Whole-genome analysis of Escherichia coli isolated from wild Amur tiger (Panthera tigris altaica) and North China leopard (Panthera pardus japonensis).

Background: Escherichia coli is an important intestinal flora, of which pathogenic E. coli is capable of causing many enteric and extra-intestinal diseases. Antibiotics are essential for the treatment of bacterial infections caused by pathogenic E. coli; however, with the widespread use of antibiotics, drug resistance in E. coli has become particularly serious, posing a global threat to human, animal, and environmental health. While the drug resistance and pathogenicity of E. coli carried by tigers and leopards in captivity have been studied intensively in recent years, there is an extreme lack of information on E. coli in these top predators in the wild environment. Methods: Whole genome sequencing data of 32 E. coli strains collected from the feces of wild Amur tiger (Panthera tigris altaica, n = 24) and North China leopard (Panthera pardus japonensis, n = 8) were analyzed in this article. The multi-locus sequence types, serotypes, virulence and resistance genotypes, plasmid replicon types, and core genomic SNPs phylogeny of these isolates were studied. Additionally, antimicrobial susceptibility testing (AST) was performed on these E. coli isolates. Results: Among the E. coli isolates studied, 18 different sequence types were identified, with ST939 (21.9%), ST10 (15.6%), and ST3246 (9.4%) being the most prevalent. A total of 111 virulence genes were detected, averaging about 54 virulence genes per sample. They contribute to invasion, adherence, immune evasion, efflux pump, toxin, motility, stress adaption, and other virulence-related functions of E. coli. Sixty-eight AMR genes and point mutations were identified. Among the detected resistance genes, those belonging to the efflux pump family were the most abundant. Thirty-two E. coli isolates showed the highest rate of resistance to tetracycline (14/32; 43.8%), followed by imipenem (4/32; 12.5%), ciprofloxacin (3/32; 9.4%), doxycycline (2/32; 6.3%), and norfloxacin (1/32; 3.1%). Conclusions: Our results suggest that E. coli isolates carried by wild Amur tigers and North China leopards have potential pathogenicity and drug resistance.

Gut microbiota and fecal metabolites in captive and wild North China leopard (Panthera pardus japonensis) by comparsion using 16 s rRNA gene sequencing and LC/MS-based metabolomics.

BACKGROUND: Gut microbes significantly contribute to nutrient digestion and absorption, intestinal health and immunity, and are essential for the survival and environmental adaptation of wild animals. However, there are few studies on the gut microbiota of captive and wild North China leopard (Panthera pardus japonensis). RESULTS: A total of 10 mainly bacterial phyla were identified in the fecal microbiota of North China leopard, Lachnoclostridium (p = 0.003), Peptoclostridium (p = 0.005), Bacteroides (p = 0.008), Fusobacterium (p = 0.017) and Collinsella (p = 0.019) were significantly higher than those of wild North China leopard. Distinct differences in the fecal metabolic phenotypes of captive and wild North China leopard were found, such as content of l-methionine, n-acetyl-l-tyrosine, pentadecanoic acid and oleic acid. Differentially abundant gut microbes were associated with fecal metabolites, especially the bacteria in Firmicutes and Bacteroidetes, involved in the metabolism of N-acetyl-L-alanine and D-quinovose. CONCLUSION: This study reports for the first time the differences in gut microbiota abundance between captive and wild North China leopard, as well as significant differences in fecal metabolic phenotypes between two groups.

[Trichophyton mentagrophytes-from snow leopard to man : A molecular approach for uncovering the chain of infection]. / Trichophyton mentagrophytes ­ vom Schneeleoparden zum Menschen : Ein molekularer Ansatz zur Aufdeckung der Infektionskette.

Sources of infection for Trichophyton (T.) mentagrophytes-a zoophilic dermatophyte-comprise pet rodents (guinea pigs, mice, rabbits) and sometimes cats. Human infections due to dermatophytes after contact with zoo animals, however, are extreme rare. Four zoo keepers from Basel Zoo were diagnosed to suffer from tinea manus and tinea corporis due to T. mentagrophytes. The 22-year-old daughter of one zoo keeper was also infected with tinea corporis after having worked in the snow leopard section for one day. The strain of the index patient was confirmed by a direct uniplex-PCR-EIA and sequence analysis of the ribosomal internal transcribed spacer (ITS) region (18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA) as T. mentagrophytes. Three young snow leopards from Basel Zoo were identified as the origin of the fungal skin infection. The transmission occurred due to direct contact of the zoo keepers with the young snow leopards when removing hedgehog ticks (Ixodes hexagonus). Two adult snow leopards had developed focal alopecia of the facial region which was diagnosed as dermatomycoses due to T. mentagrophytes by the zoo veterinarians. By sequence analysis, both the strains from the animals and a single strain of the index patient showed 100% accordance proving transmission of T. mentagrophytes from animals to the zoo keepers. Molecular biological identification revealed a strong relationship to a strain of T. mentagrophytes from European mink (Mustela lutreola) from Finland. Treatment of patients was started using topical ointment with azole antifungals, and oral terbinafine 250 mg once daily for 4 weeks. Both adult snow leopards and the asymptomatic young animals were treated with oral itraconazole.

