Broad Bean Stain Virus: Identification, Detectability with ELISA in Faba Bean Leaves and Seeds, Occurrence in West Asia and North Africa and Possible Wild Hosts

Abstract During a survey of faba bean viruses in West Asia and North Africa a virus was identified as broad bean stain virus (BBSV) based on host reactions, electron microscopy, physical properties and serology. An antiserum to a Syrian isolate was prepared. With this antiserum both the direct double antibody sandwich ELISA (DAS-ELISA) and dot-ELISA were very sensitive in detecting BBSV in leaf extracts, ground whole seeds and germi nated embryos. Sens it i vity was not reduced when the two-day procedure was replaced by a one-day procedure. us i ng ELISA the vi rus was detected in 73 out of 589 faba bean samples with virus-like symptoms collected from Egypt (4 out of 70 samples tested), Lebanon (6/44) , Morocco (017), Sudan (19/254), Syria (36/145) and Tunisia (8/69). This is the first report of BBSV infection of faba bean in Lebanon, Sudan, Syria and Tunisia. speci es i ndi genous to Syri a were Fourteen wild legume susceptible to BBSV infection, with only two producing obvious symptoms. The virus was found to be seed transmitted ~n Vicia palaestina.

Tuberculosis epidemiology in islands: insularity, hosts and trade

Because of their relative simplicity and the barriers to gene flow, islands are ideal systems to study the distribution of biodiversity. However, the knowledge that can be extracted from this peculiar ecosystem regarding epidemiology of economically relevant diseases has not been widely addressed. We used information available in the scientific literature for 10 old world islands or archipelagos and original data on Sicily to gain new insights into the epidemiology of the Mycobacterium tuberculosis complex (MTC). We explored three nonexclusive working hypotheses on the processes modulating bovine tuberculosis (bTB) herd prevalence in cattle and MTC strain diversity: insularity, hosts and trade. Results suggest that bTB herd prevalence was positively correlated with island size, the presence of wild hosts, and the number of imported cattle, but neither with isolation nor with cattle density. MTC strain diversity was positively related with cattle bTB prevalence, presence of wild hosts and the number of imported cattle, but not with island size, isolation, and cattle density. The three most common spoligotype patterns coincided between Sicily and mainland Italy. However in Sicily, these common patterns showed a clearer dominance than on the Italian mainland, and seven of 19 patterns (37%) found in Sicily had not been reported from continental Italy. Strain patterns were not spatially clustered in Sicily. We were able to infer several aspects of MTC epidemiology and control in islands and thus in fragmented host and pathogen populations. Our results point out the relevance of the intensity of the cattle commercial networks in the epidemiology of MTC, and suggest that eradication will prove more difficult with increasing size of the island and its environmental complexity, mainly in terms of the diversity of suitable domestic and wild MTC hosts.

Infecção experimental de Amblyomma spp. (Acari: Ixodidae) com Hepatozoon canis (Apicomplexa: Hepatozoidae) de cães naturalmente infectados

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ecologia química no controle biológico de fitófagos e respectivos predadores e parasitoides

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The Significance of Wild Plants in the Evolutionary Ecology of Three Major Viruses Infecting Cultivated Sweetpotato in Uganda

