Hemoplasmas in wild canids and felids in Brazil

Hemotropic mycoplasmas, epicellular erythrocytic bacterial parasites lacking a cell wall, are the causative agents of infectious anemia in numerous mammalian species. The presence of hemotropic mycoplasmas in blood samples of neotropical and exotic wild canids and felids from Brazilian zoos were recorded using molecular techniques. Blood samples were collected from 146 Brazilian wild felids, 19 exotic felids, 3 European wolves (Canis lupus), and from 97 Brazilian wild canids from zoos in the Brazilian states of S?o Paulo and Mato Grosso and the Federal District. Using conventional polymerase chain reaction (PCR), this work found 22 (13%) wild felids positive to Candidatus Mycoplasma haemominutum [4 jaguars (Panthera onca); 3 pumas (Puma concolor); 10 ocelots (Leopardus pardalis); 2 jaguarondis (Puma yagouaroundi); and 3 little spotted cats (Leopardus tigrinus)]. Only one little spotted cat (Leopardus tigrinus) was positive to Mycoplasma haemofelis, and none was positive to Candidatus Mycoplasma turicensis. Two bush dogs (Speothos venaticus) were positive for a Mycoplasma sp. closely related to Candidatus Mycoplasma haematoparvum, and two European wolves were positive for a Mycoplasma sp. closely related to Candidatus Mycoplasma haemominutum. This is the first study regarding the molecular detection of hemotropic mycoplasmas in wild canids.     

Molecular characterization of Hepatozoon sp. from Brazilian dogs and its phylogenetic relationship with other Hepatozoon spp

To characterize phylogenetically the species which causes canine hepatozoonosis at two rural areas of Rio de Janeiro State, Brazil, we used universal or Hepatozoon spp. primer sets for the 18S SSU rRNA coding region. DNA extracts were obtained from blood samples of thirteen dogs naturally infected, from four experimentally infected, and from five puppies infected by vertical transmission from a dam, that was experimentally infected. DNA of sporozoites of Hepatozoon americanum was used as positive control. The amplification of DNA extracts from blood of dogs infected with sporozoites of Hepatozoon spp. was observed in the presence of primers to 18S SSU rRNA gene of Hepatozoon spp., whereas DNA of H. americanum sporozoites was amplified in the presence of either universal or Hepatozoon spp.-specific primer sets; the amplified products were approximately 600bp in size. Cloned PCR products obtained from DNA extracts of blood from two dogs experimentally infected with Hepatozoon sp. were sequenced. The consensus sequence, derived from six sequence data sets, were blasted against sequences of 18S SSU rRNA of Hepatozoon spp. available at GenBank and aligned to homologous sequences to perform the phylogenetic analysis. This analysis clearly showed that our sequence clustered, independently of H. americanum sequences, within a group comprising other Hepatozoon canis sequences. Our results confirmed the hypothesis that the agent causing hepatozoonosis in the areas studied in Brazil is H. canis, supporting previous reports that were based on morphological and morphometric analyses.     

Diagnosis of canine Hepatozoon spp. infection by quantitative PCR

Hepatozoon (H.) americanum and H. canis are the etiological agents of canine hepatozoonosis, a disease that is found worldwide and is also prevalent in the southeastern United States. Current laboratory diagnosis of canine hepatozoonosis caused by H. americanum is usually dependent on visual identification of Hepatozoon "onion skin cysts" in muscle biopsies, an approach that requires invasive sampling and can result in false negatives. We have developed a diagnostic method for detection of Hepatozoon spp. DNA that integrates nucleic acid extraction with extensive agitation to maximize DNA extraction efficiency. The DNA extracted from canine EDTA-whole blood is subjected to real-time PCR, and fluorescence resonance energy transfer (FRET) probes detect a signature polymorphism in the amplified DNA. This PCR method amplifies a fragment of the Hepatozoon 18S rDNA gene, detects as few as 7 genomic copies of Hepatozoon spp. per ml of blood with high specificity, and differentiates between H. americanum and H. canis amplicons. A surprising 300-fold increase of H. americanum 18S rDNA targets occurred during 3-0 days of storage of positive blood specimens. Examination of 614 EDTA-blood samples submitted mostly from the southeastern Unites States from dogs with suspected hepatozoonosis identified H. americanum in 167 samples (27.2%). An additional 14 samples (2.3%) were positive for H. canis, and 14 samples (2.3%) were positive for both H. americanum and H. canis. These results suggest that the Hepatozoon spp. 18S rDNA quantitative PCR may be a valuable tool that can improve diagnosis and therapy of canine hepatozoonosis.     

Parasites of free-ranging small canids and felids in the Bolivian Chaco.

