Human brucellosis in South Africa: A review for medical practitioners.

Brucellosis is recognised as a neglected zoonotic tropical disease of global health and economic importance. Medical practitioner unawareness of the disease is reported to contribute to the overall neglect. In South Africa (SA), human brucellosis is a notifiable medical condition and bovine brucellosis is a controlled animal disease. The overall aim of this review article is to increase medical practitioner capacity to detect, diagnose and treat brucellosis in the SA context. A brief review of the literature on human brucellosis in SA is presented, together with a discussion of current issues related to medical detection, treatment and management of brucellosis, applicable to the SA context.

The Incursion and Spread of Highly Pathogenic Avian Influenza H5N8 Clade 2.3.4.4 Within South Africa.

The report of a mass die-off of white-winged terns (Chlidonias leucopterus) along the shores of Lake Victoria in Uganda in January 2017 was a warning that highly pathogenic avian influenza (HPAI) H5N8 clade 2.3.4.4 had entered the avian populations of the African Rift Valley. In early June 2017, Zimbabwe reported an outbreak of the virus in commercial breeder chickens near Harare, and on June 19, 2017, the first case of HPAI H5N8 was confirmed in a broiler breeder operation near Villiers, Mpumalanga Province, South Africa, representing the first ever notifiable influenza in gallinaceous poultry in South Africa. Forty viruses were isolated from wild birds, backyard hobby fowl, zoo collections, commercial chickens, and commercial ostriches over the course of the outbreak and full genomes were sequenced and compared to determine the epidemiologic events in the introduction and spread of clade 2.3.4.4 H5N8 across the country. We found that multiple virus variants were involved in the primary outbreaks in the north-central regions of South Africa, but that a single variant affected the southernmost regions of the continent. By November 2017 only two of the nine provinces in South Africa remained unaffected, and the layer chicken industry in Western Cape Province was all but decimated. Two distinct variants, suggesting independent introductions, were responsible for the first two index cases and were not directly related to the virus involved in the Zimbabwe outbreak. The role of wild birds in the incursion and spread was demonstrated by shared recent common ancestors with H5N8 viruses from West Africa and earlier South African aquatic bird low pathogenicity avian influenza viruses. Improved wild bird surveillance will play a more critical role in the future as an early warning system.

Incursión y propagación del virus de la influenza aviar altamente patógena H5N8 clado 2.3.4.4 en Sudáfrica. El informe de una muerte masiva de fumareles aliblancos (Chlidonias leucopterus) a lo largo de las orillas del lago Victoria en Uganda en enero del 2017 fue una advertencia de que la influenza aviar de alta patogenicidad (HPAI) H5N8, clado 2.3.4.4 había ingresado en las poblaciones de aves del Valle del Rift Africano. A principios de junio del 2017, Zimbabwe reportó un brote del virus en pollos reproductores comerciales cerca de Harare, y el 19 de junio del 2017, el primer caso de influenza aviar de alta patogenicidad H5N8 se confirmó en una operación de pollos de engorde en la provincia de Mpumalanga cerca de Villiers, Sudáfrica, que representa el primer caso de influenza notificable en aves gallináceas en Sudáfrica. Se aislaron cuarenta virus de aves silvestres, aves de traspatio, colecciones de zoológicos, pollos comerciales y avestruces comerciales durante el transcurso del brote. Se secuenciaron los genomas completos y se compararon para determinar los eventos epidemiológicos en la introducción y propagación del subtipo H5N8 clado 2.3.4.4 a través del país. Se encontró que múltiples variantes del virus estaban involucradas en los brotes primarios en las regiones centro y norte de Sudáfrica, pero que una sola variante afectaba a las regiones más al sur del continente. En noviembre de 2017, solo dos de las nueve provincias de Sudáfrica permanecían sin afectarse y la industria de pollos en la Provincia de Cabo Occidental resultó casi diezmada. Dos variantes distintas, que sugieren introducciones independientes, fueron responsables de los dos primeros casos índices y no estuvieron directamente relacionados con el virus involucrado en el brote de Zimbabwe. El papel de las aves silvestres en la incursión y diseminación fue demostrado por los ancestros comunes compartidos con los virus H5N8 de África Occidental y los virus de la influenza aviar de baja patogenicidad de aves acuáticas de Sudáfrica detectados anteriormente. La mejora de la vigilancia de aves silvestres jugará un papel más crítico en el futuro como un sistema de alerta temprana.

Plasmodium falciparum Cyclic GMP-Dependent Protein Kinase Interacts with a Subunit of the Parasite Proteasome.

