Epitope Peptide-Based Predication and Other Functional Regions of Antigenic F and HN Proteins of Waterfowl and Poultry Avian Avulavirus Serotype-1 Isolates From Uganda.

Uganda is a Newcastle disease (ND) endemic country where the disease is controlled by vaccination using live LaSota (genotype II) and I2 (genotype I) vaccine strains. Resurgent outbreak episodes call for an urgent need to understand the antigenic diversity of circulating wild Avian Avulavirus serotype-1 (AAvV-1) strains. High mutation rates and the continuous emergence of genetic and antigenic variants that evade immunity make non-segmented RNA viruses difficult to control. Antigenic and functional analysis of the key viral surface proteins is a crucial step in understanding the antigen diversity between vaccine lineages and the endemic wild ND viruses in Uganda and designing ND peptide vaccines. In this study, we used computational analysis, phylogenetic characterization, and structural modeling to detect evolutionary forces affecting the predicted immune-dominant fusion (F) and hemagglutinin-neuraminidase (HN) proteins of AAvV-1 isolates from waterfowl and poultry in Uganda compared with that in LaSota vaccine strain. Our findings indicate that mutational amino acid variations at the F protein in LaSota strain, 25 poultry wild-type and 30 waterfowl wild-type isolates were distributed at regions including the functional domains of B-cell epitopes or N-glycosylation sites, cleavage site, fusion site that account for strain variations. Similarly, conserved regions of HN protein in 25 Ugandan domestic fowl isolates and the representative vaccine strain varied at the flanking regions and potential linear B-cell epitope. The fusion sites, signal peptides, cleavage sites, transmembrane domains, potential B-cell epitopes, and other specific regions of the two protein types in vaccine and wild viruses varied considerably at structure by effective online epitope prediction programs. Cleavage site of the waterfowl isolates had a typical avirulent motif of 111GGRQGR'L117 with the exception of one isolate which showed a virulent motif of 111GGRQKR'F117. All the poultry isolates showed the 111GRRQKR'F117 motif corresponding to virulent strains. Amino acid sequence variations in both HN and F proteins of AAvV-1 isolates from poultry, waterfowl, and vaccine strain were distributed over the length of the proteins with no detectable pattern, but using the experimentally derived 3D structure data revealed key-mapped mutations on the surfaces of the predicted conformational epitopes encompassing the experimental major neutralizing epitopes. The phylogenic tree constructed using the full F gene and partial F gene sequences of the isolates from poultry and waterfowl respectively, showed that Ugandan ND aquatic bird and poultry isolates share some functional amino acids in F sequences yet do remain unique at structure and the B-cell epitopes. Recombination analyses showed that the C-terminus and the rest of the F gene in poultry isolates originated from prevalent velogenic strains. Altogether, these could provide rationale for antigenic diversity in wild ND isolates of Uganda compared with the current ND vaccine strains.

Genotypic characterisation of Avian paramyxovirus type-1 viruses isolated from aquatic birds in Uganda.

Avian paramyxovirus type-1 (APMV-1) viruses of the lentogenic pathotypes are often isolated from wild aquatic birds and may mutate to high pathogenicity when they cross into poultry and cause debilitating Newcastle disease. This study characterised AMPV-1 isolated from fresh faecal droppings from wild aquatic birds roosting sites in Uganda. Fresh faecal samples from wild aquatic birds at several waterbodies in Uganda were collected and inoculated into 9-10-day-old embryonated chicken eggs. After isolation, the viruses were confirmed as APMV-1 by APMV-1-specific polymerase chain reaction (PCR). The cleavage site of the fusion protein gene for 24 representative isolates was sequenced and phylogenetically analysed and compared with representative isolates of the different APMV-1 genotypes in the GenBank database. In total, 711 samples were collected from different regions in the country from which 72 isolates were recovered, giving a prevalence of 10.1%. Sequence analysis of 24 isolates revealed that the isolates were all lentogenic, with the typical 111GGRQGR'L117 avirulent motif. Twenty-two isolates had similar amino acid sequences at the cleavage site, which were different from the LaSota vaccine strain by a silent nucleotide substitution T357C. Two isolates, NDV/waterfowl/Uganda/MU150/2011 and NDV/waterfowl/Uganda/MU186/2011, were different from the rest of the isolates in a single amino acid, with aspartate and alanine at positions 124 and 129, respectively. The results of this study revealed that Ugandan aquatic birds indeed harbour APMV-1 that clustered with class II genotype II strains and had limited genetic diversity.

