Comparison of interferon gamma release assay and CXCL9 gene expression assay for the detection of Mycobacterium bovis infection in African lions (Panthera leo).

Mycobacterium bovis (M. bovis) infection has been identified in both domestic and wild animals and may threaten the conservation of vulnerable species including African lions (Panthera leo). There is a need to develop accurate ante-mortem tools for detection of M. bovis infection in African big cat populations for wildlife management and disease surveillance. The aim of this study was to compare the performances of two immunological assays, the QuantiFERON®-TB Gold Plus (QFT) Mabtech Cat interferon gamma release assay (IGRA) and QFT CXCL9 gene expression assay (GEA), which have both shown diagnostic potential for M. bovis detection in African lions. Lion whole blood (n=47), stimulated using the QFT platform, was used for measuring antigen-specific CXCL9 expression and IFN-γ production and to assign M. bovis infection status. A subset (n=12) of mycobacterial culture-confirmed M. bovis infected and uninfected African lions was used to compare the agreement between the immunological diagnostic assays. There was no statistical difference between the proportions of test positive African lions tested by the QFT Mabtech Cat IGRA compared to the QFT CXCL9 GEA. There was also a moderate association between immunological diagnostic assays when numerical results were compared. The majority of lions had the same diagnostic outcome using the paired assays. Although the QFT Mabtech Cat IGRA provides a more standardized, commercially available, and cost-effective test compared to QFT CXCL9 GEA, using both assays to categorize M. bovis infection status in lions will increase confidence in results.

Endemic infection can shape exposure to novel pathogens: Pathogen co-occurrence networks in the Serengeti lions.

Pathogens are embedded in a complex network of microparasites that can collectively or individually alter disease dynamics and outcomes. Endemic pathogens that infect an individual in the first years of life, for example, can either facilitate or compete with subsequent pathogens thereby exacerbating or ameliorating morbidity and mortality. Pathogen associations are ubiquitous but poorly understood, particularly in wild populations. We report here on 10 years of serological and molecular data in African lions, leveraging comprehensive demographic and behavioural data to test if endemic pathogens shape subsequent infection by epidemic pathogens. We combine network and community ecology approaches to assess broad network structure and characterise associations between pathogens across spatial and temporal scales. We found significant non-random structure in the lion-pathogen co-occurrence network and identified both positive and negative associations between endemic and epidemic pathogens. Our results provide novel insights on the complex associations underlying pathogen co-occurrence networks.

Detection of Simonsiella spp. in the Vagina of Lions and Leopard in Oestrus.

Reports of the vaginal flora of wild cats such as lions or leopards are scarce. The micro-organisms most commonly found in the vagina of clinically healthy cats are aerobic bacteria such as coagulase-negative Staphylococcus, Streptococcus canis, and Escherichia coli. Simonsiella spp are large Gram-negative bacteria belonging to the Neisseriaceae family, typically found in the oral cavity and upper respiratory tract of many species. To date, there are no reports of the detection of Simonsiella spp in the vaginal flora of any felid. For a period of six months, daily behaviour monitoring was performed on six captive lionesses at a South African conservation centre, in parallel with the collection of vaginal swabs and interpretation of the resultant vaginal cytologies every other day. Oestrus was identified by typical female reproductive behaviours, as well as by enlarged and separated vulvar lips, and a predominant proportion of superficial cornified cells, clearing of the background, and high bacterial presence in the vaginal smear. Simonsiella spp were identified by their characteristic morphology in 58% (60 of 103) of the vaginal samples collected during oestrus. They were also found in oral swabs of three out of three lions tested. Additionally, Simonsiella spp were opportunistically found in a vaginal smear from a zoo housed female Sri Lankan leopard in oestrus, during a routine reproduction assessment. The finding of Simonsiella spp may be more common than previously suspected, transitory, and without detectable clinical relevance. A connection between occurrence of these bacteria and oestrus was apparent.

Population Genomics of Francisella tularensis subsp. holarctica and its Implication on the Eco-Epidemiology of Tularemia in Switzerland.