The studies presented in this thesis contribute to the understanding of evolutionary ecology of three major viruses threatening cultivated sweetpotato (Ipomoea batatas Lam) in East Africa: Sweet potato feathery mottle virus (SPFMV; genus Potyvirus; Potyviridae), Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus; Closteroviridae) and Sweet potato mild mottle virus (SPMMV; genus Ipomovirus; Potyviridae). The viruses were serologically detected and the positive results confirmed by RT-PCR and sequencing. SPFMV was detected in 24 wild plant species of family Convolvulacea (genera Ipomoea, Lepistemon and Hewittia), of which 19 species were new natural hosts for SPFMV. SPMMV and SPCSV were detected in wild plants belonging to 21 and 12 species (genera Ipomoea, Lepistemon and Hewittia), respectively, all of which were previously unknown to be natural hosts of these viruses. SPFMV was the most abundant virus being detected in 17% of the plants, while SPMMV and SPCSV were detected in 9.8% and 5.4% of the assessed plants, respectively. Wild plants in Uganda were infected with the East African (EA), common (C), and the ordinary (O) strains, or co-infected with the EA and the C strain of SPFMV. The viruses and virus-like diseases were more frequent in the eastern agro-ecological zone than the western and central zones, which contrasted with known incidences of these viruses in sweetpotato crops, except for northern zone where incidences were lowest in wild plants as in sweetpotato. The NIb/CP junction in SPMMV was determined experimentally which facilitated CP-based phylogenetic and evolutionary analyses of SPMMV. Isolates of all the three viruses from wild plants were genetically similar to those found in cultivated sweetpotatoes in East Africa. There was no evidence of host-driven population genetic structures suggesting frequent transmission of these viruses between their wild and cultivated hosts. The p22 RNA silencing suppressor-encoding sequence was absent in a few SPCSV isolates, but regardless of this, SPCSV isolates incited sweet potato virus disease (SPVD) in sweetpotato plants co-infected with SPFMV, indicating that p22 is redundant for synergism between SCSV and SPFMV. Molecular evolutionary analysis revealed that isolates of strain EA of SPFMV that is largely restricted geographically in East Africa experience frequent recombination in comparison to isolates of strain C that is globally distributed. Moreover, non-homologous recombination events between strains EA and C were rare, despite frequent co-infections of these strains in wild plants, suggesting purifying selection against non-homologous recombinants between these strains or that such recombinants are mostly not infectious. Recombination was detected also in the 5 - and 3 -proximal regions of the SPMMV genome providing the first evidence of recombination in genus Ipomovirus, but no recombination events were detected in the characterized genomic regions of SPCSV. Strong purifying selection was implicated on evolution of majority of amino acids of the proteins encoded by the analyzed genomic regions of SPFMV, SPMMV and SPCSV. However, positive selection was predicted on 17 amino acids distributed over the whole the coat protein (CP) in the globally distributed strain C, as compared to only 4 amino acids in the multifunctional CP N-terminus (CP-NT) of strain EA largely restricted geographically to East Africa. A few amino acid sites in the N-terminus of SPMMV P1, the p7 protein and RNA silencing suppressor proteins p22 and RNase3 of SPCSV were also submitted to positive selection. Positively selected amino acids may constitute ligand-binding domains that determine interactions with plant host and/or insect vector factors. The P1 proteinase of SPMMV (genus Ipomovirus) seems to respond to needs of adaptation, which was not observed with the helper component proteinase (HC-Pro) of SPMMV, although the HC-Pro is responsible for many important molecular interactions in genus Potyvirus. Because the centre of origin of cultivated sweetpotato is in the Americas from where the crop was dispersed to other continents in recent history (except for the Australasia and South Pacific region), it would be expected that identical viruses and their strains occur worldwide, presuming virus dispersal with the host. Apparently, this seems not to be the case with SPMMV, the strain EA of SPFMV and the strain EA of SPCSV that are largely geographically confined in East Africa where they are predominant and occur both in natural and agro-ecosystems. The geographical distribution of plant viruses is constrained more by virus-vector relations than by virus-host interactions, which in accordance of the wide range of natural host species and the geographical confinement to East Africa suggest that these viruses existed in East African wild plants before the introduction of sweetpotato. Subsequently, these studies provide compelling evidence that East Africa constitutes a cradle of SPFMV strain EA, SPCSV strain EA, and SPMMV. Therefore, sweet potato virus disease (SPVD) in East Africa may be one of the examples of damaging virus diseases resulting from exchange of viruses between introduced crops and indigenous wild plant species. Keywords: Convolvulaceae, East Africa, epidemiology, evolution, genetic variability, Ipomoea, recombination, SPCSV, SPFMV, SPMMV, selection pressure, sweetpotato, wild plant species Author s Address: Arthur K. Tugume, Department of Agricultural Sciences, Faculty of Agriculture and Forestry, University of Helsinki, Latokartanonkaari 7, P.O Box 27, FIN-00014, Helsinki, Finland. Email: tugume.arthur@helsinki.fi Author s Present Address: Arthur K. Tugume, Department of Botany, Faculty of Science, Makerere University, P.O. Box 7062, Kampala, Uganda. Email: aktugume@botany.mak.ac.ug, tugumeka@yahoo.com

