General insights on obstacles to dog vaccination in Chad on community and institutional level

Domestic dogs are responsible for 95% of all human rabies cases worldwide and continue to be the main reservoir for this fatal virus in African and Asian countries. Interrupting the spread of the disease in the domestic dog population is therefore necessary for long-term, sustainable rabies control. Chad has been recognized as a rabies-endemic country since 1961, but no national control strategy is in place to date and dog vaccination coverage is very low. This qualitative, descriptive study aims to describe the main barriers to dog vaccination on both the community and the institutional level from a socio-anthropological point of view in Chad. The study was embedded in an overall project conducted from 2016 to 2018, to determine rabies burden and vaccine demand in West and Central Africa, funded by GAVI, the vaccine alliance. Data collection was conducted on the occasion of the project's closing workshops with stakeholders organized between August to September 2018 in the four (4) project areas: Logone Occidental, Ouaddai, Hadjer Lamis and Chari Baguirmi. We conducted interviews and focus group discussions (FGD) among veterinary officers and dog owners. Participants were selected purposively based on their place of residence (dog owners) or work place (veterinary officers) and their previous contact with the project through reporting (dog owner) or management (veterinary officers) of a suspect dog rabies case. In each region, one FGD was organized with dog owners, and one FGD with heads of veterinary posts. At the end of the FGDs, a few participants were randomly selected for interviews. In addition, in each region an interview was conducted with the head of the livestock sector, the chief district medical officers and the head of a civil society association. The identified barriers to dog vaccination access are grouped into three main aspects: the economic, the socio-cultural and the institutional level. Economic constraints encountered relate to the cost of the vaccine itself and the expenses for transporting the dogs to the vaccination site. The cultural belief that the vaccine will have an impact on the therapeutic properties of dog meat for consumers (observed in Southern Chad), and the fact that dogs are considered impure animals in Muslim faith, which prohibits handling of dogs, are obstacles identified on the sociocultural level. At the institutional level, the unavailability of vaccines in veterinary services, the lack of communication about the law on dog vaccination, the absence of rabies in the training curricula of veterinary agents, and the lack of intersectoral collaboration limit vaccination coverage. In order to improve vaccination coverage and rabies surveillance with a view to eradicate rabies by 2030, communication strategies that are adapted to the context and that take cultural obstacles into account must be put in place in a synergy of interdisciplinary action. In addition, factors such as affordability, geographical access and availability of dog rabies vaccines needs to be addressed throughout the country. Although our study design did not allow a detailed analysis of obstacles related to socio-economic level, gender and age the broad insights gained can provide general guidance for future interventions in Chad and similar countries

The potential effect of improved provision of rabies post-exposure prophylaxis in Gavi-eligible countries: a modelling study

Background: Tens of thousands of people die from dog-mediated rabies annually. Deaths can be prevented through post-exposure prophylaxis for people who have been bitten, and the disease eliminated through dog vaccination. Current post-exposure prophylaxis use saves many lives, but availability remains poor in many rabies-endemic countries due to high costs, poor access, and supply. Methods: We developed epidemiological and economic models to investigate the effect of an investment in post-exposure prophylaxis by Gavi, the Vaccine Alliance. We modelled post-exposure prophylaxis use according to the status quo, with improved access using WHO-recommended intradermal vaccination, with and without rabies immunoglobulin, and with and without dog vaccination. We took the health provider perspective, including only direct costs. Findings: We predict more than 1 million deaths will occur in the 67 rabies-endemic countries considered from 2020 to 2035, under the status quo. Current post-exposure prophylaxis use prevents approximately 56 000 deaths annually. Expanded access to, and free provision of, post-exposure prophylaxis would prevent an additional 489 000 deaths between 2020 and 2035. Under this switch to efficient intradermal post-exposure prophylaxis regimens, total projected vaccine needs remain similar (about 73 million vials) yet 17·4 million more people are vaccinated, making this an extremely cost-effective method, with costs of US$635 per death averted and$33 per disability-adjusted life-years averted. Scaling up dog vaccination programmes could eliminate dog-mediated rabies over this time period; improved post-exposure prophylaxis access remains cost-effective under this scenario, especially in combination with patient risk assessments to reduce unnecessary post-exposure prophylaxis use. Interpretation: Investing in post-exposure vaccines would be an extremely cost-effective intervention that could substantially reduce disease burden and catalyse dog vaccination efforts to eliminate dog-mediated rabies. Funding: World Health Organization