Parasite surveys of free-ranging wildlife provide important information for monitoring population health. Between March 2001 and March 2003, we sampled 10 ocelots (Leopardus pardalis), eight Geoffroy's cats (Oncifelis geoffroyi), a jaguarundi (Herpailurus yaguarondi), five pampas foxes (Pseudalopex gymnocercus), and three crab-eating foxes (Cerdocyon thous) at three sites in the Bolivian Chaco. The objective of the study was to survey the parasite fauna of these carnivores and compare prevalence of parasites among the sites. The parasite community of these carnivores was diverse, with representatives from eight genera of nematodes, two families of cestodes, two protozoan species, and six arthropod species. Fecal parasites identified from 12 of the 13 felids and five of the six canids examined included Aelurostrongylus abstrusus, Ancylostoma tubaeforme, Uncinaria sp., Crenosoma sp., Toxocara cati, Spirurida, Capillaria aerophila, Spirometra sp., Taeniidae, and Cystoisospora sp. Four tick species, Amblyomma parvum, A. tigrinum, A. ovale, and A. cajennense, and two flea species, Pulex irritans and Delostichus phyllotis, were identified. Two crab-eating foxes had serologic evidence of heartworm disease (HWD). Antibodies against Toxoplasma gondii were found in 15 of 26 animals. Although HWD was found only in canids inside the national park, parasite prevalence did not appear to differ among sites, and no evidence was found of parasite spillover from domestic to wild carnivores.     

Yohimbine antagonizes the anaesthetic effects of ketamine–xylazine in captive Indian wild felids

ObjectiveTo determine the effectiveness of yohimbine as an antagonist of ketamine-xylazine anaesthesia in captive Asiatic lions (Panthera leo persica), tigers (Panthera tigris) and leopards (Panthera pardus).Study designProspective clinical trial.AnimalsFifty-two healthy adult lions, 55 adult leopards and 16 adult male tigers.MethodsCaptive wild felids in Indian zoos were anaesthetized with a combination of ketamine (2.2-2.6 mg kg^?1) and xylazine (1.1-1.3 mg kg^?1) using a dart propelled from a blowpipe. Time to onset of anaesthesia, lateral recumbency and induction time were measured, and physiological variables (respiration, heart rate and rectal temperature) were recorded once after the onset of complete anaesthesia. Anaesthesia was antagonized at various time periods with an intravenous administration of either 0.1 or 0.15 mg kg^?1 yohimbine. Onset of arousal and time to complete anaesthetic recovery were recorded.ResultsA total of 123 immobilizations were conducted between 2000 and 2005. Anaesthetic induction was achieved in 15-25 minutes in all animals. Incidents of sudden recovery or life-threatening effects associated with immobilizations were not observed. Yohimbine effectively antagonized anaesthesia in all animals within 10 minutes without any excitatory behaviour compared to control animals. No adverse reactions/side effects to yohimbine were recorded except that a few leopards exhibited seizure-like signs for a short period immediately after yohimbine administration. The duration of anaesthesia had no significant effect on the recovery time in any of the animals.Conclusion and clinical relevanceYohimbine antagonized the xylazine portion of ketamine-xylazine anaesthesia and thereby hastened recovery from anaesthesia in Asiatic lions, tigers and leopards.     

Molecular detection of Babesia rossi and Hepatozoon sp. in African wild dogs (Lycaon pictus) in South Africa

Blood specimens from wild dogs (n=301) were obtained from De Wildt Cheetah and Wildlife Centre (Pretoria) and five game reserves (4 in the North-West Province and 1 in Limpopo Province), South Africa. Specimens were screened for Babesia, Theileria, Hepatozoon and Ehrlichia/Anaplasma species using PCR and Reverse Line Blot (RLB) assays. Positive results were obtained in 18 (6%) wild dogs. Sixteen specimens were found positive for Babesia rossi and two dogs were Hepatozoon sp. positive. It appears that these tick-borne pathogens are not widely distributed in wild dog populations.     

The first report of Hepatozoon sp. (Apicomplexa: Hepatozoidae) in neotropical felids from Brazil

In order to investigate the occurrence of Hepatozoon infection in Neotropical felids from Brazil, blood from the jugular or cephalic vein was taken from 29 non-domestic felids including ocelot (Leopardus pardalis), little spotted cat (Leopardus tigrinus), margay (Leopardus wiedii), and jaguarondi (Puma yagouaroundi) from the Northeast region of Brazil. Hepatozoon infection was confirmed by light microscopy and molecular techniques. The results showed five naturally infected felids. Partial sequences of the 18S rRNA gene of the Hepatozoon sp. from these felids were further analyzed. Sequences revealed that the isolates found are closely related to Hepatozoon sp. from domestic cats in Spain. Hepatozoon species from Neotropical felids were identified molecularly and characterized for the first time. This is also the first report of Hepatozoon infection in a little spotted cat.     