Immunogenicity and protection efficacy evaluation of avian paramyxovirus serotype-1 (APMV-1) isolates in experimentally infected chickens.

Vaccine failures after Newcastle disease vaccination with the current commercial vaccines have been reported and are associated with many factors, including genotypic and antigenic differences between vaccine and outbreak strains, although all APMV-1 members belong to one serotype. We assessed the immunoprotection ability of four thermostable, low-virulent Newcastle disease-virus isolates from Ugandan waterfowl against challenge with a virulent strain (MDT = 36.8 h, ICPI = 1.78) isolated from morbid chicken. Six-week-old commercial Leghorn layers, challenged at 21 days post immunization were used. Four isolates designated: NDV-133/UG/MU/2011, NDV-177/UG/MU/2011, NDV-178/UG/MU/2011 and NDV-173/UG/MU/2011 induced mean haemagglutinin inhibition antibody titres of log2 9.3, 8.2, 6.3 and 2.0, respectively, at 21 days post immunization. The antibody titres correlated with the protection rates (R² = 0.86, p < 0.007) of 60%, 50%, 20% and 0% of birds, respectively, against challenge at 14 days post challenge. Further evaluation of these and more low-virulent isolates might provide an alternative to the current commercial vaccine failures.

Sociocultural factors associated with fish consumption in Lake Albert fishing community: Guidelines for lead and mercury.

Fish consumption in subsistence fishing community is a life style associated with lead and mercury uptake for humans. Fish consumption is influenced by sociocultural factors, exposure and health risks. Unfortunately, no sociocultural study in the Lake Albert fishing community in light of lead and mercury exists. A cross-sectional sociocultural study was carried out between March and June 2015. A total of 270 household heads in four landing sites in Hoima district completed structured questionnaires and data analyzed using SPSS version 20. The majority of the households (74.8%) had primary education or below, 51.1% drank unboiled water, and 30% perceived lake water safe for drinking. Children under five ate soup (15%) and middle piece of the fish (29%). The Poisson general linear model predicting weekly fish consumption amounts against sociocultural factors showed that household size (p = 0.047), male child presence (p = 0.007), methods of preparation i.e. salting (p < 0.0001), fish parts consumed by adults (p < 0.0001), fish preference (p < 0.0001), awareness about the beach management unit (p < 0.0001), and income from charcoal selling (p < 0.0001) were positive predictors. The negative predictors of weekly fish consumption amounts were awareness about fish consumption benefits (p < 0.0001), eating young fish (p = 0.002), donor agency presence (p < 0.0001), and frying as the method of fish preparation (p = 0.002). In conclusion, knowledge of the sociocultural factors associated with fish consumption determines the amounts and frequency of the predominant fish eaten. Therefore, to establish and adopt fish consumption guidelines for lead and mercury in the Lake Albert, the sociocultural factors should be integrated in the message disseminated.

Disparate thermostability profiles and HN gene domains of field isolates of Newcastle disease virus from live bird markets and waterfowl in Uganda.

BACKGROUND: Uganda poultry production is still faced with frequent outbreaks of Newcastle disease (ND) in the backyard free-range systems despite the accessibility of cross protective vaccines. Live bird markets and waterfowl has long been reported as a major source of disease spread as well as potential sources of avirulent strains that may mutate to virulent strains. ND-virus has been reported enzootic in Ugandan poultry but limited studies have been conducted to ascertain thermostability phenotypes of the Ugandan ND-virus strains and to understand how these relate to vaccine strains. METHODS: This study evaluated thermostability of 168 ND-virus field isolates recovered from live bird markets and waterfowls in Uganda compared to two live commercial vaccine strains (I2 and LaSota) by standard thermostability procedures and Hemagglutinin-Neuraminidase (HN) gene domains. The known pathotypes with thermostability profiles were compared at HN amino acid sequences. RESULTS: Field isolates displayed disparate heat stability and HN gene domains. Thermolabile isolates were inactivated within 15 min, while the most thermostable isolates were inactivated in 120 min. Four thermostable isolates had more than 2 log2 heamaglutinin (HA) titers during heat treatment and the infectivity of 9.8 geometric mean of log10 EID50 % in embryonated eggs. One isolate from this study exhibited a comparable thermostability and stable infectivity titers after serial passages, to that of reference commercial vaccine was recommended for immunogenicity and protection studies. CONCLUSION: The occurrence of ND-virus strains in waterfowl and live bird markets with disparate thermostability and varying HN gene domains indicate circulation of different thermostable and thermolabile ND-virus pathotypes in the country.

