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.

Molecular identification of hookworm isolates from stray dogs, humans and selected wildlife from South Africa.

There is a paucity of information on hookworm species in humans, domestic animals and wildlife in southern Africa. Our study aimed to identify hookworm species from stray dogs, humans, and selected wildlife from South Africa. A total of 356 faecal samples were screened for the presence of hookworm-like eggs and subsequently coproculture from the positive samples was carried out to obtain larvae. Hookworm-like eggs were detected in 23.03% (82/356) of samples. Of these samples, 78/296 were from dogs, 3/50 from humans and 1/10 from wildlife. DNA was then isolated from the larvae of 55 positive samples, which were subjected to polymerase chain reaction (PCR), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing of the nuclear ribosomal internal transcribed spacer (ITS1) and 5.8S rRNA region. Presence of Ancylostoma caninum, A. braziliense and A. ceylanicum-like species was recorded in stray dogs and A. caninum was recorded in wildlife and humans, using PCR-RFLP. Although PCR-RFLP results pointed to the presence of A. ceylanicum, we did not get a sequence that matched with A. ceylanicum from GenBank. This may have been due to the low proportion of A. ceylanicum larvae in our samples. Twenty-two of the 27 positive amplicons from stray dogs matched with A. caninum, three with A. braziliense and two had mixed infections of A. braziliense and A. caninum. Sequences from a lion and three humans matched with A. caninum. This is the first confirmation of a patent A. caninum infection in humans as evidenced by the presence of eggs in faeces, with the subsequent larvae from coproculture being identified as A. caninum.

Molecular and Morphologic Identification of Spirometra ranarum Found in the Stool of African Lion, Panthera leo in the Serengeti Plain of Tanzania.

The present study was performed with morphological and molecular analysis (cox1 and nad1 mitochondrial genes) to identify the proglottids of spirometrid tapeworm found in the stool of an African lion, Panthera leo, in the Serengeti plain of Tanzania. A strand of tapeworm strobila, about 75 cm in length, was obtained in the stool of a male African lion in the Serengeti National Park (34˚ 50' E, 02˚ 30' S), Tanzania, in February 2012. The morphological features of the adult worm examined exhibited 3 uterine coils with a bow tie appearance and adopted a diagonal direction in the second turn. The posterior uterine coils are larger than terminal uterine ball and the feature of uteri are swirling rather than spirally coiling. The sequence difference between the Spirometra species (Tanzania origin) and S. erinaceieuropaei (GenBank no. KJ599680) was 9.4% while those of S. decipiens (GenBank no. KJ599679) differed by 2.1% in the cox1 and nad1 genes. Phylogenetic tree topologies generated using the 2 analytic methods were identical and presented high level of confidence values for the 3 major branches of the 3 Spirometra species in the cox1 gene. The morphological and molecular findings obtained in this study were nearly coincided with those of S. ranarum. Therefore, we can know for the first time that the African lion, Panthera leo, is to the definitive host of this tapeworm.

RETROSPECTIVE EVALUATION OF A NOVEL SUSTAINED-RELEASE IVERMECTIN VARNISH FOR TREATMENT OF WOUND MYIASIS IN ZOO-HOUSED ANIMALS.

Myiasis is a major disease condition in human and veterinary medicine. Domestic, free-ranging, and zoo-housed animals can be severely affected by myiasis. Depending on case severity, multiple treatment episodes may be indicated and can lead to recurrent capturing, handling stress, and anesthetics, all of which increase the risk of adverse responses (including death) individually and also in the herd. As an insecticide, ivermectin is often used for larval control. A total of 28 individual myiasis cases were retrospectively evaluated, out of which 11 cases were also treated using an ivermectin sustained-release varnish (SRV). The clinical outcome of all cases was assessed and the results suggest that the use of a topical ivermectin SRV (with or without concurrent injectable ivermectin) can reduce handling and treatments, has no adverse effects, and has minimal recurrence of the disease when compared with cases treated without it.

SURVEILLANCE FOR VIRAL AND PARASITIC PATHOGENS IN A VULNERABLE AFRICAN LION (PANTHERA LEO) POPULATION IN THE NORTHERN TULI GAME RESERVE, BOTSWANA.