Whole genome sequencing (WGS) methods provide new possibilities in the field of molecular epidemiology. This is particularly true for monomorphic organisms where the discriminatory power of traditional methods (e.g., restriction enzyme length polymorphism typing, multi locus sequence typing etc.) is inadequate to elucidate complex disease transmission patterns, as well as resolving the phylogeny at high resolution on a micro-geographic scale. In this study, we present insights into the population structure of Francisella tularensis subsp. holarctica, the causative agent of tularemia in Switzerland. A total of 59 Fth isolates were obtained from castor bean ticks (Ixodes ricinus), animals and humans and a high resolution phylogeny was inferred using WGS methods. The majority of the Fth population in Switzerland belongs to the west European B.11 clade and shows an extraordinary genetic diversity underlining the old evolutionary history of the pathogen in the alpine region. Moreover, a new B.11 subclade was identified which was not described so far. The combined analysis of the epidemiological data of human tularemia cases with the whole genome sequences of the 59 isolates provide evidence that ticks play a pivotal role in transmitting Fth to humans and other vertebrates in Switzerland. This is further underlined by the correlation of disease risk estimates with climatic and ecological factors influencing the survival of ticks.

Progenitor strain introduction of Mycobacterium bovis at the wildlife-livestock interface can lead to clonal expansion of the disease in a single ecosystem.

Mycobacterium bovis infects multiple wildlife species and domesticated cattle across South Africa, and negatively impacts on livestock trade and movement of wildlife for conservation purposes. M. bovis infection was first reported in the Kruger National Park (KNP) in South Africa during the 1990s, and has since spread to infect numerous animal host species throughout the park and across South Africa. Whole genome sequencing data of 17 M. bovis isolates were analyzed to investigate the genomic diversity among M. bovis isolates causing disease in different animal host species from various locations in South Africa. M. bovis strains analyzed in this study are geographic rather than host species-specific. The clonal expansion of M. bovis in the KNP highlights the effect of an introduction of a transmissible infectious disease leading to a rising epidemic in wildlife, and emphasizes the importance of disease control and movement restriction of species that serve as disease reservoirs. In conclusion, the point source introduction of a single M. bovis strain type in the KNP ecosystem lead to an M. bovis outbreak in this area that affects various host species and poses an infection risk in neighboring rural communities where HIV prevalence is high.

Prevalence and Risk Factors for Mycobacterium bovis Infection in African Lions ( Panthera leo ) in the Kruger National Park.

Mycobacterium bovis, the causative agent of bovine tuberculosis (BTB), is endemic in the Kruger National Park (KNP), South Africa. African lions ( Panthera leo ) are susceptible to BTB, but the impact of the disease on lion populations is unknown. In this study, we used a novel gene expression assay for chemokine (C-X-C motif) ligand 9 (CXCL9) to measure the prevalence of M. bovis infection in 70 free-ranging lions that were opportunistically sampled in the southern and central regions of the KNP. In the southern region of the KNP, the apparent prevalence of M. bovis infection was 54% (95% confidence interval [CI]=36.9-70.5%), compared with 33% (95% CI=18.0-51.8%) in the central region, an important difference (P=0.08). Prevalence of M. bovis infection in lions showed similar patterns to estimated BTB prevalence in African buffaloes ( Syncerus caffer ) in the same areas. Investigation of other risk factors showed a trend for older lions, males, or lions with concurrent feline immunodeficiency virus infection to have a higher M. bovis prevalence. Our findings demonstrate that the CXCL9 gene expression assay is a useful tool for the determination of M. bovis status in free-ranging lions and identifies important epidemiologic trends for future studies.

Development of a Gene Expression Assay for the Diagnosis of Mycobacterium bovis Infection in African Lions (Panthera leo).