Malaria is caused by the protozoan parasite Plasmodium, which undergoes a complex life cycle in a human host and a mosquito vector. The parasite's cyclic GMP (cGMP)-dependent protein kinase (PKG) is essential at multiple steps of the life cycle. Phosphoproteomic studies in Plasmodium falciparum erythrocytic stages and Plasmodium berghei ookinetes have identified proteolysis as a major biological pathway dependent on PKG activity. To further understand PKG's mechanism of action, we screened a yeast two-hybrid library for P. falciparum proteins that interact with P. falciparum PKG (PfPKG) and tested peptide libraries to identify its phosphorylation site preferences. Our data suggest that PfPKG has a distinct phosphorylation site and that PfPKG directly phosphorylates parasite RPT1, one of six AAA+ ATPases present in the 19S regulatory particle of the proteasome. PfPKG and RPT1 interact in vitro, and the interacting fragment of RPT1 carries a PfPKG consensus phosphorylation site; a peptide carrying this consensus site competes with the RPT1 fragment for binding to PfPKG and is efficiently phosphorylated by PfPKG. These data suggest that PfPKG's phosphorylation of RPT1 could contribute to its regulation of parasite proteolysis. We demonstrate that proteolysis plays an important role in a biological process known to require Plasmodium PKG: invasion by sporozoites of hepatocytes. A small-molecule inhibitor of proteasomal activity blocks sporozoite invasion in an additive manner when combined with a Plasmodium PKG-specific inhibitor. Mining the previously described parasite PKG-dependent phosphoproteomes using the consensus phosphorylation motif identified additional proteins that are likely to be direct substrates of the enzyme.

Pre-freezing Treatment with Butylated Hydroxytoluene and Cholesterol-loaded Methyl-ß-cyclodextrin Improves Quality of Cryopreserved Boar Semen.

BACKGROUND: Pre-freezing treatment of boar sperm with additives improves the quality of post-thaw sperms. OBJECTIVES: The study aimed to determine the efficacy of butylated hydroxy-toluene (BHT) and cholesterol-loaded methyl-ß-cyclodextrin (CLC) for the improvement of the frozen-thawed boar sperm quality. METHODS: Split samples of 30 ejaculates from six boars were cryopreserved in lactose-egg yolk-glycerol extender containing BHT (0.2 mM), CLC (5 mg/ 200-240 x 106 sperm) or BHT (0.2 mM) plus CLC (5 mg per 200-240 x 106 sperm). Semen samples were evaluated for motility, membrane integrity, acrosomal status, mitochondrial membrane potential (MMP), DNA integrity and lipid peroxidation after equilibration and after freezing. RESULTS: The addition of BHT and CLC into the extender significantly improved (P<0.05) plasma membrane integrity and decreased (P<0.05) lipid peroxidation after freezing. Post-thaw motility and live intact acrosome were significantly (P<0.05) higher in the extenders with BHT or BHT plus CLC. The post-thaw MMP of viable spermatozoa and DNA integrity were not affected. BHT plus CLC showed a significant (P<0.05) improvement on motility as compared to BHT and CLC alone. CONCLUSION: Treatment of boar spermatozoa with BHT and CLC improved post-thaw sperm quality.

Effect of Reduced Glutathione, Water Soluble Vitamin E Analogue and Butylated Hydroxytoluene on the Post Thaw Characteristics of Boar Spermatozoa.

BACKGROUND: Antioxidant in freezing extender of boar semen improved post thaw sperm function. OBJECTIVE: The study compared the effects of reduced glutathione (GSH), water soluble vitamin E analogue Trolox and butylated hydroxytoluene (BHT) on quality of cryopreserved boar spermatozoa. MATERIALS AND METHODS: Using split sample technique three different antioxidants namely, GSH (1 mM), vitamin E (0.2 mM) and BHT (0.2 mM) were added to the freezing medium of lactose-egg yolk-glycerol extender, and samples were frozen using controlled freezing rate of 40°C/min from -6 to -140°C. Samples were evaluated for sperm motility, acrosomal status, plasma membrane integrity, mitochondrial membrane potential, lipid peroxidation and sperm DNA integrity after equilibration and after freezing. RESULTS: The supplementation of GSH, vitamin E and BHT resulted in significantly higher post thaw motility, live intact acrosome and plasma membrane intact sperm. The incidence of post thaw sperm lipid peroxidation was significantly reduced after addition of antioxidants. However, antioxidants treatment neither significantly improved mitochondrial membrane potential of live sperm sub-population nor sperm DNA integrity after freezing. There was no significant difference of the post thaw sperm characteristics among three antioxidants. Protective effect of GSH, vitamin E and BHT are comparable on cryopreserved boar spermatozoa.

Invasion of hepatocytes by Plasmodium sporozoites requires cGMP-dependent protein kinase and calcium dependent protein kinase 4.

Invasion of hepatocytes by sporozoites is essential for Plasmodium to initiate infection of the mammalian host. The parasite's subsequent intracellular differentiation in the liver is the first developmental step of its mammalian cycle. Despite their biological significance, surprisingly little is known of the signalling pathways required for sporozoite invasion. We report that sporozoite invasion of hepatocytes requires signalling through two second-messengers - cGMP mediated by the parasite's cGMP-dependent protein kinase (PKG), and Ca2+ , mediated by the parasite's calcium-dependent protein kinase 4 (CDPK4). Sporozoites expressing a mutated form of Plasmodium berghei PKG or carrying a deletion of the CDPK4 gene are defective in invasion of hepatocytes. Using specific and potent inhibitors of Plasmodium PKG and CDPK4, we demonstrate that PKG and CDPK4 are required for sporozoite motility, and that PKG regulates the secretion of TRAP, an adhesin that is essential for motility. Chemical inhibition of PKG decreases parasite egress from hepatocytes by inhibiting either the formation or release of merosomes. In contrast, genetic inhibition of CDPK4 does not significantly decrease the number of merosomes. By revealing the requirement for PKG and CDPK4 in Plasmodium sporozoite invasion, our work enables a better understanding of kinase pathways that act in different Plasmodium stages.