African lion ( Panthera leo ) numbers are decreasing rapidly and populations are becoming smaller and more fragmented. Infectious diseases are one of numerous issues threatening free-ranging lion populations, and low-density populations are particularly at risk. We collected data on the prevalence and diversity of viral and parasitic pathogens in a small lion population in eastern Botswana. During 2012 and 2014, blood samples were collected from 59% (n=13) of the adult-subadult lions in the Northern Tuli Game Reserve in eastern Botswana. One lion had antibodies to feline panleukopenia virus, two had antibodies to canine distemper virus, and two had feline calicivirus antibodies. Ten of the 13 had antibodies to feline immunodeficiency virus and 11 had feline herpesvirus antibodies. All lions were negative for antibodies to feline coronavirus. Blood samples from all lions were negative for Trypanosoma, Anaplasma, Theileria, and Ehrlichia spp. by molecular testing; however, all lions were positive for Babesia spp. by reverse line blot hybridization assay. Sequencing of amplicons from four lions revealed four groups of Babesia spp. including several genetic variants of Babesia felis , Babesia lengau, and Babesia canis and a group of novel Babesia sequences which were only 96% similar to other Babesia spp. Six lions were infested with four species of ticks (Rhipicentor nuttalli, Rhipicephalus simus, Rhipicephalus sulcatus, and Rhipicephalus appendiculatus). These data provide the first health assessment of this population and can be used to identify management and conservation strategies to decrease the impact of pathogens on this population. This is particularly important as there is an initiative to incorporate this population into a larger metapopulation of lions from adjacent South Africa and Zimbabwe.

Aelurostrongylus abstrusus in wild felids of South Africa.

The increasing interest on respiratory nematodes of domestic cats (Felis silvestris catus) has recently stimulated several studies on their epidemiology and geographic distribution. At the same time, there are still important gaps in our knowledge of the infections caused by respiratory nematodes in wild felids. The present study investigated the occurrence of pulmonary parasites in wild hosts housed in sanctuaries and protected areas of South Africa. Faecal samples collected from seven species of wild felids living in three study sites were copromicroscopically and genetically examined. Of twenty-one samples six, i.e. three from caracals (Caracal caracal), two from lions (Panthera leo) and one from a serval (Leptailurus serval), scored positive for the metastrongyloid Aelurostrongylus abstrusus at copromicroscopic and/or molecular tests. No other lungworms were recorded. The occurrence of the cosmopolitan cat lungworm A. abstrusus in wild hosts has been so far questioned. Importantly, the present findings represent an unequivocal evidence of the capability of A. abstrusus to infect some species of wild felids. Further studies are warranted to understand the epidemiological patterns of lungworms in wild and domestic felids, and to better investigate the impact of these parasitoses on health and welfare of wild animals.

Further studies on Toxascaris leonina (Linstow, 1902) (Ascaridida: Ascarididae) from Felis lynx (Linnaeus) and Panthera leo (Linnaeus) (Carnivora: Felidae).

Toxascaris leonina (Linstow, 1902) is a most common intestinal parasite of various animals in Felidae and Canidae. In the present paper, light and scanning electron microscopy (SEM) were used to study the morphological aspects of adult worms of this nematode in detail, based on the material collected from Panthera leo (Linnaeus) and Felis lynx (Linnaeus) (Carnivora: Felidae) in China. The results showed that there were some morphometric differences between the present material and the previous studies, including the body size, the width and length of cervical alae, the number of denticles on each lip and the tail length of the female. Previously unreported morphological features were also revealed. These supplementary morphological and morphometric data contributed to a more accurate identification of this worldwide distributed ascarid nematode.

Occurrence, prevalence and intensity of internal parasite infections of African lions (Panthera leo) in enclosures at a recreation park in Zimbabwe.

A coprological survey was conducted to determine the types, prevalence, and intensity of infection of internal parasites in a population of captive African lions (Panthera leo) at a recreational game park in Zimbabwe. Individual fecal samples were collected on three occasions over a 4-month period from each of 30 lions (55%) out of 55 animals held. The samples were examined using flotation and sedimentation techniques to assess the presence and count of parasite eggs, oocysts, and cysts per gram of feces as well as larvae identification. The overall prevalence of helminth infections was 100% (30/30), and 80% (24/30) of fecal samples also were positive for protozoan parasite forms. Eggs of Ancylostoma spp. were found in the feces of 23 (76.7%) lions, Physaloptera sp. in 14 (46.7%) lions, Toxascaris leonina in 13 (43.3%) lions, Toxocara cati in 12 (40%) lions, and Gnathostoma spinigerum and Toxocara canis in 2 (6.7%) lions. Furthermore, eggs of Cylicospirura subequalis, Gnathostoma spp., Lagochilascaris major, Acanthocephalan and Linguatula spp. as well as larvae of Aelurostrongylus sp. were identified in the feces of one lion. Oocysts of five apicomplexan parasites and cysts of one mastigophoran protozoan parasite were recorded, namely, Cystoisospora leonina in 11 (36.7%) lions' feces, Cystoisospora spp. in 9 (30.0%) lions, Cystoisospora felis in 5 (16.7%) lions; Toxoplasma-like spp. in 5 (16.7 %) lions, and Giardia spp. in 8 (26.7%) lions. The majority of lions (28/30) showed mixed infections with different internal parasites, whereas only two animals had single parasite infections. The intensity of infection was relatively low. Some parasite forms observed and identified, such as Eimeria spp. oocysts, were spurious and probably originated from the prey species for the lions. Among the parasites identified were some of zoonotic importance that have health implications for at-risk personnel and visitors who get into contact with the animals.

