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.

Are open access article processing charges affordable for otolaryngologists in low-income and middle-income countries?

PURPOSE OF REVIEW: Open access articles are more frequently read and cited, and hence promote access to knowledge and new advances in healthcare. Unaffordability of open access article processing charges (APCs) may create a barrier to sharing research. We set out to assess the affordability of APCs and impact on publishing for otolaryngology trainees and otolaryngologists in low-income and middle-income countries (LMICs). RECENT FINDINGS: A cross-sectional online survey was conducted among otolaryngology trainees and otolaryngologists in LMICs globally. Seventy-nine participants from 21 LMICs participated in the study, with the majority from lower middle-income status (66%). Fifty-four percent were otolaryngology lecturers while 30% were trainees. Eighty-seven percent of participants received a gross monthly salary of less than USD 1500. Fifty-two percent of trainees did not receive a salary. Ninety-one percent and 96% of all study participants believed APCs limit publication in open access journals and influence choice of journal for publication, respectively. Eighty percent and 95% believed APCs hinder career progression and impede sharing of research that influences patient care, respectively. SUMMARY: APCs are unaffordable for LMIC otolaryngology researchers, hinder career progression and inhibit the dissemination of LMIC-specific research that can improve patient care. Novel models should be developed to support open access publishing in LMICs.

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.

Stapler-Assisted Pharyngeal Closure After Total Laryngectomy: A Systematic Review and Meta-Analysis.

INTRODUCTION: Pharyngocutaneous fistula (PCF) remains the most frequent complication following total laryngectomy (TL). Pharyngeal closure with a surgical stapler (SAPC) has been proposed as an effective closure technique that decreases the rate of PCF, reduces surgical time, decreases the length of hospital stay, and shortens the time required before safely initiating oral feeding. METHODS: This study involved a systematic review and meta-analysis of patients with laryngeal cancer who underwent TL and with subsequent stapler pharyngeal closure, in order to analyse the current literature regarding the role of SAPC after TL. RESULTS: The incidence of PCF in the stapler-assisted suture group (SASG) was 9.5% (95% CI 8.2-15.9%), with a mean absolute deviation of 1.12, while in the hand-suture group (HSG) group the incidence was 23.4% (95% CI 23-26.1%), with a mean absolute deviation of 5.71 (p = 0.01). CONCLUSIONS: SAPC may decrease the risk of PCF in patients following TL. Based on the current data, SAPC appears to shorten the surgical time and the length of hospital stay. Nevertheless, prospective randomized trials are required to validate these findings.

Management of oral feeding following total laryngectomy around the world: YO-IFOS international study.

BACKGROUND: To analyze worldwide practices regarding the initiation of oral feeding after total laryngectomy (TL). METHODS: Online survey. RESULTS: Among the 332 responses received, 278 from 59 countries were analyzed. Our results showed that 45.6% of respondents started water and 45.1% started liquid diet between postoperative days 7 and 10. Semi-solid feeds were initiated between days 10 and 14 for 44.9% of respondents and a free diet was allowed after day 15 for 60.8% of respondents. This timing was significantly delayed in cases of laryngo-pharyngectomy and after prior radiotherapy (p < 0.001). A greater proportion of respondents in Africa and Oceania allowed early oral feeding before day 6 as compared with the rest of the world (p < 0.001). CONCLUSION: Despite increasing number of publications, there is still a lack of evidence to support early oral feeding. The majority of respondents preferred to delay its initiation until at least 7 days after surgery.

Orbito-Cranial Gunshot Injuries with Retained Sinonasal Bullets.

INTRODUCTION: Gunshot injuries to the sino-orbital region are rare. In South Africa, where gunshot injuries are common, sino-orbital gunshot injuries are encountered. Sino-orbital gunshot injuries are associated with trauma to surrounding facial and intracranial structures. Therefore, the management of these injuries may be complex and often requires an interdisciplinary approach. AIMS: To review the management of orbito-cranial gunshot injuries with retained sinonasal bullets. PATIENTS AND METHODS: Three cases of orbito-cranial gunshot injuries with retained sinonasal bullets were reviewed. Two cases were complicated by cerebrospinal fluid leaks with ensuing meningitis. The retained bullets in all three cases were successfully removed via a transnasal endoscopic approach. CONCLUSION: Sino-orbital gunshot injuries are rare, but may be encountered in areas with high frequencies of gun violence. An associated anterior skull base fracture with CSF rhinorrhoea poses a risk for meningitis and a low threshold for diagnosis and treatment of meningitis should be maintained. Retained bullets in the paranasal sinuses do not pose an immediate risk and may be removed on an elective basis.

