Development of a Plant-Expressed Subunit Vaccine against Brucellosis.

Brucellosis is an important bacterial disease of livestock and the most common zoonotic disease. The current vaccines are effective but unsafe, as they result in animal abortions and are pathogenic to humans. Virus-like particles are being investigated as molecular scaffolds for foreign antigen presentation to the immune system. Here, we sought to develop a new-generation vaccine by presenting selected Brucella melitensis T cell epitopes on the surface of Orbivirus core-like particles (CLPs) and transiently expressing these chimeric particles in Nicotiana benthamiana plants. We successfully demonstrated the assembly of five chimeric CLPs in N. benthamiana plants, with each CLP presenting a different T cell epitope. The safety and protective efficacy of three of the highest-yielding CLPs was investigated in a mouse model of brucellosis. All three plant-expressed chimeric CLPs were safe when inoculated into BALB/c mice at specific antigen doses. However, only one chimeric CLP induced protection against the virulent Brucella strain challenge equivalent to the protection induced by the commercial Rev1 vaccine. Here, we have successfully shown the assembly, safety and protective efficacy of plant-expressed chimeric CLPs presenting B. melitensis T cell epitopes. This is the first step in the development of a safe and efficacious subunit vaccine against brucellosis.

Accuracy of molecular diagnostic assays for detection of Mycobacterium bovis: A systematic review and meta-analysis.

Bovine tuberculosis (bovine TB) is a chronic wasting disease of cattle caused primarily by Mycobacterium bovis. Controlling bovine TB requires highly sensitive, specific, quick, and reliable diagnostic methods. This systematic review and meta-analysis evaluated molecular diagnostic tests for M. bovis detection to inform the selection of the most viable assay. On a per-test basis, loop-mediated isothermal amplification (LAMP) showed the highest overall sensitivity of 99.0% [95% CI: 86.2%-99.9%] and specificity of 99.8% [95% CI: 96.2%-100.00%]. Quantitative real-time polymerase chain reaction (qPCR) outperformed conventional PCR and nested PCR (nPCR) with a diagnostic specificity of 96.6% [95% CI: 88.9%-99.0%], while the diagnostic sensitivity of 70.8% [95% CI: 58.6-80.5%] was comparable to that of nPCR at 71.4% [95% CI: 60.7-80.2%]. Test sensitivity was higher with the input of milk samples (90.9% [95% CI: 56.0%-98.7%]), while specificity improved with tests based on major M. bovis antigens (97.8% [95% CI: 92.3%-99.4%]), the IS6110 insertion sequence (95.4% [95% CI: 87.6%-98.4%]), and the RD4 gene (90.7% [95% CI: 52.2%-98.9%]). The design of the currently available molecular diagnostic assays, while mostly based on nonspecific gene targets, prevents them from being accurate enough to diagnose M. bovis infections in cattle, despite their promise. Future assay development should focus on the RD4 region since it is the only target identified by genome sequence data as being distinctive for detecting M. bovis. The availability of a sufficiently accurate diagnostic test combined with the routine screening of milk samples can decrease the risk of zoonotic transmissions of M. bovis.

Accuracy of molecular diagnostic methods for the detection of bovine brucellosis: A systematic review and meta-analysis.

Background and Aim: Bovine brucellosis is a disease of global socio-economic importance caused by Brucella abortus. Diagnosis is mainly based on bacterial culture and serology. However, these methods often lack sensitivity and specificity. A range of molecular diagnostic methods has been developed to address these challenges. Therefore, this study aims to investigate the diagnostic accuracy of molecular tools, in comparison to gold standard bacterial isolation and serological assays for the diagnosis of bovine brucellosis. Materials and Methods: The systematic review and meta-analysis were conducted based on analyses of peer-reviewed journal articles published between January 1, 1990, and June 6, 2020, in the PubMed, Science Direct, Scopus, and Springer Link databases. Data were extracted from studies reporting the use of molecular diagnostic methods for the detection of B. abortus infections in animals according to Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. The quality of included journal articles was assessed using the quality assessment of diagnostic-accuracy studies assessment tool and meta-analysis was carried out using Review Manager. Results: From a total of 177 studies, only 26 articles met the inclusion criteria based on PRISMA guidelines. Data from 35 complete studies were included in the meta-analysis and used to construct 2 × 2 contingency tables. Improved diagnostic performance was observed when tissue (sensitivity 92.7% [95% confidence interval (CI) 82.0-98.0%]) and serum samples (sensitivity 91.3% [95% CI 86.0-95.0%]) were used, while the BruAb2_0168 locus was the gene of preference for optimal assay performance (sensitivity 92.3% [95% CI 87.0-96.0%] and specificity 99.3% [95% CI 98.0-100.0%]). Loop-mediated isothermal amplification (LAMP) had a higher diagnostic accuracy than polymerase chain reaction (PCR) and real-time quantitative PCR with sensitivity of 92.0% (95% CI 78.0-98.0%) and specificity of 100.0% (95% CI 97.0-100.0%). Conclusion: The findings of this study assign superior diagnostic performance in the detection of B. abortus to LAMP. However, due to limitations associated with decreased specificity and a limited number of published articles on LAMP, the alternative use of PCR-based assays including those reported in literature is recommended while the use of LAMP for the detection of bovine brucellosis gains traction and should be evaluated more comprehensively in future.