Detection of Mycoplasma conjunctivae in the eyes of healthy, free-ranging Alpine ibex: possible involvement of Alpine ibex as carriers for the main causing agent of infectious keratoconjunctivitis in wild Caprinae

Mycoplasma conjunctivae is considered the major cause of infectious keratoconjunctivitis (IKC) in Alpine ibex (Capra i. ibex) and chamois (Rupicapra r. rupicapra). While it is known that domestic sheep can act as healthy carriers for M. conjunctivae, this question has not been addressed in wild ungulates so far. In this study, bacteriological investigations and field observations were performed to assess whether free-ranging Alpine ibex can be healthy carriers of M. conjunctivae. Among 136 ibex without clinical signs of IKC, M. conjunctivae was identified 26 times (19.1%) by TaqMan PCR. To assess the potential pathogenicity of M. conjunctivae strains isolated from asymptomatic eyes, strains from three healthy ibex and from 15 IKC-ibex and IKC-chamois were analysed genetically by DNA sequence analysis of the variable part of the lppS gene. No significant differences were observed between strains from asymptomatic and clinically affected animals, reflecting the assumption that healthy ibex may act as carriers for M. conjunctivae strains that may be pathogenic for other individuals. Our results further indicate that development of IKC is associated with M. conjunctivae load in the eyes. In addition, a questionnaire survey revealed that IKC is generally less common in ibex than chamois and that infection in wild ungulates is not necessarily linked to the presence of sheep. These data support the hypothesis that apparently healthy ibex may be important in the epizootiology of IKC and indicate that host predilection may play a role in IKC development.

Interaction of myxomatosis and Rabbit Haemorrhagic Disease in wild rabbit

Increasing resistance of rabbits to myxomatosis in Australia has led to the exploration of Rabbit Haemorrhagic Disease, also called Rabbit Calicivirus Disease (RCD) as a possible control agent. While the initial spread of RCD in Australia resulted in widespread rabbit mortality in affected areas, the possible population dynamic effects of RCD and myxomatosis operating within the same system have not been properly explored. Here we present early mathematical modelling examining the interaction between the two diseases. In this study we use a deterministic compartment model, based on the classical SIR model in infectious disease modelling. We consider, here, only a single strain of myxomatosis and RCD and neglect latent periods. We also include logistic population growth, with the inclusion of seasonal birth rates. We assume there is no cross-immunity due to either disease. The mathematical model allows for the possibility of both diseases to be simultaneously present in an individual, although results are also presented for the case where co infection is not possible, since co-infection is thought to be rare and questions exist as to whether it can occur. The simulation results of this investigation show that it is a crucial issue and should be part of future field studies. A single simultaneous outbreak of RCD and myxomatosis was simulated, while ignoring natural births and deaths, appropriate for a short timescale of 20 days. Simultaneous outbreaks may be more common in Queensland. For the case where co-infection is not possible we find that the simultaneous presence of myxomatosis in the population suppresses the prevalence of RCD, compared to an outbreak of RCD with no outbreak of myxomatosis, and thus leads to a less effective control of the population. The reason for this is that infection with myxomatosis removes potentially susceptible rabbits from the possibility of infection with RCD (like a vaccination effect). We found that the reduction in the maximum prevalence of RCD was approximately 30% for an initial prevalence of 20% of myxomatosis, for the case where there was no simultaneous outbreak of myxomatosis, but the peak prevalence was only 15% when there was a simultaneous outbreak of myxomatosis. However, this maximum reduction will depend on other parameter values chosen. When co-infection is allowed then this suppression effect does occur but to a lesser degree. This is because the rabbits infected with both diseases reduces the prevalence of myxomatosis. We also simulated multiple outbreaks over a longer timescale of 10 years, including natural population growth rates, with seasonal birth rates and density dependent(logistic) death rates. This shows how both diseases interact with each other and with population growth. Here we obtain sustained outbreaks occurring approximately every two years for the case of a simultaneous outbreak of both diseases but without simultaneous co-infection, with the prevalence varying from 0.1 to 0.5. Without myxomatosis present then the simulation predicts RCD dies out quickly without further introduction from elsewhere. With the possibility of simultaneous co-infection of rabbits, sustained outbreaks are possible but then the outbreaks are less severe and more frequent (approximately yearly). While further model development is needed, our work to date suggests that: 1) the diseases are likely to interact via their impacts on rabbit abundance levels, and 2) introduction of RCD can suppress myxomatosis prevalence. We recommend that further modelling in conjunction with field studies be carried out to further investigate how these two diseases interact in the population.

