Identification of alternative hosts to citrus of “Candidatus Liberibacter africanus” amongst indigenous Rutaceae of South Africa

Citrus greening or Huanglongbing is a severe disease affecting all citrus cultivars and rootstocks and is responsible for causing severe economic losses in many citrus producing countries. It is caused by various fastidious phloem-restricted, Gram negative bacteria belonging to the “Candidatus Liberibacter” genus. Currently only three species that affects citrus have been identified, “Candidatus Liberibacter africanus” (Laf) found in Africa, Mascarene Islands and the Middle East,“Candidatus Liberibacter asiaticus” (Las) present in Asia, and only recently emerged in Florida USA, São Paulo Brazil and Cuba and a new species “Candidatus Liberibacter americanus” (Lam) found only in São Paulo Brazil. Due to the continuing spread of this serious and destructive disease into areas of South Africa previously regarded as disease-free and disease-managed, the role of alternative hosts in the transmission of the disease is questioned. The aim of this study was to identify possible alternate hosts of Laf amongst plants of Rutaceae indigenous to South Africa. Identification of these alternate hosts forms a critical part of the integrated management and control strategies of the disease. Leaf material from symptomatic and asymptomatic indigenous rutaceous and non-rutaceous plants were collected from various sites in South Africa, from different environments which included vegetation adjacent to citrus orchards, in areas with high incidence of the citrus greening disease, from natural habitats, botanical gardens and private properties. A multiplex polymerase chain reaction (PCR) was developed for the simultaneous detection of Laf DNA and ubiquitous ribulose bisphosphate carboxylase oxygenase (Rubisco) gene which served as an internal control for the presence and quality of extracted DNA and potential amplification inhibition by compounds of the DNA extract. The multiplex PCR oligonucleotide primers were used to amplify fragments of 669bp from the β ribosomal operon of Laf and 179bp of Rubisco gene. Petioles and/or leaf midrib tissues were tested for the presence of Laf using the newly developed multiplex PCR and β operon specific primers of a published conventional PCR method. In this study seventeen Calodendrum capense Thunb. (Cape chestnut) plants from various geographic areas of South Africa were identified to be naturally infected with Liberibacter DNA. Sequence analysis revealed the DNA to be that of “Candidatus Liberibacter africanus subspecies capensis” (LafC). Although this subspecies of Liberibacter has previously been detected on C. capense in the Stellenbosch area of the Western Cape Province, this is the first report indicating the natural occurrence of LafC in C. capense in Gauteng, Limpopo and Mpumalanga Provinces of South Africa. The geographic distribution of the LafC bacterium associated with Cape chestnut appears to be more widespread than initially expected. No Laf DNA was detected from C.capense or any other indigenous plants surveyed. CopyrightDissertation (MSc)--University of Pretoria, 2010.Microbiology and Plant Pathologyunrestricte

Widespread occurrence of "Candidatus liberibacter africanus subspecies capensis" in Calodendrum capense in South Africa

Recent studies in citrus orchards confirmed that Citrus Greening, a heat sensitive citrus disease, similar to Huanglongbing (HLB), is associated with the presence of “Candidatus Liberibacter africanus” (Laf) in South Africa. Neither “Candidatus Liberibacter asiaticus” (Las), associated with HLB, “Candidatus Liberibacter americanus”, nor “Candidatus Liberibacter africanus ssp. capensis” (LafC), previously detected in the Western Cape, South Africa on an indigenous Rutaceous species, Calodendrum capense (L. f.) Thunb. (Cape Chestnut), were detected in citrus. The current study aims to determine the potential role of C. capense in the epidemiology of Citrus Greening in South Africa and whether LafC poses a risk to citriculture. A total of 278C. capense samples were collected throughout South Africa and tested for Liberibacters using realtime PCR. While LafC was found in 100 samples, distributed from all areas where collected, no evidence of Laf infection in any sample was found . The identity of the LafC present was confirmed by sequencing the amplicon derived from conventional PCR of the ßoperon of the ribosomal protein gene region of the first 17 infected trees found and of a representative sample from each district. The Liberibacter status of 44C. capense and 272 citrus (Midnight Valencia) trees growing in close proximity to each other for over 15 years was determined. Out of 44C. capense specimens, 43 were infected with LafC, but none of the citrus trees were infected with LafC. Based on the results of this it appears that natural spread of LafC to citrus does not occur.Citrus Research International (CRI), Nelspruithttp://www.springerlink.com /content/100265

Spatio-temporal epidemiology of animal and human rabies in northern South Africa between 1998 and 2017

