Katja V. Goller

A HEPATOZOON SPECIES GENETICALLY DISTINCT FROM H. CANIS INFECTING SPOTTED HYENAS IN THE SERENGETI ECOSYSTEM, TANZANIA

Health monitoring of spotted hyenas (Crocuta crocuta) in the Serengeti ecosystem, Tanzania, revealed Hepatozoon infection in all of 11 immature individuals examined following death from natural causes. Hepatozoon infection was probably an important factor contributing to mortality in two cases that exhibited clinical signs of ataxia, lethargy, ocular discharge, retching, and labored breathing before death. Whether Hepatozoon infection contributed to six deaths from fire, probable lion predation and unknown causes could not be determined. Four deaths from infanticide and starvation were unlikely to be associated with Hepatozoon infection. Histologic examination revealed lung tissue infected with cyst-like structures containing protozoan stages in all eight cases examined and interstitial pneumonia in most cases. Systemic spread of infection to several organs was found in three cases. Alignment of a 426 bp sequence from the parasites 18s rRNA gene revealed a Hepatozoon species identical to that recently described from two domestic cats in Spain and only 7 bp substitutions when a 853 bp sequence was aligned to this cat Hepatozoon species. Previous reports of infection of wild carnivores in eastern and southern Africa with an unspecified Hepatozoon species similar in appearance to H. canis may have involved the species described in this study.

Genetic variability and distribution of Classical swine fever virus.

Abstract Classical swine fever is a highly contagious disease that affects domestic and wild pigs worldwide. The causative agent of the disease is Classical swine fever virus (CSFV), which belongs to the genus Pestivirus within the family Flaviviridae. On the genome level, CSFV can be divided into three genotypes with three to four sub-genotypes. Those genotypes can be assigned to distinct geographical regions. Knowledge about CSFV diversity and distribution is important for the understanding of disease dynamics and evolution, and can thus help to design optimized control strategies. For this reason, the geographical pattern of CSFV diversity and distribution are outlined in the presented review. Moreover, current knowledge with regard to genetic virulence markers or determinants and the role of the quasispecies composition is discussed.

Out of the Reservoir: Phenotypic and Genotypic Characterization of a Novel Cowpox Virus Isolated from a Common Vole

ABSTRACT The incidence of human cowpox virus (CPXV) infections has increased significantly in recent years. Serological surveys have suggested wild rodents as the main CPXV reservoir. We characterized a CPXV isolated during a large-scale screening from a feral common vole. A comparison of the full-length DNA sequence of this CPXV strain with a highly virulent pet rat CPXV isolate showed a sequence identity of 96%, including a large additional open reading frame (ORF) of about 6,000 nucleotides which is absent in the reference CPXV strain Brighton Red. Electron microscopy analysis demonstrated that the vole isolate, in contrast to the rat strain, forms A-type inclusion (ATI) bodies with incorporated virions, consistent with the presence of complete ati and p4c genes. Experimental infections showed that the vole CPXV strain caused only mild clinical symptoms in its natural host, while all rats developed severe respiratory symptoms followed by a systemic rash. In contrast, common voles infected with a high dose of the rat CPXV showed severe signs of respiratory disease but no skin lesions, whereas infection with a low dose led to virus excretion with only mild clinical signs. We concluded that the common vole is susceptible to infection with different CPXV strains. The spectrum ranges from well-adapted viruses causing limited clinical symptoms to highly virulent strains causing severe respiratory symptoms. In addition, the low pathogenicity of the vole isolate in its eponymous host suggests a role of common voles as a major CPXV reservoir, and future research will focus on the correlation between viral genotype and phenotype/pathotype in accidental and reservoir species. IMPORTANCE We report on the first detection and isolation of CPXV from a putative reservoir host, which enables comparative analyses to understand the infection cycle of these zoonotic orthopox viruses and the relevant genes involved. In vitro studies, including whole-genome sequencing as well as in vivo experiments using the Wistar rat model and the vole reservoir host allowed us to establish links between genomic sequences and the in vivo properties (virulence) of the novel vole isolate in comparison to those of a recent zoonotic CPXV isolated from pet rats in 2009. Furthermore, the role of genes present only in a reservoir isolate can now be further analyzed. These studies therefore allow unique insights and conclusions about the role of the rodent reservoir in CPXV epidemiology and transmission and about the zoonotic threat that these viruses represent.