First molecular characterization of poxviruses in cattle, sheep, and goats in Botswana.

BACKGROUND: Poxviruses within the Capripoxvirus, Orthopoxvirus, and Parapoxvirus genera can infect livestock, with the two former having zoonotic importance. In addition, they induce similar clinical symptoms in common host species, creating a challenge for diagnosis. Although endemic in the country, poxvirus infections of small ruminants and cattle have received little attention in Botswana, with no prior use of molecular tools to diagnose and characterize the pathogens. METHODS: A high-resolution melting (HRM) assay was used to detect and differentiate poxviruses in skin biopsy and skin scab samples from four cattle, one sheep, and one goat. Molecular characterization of capripoxviruses and parapoxviruses was undertaken by sequence analysis of RPO30 and GPCR genes. RESULTS: The HRM assay revealed lumpy skin disease virus (LSDV) in three cattle samples, pseudocowpox virus (PCPV) in one cattle sample, and orf virus (ORFV) in one goat and one sheep sample. The phylogenetic analyses, based on the RPO30 and GPCR multiple sequence alignments showed that the LSDV sequences of Botswana were similar to common LSDV field isolates encountered in Africa, Asia, and Europe. The Botswana PCPV presented unique features and clustered between camel and cattle PCPV isolates. The Botswana ORFV sequence isolated from goat differed from the ORFV sequence isolated from sheep. CONCLUSIONS: This study is the first report on the genetic characterization of poxvirus diseases circulating in cattle, goats, and sheep in Botswana. It shows the importance of molecular methods to differentially diagnose poxvirus diseases of ruminants.

Large-Scale International Validation of an Indirect ELISA Based on Recombinant Nucleocapsid Protein of Rift Valley Fever Virus for the Detection of IgG Antibody in Domestic Ruminants.

Diagnostic performance of an indirect enzyme-linked immunosorbent assay (I-ELISA) based on a recombinant nucleocapsid protein (rNP) of the Rift Valley fever virus (RVFV) was validated for the detection of the IgG antibody in sheep (n = 3367), goat (n = 2632), and cattle (n = 3819) sera. Validation data sets were dichotomized according to the results of a virus neutralization test in sera obtained from RVF-endemic (Burkina Faso, Democratic Republic of Congo, Mozambique, Senegal, Uganda, and Yemen) and RVF-free countries (France, Poland, and the USA). Cut-off values were defined using the two-graph receiver operating characteristic analysis. Estimates of the diagnostic specificity of the RVFV rNP I-ELISA in animals from RVF-endemic countries ranged from 98.6% (cattle) to 99.5% (sheep) while in those originating from RVF-free countries, they ranged from 97.7% (sheep) to 98.1% (goats). Estimates of the diagnostic sensitivity in ruminants from RVF-endemic countries ranged from 90.7% (cattle) to 100% (goats). The results of this large-scale international validation study demonstrate the high diagnostic accuracy of the RVFV rNP I-ELISA. Standard incubation and inactivation procedures evaluated did not have an adverse effect on the detectable levels of the anti-RVFV IgG in ruminant sera and thus, together with recombinant antigen-based I-ELISA, provide a simple, safe, and robust diagnostic platform that can be automated and carried out outside expensive bio-containment facilities. These advantages are particularly important for less-resourced countries where there is a need to accelerate and improve RVF surveillance and research on epidemiology as well as to advance disease control measures.

Inflammatory leiomyosarcoma of the head and neck: Case report.

INTRODUCTION AND IMPORTANCE: Primary sarcomas in the head and neck region are rare. Inflammatory leiomyosarcoma was first described in 1995. The case reported herein is the first reported inflammatory leiomyosarcoma occurring in the head and neck. PRESENTATION OF CASE: A 37-year-old male presented with a long history of an asymptomatic slowly enlarging neck mass. Examination revealed a firm mass in the lower third of the right sternocleidomastoid muscle. Computerized tomography and magnetic resonance imaging showed a lobulated, well-circumscribed tumour with malignant features. A wide local excision was performed and histopathological examination confirmed an inflammatory leiomyosarcoma. DISCUSSION: Inflammatory leiomyosarcoma is a recently described peculiar soft tissue tumour with histological features overlapping conventional leiomyosarcoma, and dense lymphocytic inflammation and immunohistochemical reactivity for both smooth and skeletal muscle markers. These are indolent tumours and wide local excision is curative. CONCLUSION: This case highlights the importance of considering primary sarcomas in the differential diagnosis of asymptomatic head and neck masses.