Mycobacterium bovis infection, the cause of bovine tuberculosis (BTB), is endemic in wildlife in the Kruger National Park (KNP), South Africa. In lions, a high infection prevalence and BTB mortalities have been documented in the KNP; however, the ecological consequences of this disease are currently unknown. Sensitive assays for the detection of this infection in this species are therefore required. Blood from M. bovis-exposed, M. bovis-unexposed, M. tuberculosis-exposed and M. bovis-infected lions was incubated in QuantiFERON® -TB Gold (QFT) tubes containing either saline or ESAT-6/CFP-10 peptides. Using qPCR, selected reference genes were evaluated for expression stability in these samples and selected target genes were evaluated as markers of antigen-dependent immune activation. The abundance of monokine induced by gamma interferon (MIG/CXCL9) mRNA, measured in relation to that of YWHAZ, was used as a marker of ESAT-6/CFP-10 sensitization. The gene expression assay results were compared between lion groups, and lenient and stringent diagnostic cut-off values were calculated. This CXCL9 gene expression assay combines a highly specific stimulation platform with a sensitive diagnostic marker that allows for discrimination between M. bovis-infected and M. bovis-uninfected lions.

Piometra em uma leoa (Panthera leo): relato de caso / Pyometra in a lion (Panthera leo): case report

A piometra é uma infecção aguda ou crônica do útero que ocorre frequentemente em cadelas não castradas, podendo também ocorrer em gatas domésticas e selvagens, sendo poucos os estudos relacionados à piometra em grandes felídeos. O objetivo deste relato foi descrever um caso de piometra em uma leoa (Panthera leo) de cativeiro, as lesões de necropsia e histológicas, bem como os resultados da análise microbiológica. Uma leoa com aproximadamente 23 anos, pertencente a um criadouro conservacionista de Santa Maria-RS, foi encontrada morta pela manhã em seu recinto. Após coleta de dados, procedeu-se à necropsia e à coleta de material para análise histopatológica e bacteriológica. A análise microbiológica revelou predomínio das bactérias Streptococcus sp. e Escherichia coli no conteúdo purulento do útero, caracterizando como piometra, e a bactéria predominante em plasma, fígado e medula óssea foi E. coli. De acordo com o laudo histopatológico, as alterações observadas nessa leoa sugerem um quadro de septicemia grave, sendo a origem do foco infeccioso bacteriano, provavelmente, a piometra. Considera-se importante chamar a atenção dos médicos veterinários de animais selvagens para um diagnóstico precoce dessa doença, que é comum em cadelas, mas que pode acometer também felídeos selvagens e levá-los à morte.

Pyometra is an acute or chronic uterus infection that occurs often in not spayed dogs, but may also occur in wild and domestic cats, and there are few studies related to pyometra in big cats. The aim of this report was to describe a case of pyometra in a lion (Panthera leo) in captivity, the gross and microscopic lesions found at necropsy, and the results of the microbiological analysis. A female with approximately 23 years of age, belonging to a Conservationist Breeding Center located in Santa Maria-RS-Brazil was found dead in her enclosure in the morning. After data collection, we proceeded to the autopsy and collection of material for histopathological and bacteriological analyzes. Microbiological analysis revealed a predominance of the bacteria Streptococcus sp and Escherichia coli in the uterus content, characterized as pyometra and the predominant bacterium in the plasma, liver and bone marrow was Escherichia coli. According to histopathology, the changes observed in this lioness suggest a framework of severe septicemia, being the source of the bacterial infection, probably, pyometra. It is considered important to draw the attention of zoo and wildlife veterinarians for an early diagnosis of this common disease in dogs, which can also affect large wild cats and lead them to death.

First evidence of hemoplasma infection in free-ranging Namibian cheetahs (Acinonyx jubatus).

Infections with feline hemotropic mycoplasmas (hemoplasmas) have been documented in domestic cats and free-ranging feline species with high prevalences in Iberian lynxes (Lynx pardinus), Eurasian lynxes (Lynx lynx), European wildcats (Felis silvestris silvestris), African lions (Panthera leo) in Tanzania and domestic cats in South Africa. The prevalence of hemoplasmas has not yet been investigated in free-ranging felids in southern Africa. In this study we screened 73 blood samples from 61 cheetahs in central Namibia for the presence of hemoplasmas using quantitative real-time PCR. One of the cheetahs tested PCR-positive. Phylogenetic analysis based on partial sequencing of the 16S rRNA and RNAse P genes revealed that the isolate belongs to the Mycoplasma haemofelis/haemocanis group. This is the first molecular evidence of a hemoplasma infection in a free-ranging cheetah.