The impact of a pathogenic bacterium on a social carnivore population

Summary 1. The long‐term ecological impact of pathogens on group‐living, large mammal populations is largely unknown. We evaluated the impact of a pathogenic bacterium, Streptococcus equi ruminatorum, and other key ecological factors on the dynamics of the spotted hyena Crocuta crocuta population in the Ngorongoro Crater, Tanzania. 2. We compared key demographic parameters during two years when external signs of bacterial infection were prevalent (‘outbreak’) and periods of five years before and after the outbreak when such signs were absent or rare. We also tested for density dependence and calculated the basic reproductive rate R 0 of the bacterium. 3. During the five pre‐outbreak years, the mean annual hyena mortality rate was 0·088, and annual population growth was relatively high (13·6%). During the outbreak, mortality increased by 78% to a rate of 0·156, resulting in an annual population decline of 4·3%. After the outbreak, population size increased moderately (5·1%) during the first three post‐outbreak years before resuming a growth similar to pre‐outbreak levels (13·9%). We found no evidence that these demographic changes were driven by density dependence or other ecological factors. 4. Most hyenas showed signs of infection when prey abundance in their territory was low. During the outbreak, mortality increased among adult males and yearlings, but not among adult females – the socially dominant group members. These results suggest that infection and mortality were modulated by factors linked to low social status and poor nutrition. During the outbreak, we estimated R 0 for the bacterium to be 2·7, indicating relatively fast transmission. 5. Our results suggest that the short‐term ‘top–down’ impact of S. equi ruminatorum during the outbreak was driven by ‘bottom–up’ effects on nutritionally disadvantaged age–sex classes, whereas the longer‐term post‐outbreak reduction in population growth was caused by poor survival of juveniles during the outbreak and subsequent poor recruitment of breeding females. These results suggest synergistic effects of ‘bottom–up’ and ‘top–down’ processes on host population dynamics.

Canine distemper virus in the Serengeti ecosystem: molecular adaptation to different carnivore species.

Was the 1993/1994 fatal canine distemper virus (CDV) epidemic in lions and spotted hyaenas in the Serengeti ecosystem caused by the recent spillover of a virulent domestic dog strain or one well adapted to these noncanids? We examine this question using sequence data from 13 ‘Serengeti’ strains including five complete genomes obtained between 1993 and 2011. Phylogenetic and haplotype network analyses reveal that strains from noncanids during the epidemic were more closely related to each other than to those from domestic or wild canids. All noncanid ‘Serengeti’ strains during the epidemic encoded: (1) one novel substitution G134S in the CDV‐V protein; and (2) the rare amino acid combination 519I/549H at two sites under positive selection in the region of the CDV‐H protein that binds to SLAM (CD 150) host cell receptors. Worldwide, only a few noncanid strains in the America II lineage encode CDV‐H 519I/549H. All canid ‘Serengeti’ strains during the epidemic coded CDV‐V 134G, and CDV‐H 519R/549Y, or 519R/549H. A functional assay of cell entry revealed the highest performance by CDV‐H proteins encoding 519I/549H in cells expressing lion SLAM receptors, and the highest performance by proteins encoding 519R/549Y, typical of dog strains worldwide, in cells expressing dog SLAM receptors. Our findings are consistent with an epidemic in lions and hyaenas caused by CDV variants better adapted to noncanids than canids, but not with the recent spillover of a dog strain. Our study reveals a greater complexity of CDV molecular epidemiology in multihost environments than previously thought.

Divergent Sapovirus Strains and Infection Prevalence in Wild Carnivores in the Serengeti Ecosystem: A Long-Term Study

The genus Sapovirus, in the family Caliciviridae, includes enteric viruses of humans and domestic animals. Information on sapovirus infection of wildlife is limited and is currently lacking for any free-ranging wildlife species in Africa. By screening a large number of predominantly fecal samples (n = 631) obtained from five carnivore species in the Serengeti ecosystem, East Africa, sapovirus RNA was detected in the spotted hyena (Crocuta crocuta, family Hyaenidae), African lion (Panthera leo, family Felidae), and bat-eared fox (Otocyon megalotis, family Canidae), but not in golden or silver-backed jackals (Canis aureus and C. mesomelas, respectively, family Canidae). A phylogenetic analysis based on partial RNA-dependent RNA polymerase (RdRp) gene sequences placed the sapovirus strains from African carnivores in a monophyletic group. Within this monophyletic group, sapovirus strains from spotted hyenas formed one independent sub-group, and those from bat-eared fox and African lion a second sub-group. The percentage nucleotide similarity between sapoviruses from African carnivores and those from other species was low (< 70.4%). Long-term monitoring of sapovirus in a population of individually known spotted hyenas from 2001 to 2012 revealed: i) a relatively high overall infection prevalence (34.8%); ii) the circulation of several genetically diverse variants; iii) large fluctuations in infection prevalence across years, indicative of outbreaks; iv) no significant difference in the likelihood of infection between animals in different age categories. The likelihood of sapovirus infection decreased with increasing hyena group size, suggesting an encounter reduction effect, but was independent of socially mediated ano-genital contact, or the extent of the area over which an individual roamed.

