Kathryn J. Allan

Identification and characterization of a novel non-structural protein of bluetongue virus.

Bluetongue virus (BTV) is the causative agent of a major disease of livestock (bluetongue). For over two decades, it has been widely accepted that the 10 segments of the dsRNA genome of BTV encode for 7 structural and 3 non-structural proteins. The non-structural proteins (NS1, NS2, NS3/NS3a) play different key roles during the viral replication cycle. In this study we show that BTV expresses a fourth non-structural protein (that we designated NS4) encoded by an open reading frame in segment 9 overlapping the open reading frame encoding VP6. NS4 is 77–79 amino acid residues in length and highly conserved among several BTV serotypes/strains. NS4 was expressed early post-infection and localized in the nucleoli of BTV infected cells. By reverse genetics, we showed that NS4 is dispensable for BTV replication in vitro, both in mammalian and insect cells, and does not affect viral virulence in murine models of bluetongue infection. Interestingly, NS4 conferred a replication advantage to BTV-8, but not to BTV-1, in cells in an interferon (IFN)-induced antiviral state. However, the BTV-1 NS4 conferred a replication advantage both to a BTV-8 reassortant containing the entire segment 9 of BTV-1 and to a BTV-8 mutant with the NS4 identical to the homologous BTV-1 protein. Collectively, this study suggests that NS4 plays an important role in virus-host interaction and is one of the mechanisms played, at least by BTV-8, to counteract the antiviral response of the host. In addition, the distinct nucleolar localization of NS4, being expressed by a virus that replicates exclusively in the cytoplasm, offers new avenues to investigate the multiple roles played by the nucleolus in the biology of the cell.

Reassortment between Two Serologically Unrelated Bluetongue Virus Strains Is Flexible and Can Involve any Genome Segment

ABSTRACT Coinfection of a cell by two different strains of a segmented virus can give rise to a “reassortant” with phenotypic characteristics that might differ from those of the parental strains. Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) segmented virus and the cause of bluetongue, a major infectious disease of livestock. BTV exists as at least 26 different serotypes (BTV-1 to BTV-26). Prompted by the isolation of a field reassortant between BTV-1 and BTV-8, we systematically characterized the process of BTV reassortment. Using a reverse genetics approach, our study clearly indicates that any BTV-1 or BTV-8 genome segment can be rescued in the heterologous “backbone.” To assess phenotypic variation as a result of reassortment, we examined viral growth kinetics and plaque sizes in in vitro experiments and virulence in an experimental mouse model of bluetongue disease. The monoreassortants generated had phenotypes that were very similar to those of the parental wild-type strains both in vitro and in vivo. Using a forward genetics approach in cells coinfected with BTV-1 and BTV-8, we have shown that reassortants between BTV-1 and BTV-8 are generated very readily. After only four passages in cell culture, we could not detect wild-type BTV-1 or BTV-8 in any of 140 isolated viral plaques. In addition, most of the isolated reassortants contained heterologous VP2 and VP5 structural proteins, while only 17% had homologous VP2 and VP5 proteins. Our study has shown that reassortment in BTV is very flexible, and there is no fundamental barrier to the reassortment of any genome segment. Given the propensity of BTV to reassort, it is increasingly important to have an alternative classification system for orbiviruses.

Epidemiology of Leptospirosis in Africa: A Systematic Review of a Neglected Zoonosis and a Paradigm for ‘One Health’ in Africa

Background Leptospirosis is an important but neglected bacterial zoonosis that has been largely overlooked in Africa. In this systematic review, we aimed to summarise and compare current knowledge of: (1) the geographic distribution, prevalence, incidence and diversity of acute human leptospirosis in Africa; and (2) the geographic distribution, host range, prevalence and diversity of Leptospira spp. infection in animal hosts in Africa. Methods Following Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines, we searched for studies that described (1) acute human leptospirosis and (2) pathogenic Leptospira spp. infection in animals. We performed a literature search using eight international and regional databases for English and non-English articles published between January 1930 to October 2014 that met out pre-defined inclusion criteria and strict case definitions. Results and Discussion We identified 97 studies that described acute human leptospirosis (n = 46) or animal Leptospira infection (n = 51) in 26 African countries. The prevalence of acute human leptospirosis ranged from 2 3% to 19 8% (n = 11) in hospital patients with febrile illness. Incidence estimates were largely restricted to the Indian Ocean islands (3 to 101 cases per 100,000 per year (n = 6)). Data from Tanzania indicate that human disease incidence is also high in mainland Africa (75 to 102 cases per 100,000 per year). Three major species (Leptospira borgpetersenii, L. interrogans and L. kirschneri) are predominant in reports from Africa and isolates from a diverse range of serogroups have been reported in human and animal infections. Cattle appear to be important hosts of a large number of Leptospira serogroups in Africa, but few data are available to allow comparison of Leptospira infection in linked human and animal populations. We advocate a ‘One Health’ approach to promote multidisciplinary research efforts to improve understanding of the animal to human transmission of leptospirosis on the African continent.