Hepatozoon species infection in wild red squirrels (Sciurus vulgaris) on the Isle of Wight

Postmortem examinations of 49 red squirrels (Sciurus vulgaris) found dead on the Isle of Wight revealed the presence of a Hepatozoon species in 18 of them (37 per cent). The prevalence of infection was highest in subadult animals and no juveniles were infected. The prevalence was higher in the squirrels dying from natural causes (nine of 12) than in squirrels killed in road accidents (seven of 27). The weight of infection varied, and there were heavy infections in squirrels dying from toxoplasmosis and bacterial pneumonia. A PCR-based assay was used to identify the presence of Hepatozoon species DNA in the lungs, and immunoperoxidase staining was used to confirm the identity of schizonts observed in histological sections. The nucleotide base sequence of the PCR products indicated that the organism was a novel species closely related to, but distinct from, Hepatozoon erhardovae of bank voles (Clethrionomys glareolus).     

Diagnosis of Hepatozoon spp. in Amblyomma ovale and its experimental transmission in domestic dogs in Brazil

Transmission of Hepatozoon spp. to dogs was investigated using four species of ixodid ticks: Rhipicephalus sanguineus, Amblyomma aureolatum, Amblyomma ovale and Amblyomma cajennense. We collected completely or partially engorged adult ticks of these species from dogs that were naturally infested and positive for Hepatozoon spp. We selected some of these ixodids and inoculated them orally in four negative dogs. The other ticks were dissected and examined for oocysts. Of all dogs inoculated orally with R. sanguineus, A. aureolatum, A. cajennense and A. ovale, only the animal that received the macerate of A. ovale was positive; evidence (gametocytes in peripheral blood) of infection was found 63 days after inoculation. Among all dissected ticks, we found only two oocysts; these were similar to those of Hepatozoon canis, and both were recovered from a single A. ovale specimen. We inoculated sporozoites recovered from the oocysts intraperitoneally into a Hepatozoon spp. negative dog, and circulating gametocytes were detected 84 days later. Our study demonstrated that A. ovale can be a vector of Hepatozoon spp. in Brazil.     

Low seroprevalence of antibodies to Neospora caninum in wild canids in Israel

The role of domestic dogs in the epidemiology of Neospora caninum as well as the relationship between N. caninum infection of farm dogs and cattle were demonstrated, however, evidence is scarce regarding the role of wild canids in domestic animal neosporosis. The present study was undertaken to evaluate the role of wild canids in the epidemiology of bovine neosporosis in Israel by analyzing the prevalence of antibodies to N. caninum in wild canids. Sera samples were collected from 114 free ranging wild golden jackals (Canis aureus), 24 red foxes (Vulpes vulpes) and nine wolves (Canis lupus), which were collected in Israel during the years 1999-2004. Of a total of 147 wild canids tested antibodies to N. caninum were only found in two golden jackals with IFAT titers of 1:50, and in one red fox and one wolf with IFAT titer of 1:400. The low seroprevalence found in this study (2.7%) indicated that wild canids probably do not have an important role in the epidemiology of N. caninum in Israel. However, since the diet of different species of wild canids and even diverse populations of the same canid species vary, it is possible that other results might be obtained from specific wild canids populations, which scavenge in the vicinity of infected bovines.     

Dirofilariasis in wild canids from the Gulf coastal prairies of Texas and Louisiana,U.S.A.

Heartworms, Dirofilaria immitis, were recovered from 17 out of 24 (71%) coyotes, Canis latrans, 38 out of 46 (83%) coyote × red wolf hybrids and all of 8 (100%) red wolves, Canis rufus gregoryi, collected from the Gulf coastal prairies of southeast Texas and southwest Louisiana. Intensities of infection ranged from 1 to 176 ($\text{x}\text{=25}$) worms per host. There was a significantly (P<0.05) higher intensity of infection in red wolves. Prevalence of heartworms increased significantly with increasing age. There were no significant differences between coyotes, hybrids and red wolves or between different host sexes in terms of prevalence. The female to male ratio of heartworms was close to unity (1.2:1) and was not correlated with worm burdens. Nematodes were primarily localized in the right heart, frequently extending into the pulmonary artery and the pulmonary arterial tree in the lungs. In 13 instances, 1–4 adult heartworms were recovered from the venae cavae. Pathological responses in the right heart were variable, depending on the intensity of infection. In severe infections, there were small areas of infarctive necrosis with mild to severe interstitial edema in the myocardium. Lesions in the pulmonary artery and pulmonary arterial trunk varied from mild focal hyperplastic intimal changes to extensive exudative villous endarteritis. The latter was characterized by a hyperplastic collagenous stroma with numerous histiocytes, plasma cells and eosinophils. Lung pathology varied from patchy to extensive areas of congestion, edema, hemorrhage, interstitial pneumonitis and infarction. In cases with heartworms in the venae cavae, hepatic changes were minimal and associated with liver changes such as passive congestion and centrolobular necrosis seen in cases without adult worms in the venae cavae. In heavily infected animals, hemosiderosis of the liver, spleen and kidneys was pronounced. A microfilaremia was noted in 46% of the infected wild canids. Microfilariae were observed in tissue sections of the myocardium, lungs, liver, kidney, spleen, lymph nodes, pancreas and appendix. Wild canids from this area are regarded as natural definitive hosts and primary reservoirs for heartworms and it appears that this infection is an important factor in the morbidity and mortality of these hosts.     