Survival of bottlenose dolphin (tursiops sp.) calves at a wild dolphin provisioning program, Tangalooma, Australia

Mortality of calves born to provisioned mothers is identified in the literature as an issue of concern in dolphin provisioning programs. Wild dolphin provisioning at Tangalooma, Moreton Island, Australia has been occurring since 1992. Each evening, up to eight dolphins are provided with fish in a regulated provisioning program. In this paper, calf survival at the Tangalooma provisioning program is reported and contrasted with that from wild populations and from a similar provisioning program at Monkey Mia, Western Australia. At Tangalooma, the calf survival rate is 100%, including both orphaned and first-born calves, both of which are expected to have relatively low survival rates. Possible explanations for the high calf survival rate are explored. These include site attributes such as isolated location and high water quality, aspects of foraging ecology likely to benefit calves of provisioned mothers, and the management regime used in the provisioning program (e.g., duration and timing of provisioning; quality of provisioned fish).

Plasma biochemistry and hematologic values for wild-caught flying foxes (Pteropus giganteus) in India

Although bats of the genus Pteropus are important ecologically as pollinators and natural hosts for zoonotic pathogens, little is known about their basic physiology. Hematology and plasma biochemistries were determined from wild-caught flying foxes (Pteropus giganteus) in northern India (n = 41). Mean lymphocyte differential count was higher for juveniles than adults. Mean platelet count was lower than previously reported. No hemoparasites were observed. No differences were observed between plasma biochemistry values of male and female bats, juveniles and adults, or lactating and nonlactating females. Variation in aspartate aminotransferase (AST) was seen based on body condition score. Blood urea nitrogen and cholesterol concentrations were lower in P. giganteus than other mammalian groups, but were consistent with those reported from other Pteropus species. Alanine aminotransferase and AST concentrations were higher than those reported for Pteropus vampyrus, a closely related species. This study provides basic physiologic information that can be used in future health and disease studies of Indian flying foxes.

Plant hosts of the phytoplasmas and rickettsia-like-organisms associated with strawberry lethal yellows and green petal diseases

Candidatus Phytoplasma australiense (Ca. P. australiense) is associated with the plant diseases strawberry lethal yellows (SLY), strawberry green petal (SGP), papaya dieback (PDB), Australian grapevine yellows (AGY) and Phormium yellow leaf (PYL; New Zealand). Strawberry lethal yellows disease is also associated with a rickettsia-like-organism (RLO) or infrequently with the tomato big bud (TBB) phytoplasma, the latter being associated with a wide range of plant diseases throughout Australia. In contrast, the RLO has been identified only in association with SLY disease, and Ca. P. australiense has been detected only in a limited number of plant host species. The aim of this study was to identify plant hosts that are possible reservoirs of Ca. P. australiense and the SLY RLO. Thirty-one plant species from south-east Queensland were observed with disease between 2001 and 2003 and, of these, 18 species tested positive using phytoplasma-specific primers. The RLO was detected in diseased Jacksonia scoparia and Modiola caroliniana samples collected at Stanthorpe. The TBB phytoplasma was detected in 16 different plant species and Ca. P. australiense Australian grapevine yellows strain was detected in six species. The TBB phytoplasma was detected in plants collected at Nambour, Stanthorpe, Warwick and Brisbane. Ca. P. australiense was detected in plants collected at Nambour, Stanthorpe, Gatton and Allora. All four phytoplasmas were detected in diseased Gomphocarpus physocarpus plants collected at Toowoomba, Allora, Nambour and Gatton. These results indicated that the vector(s) of Ca. P. australiense are distributed throughout south-east Queensland and the diversity of phytoplasmas detected in G. physocarpus suggests it is a feeding source for phytoplasma insect vectors or it has a broad susceptibility to a range of phytoplasmas.