Helicobacter pylori among patients with symptoms of gastroduodenal ulcer disease in rural Uganda.

INTRODUCTION: To meet key millennium development goals, the rural population needs to be reached for health assessment and service delivery. Gastroduodenal ulcer disease is a common ailment affecting the health of people in Uganda. A cross-sectional study was conducted at Bwera Hospital in Kasese district of western Uganda, to establish the prevalence and predisposing factors of Helicobacter pylori among gastroduodenal ulcer disease patients. METHODS: A sample of 174 patients with symptoms of gastroduodenal ulcer disease was purposively obtained. Using two laboratory test methods, the prevalence of H. pylori among these patients was determined. A structured questionnaire was administered to participants to establish their demographic background and selected aspects of their lifestyle. Finally, the results obtained by enzyme-linked immunosorbent assay (ELISA) and immunochromatographic rapid test (IRT) were compared. RESULTS: We established the prevalence of H. pylori as 29.9% (52/174) by ELISA and 37.4% (65/174) by IRT. Cigarette smoking, poor sanitation, and lack of formal education were the significant predisposing factors with p-values <0.05. The two tests gave identical results in 87.9% of the patients. DISCUSSION: The prevalence of H. pylori by IRT and ELISA test methods was similar to what has been reported elsewhere in developed countries; but was lower than previously reported in developing countries including Uganda. The previous studies in Uganda were carried out in the urban population and on young children; and some used antibody-detection methods only, therefore leading to different prevalence as a result of difference in study population and methods.

In vitro Antibacterial Activity of Ocimum suave Essential Oils against Uropathogens Isolated from Patients in Selected Hospitals in Bushenyi District, Uganda.

AIMS: To determine antibacterial activity of Ocimum suave essential oils against bacterial uropathogens. STUDY DESIGN: A cross sectional and experimental study. PLACE AND DURATION OF STUDY: Six selected hospitals in Bushenyi District, Uganda between June 2012 and July 2013. METHODOLOGY: Clean catch midstream urine samples were collected and inoculated on Cystine Lysine Electrolyte Deficient (CLED) agar. The plates were incubated at 37°C for 24hrs to 48hrs. The O. suave essential oils were extracted by hydrodistillation of leaves for 4hrs using a Clevenger apparatus. The oil was collected and dried over anhydrous sodium sulphate (Na2SO4) and kept at 4°C till further use. The antimicrobial activity of O. suave essential oils against isolates was determined by agar well method. The MIC of O. suave essential oil extract was carried out by microbroth dilution method. RESULTS: Of the three hundred (300) midstream urine samples collected, 67(22.33%) had significant bacterial growth. Escherichia coli is the most common isolate (61.19%, n = 41). The essential oil from O. suave showed activity against isolates of E. coli, K. pneumoniae, S. aureus, E. feacalis, M. morganii, Citrobacter species, Enterobacter species and P. aeruginosa with mean zone of inhibition (ZI) ranging from 10-22 mm. The essential oils had no inhibitory activity on Acinetobacter species. The minimum inhibitory concentration (MIC) for O. suave essential oils ranged from 0.78 to 22 µg/ml. This study showed that O. suave essential oils had MIC value of 0.78 µg/ml against S. aureus and MIC values ranging from 3 to 22 µg/ml against the other tested isolates. CONCLUSION: The most common uropathogen was E. coli (61.19% n = 41). O. suave essential oils exhibited antibacterial activity against majority of the uropathogens, except Acinetobacter species, mean ZI of 10-22 mm and MIC of 0.78 - 22 µg/ml.

High pathogenicity and low genetic evolution of avian paramyxovirus type I (Newcastle disease virus) isolated from live bird markets in Uganda.

BACKGROUND: Newcastle disease is still a serious disease of poultry especially in backyard free-range production systems despite the availability of cross protective vaccines. Healthy-looking poultry from live bird markets have been suspected as a major source of disease spread although limited studies have been conducted to ascertain the presence of the virulent strains in the markets and to understand how they are related to outbreak strains. METHODS: This study evaluated the occurrence of Newcastle disease virus in samples collected from poultry in live bird markets across Uganda. The isolates were pathoyped using standard methods (mean death time (MDT), intracelebral pathogenicity index (ICPI), and sequencing of the fusion protein cleavage site motif) and also phylogenetically analysed after sequencing of the full fusion and hemagglutin-neuraminidase genes. The isolates were classified into genotypes and subgenotypes based on the full fusion protein gene classification system and compared with other strains in the region and world-wide. RESULTS: Virulent avian paramyxovirus type I (APMV-1) (Newcastle disease virus) was isolated in healthy-looking poultry in live bird markets. The viruses belonged to a new subgenotype, Vd, in genotype V, and clustered together with Tanzania and Kenya strains. They harbored low genetic diversity. CONCLUSION: The occurrence of virulent AMPV-1 strains in live bird markets may serve as sources of Newcastle disease outbreaks in non-commercial farms.