Urban leptospirosis in Africa: a cross-sectional survey of Leptospira infection in rodents in the Kibera urban settlement, Nairobi, Kenya.

Leptospirosis is a widespread but under-reported cause of morbidity and mortality. Global re-emergence of leptospirosis has been associated with the growth of informal urban settlements in which rodents are thought to be important reservoir hosts. Understanding the multi-host epidemiology of leptospirosis is essential to control and prevent disease. A cross-sectional survey of rodents in the Kibera settlement in Nairobi, Kenya was conducted in September–October 2008 to demonstrate the presence of pathogenic leptospires. A real-time quantitative polymerase chain reaction showed that 41 (18.3%) of 224 rodents carried pathogenic leptospires in their kidneys, and sequence data identified Leptospira interrogans and L. kirschneri in this population. Rodents of the genus Mus (37 of 185) were significantly more likely to be positive than those of the genus Rattus (4 of 39; odds ratio = 15.03). Questionnaire data showed frequent contact between humans and rodents in Kibera. This study emphasizes the need to quantify the public health impacts of this neglected disease at this and other urban sites in Africa.

Endemic zoonoses in the tropics: a public health problem hiding in plain sight

Zoonotic diseases are a significant burden on animal and human health, particularly in developing countries. Despite recognition of this fact, endemic zoonoses often remain undiagnosed in people, instead being mistaken for febrile diseases such as malaria. Here, as part of Veterinary Records ongoing series of articles on One Health, a multidisciplinary team of researchers from Scotland, Tanzania and New Zealand argues that a One Health approach is needed to effectively combat these diseases

Meningeal cholesterol granulomas in two meerkats (Suricata suricatta)