How can warren destruction by ripping control European wild rabbits (Oryctolagus cuniculus) on large properties in the Australian arid zone?

Context: For over 100 years, control efforts have been unable to stop rabbits causing damage to cattle production and native plants and animals on large properties in arid parts of Australia. Warren destruction by ripping has shown promise, but doubts about long-term success and the perceived expense of treating vast areas have led to this technique not being commonly used. Aims: This study measured the long-term reduction in rabbit activity and calculated the potential cost saving associated with treating just the areas where rabbits are believed to survive drought. Wealso considered whether ripping should be used in a full-scale rabbit control program on a property where rabbits have been exceptionally resilient to the influence of biological and other control measures. Methods: Rabbits were counted along spotlight transects before warrens were ripped and during the two years after ripping, in treated and untreated plots. Rabbit activity was recorded to determine the immediate and long-term impact of ripping, up to seven years after treatment. The costs of ripping warrens within different distances from drought refuge areas were calculated. Key results: Destroying rabbit warrens by ripping caused an immediate reduction in rabbit activity and there were still 98% fewer rabbits counted by spotlight in ripped plots five months after ripping. Seven years after ripping no active warrens were found in ripped plots, whereas 57% of warrens in unripped plots showed signs of rabbit activity. The cost of ripping only the areas where rabbits were likely to seek refuge from drought was calculated to be less than 4%of the cost of ripping all warrens on the property. Conclusions: Destroying rabbit warrens by ripping is a very effective way of reducing rabbit numbers on large properties in arid Australia. Ripping should commence in areas used by rabbits to survive drought. It is possible that no further ripping will be required. Implications: Strategic destruction of warrens in drought refuge areas could provide an alternative to biological control for managing rabbits on large properties in the Australian arid zone.

Increased virulence of rabbit haemorrhagic disease virus associated with genetic resistance in wild Australian rabbits (Oryctolagus cuniculus)

The release of myxoma virus (MYXV) and Rabbit Haemorrhagic Disease Virus (RHDV) in Australia with the aim of controlling overabundant rabbits has provided a unique opportunity to study the initial spread and establishment of emerging pathogens, as well as their co-evolution with their mammalian hosts. In contrast to MYXV, which attenuated shortly after its introduction, rapid attenuation of RHDV has not been observed. By studying the change in virulence of recent field isolates at a single field site we show, for the first time, that RHDV virulence has increased through time, likely because of selection to overcome developing genetic resistance in Australian wild rabbits. High virulence also appears to be favoured as rabbit carcasses, rather than diseased animals, are the likely source of mechanical insect transmission. These findings not only help elucidate the co-evolutionary interaction between rabbits and RHDV, but reveal some of the key factors shaping virulence evolution.

Detection, epidemiology and host spectrum of cowpox and Borna disease virus infections