Serological evaluation of virus exposure in Beira antelopes (Dorcatragus megalotis) and Speke's gazelles. (Gazella spekei) at Al Wabra Wildlife Preservation (AWWP), Qatar

A retrospective evaluation of data on antibodies to respiratory viruses in Beira antelopes was conducted due to the occurrence of chronic “Fibrinous Pleuropneumonia Syndrome” (FPPS) in Beira antelopes at Al Wabra Wildlife Preservation, to detect an indication for a virological problem contributing to the susceptibility of Beiras to infection with Mycoplasma spp. Data on Speke’s gazelles at AWWP were evaluated for comparison. From the 5 tested respiratory viruses BHV-1, BVD, ADENO-3, PI-3 and BRSV, significant antibody titres were only found for BRSV and PI-3. Speke‘s gazelles were found to have higher antibody titres for both viruses than the Beira antelope, with respiratory signs noted in accordance with virological findings In Beira, the often fatal respiratory syndrome appears unlikely to be related to any of these viruses

Fast and reliable PCR/sequencing/RFLP assay for identification of fungi in onychomycoses

Fusarium spp. and other non-dermatophyte fungi are repeatedly isolated from abnormal nails. To investigate whether these fungi are the aetiological agents of infection or simply transient contaminants, a PCR/sequencing/RFLP assay was developed for direct and routine identification of the infecting fungi in onychomycosis. Fungal DNA was readily extracted using a commercial kit after dissolving nail fragments in a Na2S solution. Amplification of part of the 28S rDNA by PCR was performed with universal primers and the fungal species were identified by sequencing. The PCR/sequencing results were comparable with microbiological identification from the same nail sample. In addition to dermatophytes, Fusarium spp. and other less frequently isolated non-dermatophyte fungi were identified as single fungal agents in onychomycosis. Moreover, mixed infections were clearly demonstrated in 10% of cases by RFLP analysis of PCR products. Identification of infectious agents could be obtained in 2 days, whilst results from fungal cultures take 1-3 weeks. Rapid and reliable molecular identification of the infectious fungus expedites the choice of appropriate antifungal therapy, thereby improving the cure rate of onychomycosis

Secreted proteases from pathogenic fungi

Many species of human pathogenic fungi secrete proteases in vitro or during the infection process. Secreted endoproteases belong to the aspartic proteases of the pepsin family, serine proteases of the subtilisin family, and metalloproteases of two different families. To these proteases has to be added the non-pepsin-type aspartic protease from Aspergillus niger and a unique chymotrypsin-like protease from Coccidioides immitis. Pathogenic fungi also secrete aminopeptidases, carboxypeptidases and dipeptidyl-peptidases. The function of fungal secreted proteases and their importance in infections vary. It is evident that secreted proteases are important for the virulence of dermatophytes since these fungi grow exclusively in the stratum corneum, nails or hair, which constitutes their sole nitrogen and carbon sources. The aspartic proteases secreted by Candida albicans are involved in the adherence process and penetration of tissues, and in interactions with the immune system of the infected host. For Aspergillus fumigatus, the role of proteolytic activity has not yet been proved. Although the secreted proteases have been intensively investigated as potential virulence factors, knowledge on protease substrate specificities is rather poor and few studies have focused on the research of inhibitors. Knowledge of substrate specificities will increase our understanding about the action of each protease secreted by pathogenic fungi and will help to determine their contribution to virulence

Production and Characterization of Recombinant Aspergillus fumigatus Cu,Zn Superoxide Dismutase and Its Recognition by Immune Human Sera