Classical swine fever virus marker vaccine strain CP7_E2alf: genetic stability in vitro and in vivo

Recently, CP7_E2alf (Suvaxyn®CSF Marker), a live marker vaccine against classical swine fever virus, was licensed through the European Medicines Agency. For application of such a genetically engineered virus under field conditions, knowledge about its genetic stability is essential. Here, we report on stability studies that were conducted to assess and compare the mutation rate of CP7_E2alf in vitro and in vivo. Sequence analyses upon passaging confirmed the high stability of CP7_E2alf, and no recombination events were observed in the experimental setup. The data obtained in this study confirm the genetic stability of CP7_E2alf as an important safety component.

Does the Vastness of the Serengeti Limit Human–Wildlife Conflicts?

In this chapter we discuss whether the vastness of the Serengeti National Park (SNP) in northern Tanzania and associated protected areas minimises three forms of human–wildlife conflict: livestock predation by carnivores, bushmeat hunting and pathogen transmission between domestic animals and wildlife. The SNP covers 14,763 km2 and is surrounded by protected areas that form buffer-zones between the park and local people. This vast network of protected areas covers the majority of the 25,500 km2 Serengeti ecosystem; an area defined by the migratory movements of over one million herbivores. None of the protected areas are fenced. Fences would be detrimental to the ecosystem. We begin by discussing livestock predation in rural areas west of the SNP. Current low levels of livestock predation do not generate strong resentment among farmers. Predation levels will probably remain low provided there is adequate protection of migratory herbivore species. Next we detail the illegal harvest of bushmeat for home consumption and trade. Currently, hunters mostly work on foot; thus, buffer-zones form an effective distance barrier to wildlife deep within the SNP. This barrier will be diminished if hunters start using vehicles and if a proposed road through the north of the SNP is constructed. Finally, we consider disease transmission between domestic animals and wildlife and the efficacy of non-physical vaccine-induced barriers of immunity against disease spread. Mass vaccination of cattle against rinderpest has successfully eliminated this disease in wild ungulate species in the SNP. Although, vaccine-induced immunity barriers against rabies in domestic dogs recently failed to prevent epidemic rabies in domestic dogs surrounding the ecosystem, rabies did not spread to wild carnivores inside the SNP, suggesting that demographic and ecological factors within the SNP prevented the spread of epidemic rabies from domestic dogs into the park. Recent “silent” waves of canine distemper virus (CDV) infection in wild carnivores inside the SNP suggest that mass vaccination of domestic dogs have either failed to prevent CDV spreading to wildlife, or have controlled CDV in the surrounding domestic dog population but have not controlled cycles of CDV inside the SNP, suggesting that CDV is maintained in wild carnivore species and not in domestic dogs outside the park.

Development of a duplex lateral flow assay for simultaneous detection of antibodies against African and Classical swine fever viruses

Classical swine fever (CSF) and African swine fever (ASF) are both highly contagious diseases of domestic pigs and wild boar and are clinically indistinguishable. For both diseases, antibody detection is an integral and crucial part of prevention and control measures. The purpose of our study was to develop and initially validate a duplex pen-side test for simultaneous detection and differentiation of specific antibodies against CSF virus (CSFV) and ASF virus (ASFV). The test was based on the major capsid protein VP72 of ASFV and the structural protein E2 of CSFV, both considered the most immunogenic proteins of these viruses. The performance of the pen-side test was evaluated using a panel of porcine samples consisting of experimental, reference, and field sera, with the latter collected from European farms free of both diseases. The new lateral flow assay was able to detect specific antibodies to ASFV or CSFV, showing good levels of sensitivity and specificity. These preliminary data indicate the potential of the newly developed pen-side test for rapid differential detection of antibodies found in the 2 diseases, which is of particular importance in the field and in front-line laboratories where equipment and skilled personnel are limited and control of ASF and CSF is crucial.