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.

First detection of feline hemoplasmas in free-ranging jaguars (Panthera onca).

Species of hemoplasmas have been described worldwide, but little information is available for wild felids. Between February 2000 and January 2010, blood samples were collected from 30 jaguars (Panthera onca) and 22 domestic cats (Felis catus) from the Cerrado, Pantanal and Amazon biomes of Brazil. In all samples molecular tests were performed for Mycoplasma haemofelis/Mycoplasma haemocanis (Mhf/Mhc), 'Candidatus Mycoplasma haemominutum' (CMhm) and 'Candidatus Mycoplasma turicensis' (CMt). Twenty-two (73.4%) jaguars and four domestic cats (18.2%) tested positive for infection with at least one feline hemoplasma: 73.4% jaguars from the three areas were positive for CMhm, 13.6% jaguars from the Pantanal and 50.0% from the Amazon were positive for Mhf/Mhc, and 9.1% of individuals from the Pantanal tested positive for CMt. Domestic cats from the Cerrado (28.6%) and the Pantanal (30.0%) were positive for feline hemoplasma. All but one jaguar from the three sites are healthy. One female adult jaguar showed low body weight and dehydration. This is the first record of feline hemoplasmas in free-ranging jaguars. The high prevalence of CMhm suggest the participation of jaguars in the maintenance of this hemoplasma in nature. Although susceptible to Mhf/Mhc and CMt, jaguars did not appear to participate in the maintenance of these agents in the environment. The involvement of domestic cats in the transmission of any of these hemoplasmas cannot be excluded.

Detection of Chlamydophila felis and Feline Herpesvirus Type-1 in non-domestic felids in Brazil / Detecção de Chlamydophila felis e Herpesvirus felino tipo 1 em felídeo não doméstico no Brasil

Little is known about the occurrence of feline upper respiratory tract disease agents, namely Feline Herpesvirus type 1 (FHV-1) and Chlamydophila felis, and co-infection of these agents with Feline Immunodeficiency virus (FIV) and Feline Leukemia Virus (FeLV) in non-domestic felids in Brazil. Between 2009 and 2010, 72 conjunctival swab and serum samples were collected from eight non-domestic felid species (Leopardus pardalis, Leopardus tigrinus, Panthera leo, Panthera tigris, Puma concolor, Puma yagouaroundi, Oncifelis colocolo, and Panthera onca) maintained in captivity in Brazilian zoos. DNA extracted from conjunctival swabs were used in PCR assays for the detection of Chlamydophila sp, FHV-1, and retrovirus DNA, respectively. Antibodies to FIV and FeLV antigen were detected in non-domestic felid serum samples using a commercial ELISA kit. Antibodies to FIV were found only in five (6.9%) felids. No sampled non-domestic felid was positive for FeLV antigen detection. One (1.3%) out of 72 non-domestic felid conjunctival swab samples was positive for Chlamydophilasp. and Feline Herpesvirus-1 in PCR. This felid was an ocelot and was negative for FIV and FeLV. The results of this survey showed the occurrence of co-infection with C. felis and FHV-1 in an ocelot (Leopardus pardalis) in Brazil...

Poucos trabalhos descrevem a ocorrência dos agentes do complexo respiratório felino, Herpesvírus Felino tipo 1 (FHV-1) e Chlamydophila felis, e a coinfecção com o vírus da imunodeficiência felina (FIV) e leucemia viral felina (FeLV) em felinos não domésticos no Brasil. Entre 2009 e 2010, 72 amostras de swab de conjuntiva e de soro foram coletados de oito espécies de felinos não domésticos (Leopardus pardalis, Leopardus tigrinus, Panthera leo, Panthera tigris, Puma concolor, Puma yagouaroundi, Oncifelis colocolo, and Panthera onca) mantidos em cativeiro em zoológicos brasileiros. O DNA foi extraído das amostras de swab de conjuntiva para detecção de Chlamydophila sp e FHV-1 pela PCR. Anticorpos para FIV e antígeno para FeLV foram determinados pelo kit comercial de ELISA. Anticorpos para FIV foram detectados em cinco felídeos (6,9%). Nenhuma amostra foi positiva para a presença de antígeno de FeLV. Um (1,3%) dos 72 felinos não domésticos apresentou fragmentos de DNA de Chlamydophila sp e FHV-1 pela PCR. Este felino era uma jaguatirica que não apresentou anticorpos para FIV e nem antígeno para FelV. Estes resultados demonstram a ocorrência de coinfecção de C. felis e FHV-1 em uma jaguatirica (Leopardus pardalis) no Brasil...