Several orthopoxviruses (OPV) and Borna disease virus (BDV) are enveloped, zoonotic viruses with a wide geographical distribution. OPV antibodies cross-react, and former smallpox vaccination has therefore protected human populations from another OPV infection, rodent-borne cowpox virus (CPXV). Cowpox in humans and cats usually manifests as a mild, self-limiting dermatitis and constitutional symptoms, but it can be severe and even life-threatening in the immunocompromised. Classical Borna disease is a progressive meningoencephalomyelitis in horses and sheep known in central Europe for centuries. Nowadays the virus or its close relative infects humans and also several other species in central Europe and elsewhere, but the existence of human Borna disease with its suspected neuropsychiatric symptoms is controversial. The epidemiology of BDV is largely unknown, and the present situation is even more intriguing following the recent detection of several-million-year-old, endogenized BDV genes in primate and various other vertebrate genomes. The aims of this study were to elucidate the importance of CPXV and BDV in Finland and in possible host species, and particularly to 1) establish relevant methods for the detection of CPXV and other OPVs as well as BDV in Finland, 2) determine whether CPXV and BDV exist in Finland, 3) discover how common OPV immunity is in different age groups in Finland, 4) characterize possible disease cases and clarify their epidemiological context, 5) establish the hosts and possible reservoir species of these viruses and their geographical distribution in wild rodents, and 6) elucidate the infection kinetics of BDV in the bank vole. An indirect immunofluorescence assay and avidity measurement were established for the detection, timing and verification of OPV or BDV antibodies in thousands of blood samples from humans, horses, ruminants, lynxes, gallinaceous birds, dogs, cats and rodents. The mostly vaccine-derived OPV seroprevalence was found to decrease gradually according to the year of birth of the sampled human subjects from 100% to 10% in those born after 1977. On the other hand, OPV antibodies indicating natural contact with CPXV or other OPVs were commonly found in domestic and wild animals: the horse, cow, lynx, dog, cat and, with a prevalence occasionally even as high as 92%, in wild rodents, including some previously undetected species and new regions. Antibodies to BDV were detected in humans, horses, a dog, cats, and for the first time in wild rodents, such as bank voles (Myodes glareolus). Because of the controversy within the human Borna disease field, extra verification methods were established for BDV antibody findings: recombinant nucleocapsid and phosphoproteins were produced in Escherichia coli and in a baculovirus system, and peptide arrays were additionally applied. With these verification assays, Finnish human, equine, feline and rodent BDV infections were confirmed. Taken together, wide host spectra were evident for both OPV and BDV infections based on the antibody findings, and OPV infections were found to be geographically broadly distributed. PCR amplification methods were utilised for hundreds of blood and tissue samples. The methods included conventional, nested and real-time PCRs with or without the reverse transcription step and detecting four or two genes of OPVs and BDV, respectively. OPV DNA could be amplified from two human patients and three bank voles, whereas no BDV RNA was detected in naturally infected individuals. Based on the phylogenetic analyses, the Finnish OPV sequences were closely related although not identical to a Russian CPXV isolate, and clearly different from other CPXV strains. Moreover, the Finnish sequences only equalled each other, but the short amplicons obtained from German rodents were identical to monkeypox virus, in addition to German CPXV variants. This reflects the close relationship of all OPVs. In summary, RNA of the Finnish BDV variant could not be detected with the available PCR methods, but OPV DNA infrequently could. The OPV species infecting the patients of this study was proven to be CPXV, which is most probably also responsible for the rodent infections. Multiple cell lines and some newborn rodents were utilised in the isolation of CPXV and BDV from patient and wildlife samples. CPXV could be isolated from a child with severe, generalised cowpox. BDV isolation attempts from rodents were unsuccessful in this study. However, in parallel studies, a transient BDV infection of cells inoculated with equine brain material was detected, and BDV antigens discovered in archival animal brains using established immunohistology. Thus, based on several independent methods, both CPXV and BDV (or a closely related agent) were shown to be present in Finland. Bank voles could be productively infected with BDV. This experimental infection did not result in notable pathological findings or symptoms, despite the intense spread of the virus in the central and peripheral nervous system. Infected voles commonly excreted the virus in urine and faeces, which emphasises their possible role as a BDV reservoir. Moreover, BDV RNA was regularly reverse transcribed into DNA in bank voles, which was detected by amplifying DNA by PCR without reverse transcription, and verified with nuclease treatments. This finding indicates that BDV genes could be endogenized during an acute infection. Although further transmission studies are needed, this experimental infection demonstrated that the bank vole can function as a potential BDV reservoir. In summary, multiple methods were established and applied in large panels to detect two zoonoses novel to Finland: cowpox virus and Borna disease virus. Moreover, new information was obtained on their geographical distribution, host spectrum, epidemiology and infection kinetics.