Sublingual papillomas of cheetahs in southern Africa.

Nine distinct papillomaviruses (Lambdapapillomavirus) have been described in domestic and nondomestic cats, but not in cheetahs. These viruses have been associated with cutaneous papillomas or plaques, bowenoid in situ carcinomas, feline cutaneous squamous cell carcinomas (SCC), feline sarcoids, and oral (often sublingual) papillomas. Fourteen cheetahs from the AfriCat foundation (Namibia) and one from the Ann van Dyk Cheetah center (South Africa) presented with sublingual lesions reminiscent of sublingual papillomas. Two animals were biopsied and the histopathology revealed benign proliferative epithelial lesions with prominent thickening of the overlying squamous epithelium. Throughout the squamous epithelial layers were cells with nuclear enlargement, irregularity of the nuclear membranes and cell contours, focal hyperchromasia of the nuclei, and perinuclear halos, reminiscent of a virus-associated process as seen in papillomavirus infections. Thirteen more cheetahs were sampled and the tissue snap frozen for molecular characterization. Amplification and sequencing of the papillomavirus L1, E6, E7, and E1 gene regions was achieved with modified primers. Maximum likelihood phylogenetic analyses revealed all 15 cheetah papilloma samples were 99.99% genetically similar and closely related to, but genetically distinct from any known felinepapillomaviruses. All cheetahs were FIV and FeLV negative. The results suggest the samples identified in this study can be considered a previously undescribed or novel feline papillomavirus and the authors propose "Acinonyx jubatus papillomavirus type 1" (AjPV-1), within the Lambdapapillomavirus 1 genus (Family: Papillomaviridae).

Patents and technology transfer in CRISPR technology.

CRISPR technology has revolutionized biological research in the last decade and many academic institutions and companies have patented CRISPR systems and applications. Several patents have been filed for various applications of CRISPR in different industries such as agriculture, synthetic biology, bio-nanotechnology and precision medicine. Despite tremendous pressure on the technology transfer teams, several startups and spin-out companies are already using CRISPR technologies for commercial applications. In this chapter, we discuss the different CRISPR nucleases and their applications. Secondly, we detail our current opinion and perspective on the CRISPR patent and technology landscape for non-mammalian systems. We present two case-studies on CRISPR diagnostics companies, SHERLOCK and Mammoth Biosciences, who are currently at the forefront of establishing diagnostics platforms for coronavirus (SARS-CoV-2) detection. Finally, our chapter identifies future advancements and possible challenges that CRISPR technology might face in non-mammalian systems.

The complete mitochondrial genome of Gyps coprotheres (Aves, Accipitridae, Accipitriformes): phylogenetic analysis of mitogenome among raptors.

Three species of Old World vultures on the Asian peninsula are slowly recovering from the lethal consequences of diclofenac. At present the reason for species sensitivity to diclofenac is unknown. Furthermore, it has since been demonstrated that other Old World vultures like the Cape (Gyps coprotheres; CGV) and griffon (G. fulvus) vultures are also susceptible to diclofenac toxicity. Oddly, the New World Turkey vulture (Cathartes aura) and pied crow (Corvus albus) are not susceptible to diclofenac toxicity. As a result of the latter, we postulate an evolutionary link to toxicity. As a first step in understanding the susceptibility to diclofenac toxicity, we use the CGV as a model species for phylogenetic evaluations, by comparing the relatedness of various raptor species known to be susceptible, non-susceptible and suspected by their relationship to the Cape vulture mitogenome. This was achieved by next generation sequencing and assembly. The Cape vulture mitogenome had a genome size of 16,908 bp. The mitogenome phylogenetic analysis indicated a close evolutionary relationship between Old World vultures and other members of the Accipitridae as indicated by bootstrap value of 100% on the phylogenetic trees. Based on this, we postulate that the other species could also be sensitive to the toxic effects of diclofenac. This warrants further investigations.