Molecular detection and characterization of potentially new Babesia and Theileria species/variants in wild felids from Kenya.

Piroplasms frequently infect domestic and wild carnivores. At present, there is limited information on the occurrence and molecular identity of these tick-borne parasites in wild felids in Kenya. In 2009, a pair of captive lions (Panthare leo) was diagnosed with suspected babesiosis and mineral deficiency at an animal orphanage on the outskirts of Nairobi, Kenya. Blood smears indicated presences of haemoparasites in the erythrocytes, however, no further investigations were conducted to identify the infecting agent. The animals recovered completely following diet supplementation and treatment with anti-parasite drug. In this report, we extracted and detected parasite DNA from the two lions and seven other asymptomatic feline samples; two leopards (Panthera pardus) and five cheetahs (Acinonyx jubatus). Reverse line blot with probes specific for Babesia spp. of felines indicated the presence of new Babesia species or genotypes in the lions and leopards, and unknown Theileria sp. in the cheetahs. Phylogenetic analyses using partial sequences of 18S ribosomal RNA (18S rRNA) gene showed that the parasite infecting the lions belong to the Babesia canis complex, and the parasite variant detected in the leopards clusters in a clade bearing other Babesia spp. reported in wild felids from Africa. The cheetah isolates falls in the Theileria sensu stricto group. Our findings indicate the occurrence of potentially new species or genotypes of piroplams in all three feline species.

The first case report of Leishmania (leishmania) chagasi in Panthera leo in Brazil.

We reported here the first known case of natural infection of a lion (Panthera leo-Linnaeus, 1758) with Leishmania (Leishmania) chagasi (L. chagasi) in Brazil. The specimen was created by a circus handler in the state of Mato Grosso and was donated to the zoological park of the Federal University of Mato Grosso. Infection by L. chagasi was detected using a PCR-RFLP test. It was known that the domestic felids can act as reservoir of infection of L. chagasi in endemic areas, making it important that studies demonstrate their participation in the epidemiological chain. We demonstrate in this work that wild animals can have an important role in the epidemiological chain and must be considered in order to plan methods of control of this zoonosis.

Trichinella zimbabwensis in a naturally infected mammal.

Trichinella zimbabwensis has been detected in wild and farmed Nile crocodiles (Crocodylus niloticus) and in wild monitor lizards (Varanus niloticus) of several African countries, but it has never been detected in mammals in nature, in spite of its infectivity to rodents, pigs, foxes and monkeys under laboratory conditions. The aim of this work was to describe the first detection of T. zimbabwensis in a naturally infected lion (Panthera leo) of the Kruger National Park (KNP) of South Africa. The sequence of the expansion segment V, a highly variable non-coding sequence of the large subunit ribosomal RNA of the genus Trichinella, of larvae from the lion was identical to that of larvae of T. zimbabwensis collected from a Nile crocodile originating from the same locality as the lion, suggesting a possible transmission of this parasite between mammals and reptiles. The KNP proves to be a very interesting area for parasites of the genus Trichinella since three taxa (Trichinella nelsoni, Trichinella T8 and T. zimbabwensis) circulate among the wildlife of this protected area.

Echinococcus species in African wildlife.

Cystic echinococcosis, caused by different species of the Echinococcus granulosus complex, is an important zoonotic disease with a particular impact on pastoralist societies. In addition to the widespread taxa with synanthropic transmission, a number of Echinococcus species were described from African wild carnivores early in the 20th century. For lack of study material, most of these were later tentatively synonymized with E. granulosus. Early infection experiments with wildlife isolates gave ambiguous results due to the use of unspecified parasite material, and only recently molecular methods provided the opportunity to shed light on the confusing scenery e.g. by characterizing E. felidis from the African lion. Here we will summarize the convoluted history of Echinococcus research in sub-Saharan Africa and highlight the necessity of molecular surveys to establish the life cycles and estimate the zoonotic potential of these parasites.

A survey of Echinococcus species in wild carnivores and livestock in East Africa.