Serosurvey of Smooth Brucella, Leptospira spp. and Toxoplasma gondii in Free-Ranging Jaguars (Panthera onca) and Domestic Animals from Brazil.

This study investigated the exposure of jaguar populations and domestic animals to smooth Brucella, Leptospira spp. and Toxoplasma gondii in the Cerrado, Pantanal and Amazon biomes of Brazil. Between February 2000 and January 2010, serum samples from 31 jaguars (Panthera onca), 1,245 cattle (Bos taurus), 168 domestic dogs (Canis lupus familiaris) and 29 domestic cats (Felis catus) were collected and analysed by rose bengal test for smooth Brucella, microscopic agglutination test for Leptospira spp. and modified agglutination test for T. gondii. Cattle populations from all sites (9.88%) were exposed to smooth Brucella, but only one jaguar from Cerrado was exposed to this agent. Jaguars captured in the Cerrado (60.0%) and in the Pantanal (45.5%) were seropositive for different serovars of Leptospira spp., cattle (72.18%) and domestic dogs (13.1%) from the three sites and one domestic cat from Pantanal were also seropositive for the agent. The most prevalent serotype of Leptospira spp. identified in jaguars from the Cerrado (Grippotyphosa) and the Pantanal (Pomona) biomes were distinct from those found in the domestic animals sampled. Jaguars (100%), domestic dogs (38.28%) and domestic cats (82.76%) from the three areas were exposed to T. gondii. Our results show that brucellosis and leptospirosis could have been transmitted to jaguars by domestic animals; and jaguars probably play an important role in the maintenance of T. gondii in nature.

Detection of Leptospira spp. and Brucella abortus antibodies in free-living jaguars (Panthera onca) in two protected areas of northern Pantanal, Brazil.

This study aimed to assess the exposure of free-living jaguars (Panthera onca) to Leptospira spp. and Brucella abortus in two conservation units in the Pantanal of Mato Grosso, Brazil. The presence of antibodies in blood samples of eleven jaguars was investigated using autochthonous antigens isolated in Brazil added to reference antigen collection applied to diagnosis of leptospirosis by Microscopic Agglutination Test (MAT). The Rose Bengal test was applied for B. abortus antibodies. Two (18.2%) jaguars were seroreactive for the Leptospira spp. antigen and the serovar considered as most infective in both animals was a Brazilian isolate of serovar Canicola (L01). All jaguars were seronegative for B. abortus. These data indicate that the inclusion of autochthonous antigens in serological studies can significantly increase the number of reactive animals, as well as modify the epidemiological profile of Leptospira spp. infection.

Microbiologia de swabs retais e otológicos em carnívoros silvestres do zoológico do Parque Estadual de Dois Irmãos, Pernambuco / Microbiology of rectal and otologic swabs of wild carnivores from the Zoo of the Parque Estadual de Dois Irmãos, Pernambuco, Brazil

O estudo epidemiológico de patógenos em populações selvagens no ambiente in situ e ex situ é fundamental para implementação de programas de prevenção, controle e monitoramento de enfermidades e para elaboração de políticas públicas de saúde pública e animal. O estudo foi realizado no zoológico do Parque Estadual de Dois Irmãos no período de janeiro a julho de 2011 onde foram coletados swabs anais e otológicos referentes a 29 carnívoros silvestres cativos do zoológico. Dos swabs otológicos analisados, 1/29 (3,4%) foi positivo para Malassezia pachy dermatis no exame direto. No exame microbiológico dos swabs otológicos, observou-se maior freqüência para bactérias do gênero Bacillus 16/29 (55,2%), seguida de Sta phylococcus 15/29 (51,7%), Escherichia coli 7/29 (24,1%), Streptobacillus 1/29 (3,4%), Micrococcus 1/29 (3,4%) e Klebsiella 1/29 (3,4%). Com relação ao exame presuntivo para o gênero Salmonella a partir das amostras de swab retal, observou-se positividade para seis raposas (Cerdocyon thous) e um guaxinim (Procyon cancrivorus). O isolamento de Salmonella spp. em C. thous e em P. cancrivorus indica um risco à saúde pública, principalmente para os profissionais do zoológico que trabalham diretamente com esses animais. Essa pesquisa reforça a importância da criação de estratégias de vigilância epidemiológica voltadas para a prevenção, controle e monitoramento de potenciais reservatórios de agentes etiológicos de doenças infecciosas e parasitárias no ambiente dos zoológicos.

