A novel multiplex qPCR­HRM assay for the simultaneous detection of four abortive zoonotic agents in cattle, sheep, and goats.

Abortifacient pathogens induce substantial economic losses in the livestock industry worldwide, and many of these pathogens are zoonotic, impacting human health. As Brucella spp., Coxiella burnetii, Leptospira spp., and Listeria monocytogenes cause abortion, rapid differential molecular diagnostic tests are needed to facilitate early and accurate detection of abortion to establish effective control measures. However, the available molecular methods are laborious, time-consuming, or costly. Therefore, we developed and validated a novel multiplex real-time polymerase chain reaction (qPCR) method based on high-resolution melting (HRM) curve analysis to simultaneously detect and differentiate four zoonotic abortifacient agents in cattle, goats, and sheep. Our HRM assay generated four well-separated melting peaks allowing the differentiation between the four zoonotic abortifacients. Out of 216 DNA samples tested, Brucella spp. was detected in 45 samples, Coxiella burnetii in 57 samples, Leptospira spp. in 12 samples, and Listeria monocytogenes in 19 samples, co-infection with Brucella spp. and Coxiella burnetii in 41 samples, and 42 samples were negative. This assay demonstrated good analytical sensitivity, specificity, and reproducibility. This is a valuable rapid, cost-saving, and reliable diagnostic tool for detecting individual and co-infections for zoonotic abortifacient agents in ruminants.

Advances in Irradiated Livestock Vaccine Research and Production Addressing the Unmet Needs for Farmers and Veterinary Services in FAO/IAEA Member States.

The Animal Production and Health section (APH) of the Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture at the International Atomic Energy Agency has over the last 58 years provided technical and scientific support to more than 100 countries through co-ordinated research activities and technical co-operation projects in peaceful uses of nuclear technologies. A key component of this support has been the development of irradiated vaccines targeting diseases that are endemic to participating countries. APH laboratories has over the last decade developed new techniques and has put in place a framework that allows researchers from participating member states to develop relevant vaccines targeting local diseases while using irradiation as a tool for improving livestock resources.

Molecular characterization of avipoxviruses circulating in Mozambique, 2016-2018.

Samples from 45 chickens, two turkeys, one peacock and one quail with symptoms of fowlpox were collected in Mozambique between November 2016 and January 2018. Phylogenetic analysis revealed that the samples contained avipoxviruses belonging to both clade A1 and clade A2. In addition, all of the Clade A1 viruses were positive by PCR for the integration of reticuloendotheliosis virus, while the clade A2 avipoxvirus samples were negative. This study confirms the circulation of clade A1 avipoxviruses in Mozambique in addition to identifying clade A2 for the first time in the country.

Identification of Clade E Avipoxvirus, Mozambique, 2016.

Analysis of scab samples collected from poultry during outbreaks of fowlpox in Mozambique in 2016 revealed the presence of clade E avipoxviruses. Infected poultry were from flocks that had been vaccinated against fowlpox virus. These findings require urgent reevaluation of the vaccine formula and control strategies in this country.

Developing a Progressive Control Pathway for African Animal Trypanosomosis.

Progressive control pathways (PCPs) are stepwise approaches for the reduction, elimination, and eradication of human and animal diseases. They provide systematic frameworks for planning and evaluating interventions. Here we outline a PCP for tsetse-transmitted animal trypanosomosis, the scourge of poor livestock keepers in tropical Africa. Initial PCP stages focus on the establishment of national coordination structures, engagement of stakeholders, development of technical capacities, data collection and management, and pilot field interventions. The intermediate stage aims at a sustainable and economically profitable reduction of disease burden, while higher stages target elimination. The mixed-record of success and failure in past efforts against African animal trypanosomosis (AAT) makes the development of this PCP a high priority.

Phylogenetic analysis of Newcastle disease viruses isolated from commercial poultry in Mozambique (2011-2016).

The complete sequence of the fusion (F) protein gene from 11 Newcastle disease viruses (NDVs) isolated from commercial poultry in Mozambique between 2011 and 2016 has been generated. The F gene cleavage site motif for all 11 isolates was (112)RRRKRF(117) indicating that the viruses are virulent. A phylogenetic analysis using the full F gene sequence revealed that the viruses clustered within genotype VIIh and showed a higher similarity to NDVs from South Africa, China and Southeast Asia than to viruses previously described in Mozambique in 1994, 1995 and 2005. The identification of these new NDVs has important implications for Newcastle disease management and control in Mozambique.

The role of nuclear technologies in the diagnosis and control of livestock diseases--a review.