Characterizing the next-generation matrix and basic reproduction number in ecological epidemiology.

We address the interaction of ecological processes, such as consumer-resource relationships and competition, and the epidemiology of infectious diseases spreading in ecosystems. Modelling such interactions seems essential to understand the dynamics of infectious agents in communities consisting of interacting host and non-host species. We show how the usual epidemiological next-generation matrix approach to characterize invasion into multi-host communities can be extended to calculate R0, and how this relates to the ecological community matrix. We then present two simple examples to illustrate this approach. The first of these is a model of the rinderpest, wildebeest, grass interaction, where our inferred dynamics qualitatively matches the observed phenomena that occurred after the eradication of rinderpest from the Serengeti ecosystem in the 1980s. The second example is a prey-predator system, where both species are hosts of the same pathogen. It is shown that regions for the parameter values exist where the two host species are only able to coexist when the pathogen is present to mediate the ecological interaction.

Development of a lion-specific interferon-gamma assay.

The ongoing spread of bovine tuberculosis (BTB) in African free-ranging lion populations, for example in the Kruger National Park, raises the need for diagnostic assays for BTB in lions. These, in addition, would be highly relevant for zoological gardens worldwide that want to determine the BTB status of their lions, e.g. for translocations. The present study concerns the development of a lion-specific IFN-γ assay, following the production and characterization of monoclonal antibodies specific for lion interferon-gamma (IFN-γ). Recombinant lion IFN-γ (rLIFN-γ) was produced in mammalian cells and used to immunize mice to establish hybridoma cell lines producing monoclonal antibodies. These were used to develop a sensitive, lion IFN-γ-specific capture ELISA, able to detect rLIFN-γ to the level of 160 pg/ml. Recognition of native lion IFN-γ was shown in an initial assessment of supernatants of mitogen stimulated whole blood cultures of 11 known BTB-negative lions. In conclusion, the capture ELISA shows potential as a diagnostic assay for bovine tuberculosis in lions. Preliminary results also indicate the possible use of the test for other (feline) species.

Assessing the impact of feline immunodeficiency virus and bovine tuberculosis co-infection in African lions.

Bovine tuberculosis (BTB), caused by Mycobacterium bovis, is a disease that was introduced relatively recently into the Kruger National Park (KNP) lion population. Feline immunodeficiency virus (FIV(ple)) is thought to have been endemic in lions for a much longer time. In humans, co-infection between Mycobacterium tuberculosis and human immunodeficiency virus increases disease burden. If BTB were to reach high levels of prevalence in lions, and if similar worsening effects would exist between FIV(ple) and BTB as for their human equivalents, this could pose a lion conservation problem. We collected data on lions in KNP from 1993 to 2008 for spatio-temporal analysis of both FIV(ple) and BTB, and to assess whether a similar relationship between the two diseases exists in lions. We found that BTB prevalence in the south was higher than in the north (72 versus 19% over the total study period) and increased over time in the northern part of the KNP (0-41%). No significant spatio-temporal differences were seen for FIV(ple) in the study period, in agreement with the presumed endemic state of the infection. Both infections affected haematology and blood chemistry values, FIV(ple) in a more pronounced way than BTB. The effect of co-infection on these values, however, was always less than additive. Though a large proportion (31%) of the lions was co-infected with FIV(ple) and M. bovis, there was no evidence for a synergistic relation as in their human counterparts. Whether this results from different immunopathogeneses remains to be determined.

Inbreeding depression increases susceptibility to bovine tuberculosis in lions: an experimental test using an inbred-outbred contrast through translocation.