We examined 71 faecal samples of carnivores from Queen Elizabeth National Park (QENP), Uganda, for eggs of Echinococcus species. Thirty-nine faecal samples contained taeniid eggs. For species diagnosis, DNA was isolated from a total of 1984 individual taeniid eggs. To differentiate eggs of Echinococcus felidis from other taeniid taxa (including the closely related Echinococcus granulosus sensu stricto), a restriction fragment length polymorphism (RFLP)-PCR of the mitochondrial nad1 gene was developed. As the faecal samples were taken from the environment, the host species was determined for all samples, except for one, by RFLP-PCR of the cob gene. Seven hundred and ninety-one of the 1984 eggs yielded a suitable PCR product. E. felidis was present in 34 of 47 samples from lions, none of 18 samples from leopards, and one of five samples from spotted hyenas. No Echinococcus taxon other than E. felidis was found, but three samples from lions contained eggs of Taenia regis. Two hydatid cysts of warthog origin from QENP were available for this study; molecular examination showed that one belonged to E. felidis, the other to E. granulosus (G1 strain). As a comparison of methods demonstrated that molecular diagnostic tools used for previous surveys of Echinococcus isolates in eastern Africa are not suitable to discriminate between E. felidis and E. granulosus sensu stricto, we re-examined 412 hydatid cyst samples of human, sheep, cattle, camel and goat origin from Kenya. Previous results were confirmed, as E. granulosus sensu stricto and Echinococcus canadensis G6/7 strain, but no E. felidis was found among these samples. In conclusion, we provide evidence that E. felidis is a frequent parasite of lions in Uganda, and possibly also occurs in hyenas. Additionally, we show that warthogs interact as intermediate hosts for E. felidis. We did not find evidence that E. felidis is present in eastern Africa outside conservation areas.

Trichinella nelsoni and Trichinella T8 mixed infection in a lion (Panthera leo) of the Kruger National Park (South Africa).

In South Africa, Trichinella sp. was first discovered in 1966 in the wildlife of the Kruger National Park (KNP). Since then, both Trichinella T8 and Trichinella nelsoni have been detected in the KNP, leading to a debate on the existence of a gene flow between the two taxa. In 2006-2008, four lions were killed in the Manyeleti Game Reserve, the Mthethomusha Nature Reserve, Numbi Gate, and Skukuza, which border the KNP. Larvae were isolated from muscles by artificial digestion. The molecular identification of single larva by multiplex PCR, followed by a specific PCR to distinguish between Trichinella T8 and Trichinella britovi, revealed Trichinella T8 in the lions from Manyeleti and Skukuza, a mixed infection with T. nelsoni and Trichinella T8 in the lion from Mthethomusha, and T. nelsoni in the lion from Numbi. No larva with a hybrid pattern between the two taxa was observed. No hybrid offspring resulted when crossing single males and females of T. nelsoni and Trichinella T8 in both directions, whereas hybrid offspring were obtained when crossing T. britovi and Trichinella T8 in both directions. This is the first report of a mixed infection with two Trichinella taxa in a host from the KNP, where both Trichinella T8 and T. nelsoni circulate among wildlife. Despite the sympatry status of these two taxa, field and laboratory data seem to exclude the possibility of gene flow, confirming their evolutive separation.

Amblyomma triste Koch, 1844 (Acari: Ixodidae): hosts and seasonality of the vector of Rickettsia parkeri in Uruguay.

Amblyomma triste Koch, 1844, the tick species most frequently involved in human bites in Uruguay, has been implicated as a vector of human rickettsiosis. Seasonal abundance of adult A. triste was examined by standard flagging at three sites where human tick bites and cases of the disease have been reported. Adult tick activity occurred from August to February (end of winter to mid summer in the southern hemisphere) with a peak in spring. Activity declined in step with decreasing temperatures and photoperiod during winter. This period of activity coincides with seasonal outbreaks of human rickettsiosis in the region. In a small mammal survey, the Sigmodontinae rodents Scapteromys tumidus (Waterhouse, 1837) and Oxymycterus nasutus (Waterhouse, 1837) and the small marsupial Monodelphis dimidiata (Wagner, 1847) were the main hosts for immature A. triste. Immature ticks were observed on hosts in November, well within the period of peak adult abundance. In stored collections, immature ticks were most often collected from January to March. These data suggest that one generation might be completed in 1 year. The main animal host for adult A. triste at our study sites was the domestic dog. Humans were afflicted by the tick in rural and suburban settlements where other host animals are scarce or extinct and where dogs are common.