CHOLESTEROL granulomas form as a result of an inflammatory foreign body response to the accumulation of cholesterol crystals within tissues (Leon and others 2002). In human beings, cholesterol granulomas are an important intracranial lesion, and occur most commonly in association with chronic middle ear disease. Although they are reported in the choroid plexus of the lateral and fourth ventricles in up to 20 per cent of horses at postmortem examination (Jackson and others 1994), cholesterol granulomas are rare findings in other animals. In dogs, they have been reported in the middle ear, maxillary sinus and fourth ventricle and cerebellopontine angle of the brain (Mould 1990, O’Brien and others 1990, Cox and Payne-Johnson 1995). A cholesterol granuloma has been described in association with meningioma in a cat (Schulman 2005). Cholesterol granulomas have also been reported in the intracranial meninges of two meerkats (Suricata suricatta; family Herpestidae) at separate zoos in the USA (Schulman 1997, Sladsky and others 2000). This short communication describes two cases of meningeal cholesterol granuloma in meerkats from collections in the UK. Two meerkats were submitted for postmortem examination to the Royal Veterinary College within a period of 17 months. Case 1, a male meerkat aged three years and five months, was submitted in May 2003 from a private collection in Hertfordshire. The animal was described as off-colour before its death but no blood samples had been taken. Case 2 was submitted in October 2004 from a small zoo in Wales. This adult male meerkat had initially presented two months before its death with marked ataxia, which slowly improved but did not resolve. This animal’s serum cholesterol level had been 11·9 mmol/l in March 2003. An intracranial cholesterolbased mass was suspected, and steps were taken to reduce the level of cholesterol in the animal’s diet. Despite this, the animal developed left-sided weakness 10 days before death, which progressed to hemiplegia; it was humanely euthanased. In both cases, there was a large (3·5 x 1·5 x 1·0 to 1·5 cm and 3·0 x 3·5 x 1 to 1·5 cm), adherent, brown, granular meningeal mass occupying most of the dorsal surfaces of the cerebral hemispheres (Fig 1). Examination of cytological smears confirmed the presence of cholesterol crystals. Brain sections showed compression of the cerebral parenchyma, and in case 2 the ventricular spaces of the brain were dilated. Representative specimens from the brains and other tissues were fixed in 10 per cent neutral-buffered formalin, processed by routine methods for histology and embedded in paraffin wax. Glass slide-mounted sections (5 μm) were stained with haematoxylin and eosin, and further slides were stained for iron using Perl’s Prussian blue. Formalin-fixed specimens of the meningeal mass from case 2 were snap-frozen, cryosectioned and stained with Schultz stain for cholesterol. In both animals the cerebral leptomeninges were markedly expanded by a large, irregularly shaped mass composed of densely packed clusters of cholesterol clefts surrounded by foreign body-type multinucleate histiocytic giant cells and foamy macrophages (Fig 2); many of the macrophages contained haemosiderin, revealed in sections stained with Perl’s Prussian blue. The Schultz reaction for cholesterol was positive in case 2. There was marked cerebral compression and distortion, with multifocal invasion of the Virchow-Robin spaces. Multifocal haemorrhage was noted inside the granulomas, and within the superficial cerebral cortex in case 2. A section of the calvarium from case 2 showed marked invasion and disruption of bone, with mild reactive bone formation. In case 1, smaller, similar masses expanded the pericardium. Other postmortem findings in this animal included intestinal salmonellosis, hepatic lipidosis, mineralisation of the bronchiolar walls and lymphoid hyperplasia of the mandibular lymph node. In case 2, glomerulonephritis, glossitis, gastritis, hepatic lipidosis and lymphocytic thyroiditis were also noted. Based on the gross and histopathological findings, meningeal cholesterol granuloma was diagnosed in both animals. In human beings, one person out of every two to three million is affected by intracranial cholesterol granulomas per year (Francis and others 2005). Cholesterol granulomas have been found in the intracranial dura in human patients, but the incidence in the meninges of any species is not known as they have rarely been reported (Leon and others 2002). The typical histological appearance of densely packed cholesterol clefts with an associated granulomatous inflammatory reaction is diagnostic (Leon and others 2002).Ventricular dilation (as seen in case 2) and hydrocephalus may result from increased intracranial pressure and obstructed flow of cerebrospinal fluid as the mass develops within the skull (Johnson and others 1993). Adjacent bone erosion was noted in case 2 and in one meerkat in a previous report (Sladsky and others 2000), and is often seen in human beings suffering from cholesterol granulomas in the sinus cavities (Leon and others 2002). In the USA, meningeal cholesterol granulomas have been reported in a meerkat from the North Carolina Zoological Park (NCZP) and a second animal in a Californian collection, from which tissues were submitted to the Armed Forces Institute of Pathology (Schulman 1997, Sladsky and others 2000). In the UK there was an unreported case in an adult male meerkat at Cotswold Wildlife Park in 2004 (P. Kettlewell, personal communication). Neurological signs were noted in all the previous cases of meningeal cholesterol granuloma in meerkats but not in case 1 in the present report, which may have been due to poor observation. Other tissues reported to Veterinary Record (2006) 158, 636-637

One Health contributions towards more effective and equitable approaches to health in low- and middle-income countries

Emerging zoonoses with pandemic potential are a stated priority for the global health security agenda, but endemic zoonoses also have a major societal impact in low-resource settings. Although many endemic zoonoses can be treated, timely diagnosis and appropriate clinical management of human cases is often challenging. Preventive ‘One Health’ interventions, e.g. interventions in animal populations that generate human health benefits, may provide a useful approach to overcoming some of these challenges. Effective strategies, such as animal vaccination, already exist for the prevention, control and elimination of many endemic zoonoses, including rabies, and several livestock zoonoses (e.g. brucellosis, leptospirosis, Q fever) that are important causes of human febrile illness and livestock productivity losses in low- and middle-income countries. We make the case that, for these diseases, One Health interventions have the potential to be more effective and generate more equitable benefits for human health and livelihoods, particularly in rural areas, than approaches that rely exclusively on treatment of human cases. We hypothesize that applying One Health interventions to tackle these health challenges will help to build trust, community engagement and cross-sectoral collaboration, which will in turn strengthen the capacity of fragile health systems to respond to the threat of emerging zoonoses and other future health challenges. One Health interventions thus have the potential to align the ongoing needs of disadvantaged communities with the concerns of the broader global community, providing a pragmatic and equitable approach to meeting the global goals for sustainable development and supporting the global health security agenda. This article is part of the themed issue ‘One Health for a changing world: zoonoses, ecosystems and human well-being’.