Disease can dramatically influence the dynamics of endangered wildlife populations, especially when they are small and isolated, with increased risk of inbreeding. In Hluhluwe-iMfolozi Park (HiP), a small, enclosed reserve in South Africa, a large lion (Panthera leo) population arose from a small founder group in the 1960s and started showing conspicuous signs of inbreeding. To restore the health status of the HiP lion population, outbred lions were translocated into the existing population. In this study, we determined the susceptibility to bovine tuberculosis (bTB), and the prevalence of antibody to feline viruses of native lions, and compared the findings with those from translocated outbred lions and their offspring. Antibodies to feline herpesvirus, feline calicivirus, feline parvovirus, and feline coronavirus were present in the lion population, but there was no significant difference in antibody prevalence between native and translocated lions and their offspring, and these feline viruses did not appear to have an effect on the clinical health of HiP lions. However, feline immunodeficiency virus (FIV), which was previously absent from HiP, appears to have been introduced into the lion population through translocation. Within 7 yr, the prevalence of antibody to FIV increased up to 42%. Bovine tuberculosis posed a major threat to the inbred native lion population, but not to translocated lions and their offspring. More than 30% of the native lion population died from bTB or malnutrition compared with <2% of the translocated lions and their offspring. We have demonstrated that management of population genetics through supplementation can successfully combat a disease that threatens population persistence. However, great care must be taken not to introduce new diseases into populations through translocation.

Persistence of Yersinia pestis in soil under natural conditions.

As part of a fatal human plague case investigation, we showed that the plague bacterium, Yersinia pestis, can survive for at least 24 days in contaminated soil under natural conditions. These results have implications for defining plague foci, persistence, transmission, and bioremediation after a natural or intentional exposure to Y. pestis.

Properties of corynephage attachment site and molecular epidemiology of Corynebacterium ulcerans isolated from humans and animals in Japan.

Sporadic reports of Corynebacterium ulcerans infection in humans and animals have become increasingly common throughout the world. Between 2001 and 2006, five human cases, in addition to isolation of the bacterium from the carcasses of Orcinus orca and Panthera leo, were reported in Japan. While an isolate from P. leo generated only phospholipase D (PLD), the other isolates produced both PLD and diphtheria-like toxin (DLT). Pulsed-field gel electrophoresis analysis showed that isolates from P. leo and humans were genetically homologous. Southern blotting found that a human isolate was lysogenized by two corynephages coding DLT. Sequence analysis of the region of the DLT gene revealed that the integration in C. ulcerans occurred in the same manner as that in C. diphtheriae.

Molecular detection of "Candidatus Mycoplasma haemominutum" in a lion (Panthera leo) from a Brazilian zoological garden.

Although Mycoplasma haemofelis and "Candidatus Mycoplasma haemominutum" infections have been reported in wild cats from United States, their presence among native and captive wild cats in Brazil is still unknown. A 12 year old healthy male lion (Panthera leo) from the Zoological Garden of Curitiba, Brazil was anesthetized for transportation and dental evaluation. A blood sample was obtained for a complete blood cell count (CBC) and PCR analysis. DNA was extracted and fragments of Mycoplasma haemofelis and "Candidatus Mycoplasma haemominutum" 16S ribosomal RNA gene were amplified in PCR assays. CBC results were within reference intervals. A weak band of 192 pb for "Candidatus Mycoplasma haemominutum" was observed, and no band was amplified from Mycoplasma haemofelis reaction. A weak PCR band associated with normal CBC results and without visible parasitemia or clinical signs may suggest a chronic subclinical infection with "Candidatus Mycoplasma haemominutum". The lack of clinical signs may also represent the low pathogenicity of this organism; however, it is noteworthy that immune suppression caused by management and/or corticoids treatment may induce parasitemia and anemia in this animal. This detection suggests further studies in captive wild cats in Brazilian Zoological Gardens.

Molecular detection of "Candidatus Mycoplasma haemominutum" in a lion (Panthera leo) from a brazilian zoological garden

Although Mycoplasma haemofelis and "Candidatus Mycoplasma haemominutum" infections have been reported in wild cats from United States, their presence among native and captive wild cats in Brazil is still unknown. A 12 year old healthy male lion (Panthera leo) from the Zoological Garden of Curitiba, Brazil was anesthetized for transportation and dental evaluation. A blood sample was obtained for a complete blood cell count (CBC) and PCR analysis. DNA was extracted and fragments of Mycoplasma haemofelis and "Candidatus Mycoplasma haemominutum" 16S ribosomal RNA gene were amplified in PCR assays. CBC results were within reference intervals. A weak band of 192 pb for "Candidatus Mycoplasma haemominutum" was observed, and no band was amplified from Mycoplasma haemofelis reaction. A weak PCR band associated with normal CBC results and without visible parasitemia or clinical signs may suggest a chronic subclinical infection with "Candidatus Mycoplasma haemominutum". The lack of clinical signs may also represent the low pathogenicity of this organism; however, it is noteworthy that immune suppression caused by management and/or corticoids treatment may induce parasitemia and anemia in this animal. This detection suggests further studies in captive wild cats in Brazilian Zoological Gardens.