Genetic characterization and phylogenetic position of Echinococcus felidis (Cestoda: Taeniidae) from the African lion.

Echinococcus felidis had been described in 1937 from African lions, but was later included in Echinococcus granulosus as a subspecies or a strain. In the absence of any genetic characterization, most previous records of this taxon from a variety of large African mammals remained unconfirmed due to the lack of diagnostic criteria and the possible confusion with the sympatric E. granulosus sensu stricto, Echinococcus ortleppi and Echinococcus canadensis. In this study, we obtained taeniid eggs from lion feces in Uganda and amplified DNA from individual eggs. Mitochondrial and nuclear DNA sequences showed similarities with those of other Echinococcus spp., but high values of percentage divergence of mitochondrial genes indicated the presence of a distinct species. In a second step, we compared this material with the preserved specimens of adult E. granulosus felidis, which had been identified morphologically approximately 40 years ago in South Africa. All DNA fragments (<200 bp) that could be amplified from the adults showed 100% similarity with the Ugandan material. In the phylogenetic tree of Echinococcus which was constructed from the mitochondrial genes, E. felidis is positioned as a sister taxon of E. granulosus sensu stricto. The data obtained will facilitate the development of diagnostic tools necessary to study the epidemiology of this enigmatic parasite.

Fatal cytauxzoonosis in captive-reared lions in Brazil.

Fatal cytauxzoonosis is described in a captive reared lioness (Panthera leo) and its 6-month-old cub. Clinical signs in the lioness included loss of weight, depression, anaemia, loss of hair, dark discolored urine, tachypnoea, nystagmus, deaphness and staggering gait. The cub died after a short period of depression. In the lioness, laboratory examination revealed normochromic normocytic anaemia, neutrophilia, lymphopenia, monocytosis, eosinopenia, thrombocytopenia, proteinuria, pyuria, haematuria and increased. At necropsy the lioness showed marked pulmonary edema and slight gelatinous translucent edema in the mediastinum, petechiae and echymosis disseminated in the serosae, and the intestinal content was red and semiliquid. The cub presented hemothorax, endocardial and pulmonary edema, petechiae in the cardiac serosae, hepatic and splenic congestion and segments of the small intestine with blood stained fluid contents and reddish mesenteric lymph nodes. Histopathological examination of liver, spleen, heart, lungs, intestines, pancreas, mesenteric lymph nodes, kidneys, skeletal muscle, brain and skin revealed large number of intravascular macrophages with their cytoplasm filled with various schizogonic stages of a Theileriidae. Electron microscopy confirmed the presence of schizonts in endothelial-associated macrophages. The diagnosis was established by the finding of the pathognomonic schizonts in macrophages within blood vessels in several organs and tissues from both lions. This is the first report of feline cytauxzoonosis in P. leo and of a confirmed infection by Cytauxzoon felis in felidae in South America.

Faecal helminth egg and oocyst counts of a small population of African lions (Panthera leo) in the southwestern Kalahari, Namibia.

An endoparasite survey of a small pride of African lions (Panthera leo) was conducted at Intu Afrika Kalahari Game Reserve, southwestern Namibia, during winter and summer of 2003 and 2004, respectively. Overall, 23 fresh lion scats were collected opportunistically during fieldwork trials. A flotation technique was employed for the diagnosis of parasites. Three nematodes, Ancylostoma braziliense, Gnathostoma spinigerum and Uncinaria stenocephala and two coccidians, Toxoplasma gondii and Isospora felis were recorded. By using the McMaster method for quantification, a maximum number of 14866 oocysts per gram of faeces was obtained for I. felis during winter 2003. Endoparasite taxa carried by the different individuals in the pride were found to be related to their levels of association. Rates of infection were relatively low as a result of the habitat, semi-captive conditions and earlier sporadic deworming.

Babesia leo n. sp. from lions in the Kruger National Park, South Africa, and its relation to other small piroplasms.

Babesia leo, a small piroplasm isolated from lions in South Africa is described as a distinct species based on a phylogenetic analysis of the 18S rRNA gene. Intraerythrocytic trophozoite and merozoite stages of B. leo are morphologically indistinguishable from other small piroplasms of felids. Previous studies showed that B. leo was biologically and antigenically distinct from B. felis, which is known to infect wild and domestic felids in South Africa. Molecular characterization showed strong support for the phylogenetic seperation of B. leo as a distinct species from B. felis and other felid piroplasms. Phylogenetic analysis also showed that Babesia microti and all of the felid piroplasms from Africa with known 18S rRNA gene sequences available, including B. leo, formed a single, separate clade, sister to the other babesial and theilerial piroplasm parasites.