Trypanosoma spp. infection in wild rabbits (Oryctolagus cuniculus) during a restocking program in Southern Spain

The effect of parasites on managed rabbit populations may prove crucial to develop sanitary strategies during restocking programs of such key prey species. We investigated natural infection of European wild rabbits (Oryctolagus cuniculus) with Trypanosoma spp. in Spain. By fencing part of the warrens during a rabbit restocking program, we induced host variation in rabbit density across these socio-spatial units. We aimed (i) to compare Trypanosoma spp. infection spread between fenced and open warrens and (ii) to assess the relationship between body condition and infection. Trypanosoma spp. parasitaemia peaked in juveniles and decreased onwards. Adult females showed statistically higher infection rates than males. Rabbits from fenced warrens presented statistically higher infection rates than those from open ones, but did not differ in body condition. Parasite abundance negatively correlated with body condition in adults. Sex differences could resemble increased susceptibility to infection in females as a cost of reproduction and/or a higher exposition inside the warrens. Future studies should clarify whether aggregation caused enhanced exposition to intermediate hosts (fleas) and subsequent transmission of the parasite, and we stress that the study of non-lethal parasites during restocking programs provides valuable information on host contact rates and on factors affecting disease susceptibility.     

Molecular studies on Babesia,Theileria and Hepatozoon in southern Europe. Part I. Epizootiological aspects

Molecular epizootiology of piroplasmids (Babesia spp., Theileria spp.) and Hepatozoon canis was studied in mammals from southern Europe (mainly from Spain, but also from Portugal and France). Partial amplification and sequencing of the 18s rRNA gene was used for molecular diagnosis. In some particular cases (B. ovis and B. bovis) the complete 18s rRNA gene was sequenced. Blood samples were taken from domestic animals showing clinical symptoms: 10 dogs, 10 horses, 10 cows, 9 sheep and 1 goat. In addition, DNA samples were isolated from blood of 12 healthy dogs and from spleen of 10 wild red foxes (Vulpes vulpes). The results of the survey were the following: Piroplasmid infections: Approximately from 50 to 70% of wild or domestic mammals (symptomatic) were infected.Piroplasmids detected in ruminants were:COW: B. bovis, T. annulata and Theileria sp. (type C). Sheep and goat: B. ovis. Piroplasmids present in canids were: Babesia canis vogeli, Babesia canis canis, Theileria annae and B. equi. The only piroplasmid found in asymptomatic dogs was B. equi. Piroplasmids found in horse were: B. equi and B. canis canis.H. canis infections in canids: H. canis was absent of domestic dog samples, whereas all foxes studied were infected by this protozoa.Genetic analysis showed that most of piroplasmid and Hepatozoon isolates from southern Europe matched unambigously with previously described species, as demonstrated by the high level sequence identity between them, usually between 99 and 100%. Minor differences, usually detected in hypervariable regions of 18s rRNA gene are probably due to strain variations or rare genetic polymorphisms. A possible exception was B. bovis, which shows a relatively lower degree of homology (94%) with regard to other B. bovis isolates from several countries. The same is true for B. ovis, that showed a 94% identity with regard to Babesia sp. from South African cow and a 92% with rapport to B. bovis from Portugal.     

Molecular characterization and phylogenetic analysis of the causative agent of hemoplasma infection in small Indian Mongoose (Herpestes Javanicus)

Hemoplasmas are the trivial name for a group of erythrocyte-parasitizing bacteria of the genus Mycoplasma. This study is the first report of hemoplasma infection in Small Indian Mongoose (Herpestes Javanicus) based on molecular analysis of 16S rDNA. Whole blood samples were collected by sterile methods, from 14 live captured mongooses, in the south of Iran.