BACKGROUND : Rabies is a fatal zoonotic disease that is maintained in domestic dogs and wildlife populations in the Republic of South Africa. A retrospective study was conducted to improve understanding of the dynamics of rabies in humans, domestic dogs, and wildlife species, in relation to the ecology for three northern provinces of South Africa (Limpopo, Mpumalanga, and North-West) between 1998 and 2017. METHODS : A descriptive epidemiology study was conducted for human and animal rabies. Dog rabies cases were analyzed using spatio-temporal scan statistics. The reproductive number (Rt) was estimated for the identified disease clusters. A phylogenetic tree was constructed based on the genome sequences of rabies viruses isolated from dogs, jackals, and an African civet, and Bayesian evolutionary analysis using a strict time clock model. Several ecological and socio-economic variables associated with dog rabies were modeled using univariate analyses with zero-inflated negative binomial regression and multivariable spatial analyses using the integrated nested Laplace approximation for two time periods: 1998– 2002 and 2008–2012. RESULTS : Human rabies cases increased in 2006 following an increase in dog rabies cases; however, the human cases declined in the next year while dog rabies cases fluctuated. Ten disease clusters of dog rabies were identified, and utilizing the phylogenetic tree, the dynamics of animal rabies over 20 years was elucidated. In 2006, a virus strain that re-emerged in eastern Limpopo Province caused the large and persistent dog rabies outbreaks in Limpopo and Mpumalanga Provinces. Several clusters included a rabies virus variant maintained in jackals in Limpopo Province, and the other variant in dogs widely distributed. The widely distributed variant maintained in jackal populations in North-West Province caused an outbreak in dogs in 2014. The Rt was high when the disease clusters were associated with either multiple virus strains or multiple animal species. High-risk areas included Limpopo and Mpumalanga Provinces characterized by woodlands and high temperatures and precipitation. CONCLUSION : Canine rabies was maintained mainly in dog populations but was also associated with jackal species. Rural communities in Limpopo and Mpumalanga Provinces were at high risk of canine rabies originating from dogs.The 2019 Japan Society for the Promotion of Science (JSPS) and National Research Foundation (NRF) of South Africa bilateral exchange program grant.https://journals.plos.org/plosntdsdm2022Geography, Geoinformatics and MeteorologyVeterinary Tropical Disease

Rabies of canid biotype in wild dog (Lycaon pictus) and spotted hyaena (Crocuta crocuta) in Madikwe Game Reserve, South Africa in 2014–2015 : diagnosis, possible origins and implications for control

Both domestic and wild carnivore species are commonly diagnosed with rabies virus (RABV) infection in South Africa. Although the majority of confirmed rabies cases in wild carnivore species are reported from the yellow mongoose (Cynictis penicillata), the rest are from other wild carnivores including the highly endangered wild dog (Lycaon pictus). Lyssavirus infection was confirmed in two wild dogs and a spotted hyaena (Crocuta crocuta) in the Madikwe Game Reserve, North West province in South Africa, in 2014 and 2015, using a direct fluorescent antibody test and immunohistochemistry. There had been no new wild dog introductions to the Madikwe Game Reserve for many years and the wild dogs were last vaccinated against rabies approximately 11 years prior to the incident. The first euthanised wild dog was the last surviving of a break-away pack of 6, and the second was the last of a larger pack of 18, the rest of which died with no carcasses being found or carcasses too decomposed for sampling. Subsequent antigenic typing of the lyssaviruses indicated that they were canid RABVs. The RABVs originating from 22 wild carnivore species, 7 dogs, and a caprine, mostly from the North West province, were genetically characterised by targeting a partial region of the nucleoprotein gene. The nucleotide sequence analyses of these viruses and two previously characterised RABVs confirmed that the outbreak viruses were also canid rabies, phylogenetically clustering with virus isolates originating from black-backed jackals recovered between 2012 and 2015 from the North West province, and domestic dogs from neighbouring communal areas. The source(s) of the mortalities and possible reservoir host(s) for the virus could only be speculated upon from data on specific predator numbers, movements and behaviour, kills, park management and the changing environmental ecology, which were monitored closely in Madikwe over several years. The most likely rabies sources were from boundary fence contacts between wild carnivores within the park, with domestic dogs or cats and/or naturally occurring wild carnivores outside the park. The associated risk of zoonotic infection and threat to important and endangered predators may be mitigated through regional rabies control primarily in domestic dogs and cats, as well as by preventative vaccination of at-risk park employees and their pets. The importance of ongoing prophylactic rabies protection by regular vaccination of highly endangered wildlife carnivores and the submission of carcasses for rabies diagnosis of any wild or domestic animals behaving uncharacteristically or found dead is emphasised.http://www.jsava.co.zaParaclinical Science

Engineering, expression in transgenic plants and characterisation of e559, a rabies virus-neutralising monoclonal antibody.

Rabies post-exposure prophylaxis (PEP) currently comprises administration of rabies vaccine together with rabies immunoglobulin (RIG) of either equine or human origin. In the developing world, RIG preparations are expensive, often in short supply, and of variable efficacy. Therefore, we are seeking to develop a monoclonal antibody cocktail to replace RIG. Here, we describe the cloning, engineering and production in plants of a candidate monoclonal antibody (E559) for inclusion in such a cocktail. The murine constant domains of E559 were replaced with human IgG1κ constant domains and the resulting chimeric mouse-human genes were cloned into plant expression vectors for stable nuclear transformation of Nicotiana tabacum. The plant-expressed, chimeric antibody was purified and biochemically characterized, was demonstrated to neutralize rabies virus in a fluorescent antibody virus neutralization assay, and conferred protection in a hamster challenge model

Rabies in the African Civet: An Incidental Host for Lyssaviruses?