Does Thyroid Gland Preserving Total Laryngectomy Affect Oncological Control in Laryngeal Carcinoma?

INTRODUCTION: Thyroid lobectomy is recommended with total laryngectomy in the National Comprehensive Cancer Network guidelines. However, it is associated with a 32% to 89% risk of hypothyroidism, which is a problem for patients without access to thyroid hormone monitoring and replacement. A number of studies have reported a low incidence of thyroid gland involvement and recommended preserving the thyroid gland in favorable cases. Yet there are no studies that report whether thyroid preserving laryngectomy for laryngeal cancer adversely affects oncologic outcomes compared to patients who have undergone thyroid lobectomy. OBJECTIVES: To determine whether patients who underwent thyroid gland preserving laryngectomy had higher local recurrence rates or poorer disease-free survival. METHOD: A retrospective folder review of patients who underwent a total laryngectomy over a 12-year period was conducted. Local recurrence and disease-free survival were determined for patients who had both their thyroid lobes preserved and compared with those who had a thyroid lobectomy. RESULTS: Sixty-nine patients had thyroid preserving laryngectomy and 73 patients had a thyroid lobectomy. The duration of follow-up was 18 to 132 months (median, 30; IQR, 30). There was no significant difference in local recurrence rates (P = .76) or survival curves between the two groups. CONCLUSION: Thyroid preserving laryngectomy in selected patients with advanced laryngeal carcinoma does not increase local recurrence rates, nor does it negatively affect disease-free survival. Thyroid preservation is appropriate when intraoperative inspection of the larynx shows no extralaryngeal extension or when paratracheal nodal metastases are not a concern. LEVEL OF EVIDENCE: Level 4 Laryngoscope, 130:1465-1469, 2020.

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.

Advances in viral disease diagnostic and molecular epidemiological technologies.

The early and rapid detection and characterization of specific nucleic acids of medico-veterinary pathogens have proven invaluable for diagnostic purposes. The integration of amplification and signal detection systems, including online real-time devices, have increased speed and sensitivity and greatly facilitated the quantification of target nucleic acids. They have also allowed for sequence characterization using melting or hybridization curves. The newer-generation molecular diagnostic technologies offer, hitherto, unparalleled detection and discrimination methodologies, which are vital for the positive detection and identification of pathogenic agents, as well as the effects of the pathogens on the production of antibodies. The development phase of the novel technologies entails a thorough understanding of accurate diagnosis and discrimination of present and emerging diseases. The development of novel technologies can only be successful if they are transferred and used in the field with a sustainable quality-assured application to allow for the optimal detection and effective control of diseases. The aim of these new tools is to detect the presence of a pathogen agent before the onset of disease. This manuscript focuses mainly on the experiences of two World Organisation for Animal Health collaborating centers in context to molecular diagnosis and molecular epidemiology of transboundary and endemic animal diseases of viral origin, food safety and zoonoses.

Improved method for the generation and selection of homogeneous lumpy skin disease virus (SA-Neethling) recombinants.

Lumpy skin disease virus (LSDV) is being developed as a vector for recombinant vaccines against diseases of veterinary importance. A strategy for generating viral thymidine kinase (TK) gene-disrupted recombinants which are stable and homogeneous using the South African Neethling vaccine strain of LSDV as vector has been developed. To assist with the selection process, the Escherichia coli beta-galactosidase (lacZ) visual marker gene was incorporated into the constructs. However, the use of lacZ has certain limitations. An improved strategy was then devised substituting lacZ with the enhanced green fluorescent protein (EGFP) under control of the vaccinia virus (VV) P11K late promoter. The EGFP marker was found to enhance the selection process, and with the inclusion of additional sonication and filtration steps the number of passages required to select recombinants to homogeneity has been reduced. In support of the improved method for generation and selection of recombinants described, three different LSDV recombinants expressing the glycoprotein genes of bovine ephemeral fever virus, Rift Valley fever virus and rabies virus were prepared and characterised.

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.