Horizon scanning for South African biodiversity: A need for social engagement as well as science.

A horizon scan was conducted to identify emerging and intensifying issues for biodiversity conservation in South Africa over the next 5-10 years. South African biodiversity experts submitted 63 issues of which ten were identified as priorities using the Delphi method. These priority issues were then plotted along axes of social agreement and scientific certainty, to ascertain whether issues might be "simple" (amenable to solutions from science alone), "complicated" (socially agreed upon but technically complicated), "complex" (scientifically challenging and significant levels of social disagreement) or "chaotic" (high social disagreement and highly scientifically challenging). Only three of the issues were likely to be resolved by improved science alone, while the remainder require engagement with social, economic and political factors. Fortunately, none of the issues were considered chaotic. Nevertheless, strategic communication, education and engagement with the populace and policy makers were considered vital for addressing emerging issues.

Gastrointestinal Parasites of Vervet Monkeys (Chlorocebus pygerythrus) in a High Latitude, Semi-Arid Region of South Africa.

Given a changing climate and large-scale human migration, understanding infectious diseases in wildlife and the factors that drive the spread of these diseases is becoming increasingly important. Owing to the close phylogenetic relationship between nonhuman primates and humans, primate parasites are of particular interest due to the potential for zoonotic disease transmission and for the study of social transmission within gregarious social groups. There is a wide range of social and environmental factors that influence the prevalence and transmission of pathogens, and identifying these, and their effects, is crucial to understanding the population-level consequences of climate change for animals that live in obligate social groups. Here we investigated gastrointestinal parasite species richness and used fecal egg counts to estimate worm intensities in 3 vervet monkey troops (Chlorocebus pygerythrus) in a high latitude, semi-arid region of South Africa. This region is characterized by unpredictable rainfall and temperature extremes in summer and winter. We identified the gastrointestinal parasites in the population and explored potential demographic predictors, namely sex and troop membership, of parasite species richness and estimated intensity. Additionally, we assessed whether there was short-term intra-individual, inter-sample consistency in egg counts. Six species of gastrointestinal helminths were identified from 3 study troops, with egg counts ranging from 0 eggs/g to 1,100 eggs/g. Neither age nor sex predicted species richness or estimated intensity. This population had the highest prevalence of parasites with an insect vector compared with all other vervet populations studied, and distinctively high prevalences of Trichostrongylus sp. (71%) and Ternidens sp. (27%). Additionally, we found intra-individual egg count consistency in the short term (mean: 32 days).

Occurrence and diversity of avian haemosporidia in Afrotropical landbirds.

Avian haemosporidian infections are widespread and can result in the decline of wild bird populations or in some cases contribute to extinction of species. We determined the prevalence and genetic diversity of avian haemosporidia in 93 samples from 22 landbird species from South Africa (N = 76) and West Africa (N = 17), of which six are intra-African migrants and one is a Palearctic migrant. The samples were analysed for the presence of avian haemosporidian DNA using real-time quantitative PCR (qPCR) and nested PCR assays targeting specific mitochondrial genes of these parasites. The cytochrome b (cytb) gene was sequenced for all samples that tested positive and phylogenetic analysis was conducted in order to determine the relationship of the new sequences with previously published sequences from the MalAvi database. The overall prevalence of avian haemosporidiosis was 68.82% (95% CI: 56.4%-78.87%) and 82.80% (95% CI: 65.68%-86.11%) as determined by qPCR and nested PCR respectively. Eighteen (19.36%; 95% CI; 10.78%-29.97%) samples had mixed infections. Infection prevalence of all haemosporidian spp. were significantly higher (p < 0.05) in samples from West Africa. Forty-six mitochondrial sequences obtained from 14 avian species grouped into three distinct clusters of Haemoproteus (36), Leucocytozoon (8) and Plasmodium (2). These represent eight published and nine new cytb lineages. The most common lineage was Haemoproteus sp. (VIMWE1) which was identified in two bird species from West Africa and seven bird species from South Africa. This study adds to our knowledge of host-parasite relationships of avian haemosporidia of Afrotropical birds.

Fatal disseminated toxoplasmosis in a zoological collection of meerkats (Suricata suricatta).

