lung consolidation and microbiological re-isolation. The clinical parameters for each group were computed daily for 6 days post infection and the lambs which died were necropsied while the remaining lambs were killed on day 7 pi and the extent of lung consolidation was measured. Clinically, the mean scores for the M. haemolytica serovars were A1 (6.1), A2 (18.8), A6 (0.5), A7 (17.4) and A9 (8.5). The mean percent lung lesion scores for M. haemolytica serovars were A1 (12.5), A2 (66.3), A6 (5.0), A7 (51.3), A9 (33.8) and A11 (2.5). The percent mean pneumonic lung lesions recorded for groups inoculated with A2, A7 and A9 were significantly (P < 0.05) higher than the extent of lung lesions in the other groups. A statistically significant correlation was observed between clinical scores and the severity of the lung lesions (r = 0.96, P < 0.01). High titres of M. haemolytica were recovered from lung lesions, with 10 to 100 times the number of organisms inoculated being present in the lung lesions of lambs inoculated with serovars A2 and A7. These data indicate that although M. haemolytica serovars A1, A2, A6, A7, A9 and A11 are important primary lung pathogens of lambs, serovars A2, A7, and A9 are to be regarded as highly virulent strains that have a greater predilection than the other serovars for causing pneumonia in lambs.

Backbone dynamics of Tet repressor alpha8intersectionalpha9 loop.

A set of single Trp mutants of class B Tet repressor (TetR), in which Trp residues are located from positions 159 to 167, has been engineered to investigate the dynamics of the loop joining the alpha-helices 8 and 9. The fluorescence anisotropy decay of most mutants can be described by the sum of three exponential components. The longest rotational correlation time, 30 ns at 10 degrees C, corresponds to the overall rotation of the protein. The shortest two components, on the subnanosecond and nanosecond time scale, are related to internal motions of the protein. The initial anisotropy, in the 0.16-0.22 range, indicates the existence of an additional ultrafast motion on the picosecond time scale. Examination of physical models for underlying motions indicates that librational motions of the Trp side chain within the rotameric chi(1) x chi(2) potential wells contribute to the picosecond depolarization process, whereas the subnanosecond and nanosecond depolarization processes are related to backbone dynamics. In the absence of inducer, the order parameters of these motions, about 0.90 and 0.80 for most positions, indicate limited flexibility of the loop backbone. Anhydrotetracycline binding to TetR induces an increased mobility of the loop on the nanosecond time scale. This suggests that entropic factors might play a role in the mechanism of allosteric transition.

Structural investigation of Tet repressor loop 154-167: a time-resolved fluorescence study of three single Trp mutants.

We have studied the time-resolved fluorescence of three engineered Tet repressor (TetR) mutants bearing a single Trp residue at positions 162, 163, and 165 in the C-terminal part of the loop joining helices 8 and 9. Detailed analysis indicates that, at 20 degrees, the fluorescence decay of each Trp can be described as the sum of three exponential components with lifetimes in the 1-, 3-, and 6-ns range. Emission wavelength and temperature dependence studies are consistent with a model in which these components are due to the existence of three classes of Trp residues non-interconverting on the nanosecond timescale. Within the framework of the rotamer model, the weak temperature dependence of the lifetimes strongly suggests that the secondary structure of the loop, at least in the 162-165 range, is not altered with temperature. The equilibrium between the rotamers is characterized by an enthalpy-entropy compensation effect which strongly suggests the involvement of background structural regions of TetR in the thermodynamics of the process. The very high deltaH degrees and TdeltaS degrees observed (up to 18 kcal/ mol) should reflect the temperature-dependent conformational change of a large part of the protein which would alter the rotamer distribution of the Trp residues. Taken together, our results are consistent with the existence of (at least) two conformations of the loop and suggest a model for loop motion.

Spectroscopic investigation of Tet repressor tryptophan-43 upon specific and nonspecific DNA binding.