The epidemiological study of pathogens in wild populations in the environment in situ and ex situ is essential for implementation of prevention, control and monitoring of diseases and for development of public policies for public and animal health. The study was conducted at the Zoo of Parque Estadual de Dois Irmãos, Recife/PE, Brazil, from January to July 2011, where we collected anal and otological swabs from 29 Zoo's captive wild carnivores. The analysis of the otological swabs showed that 1/29 (3.4%) were positive for Malassezia pachydermatis on direct examination. In the microbiological examination of otologic swabs we found mainly bacteriae of the genus Bacillus 16/29 (55.2%), followed by Staphylococcus 15/29 (51.7%), Escherichia coli 7/29 (24.1%), Streptobacillus 1/29 (3.4%), Micrococcus 1/29 (3.4%) and Klebsiella 1/29 (3.4%). Regarding the presumptive test for the genus Sal monella from rectal swabs, positive samples were observed in six foxes (Cerdocyon thous) and a raccoon (Procyon cancrivorus). The isolation of Salmonella spp. in C. thous and P. can crivorus indicates risks to public health, especially for zoo professionals who work directly with these animals. This research reinforces the importance of establishing surveillance strategies aimed at prevention, control and monitoring of potential reservoirs of the etiologic agents of infectious and parasitic diseases in the environment of zoos.

Tick-borne bacteria in free-living jaguars (Panthera onca) in Pantanal, Brazil.

Tick-borne bacteria were investigated in 10 free-living jaguars and their ticks in the Pantanal biome, Brazil. Jaguar sera were tested by indirect fluorescent antibody assays using Rickettsia rickettsii, Rickettsia parkeri, Rickettsia amblyommii, Rickettsia rhipicephali, Rickettsia felis, Rickettsia bellii, Ehrlichia canis, and Coxiella burnetii as crude antigens. All 10 jaguar sera reacted (titer ≥ 64) to at least one Rickettsia species; 4 and 3 sera reacted with E. canis and C. burnetii, respectively. One jaguar presented antibody titer to R. parkeri at least fourfold higher than those to any of the other five Rickettsia antigens, suggesting that this animal was infected by R. parkeri. Ticks collected from jaguars included the species Amblyomma cajennense, Amblyomma triste, and Rhipicephalus (Boophilus) microplus. No Rickettsia DNA was detected in jaguar blood samples, but an A. triste specimen collected on a jaguar was shown by PCR to be infected by R. parkeri. The blood of two jaguars and samples of A. triste, A. cajennense, and Amblyomma sp. yielded Ehrlichia DNA by PCR targeting the ehrlichial genes 16S rRNA and dsb. Partial DNA sequences obtained from PCR products resulted in a new ehrlichial strain, here designated as Ehrlichia sp. strain Jaguar. A partial DNA sequence of the 16S rRNA gene of this novel strain showed to be closest (99.0%) to uncultured strains of Ehrlichia sp. from Japan and Russia and 98.7% identical to different strains of Ehrlichia ruminantium. The ehrlichial dsb partial sequence of strain jaguar showed to be at most 80.7% identical to any Ehrlichia species or genotype available in GenBank. Through phylogenetic analysis, Ehrlichia sp. strain jaguar grouped in a cluster, albeit distantly, with different genotypes of E. ruminantium. Results highlight risks for human and animal health, considering that cattle ranching and ecotourism are major economic activities in the Pantanal region of Brazil.

Purification and characterization of novel extracellular cholesterol esterase from Acinetobacter sp.