Nuclear and nuclear-related technologies have played an important role in animal health, particularly in relation to disease diagnosis and characterization of pathogenic organisms. This review focuses primarily on how and where nuclear technologies, both non-isotopic and isotopic methods, have made their impact in the past and where it might be expected they could have an impact in the future. The review outlines the extensive use of radiation attenuation in attempts to create vaccines for a multiplicity of pathogenic organisms and how the technology is being re-examined in the light of recent advances in irradiation techniques and cryopreservation/lyophilization that might obviate some of the problems of maintenance of viable, attenuate vaccines and their transport and use in the field. This approach could be used for a number of parasitic diseases where vaccination has been problematic and where investigations into the development of molecular vaccines have still failed to deliver satisfactory candidates for generating protective immune responses. Irradiation of antigens or serum samples also has its uses in diagnosis, especially when the samples need to be transported across international boundaries, or when handling the pathogens in question when carrying out a test presents serious health hazards to laboratory personnel. The present-day extensive use of enzyme immunoassays and molecular methods (e.g., polymerase chain reaction) for diagnosis and characterization of animal pathogens has its origins in the use of isotope-labeled antigens and antibodies. These isotopic techniques that included the use of 75Se, 32P, 125I, and 35S isotopes enabled a level of sensitivity and specificity that was hitherto unrealized, and it is prescient to remind ourselves of just how successful these technologies were, in spite of their infrequent use nowadays. Finally, the review looks at the potential for stable isotope analysis for a variety of applications--in the tracking of animal migrations, where the migrant are potential carriers of transboundary animal diseases, and where it would be useful to determine the origins of the carrier, e.g., Highly Pathogenic Avian Influenza and its dissemination by wild water fowl. Other applications could be in monitoring sequestered microbial culture (e.g., rinderpest virus) where in the case of accidental or deliberate release of infective culture it would be possible to identify the laboratory from which the isolate originated.

Generation and evaluation of a recombinant modified vaccinia virus Ankara vaccine for rabies.

Modified vaccinia virus Ankara (MVA) has become a vaccine vector of choice for recombinant vaccine development. A MVA-based rabies vaccine would be advantageous for use as a vaccine for dogs (and wildlife), particularly if it proves innocuous and efficacious by the oral route. Here, the generation and immunological testing of a recombinant MVA expressing a rabies virus glycoprotein gene is described. In a murine model, higher dosages of recombinant MVA were needed to induce equivocal immune responses as with Vaccinia Copenhagen or Vaccinia Western Reserve recombinants, when administered by a parenteral route. The MVA recombinant was not immunogenic or efficacious when administered per os in naïve mice. The ability of the recombinant MVA to induce anamnestic responses in dogs and raccoons was also investigated. Recombinant MVA boosted humoral immune responses in these animals when administered peripherally, but not when administered orally.

Immune responses to recombinants of the South African vaccine strain of lumpy skin disease virus generated by using thymidine kinase gene insertion.

The South African vaccine strain of lumpy skin disease virus (type SA-Neethling) is currently being developed as a vector for recombinant vaccines of economically important livestock diseases throughout Africa. In this study, the feasibility of using the viral thymidine kinase gene as the site of insertion was investigated and recombinant viruses were evaluated in animal trials. Two separate recombinants were generated and selected for homogeneity expressing either the structural glycoprotein gene of bovine ephemeral fever virus (BEFV) or the two structural glycoprotein genes of Rift Valley fever virus (RVFV). Both recombinants incorporate the enhanced green fluorescent protein (EGFP) as a visual marker and the Escherichia coli guanine phosphoribosyl transferase (gpt) gene for dominant positive selection. The LSDV-RVFV recombinant construct (rLSDV-RVFV) protected mice against virulent RVFV challenge. In a small-scale BEFV-challenge cattle trial the rLSDV-BEFV construct failed to fully protect the cattle against virulent challenge, although both a humoral and cellular BEFV-specific immune response was elicited.

Inhibition of fowlpox virus by an aqueous acetone extract from galls of Guiera senegalensis J. F. Gmel (Combretaceae).

An aqueous acetone extract from the galls of Guiera senegalensis was screened for in vitro antiviral activity against fowlpox virus (FPV). Cytopathic effect (CPE) inhibition and plaque inhibition assays were used to show presence of antiviral effects against FPV, whilst cytotoxicity assays established the relative safety of the extract for cells in vitro. Phytochemical analyses revealed the presence of phenolic compounds including flavonoids, tannins and anthocyanins as well as steroids and alkaloids. Thin-layer chromatographical (TLC) analysis also revealed the presence of quercitrin, quercetin, kaempferol, apigenin, rutin, gallic acid as well as unknown flavonoids and unknown phenolic acids. The antiviral effect of the extract was partially attributed to phenolic components including flavonoids.