The F75 Tet repressor mutant (F75 TetR) contains a single tryptophan residue located at position 43 in the operator recognition alpha-helix. Previous studies [Hansen, D., & Hillen, W. (1987) J. Biol. Chem. 262, 12269-12274] have shown that the fluorescence intensity of this residue is dramatically reduced upon operator binding. In order to determine the origin of this quenching and the role of Trp-43 in the binding mechanism, we have investigated its fluorescence properties upon F75 TetR binding to a tet operator containing 76 bp DNA fragment (specific binding) and to sheared calf thymus DNA (nonspecific binding). Trp-43 steady-state fluorescence intensity was quenched by 72% upon specific binding and by 45% upon nonspecific binding. These fluorescence intensity decreases were not accounted for by similar decreases in the respective fluorescence lifetimes. The apparent quenching calculated from the average lifetimes was about 0.33 in both binding modes. This shows the presence of a static quenching process, clearly favored upon specific binding as compared to nonspecific binding. This is consistent with stacking interactions between Trp-43 and the DNA bases, as suggested by molecular graphics [Baumeister, R., Helbl, V., & Hillen, W. (1992) J. Mol. Biol. 226, 1257-1270]. The equilibrium constant between nonfluorescent and fluorescent tryptophan residues was 5 times higher upon binding to specific DNA than to nonspecific DNA. The preferential static quenching of Trp-43 in the specific complex suggests that stacking interactions might contribute to the specific binding mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescence of a tryptophan bearing peptide from smooth muscle myosin light chain kinase upon binding to two closely related calmodulins.

We have investigated the fluorescence of a calmodulin binding peptide (AS19) based on the sequence of the calmodulin binding domain of smooth muscle myosin light chain kinase and bearing a tryptophan residue at position 5 upon binding to two closely related calmodulins. The emission maximum of peptide AS19 bound to the engineered SYNCAM calmodulin was 318 nm and a vibrational structure was clearly apparent. The emission maximum of peptide AS19 bound to chicken gizzard calmodulin (ChG CaM) was 327 nm and its spectrum was featureless. Red edge excitation effect supports the assumption that the polarity of Trp-5 environment is larger in the complex with ChG CaM than with SYNCAM, in agreement with fluorescence spectra. Time-resolved fluorescence and anisotropy measurements showed that, in both complexes, the tryptophan emitting state was 1La. The X-ray structure of the calmodulin-peptide complex has been resolved (W. E. Meador, A. R. Means, and F. A. Quiocho, 1992, Science 257, 1251-1255). The Trp binding site has been characterized. It differs by a single-point mutation between the two calmodulins: Met-144 of ChG CaM has been replaced by Val in SYNCAM. This suggests that the spectral relaxation of Trp-5 in the complex with ChG CaM as compared to SYNCAM is due to the polarizability of the sulfur atom containing Met side chain that is higher than that of Val. This provides an ideal system to investigate the origin of the Stokes shift of the indole moiety in proteins.

Protein fluorescence decay: discrete components or distribution of lifetimes? Really no way out of the dilemma?

A new methodology of fluorescence decay analysis by iterative reconvolution is presented. It is based on the recent finding that the statistics of single-photon time-correlated data are best described by a compound Poisson law and requires the recording of a sample of at least 20 decays. Application of multivariate statistical methods to the analysis of the recovered decay parameters results in improved accuracy and better estimation of the uncertainties of mono- and multiexponential decays. If it is, of course, not possible to distinguish unambiguously between discrete components and a continuous distribution of lifetimes, it is, however, possible to determine a higher limit of the width of such a distribution should it be present. With our methodology, the presence of a distribution of lifetimes with a width of approximately 20% of its center value inevitably leads to a failure in the deconvolution procedure, a fact of crucial importance in protein conformational studies, for example.

Alteration in the capacities of carcinogen metabolizing system of mouse-livers during pretreatment with various antineoplastic agents.

The effect of treatment with antineoplastic drugs on the modification of the carcinog en-metabolizing capacity was studied in mice liver microsomes at different durations as a single and as a repeated dose treatment. It is generally demonstrated that there is a commonality of influence for each specific antineoplastic group on the expression of the mixed function mono-oxygenases. The antineoplastic alkylating agents (chlorambucil, melphalan and busulfan) significantly increased the activity of carcinogen metabolizing enzymes in contrary to the effects observed for the antibiotic actinomycin-D. The antimetabolite agents (5-flourouracil and methotrexate), on the other hand, although decreased these activities when administered as a single dose, a pronounced increase was observed at the repeated dose treatment. Significant increases in the activity of N-nitrosodimethylamine demethylases I and II and aryl hydrocarbon (benzo[alpha]pyrene) hydroxylase were observed with chlorambucil when administered as a single or repeated doses. Similar effects was observed when the animals were treated with repeated doses of melphalan, busulfan and 5-flourouracil. Contrary to these effects, inhibition in the enzyme activity was exhibited when actinomycin-D was administered for either single or repeated dose treatment. The hepatic content of cytochrome P450 was significantly increased with all the administered drugs, except busulfan, when applied repeatedly. The implication of such alterations in the capacities of carcinogen metabolizing enzymes for antitumour-induced toxicity and carcinogenicity are discussed.