Two confirmed cases of fatal disseminated toxoplasmosis occurred in an urban zoological collection of meerkats (Suricata suricatta). Both cases are suspected to be the result of feral cats gaining access to the enclosure. Toxoplasmosis has rarely been documented in meerkats. Subsequent to prophylactic treatment of all the animals and structural changes being implemented within the enclosure, no new cases have been recorded to date. Very little information is available on the disease in viverrids.

Investigation of false positives associated with loop-mediated isothermal amplification assays for detection of Toxoplasma gondii in archived tissue samples of captive felids.

Toxoplasma gondii is a ubiquitous protozoan parasite that infects humans and many different animals, including felids. Many molecular and serologic tests have been developed for detection of T. gondii in a wide range of hosts. Loop-mediated isothermal amplification (LAMP) is a field-friendly technique that lacks the practical drawbacks of other molecular and serologic tests, and LAMP assays have been successfully developed for detection of T. gondii in fresh tissue samples. In the current study, both a previously published and a de-novo designed LAMP assay were compared to a quantitative real-time (q)PCR assay, for the detection of T. gondii in archived formalin-fixed, paraffin-embedded (FFPE) tissue samples from captive wildlife. The LAMP assays produced conflicting results, generating both false positives and false negatives. Furthermore, the LAMP assays were unable to positively identify samples with low levels of parasites as determined by qPCR and histopathology. Therefore, these LAMP assays may not be the most suitable assays for detection of T. gondii in archived FFPE and frozen tissue samples.

Sarcocystis cafferi n. sp. (Protozoa: Apicomplexa) from the African buffalo (Syncerus caffer).

Sarcocystis infections have been reported from the African buffalo ( Syncerus caffer ), but the species have not been named. Here we propose a new name Sarcocystis cafferi from the African buffalo. Histological examination of heart (92), skeletal muscle (36), and tongue (2) sections from 94 buffalos from the Greater Kruger National Park, South Africa, and a review of the literature revealed only 1 species of Sarcocystis in the African buffalo. Macrocysts were up to 12 mm long and 6 mm wide and were located in the neck muscles and overlying connective tissue. They were pale yellow; shaped like a lychee fruit stone or cashew nut; turgid or flaccid and oval to round (not fusiform). By light microscopy (LM) the sarcocyst wall was relatively thin. By scanning electron microscopy (SEM), the sarcocyst wall had a mesh-like structure with irregularly shaped villar protrusions (vp) that were of different sizes and folded over the sarcocyst wall. The entire surfaces of vp were covered with papillomatous structures. By transmission electron microscopy (TEM), the sarcocyst wall was up to 3.6 µm thick and had highly branched villar protrusions that were up to 3 µm long. The villar projections contained filamentous tubular structures, most of which were parallel to the long axis of the projections, but some tubules criss-crossed, especially at the base. Granules were absent from these tubules. Longitudinally cut bradyzoites were 12.1 × 2.7 µm in size, had a long convoluted mitochondrion, and only 2 rhoptries. Phylogenetic analysis of 18S rRNA and cytochrome C oxidase subunit 1 (cox1) gene sequences indicated that this Sarcocystis species is very closely related to, but distinct from, Sarcocystis fusiformis and Sarcocystis hirsuta. Thus, morphological findings by LM, SEM, and TEM together with molecular phylogenetic data (from 18S rRNA and cox1) confirm that the Sarcocystis species in the African buffalo is distinct from S. fusiformis and has therefore been named Sarcocystis cafferi.

Validation of hisH4 and cox5 reference genes for RT-qPCR analysis of gene expression in Aspergillus flavus under aflatoxin conducive and non-conducive conditions.

Aspergillus flavus is an environmental pathogen that produces highly carcinogenic aflatoxins. Biosynthesis of aflatoxins is affected by external factors such as pH, temperature, carbon source and nitrogen source. Real-Time PCR (RT-qPCR) is a powerful technique used to detect minute changes in gene expression of a target gene in comparison to one or more reference genes. Several candidate genes were analysed to determine their suitability for use as reference genes for analysing gene expression in A. flavus via RT-qPCR under various aflatoxin conducive and non-conducive conditions. BestKeeper analysis indicated that histone H4 (hisH4) and cytochrome C oxidase subunit V (cox5) were suitable reference genes for analysis of gene expression in A. flavus via RT-qPCR. This was further confirmed by REST2009 analysis of hisH4 and cox5 stability. Furthermore, REST2009 was used to predict which gene or gene combination would be the best reference gene/s for RT-qPCR expression analysis under each treatment condition tested in this study.