In South Africa, canid rabies virus (RABV) infection is maintained in domestic and wildlife species. The identification of rabies in African civets raised the question of whether this wildlife carnivore is a potential reservoir host of RABVs of direct and ancestral dog origin (dog-maintained and dog-derived origins) with an independent cycle of transmission. Genetic analyses of African civet nucleoprotein sequences for 23 African civet RABVs and historically published sequences demonstrated that RABVs from African civets have two origins related to dog and mongoose rabies enzootics. The data support observations of the interaction of civets with domestic dogs and wildlife mongooses, mostly in Northern South Africa and North-East Zimbabwe. Within each host species clade, African civet RABVs group exclusively together, implying intra-species virus transfer occurs readily. The canid RABV clade appears to support virus transfer more readily between hosts than mongoose RABVs. Furthermore, these data probably indicate short transmission chains with conspecifics that may be related to transient rabies maintenance in African civets. Hence, it is important to continue monitoring the emergence of lyssaviruses in this host. Observations from this study are supported by ongoing and independent similar cases, in which bat-eared foxes and black-backed jackal species maintain independent rabies cycles of what were once dog-maintained RABVs

Complete Coding Sequences of 23 South African Domestic and Wildlife Rabies Viruses

South African rabies viruses originating from dogs and jackals (canid viruses) are closely related and highlight cross-species transmission events between the two canine species. Rabies due to the canid lyssavirus variant is a significant public health matter in this country. The complete coding sequences of 23 canid lyssaviruses from South Africa are reported here

Rabies of canid biotype in wild dog (<i>Lycaon pictus</i>) and spotted hyaena (<i>Crocuta crocuta</i>) in Madikwe Game Reserve, South Africa in 2014–2015: Diagnosis, possible origins and implications for control

Both domestic and wild carnivore species are commonly diagnosed with rabies virus (RABV) infection in South Africa. Although the majority of confirmed rabies cases in wild carnivore species are reported from the yellow mongoose (Cynictis penicillata), the rest are from other wild carnivores including the highly endangered wild dog (Lycaon pictus). Lyssavirus infection was confirmed in two wild dogs and a spotted hyaena (Crocuta crocuta) in the Madikwe Game Reserve, North West province in South Africa, in 2014 and 2015, using a direct fluorescent antibody test and immunohistochemistry. There had been no new wild dog introductions to the Madikwe Game Reserve for many years and the wild dogs were last vaccinated against rabies approximately 11 years prior to the incident. The first euthanised wild dog was the last surviving of a break-away pack of 6, and the second was the last of a larger pack of 18, the rest of which died with no carcasses being found or carcasses too decomposed for sampling. Subsequent antigenic typing of the lyssaviruses indicated that they were canid RABVs. The RABVs originating from 22 wild carnivore species, 7 dogs, and a caprine, mostly from the North West province, were genetically characterised by targeting a partial region of the nucleoprotein gene. The nucleotide sequence analyses of these viruses and two previously characterised RABVs confirmed that the outbreak viruses were also canid rabies, phylogenetically clustering with virus isolates originating from black-backed jackals recovered between 2012 and 2015 from the North West province, and domestic dogs from neighbouring communal areas. The source(s) of the mortalities and possible reservoir host(s) for the virus could only be speculated upon from data on specific predator numbers, movements and behaviour, kills, park management and the changing environmental ecology, which were monitored closely in Madikwe over several years. The most likely rabies sources were from boundary fence contacts between wild carnivores within the park, with domestic dogs or cats and/or naturally occurring wild carnivores outside the park. The associated risk of zoonotic infection and threat to important and endangered predators may be mitigated through regional rabies control primarily in domestic dogs and cats, as well as by preventative vaccination of at-risk park employees and their pets. The importance of ongoing prophylactic rabies protection by regular vaccination of highly endangered wildlife carnivores and the submission of carcasses for rabies diagnosis of any wild or domestic animals behaving uncharacteristically or found dead is emphasised

Complete coding sequences of 23 South African domestic and wildlife rabies viruses

South African rabies viruses originating from dogs and jackals (canid viruses) are closely related and highlight cross-species transmission events between the two canine species. Rabies due to the canid lyssavirus variant is a significant public health matter in this country. The complete coding sequences of 23 canid lyssaviruses from South Africa are reported here.The National Research Foundation (NRF) of South Africa, ARC and the EU Framework Horizon 2020 Innovation Grant, European Virus Archive (EVAg).https://mra.asm.orgam2021Veterinary Tropical Disease