Advances and challenges in barcoding pathogenic and environmental Leptospira

Leptospirosis is a zoonotic bacterial disease of global importance. A large spectrum of asymptomatic animal hosts can carry the infection and contribute to the burden of human disease. Environmental sources of human contamination also point to the importance of a hydrotelluric reservoir. Leptospirosis can be caused by as many as 15 different pathogenic or intermediate Leptospira species. However, classification of these bacteria remains complicated through the use of both serological and genetic classification systems that show poor correlation. With the advent of molecular techniques, DNA-based barcoding offers a conceptual framework that can be used for leptospirosis surveillance as well as source tracking. In this review, we summarize some of the current techniques, highlight significant successes and weaknesses and point to the future opportunities and challenges to successfully establish a widely applicable barcoding scheme for Leptospira.

Comparison of the Estimated Incidence of Acute Leptospirosis in the Kilimanjaro Region of Tanzania between 2007-08 and 2012-14.

Background The sole report of annual leptospirosis incidence in continental Africa of 75–102 cases per 100,000 population is from a study performed in August 2007 through September 2008 in the Kilimanjaro Region of Tanzania. To evaluate the stability of this estimate over time, we estimated the incidence of acute leptospirosis in Kilimanjaro Region, northern Tanzania for the time period 2012–2014. Methodology and Principal Findings Leptospirosis cases were identified among febrile patients at two sentinel hospitals in the Kilimanjaro Region. Leptospirosis was diagnosed by serum microscopic agglutination testing using a panel of 20 Leptospira serovars belonging to 17 separate serogroups. Serum was taken at enrolment and patients were asked to return 4–6 weeks later to provide convalescent serum. Confirmed cases required a 4-fold rise in titre and probable cases required a single titre of ≥800. Findings from a healthcare utilisation survey were used to estimate multipliers to adjust for cases not seen at sentinel hospitals. We identified 19 (1.7%) confirmed or probable cases among 1,115 patients who presented with a febrile illness. Of cases, the predominant reactive serogroups were Australis 8 (42.1%), Sejroe 3 (15.8%), Grippotyphosa 2 (10.5%), Icterohaemorrhagiae 2 (10.5%), Pyrogenes 2 (10.5%), Djasiman 1 (5.3%), Tarassovi 1 (5.3%). We estimated that the annual incidence of leptospirosis was 11–18 cases per 100,000 population. This was a significantly lower incidence than 2007–08 (p<0.001). Conclusions We estimated a much lower incidence of acute leptospirosis than previously, with a notable absence of cases due to the previously predominant serogroup Mini. Our findings indicate a dynamic epidemiology of leptospirosis in this area and highlight the value of multi-year surveillance to understand leptospirosis epidemiology.

Renewing the momentum for leptospirosis research in Africa

Leptospirosis is one of the most widespread and pervasive zoonotic bacterial diseases worldwide. Current global estimates indicate that more than 500 000 cases of human disease occur annually and that under-reporting is likely to be substantial especially in endemic regions.1 Human leptospirosis, caused by infection with pathogenic Leptospira bacteria, can range from a mild, self-limiting or subclinical infection, to a severe, life-threatening disease.2 Patients typically present with sudden onset, non-specific febrile illness, which progresses to a severe, life-threatening disease in approximately 10% of cases. Multiple organ systems can be affected and disease manifestations include icterus, renal failure and haemorrhage (Weils disease), severe pulmonary haemorrhage syndrome (SPHS) or meningitis. The reported case fatality ratio for icteric disease ranges from 5–15% but estimates are higher (>50%) for patients with SPHS.3