Comparison of transformation frequencies among selected Streptococcus pneumoniae serotypes.

Although there are over 90 serotypes of Streptococcus pneumoniae, antimicrobial resistance is predominantly found in a limited number of serotypes/serogroups, namely 6, 9, 14, 19 and 23. There is no compelling mechanism to account for this restriction. We aimed to determine whether serotypes commonly associated with drug resistance have higher transformation frequencies than those that are susceptible to antimicrobial agents. An in vitro investigation of the genetic transformation frequency of drug-resistant serotypes compared with that of susceptible serotypes under the influence of synthetic competence-stimulating peptides was performed. The transforming DNA was genomic DNA carrying a Tn916-like transposon containing the mefE gene that confers resistance to erythromycin. It was observed that serotypes 6, 9, 14, 19 and 23, which are highly associated with drug resistance, do not exhibit a higher degree of transformation efficiency than other serotypes. These findings suggest that the association of serotype with drug resistance is likely due to prolonged exposure to transforming DNA resulting from longer nasopharyngeal carriage and to a greater selective pressure from antimicrobials, particularly in children. This is the first study to compare the transformation frequencies of pneumococcal clinical isolates using genomic DNA that carries the composite Tn916-like element.

Beauveria bassiana (Balsamo) Vuillemin as an endophyte in tissue culture banana (Musa spp.).

Beauveria bassiana is considered a virulent pathogen against the banana weevil Cosmopolites sordidus. However, current field application techniques for effective control against this pest remain a limitation and an alternative method for effective field application needs to be investigated. Three screenhouse experiments were conducted to determine the ability of B. bassiana to form an endophytic relationship with tissue culture banana (Musa spp.) plants and to evaluate the plants for possible harmful effects resulting from this relationship. Three Ugandan strains of B. bassiana (G41, S204 and WA) were applied by dipping the roots and rhizome in a conidial suspension, by injecting a conidial suspension into the plant rhizome and by growing the plants in sterile soil mixed with B. bassiana-colonized rice substrate. Four weeks after inoculation, plant growth parameters were determined and plant tissue colonization assessed through re-isolation of B. bassiana. All B. bassiana strains were able to colonize banana plant roots, rhizomes and pseudostem bases. Dipping plants in a conidial suspension achieved the highest colonization with no negative effect on plant growth or survival. Beauveria bassiana strain G41 was the best colonizer (up to 68%, 79% and 41% in roots, rhizome and pseudostem base, respectively) when plants were dipped. This study demonstrated that, depending on strain and inoculation method, B. bassiana can form an endophytic relationship with tissue culture banana plants, causing no harmful effects and might provide an alternative method for biological control of C. sordidus.

Cholera in endemic districts in Uganda during El Niño rains: 2002-2003.

BACKGROUND: El Niño phenomenon causing increased rainfall and flooding has been linked to flare ups and emergence of several disease outbreaks including cholera. The latter has been reported in many districts in Uganda in recent years. Therefore an understanding of factors influencing its pattern of occurrence is needed for effective control. OBJECTIVES: To determine cholera disease status during six months of El Niño rains and assess serotypes and antibiotic sensitivity of isolates. METHODS: A prospective study was conducted in five cholera "endemic districts" in Uganda. RESULTS: Cholera outbreaks occurred in all the study districts coincident with the onset of the El Niño rains. There were 924 cholera suspect cases reported with 95 fatalities (case fatality rate 10.3%). A total of 388 clinical specimens were analyzed by culture and of these, 168 were positive for V. cholerae. Biochemical and serological analysis identified the isolates as V. cholerae O1, biotype EL Tor serotype Ogawa. Antibiotic sensitivity revealed that isolates were 100% sensitive to ciprofloxacin, tetracycline and erythromycin, whereas sensitivity was variable for other tested antibiotics. Unlike Kampala, where the disease was contained within three months, persistence occurred in other districts only dying out with end of El Niño rains, suggesting differences in disease control. CONCLUSION: These results show that El Niño rains cause increase in the number of cholera cases in Uganda, calling for preparedness and a need to emulate Kampala response incase of outbreaks in other districts.