The Cu,Zn superoxide dismutase (SOD) of Aspergillus fumigatus has previously been purified and shown to be immunoreactive to the sera of patients with aspergillosis; however, the purification of large quantities of the enzyme for expanded immunological analysis is both difficult and time-consuming. Accordingly, a λEMBL3 A. fumigatus genomic library was screened with degenerate oligonucleotides based on N-terminal amino acid sequence data; from this initial screen a 1,400-bp fragment was identified, labelled, and used to screen an A. fumigatus λgt11 cDNA library. A full-length cDNA encoding Cu,Zn SOD was subsequently identified and cloned. The cDNA encodes a protein of 154 amino acids, which does not have a signal peptide. The A. fumigatus Cu,Zn SOD possesses the typical metal binding ligands of fungal Cu,Zn SODs (six histidines and one aspartic acid) and has significant overall homology with Cu,Zn SODs in general. A recombinant A. fumigatus Cu,Zn SOD has been expressed in Pichia pastoris, is enzymatically active, and has biochemical and biophysical properties that are similar to those of the native enzyme. A sheep polyclonal antibody raised against purified native A. fumigatus Cu,Zn SOD was reactive to the recombinant enzyme by immunoenzyme development of Western blots. Sixty percent of serum samples from patients with A. fumigatus infections were reactive against the recombinant Cu,Zn SOD via immunoenzyme development of Western blots, indicating that the recombinant protein may be useful in the serodiagnostic identification of A. fumigatus infections

Secreted subtilisin gene family in Trichophyton rubrum

Secreted proteases constitute potential virulence factors of dermatophytes. A total of seven genes encoding putative serine proteases of the subtilisin family (SUB) were isolated in Trichophyton rubrum. Based on sequence data and intron-exon structure, a phylogenetic analysis of subtilisins from T rubrum and other fungi revealed a presumed ancestral lineage comprising T rubrum SUB2 and Aspergillus SUBs. All other SUBs (SUB1, SUB3-7) are dermatophyte-specific and have apparently emerged more recently, through successive gene duplication events. We showed that two subtilisins, Sub3 and Sub4, were detected in culture supernatants of T rubrum grown in a medium containing soy protein as a sole nitrogen source. Both recombinant enzymes produced in Pichia pastoris are highly active on keratin azure suggesting that these proteases play an important role in invasion of keratinised tissues by the fungus. The set of deduced amino acid sequences of T rubrum SUB ORFs allowed the identification of orthologous Subs secreted by other dermatophyte species using proteolysis and mass spectrometry. (C) 2004 Elsevier B.V. All rights reserved

Identification of the catalytic residues of the first family of beta(1-3)glucanosyltransferases identified in fungi

A new family of glycosylphosphatidylinositol-anchored beta(1-3)glucanosyltransferases (Gelp), recently identified and characterized in the filamentous fungus Aspergillus fumigatus, showed functional similarity to the Gas/Phr/Epd protein families, which are involved in yeast morphogenesis. Sequence comparisons and hydrophobic cluster analysis (HCA) showed that all the Gas/Phr/Epd/Gel proteins belong to a new family of glycosylhydrolases, family 72. We confirmed by site-directed mutagenesis and biochemical analysis that the two conserved glutamate residues (the putative catalytic residues of this family, as determined by HCA) are involved in the active site of this family of glycosylhydrolases

Aminopeptidases and dipeptidyl-peptidases secreted by the dermatophyte Trichophyton rubrum

The nature of secreted aminopeptidases in Trichophyton rubrum was investigated by using a reverse genetic approach. T. rubrum genomic and cDNA libraries were screened with Aspergillus spp. and Saccharomyces cerevisiae aminopeptidase genes as the probes. Two leucine aminopeptidases, ruLap1 and ruLap2, and two dipeptidyl-peptidases, ruDppIV and ruDppV, were characterized and compared to orthologues secreted by Aspergillus fumigatus using a recombinant protein from Pichia pastoris. RuLap1 is a 33 kDa nonglycosylated protein, while ruLap2 is a 58-65 kDa glycoprotein. The hydrolytic activity of ruLap1, ruLap2 and A. fumigatus orthologues showed various preferences for different aminoacyl-7-amido-4-methylcoumarin substrates, and various sensitivities to inhibitors and cations. ruDppIV and ruDppV showed similar activities to A. fumigatus orthologues. In addition to endopeptidases, the four aminopeptidases ruLap1, ruLap2, ruDppIV and ruDppV were produced by T. rubrum in a medium containing keratin as the sole nitrogen source. Synergism between endo- and exopeptidases is likely to be essential for dermatophyte virulence, since these fungi grow only in keratinized tissues