CHE4-1, a bacterial strain that belongs to the genus Acinetobacter and expresses high level of inducible extracellular cholesterol esterase (CHE), was isolated from feces of carnivore Panthera pardus var. The cholesterol esterase of the strain CHE4-1 was purified by ultrafiltration followed with DEAE-Sepharose FF chromatography and Phenyl-Sepharose CL-4B chromatography, and then by Sephadex G-50 gel filtration. Different from other known microbial cholesterol esterase, the purified CHE from CHE4-1 strain is a monomer with molecular weight of 6.5 kD and has high activity to both long-chain and short-chain cholesterol ester. Enzymatic activity was enhanced in the presence of metal ion Ca(2+), Zn(2+) and boracic acid, and was not significantly affected by several detergents including sodium cholate, Triton X100 and Tween-80. The enzyme was found to be stable during long-term aqueous storage at 4 °C, indicating its potential as a clinical diagnostic reagent. To the best of our knowledge, this is the first report regarding purification and characterization of CHE from Acinetobacter sp. The results demonstrated that this particular CHE is a novel cholesterol esterase.

First evidence of feline herpesvirus, calicivirus, parvovirus, and Ehrlichia exposure in Brazilian free-ranging felids.

Serum samples from 18 pumas (Puma concolor), one ocelot (Leopardus pardalis), and two little spotted cats (Leopardus tigrinus) collected from free-ranging animals in Brazil between 1998 and 2004 were tested by indirect immunofluorescence (IFA) for antibodies to feline herpesvirus 1 (FHV 1), calicivirus (FCV), coronavirus (FCoV), parvo-virus (FPV), Ehrlichia canis, Anaplasma pha-gocytophilum, and Bartonella henselae. Serum samples also were tested, by Western blot and ELISA, for feline leukemia virus (FeLV) specific antibodies and antigen, respectively, by Western blot for antibodies to feline immunodeficiency virus (FIV), and by indirect ELISA for antibodies to puma lentivirus (PLV). Antibodies to FHV 1, FCV, FCoV, FPV, FeLV, FIV, PLV or related viruses, and to B. henselae were detected. Furthermore, high-titered antibodies to E. canis or a closely related agent were detected in a puma for the first time.

A non-surgical uterine lavage technique in large cats intended for treatment of uterine infection-induced infertility.

This paper presents the successful use of a non-surgical, transcervical uterine lavage technique for the treatment of uterine infection-induced infertility in three female large cats. We developed a non-surgical uterine lavage technique, which allowed repeated flushing of the uterine lumen and installation of therapeutic antibiotics. The entire procedure was performed under general anaesthesia (duration of anesthesia ranged from 40 to 70 min). It was successfully applied in a Sumatran tiger (Panthera tigris sumatrae), a Corbett tiger (Panthera tigris corbetti) and an Amur leopard (Panthera pardus orientalis). The tigers were treated only once, whereas the leopard received four uterine treatments, due to re-infection after mating. Decisions to conduct uterine treatments were based on detection of uterine fluid during previous transrectal ultrasound examinations. The catheter was guided into the vagina, with the aid of an endoscope, passing the urethra, and then into the uterus, with the aid of transrectal ultrasonography. Both uterine horns were separately flushed with approximately 300 mL of cell medium M199, followed by an antibiotic infusion. Upon ultrasonographic re-examination, the topical uterine treatments resulted in an apparent decline in the inflammatory and/or degenerative processes. The Corbett tiger had the most severe uterine alterations, in addition to an aseptic pyometra. As a result, she was treated 1 month prior to ovariohysterectomy (in order to reduce the surgical risk). The Sumatran tiger was artificially inseminated twice after hormone-induced estrus, and the Amur leopard expressed a spontaneous estrus and re-initiated mating behaviour.

Granulomatous pneumonia caused by Pythium insidiosum in a central American jaguar, Panthera onca.

A 7-month-old, male jaguar presented with dyspnea and leukocytosis unresponsive to antibiotic therapy. Radiographs revealed unilateral pulmonary consolidation. An exploratory thoracotomy was performed, and the left lung, which contained a large multilobular mass with extensive fibrosis and numerous caseonecrotic foci, was removed. Microscopically, eosinophilic granulomatous inflammation surrounded broad (4.4-8.3 microm) rarely septate hyphae. A diagnosis of Pythium insidiosum infection was confirmed by immunohistochemistry, immunoblot serology, culture, and polymerase chain reaction. Dyspnea recurred despite treatment, and the animal succumbed 3 weeks after surgery. Necropsy findings indicated that death resulted from occlusion of the right main stem bronchus by a fungal granuloma. The oomycete P. insidiosum typically causes granulomatous disease of the skin or gastrointestinal tract in animals and arteritis, keratitis, or cellulitis in humans. Infection is uncommon in felines, and pulmonary involvement is rare. This report details the first case of P. insidiosum infection in an exotic felid and provides the first description of primary pulmonary pythiosis in any species.