Embora a infecção por Mycoplasma haemofelis e "Candidatus Mycoplasma haemominutum" tenha sido reportada em felinos selvagens dos Estados Unidos, sua presença entre felinos selvagens de vida livre e de cativeiro no Brasil ainda é desconhecida. Um leão macho, saudável, com 12 anos de idade, residente no Zoológico de Curitiba, Brasil, foi anestesiado para transporte e avaliação dentária. Uma amostra de sangue foi coletada para a realização do hemograma completo e análise pela Reação em Cadeia da Polimerase (PCR). O DNA foi extraído e fragmentos do gene 16SrRNA do Mycoplasma haemofelis e "Candidatus Mycoplasma haemominutum" foram submetidos à metodologia da PCR. O hemograma apresentou valores normais. Uma banda de baixa intensidade de aproximadamente 192 pb do "Candidatus Mycoplasma haemominutum" foi detectada, e nenhuma banda foi observada pela PCR na detecção de Mycoplasma haemofelis. A banda de baixa intensidade, o hemograma normal e a ausência de parasitemia e sinais clínicos podem sugerir uma infecção crônica subclínica por "Candidatus Mycoplasma haemominutum". A ausência de sinais clínicos pode também indicar a baixa patogenicidade desse microrganismo; entretanto, a imunossupressão por manejo e/ou tratamento com corticóides podem levar a parasitemia e conseqüente anemia neste animal. Este achado sugere novos estudos em felinos selvagens de cativeiro em zoológicos brasileiros.

Worldwide occurrence of feline hemoplasma infections in wild felid species.

While hemoplasma infections in domestic cats are well studied, almost no information is available on their occurrence in wild felids. The aims of the present study were to investigate wild felid species as possible reservoirs of feline hemoplasmas and the molecular characterization of the hemoplasma isolates. Blood samples from the following 257 wild felids were analyzed: 35 Iberian lynxes from Spain, 36 Eurasian lynxes from Switzerland, 31 European wildcats from France, 45 lions from Tanzania, and 110 Brazilian wild felids, including 12 wild felid species kept in zoos and one free-ranging ocelot. Using real-time PCR, feline hemoplasmas were detected in samples of the following species: Iberian lynx, Eurasian lynx, European wildcat, lion, puma, oncilla, Geoffroy's cat, margay, and ocelot. "Candidatus Mycoplasma haemominutum" was the most common feline hemoplasma in Iberian lynxes, Eurasian lynxes, Serengeti lions, and Brazilian wild felids, whereas "Candidatus Mycoplasma turicensis" was the most prevalent in European wildcats; hemoplasma coinfections were frequently observed. Hemoplasma infection was associated with species and free-ranging status of the felids in all animals and with feline leukemia virus provirus-positive status in European wildcats. Phylogenetic analyses of the 16S rRNA and the partial RNase P gene revealed that most hemoplasma isolates exhibit high sequence identities to domestic cat-derived isolates, although some isolates form different subclusters within the phylogenetic tree. In conclusion, 9 out of 15 wild felid species from three different continents were found to be infected with feline hemoplasmas. The effect of feline hemoplasma infections on wild felid populations needs to be further investigated.

Investigation of Livingstone's curious point.

The explorer and missionary David Livingstone was identified after death by the appearances of his humerus which had been damaged in an encounter with a lion. In his writings Livingstone suggested that the consequences of the lion's attack were not as bad as he might have expected. He wondered if this was due to the fact that he was wearing a tartan jacket when he was attacked and suggested that this curious point should be investigated. This paper looks at some of the dyes used in tartans of the time and investigates their effect on the bacteria that might be present in the mouths of lions.