Chloramphenicol and actinomycin-d-induced modifications in the carcinogen-metabolizing capacities of mouse-liver microsomes.

The effect of antibiotics chloramphenicol (CML) and actinomycin-D (AMD) on the modification of the carcinogen metabolizing capacity was studied in vivo in mouse liver microsomes at different durations of treatment, for one day and three and six consecutive days. Following the administration of CML, a significant increase was observed in the activity of the low and high substrate levels of the hepatic microsomal N-nitrosodimethylamine demethylases (NDMAd I and II, respectively). On the contrary, AMD reduced the activity of NDMAd I and II in a time-dependent manner up to 3 days of treatment while no effect was observed when the drug was administered for 6 consecutive days. No effect was observed in the activity of the arylhydrocarbon (benzo(alpha)pyrene) hydroxylase either at the single or the repeated doses of CML only for 6 days of treatment with AMD. The expression of the hepatic content of cytochrome P450 revealed a significant induction at the various treatment intervals with CML. AMD, however, reduced the cytochrome P450 at 1 and 6 days treatment with induction at 3 days of repeated treatment.

Cofactor-induced orientation of the DNA bases in single-stranded DNA complexed with RecA protein. A fluorescence anisotropy and time-decay study.

The structure of the RecA-single-stranded DNA complex was investigated by studying the fluorescence emission of poly(deoxy-1,N6-ethenoadenylic acid (poly(d epsilon A)), a fluorescent derivative of poly(dA), under various viscosity conditions. The fluorescence intensity and average lifetime of poly(d epsilon A) are much smaller than those of nonpolymerized monoethenonucleotides (1,N6-ethenoadenosine 5'-triphosphate and 1,N6-ethenoadenine deoxyribose 5'-monophosphate) at low viscosity and reflect intramolecular base-base collisions in the polymer. They considerably increased upon RecA binding, both in the presence and absence of cofactor ATP or adenosine 5'-O-(3-thiotriphosphate). This increase, as well as the increase in fluorescence anisotropy upon RecA binding, was very similar to that which resulted from sucrose addition to free poly(d epsilon A). These observations point to a decrease in the mobility of DNA bases upon RecA binding. In the presence of cofactor, the fluorescence features became independent of viscosity. This strongly suggests the absence of base motion of significant amplitude on the time scale of the fluorescence lifetime (about 10 ns). In the absence of cofactor, however, these features remained sensitive to viscosity, implying residual local motions of the bases. Such cofactor-dependent rigid attachment of DNA bases to stiff phosphate backbone could facilitate the search for homology between two DNA molecules during recombination.

Time-resolved fluorescence study of VU-9 calmodulin, an engineered calmodulin possessing a single tryptophan residue.

An engineered calmodulin (VU-9 calmodulin), which possesses a single tryptophan residue at position 99 in calcium binding domain III, was studied by time-resolved fluorescence. At least two exponential terms are needed to describe the tryptophan fluorescence decays, either in the presence or in the absence of calcium. The characteristics of the fluorescence decays are strongly dependent upon the number of calcium ions bound per molecule of VU-9 calmodulin until half of the calcium sites are occupied, i.e., three in the absence of magnesium and two in the presence of 5 mM magnesium. A clear time-dependent spectral shift is observed in the presence of calcium. The existence of an isosbestic point in the time-resolved spectra is in agreement with a two-state model. The biexponential analysis of the 340-nm fluorescence decay during calcium titration gives parameters consistent with a two-state model in which tryptophan 99 interconverts between two different conformations, characterized by a different lifetime value, with rates altered by calcium binding. This model explains the decrease in the protein quantum yield induced by calcium binding [Kilhoffer, M. C., Roberts, D. M. Adibi, A. O., Watterson, D. M., & Haiech, J. (1989) Biochemistry (preceding paper in this issue)].

Studies of the interaction of the fluorophores harmine and harmaline with calf thymus DNA.

The binding to calf thymus DNA of the hallucinogen harmine and one of its analogues harmaline was studied by absorption spectrophotometry and fluorescence quenching analysis. Viscosity measurements were also carried out. For both molecules, quenched and unquenched sites on DNA are present. For each type of binding site, the value of the product of the number of sites times the association constant was determined. Harmine is more strongly bound than harmaline. Viscosity measurements indicate intercalation in the case of harmine only.