A serologic assessment of exposure to viral pathogens and Leptospira in an urban raccoon (Procyon lotor) population inhabiting a large zoological park.

In urban environments, raccoons (Procyon lotor) may act as reservoirs for an array of pathogenic organisms, presenting spillover risks for human, domestic animal, and captive (zoo) animal populations. Over 5 yr, 159 raccoons from a high-density raccoon population in St. Louis, Missouri (USA), were surveyed for exposure to canine distemper virus (CDV), canine adenovirus 1 (CAV-1); feline parvovirus (FPV; =feline panleukopenia), and several serovars of Leptospira interrogans. Exposure to each of the viruses and two Leptospira serovars (grippotyphosa and icterohemorrhagiae) was detected (prevalence of CDV = 54.1%; FPV = 49.7%; CAV-1 = 6.9%; L. interrogans icterohemorrhagiae = 8.9%; L. interrogans grippotyphosa = 6.3%). Eighty percent of raccoons showed evidence of exposure to at least one of the five primary pathogens, and 39% were positive for multiple species. Among the viruses, there was a significant co-occurrence of CDV and CAV-1. Longitudinal data on a subset of animals revealed that among individuals who were diagnosed as seropositive on first capture, 33-100% became seronegative for the pathogen of interest when reexamined at a later date. Thus, free-ranging urban raccoons have been exposed to multiple infectious agents, some of which may pose risks to humans and to nonvaccinated domestic and captive animal populations.     

Leptospirosis in selected wild mammals of the Florida panhandle and southwestern Georgia.

A group of 144 wild mammals, including white-tailed deer, cottontail rabbits, fox squirrels, gray squirrels, raccoons, opossums, a bobcat, and various small rodents was examined for cultural or serologic evidence of leptospiral infection. Leptospires were isolated from 1 of 25 rabbits, 1 of 27 fox squirrels, 1 of 26 gray squirrels, 4 of 18 mice and rats, 8 of 21 raccoons, 7 of 17 opossums, and a bobcat. Isolations were not made from 6 deer examined. Serotypes isolated were Leptospira interrogans, serotype grippotyphosa and L interrogans, serotype ballum. New host-serotype relationships were noticed in the following instances: bobcat: grippotyphosa, and gray squirrel: ballum. These studies further confirm the occurrence of grippotyphosa in the fox squirrel. Serologic response in these animals did not necessarily correlate with isolations, although some relationship was noticed in raccoons and opossums.     

Second passage of sheep scrapie and transmissible mink encephalopathy (TME) agents in raccoons (Procyon lotor)

To determine the transmissibility and pathogenicity of sheep scrapie and transmissible mink encephalopathy (TME) agents derived from raccoons (first passage), raccoon kits were inoculated intracerebrally with either TME (one source) or scrapie (two sources-each in separate groups of raccoons). Two uninoculated raccoon kits served as controls. All animals in the TME-inoculated group developed clinical signs of neurologic dysfunction and were euthanatized between postinoculation month (PIM) 6 and 8. Raccoons in the two scrapie-inoculated groups manifested similar clinical signs of disease, but such signs were observed much later and the animals were euthanized between PIM 12 and 18. Necropsy revealed no gross lesions in any of the raccoons. Spongiform encephalopathy was observed by use of light microscopy, and the presence of protease-resistant prion protein (PrPres) was detected by use of immunohistochemical (IHC) and Western blot analytic techniques. Results of IHC analysis indicated a distinct pattern of anatomic distribution of PrPres in the TME- and scrapie-inoculated raccoons. These findings confirm that TME and sheep scrapie are experimentally transmissible to raccoons and that the incubation periods and IHC distribution for both agents are distinct. Therefore, it may be possible to use raccoons for differentiating unknown transmissible spongiform encephalopathy (TSE) agents. Further studies, with regard to the incubation period and the pattern of PrPres deposition by use of IHC analysis in bovine spongiform encephalopathy and for other isolates of scrapie, chronic wasting disease, and TME in raccoons are needed before the model can be further characterized for differentiation of TSE agents.     

An outbreak of leptospirosis in seals (Phoca vitulina) in captivity

An outbreak of leptospirosis in seals (Phoca vitulina) in captivity is described. In a zoo in The Netherlands 5 adult seals died within 12 days. At necropsy all animals showed signs of acute septicaemia, consistent with acute leptospirosis. Serological examination of one animal was positive for antibodies against Leptospira interrogans serovar Icterohaemorrhagiae and the serologically closely related serovar Copenhageni. Polymerase chain reaction was positive in one other animal. 8 nutria (Myocastor coypus) were examined, serologically, through bacteriological culture and PCR. 81,8% (9/11) were serologically positive for Leptospira. The seals and nutria were housed in the same water system.     

Incidence of zoonoses in petting zoos and evaluation of hygiene measures to prevent the transmission to humans

In summer 2003, a study was performed in thirty Swiss petting zoos with the objective to determine the prevalence of zoonotic agents, and to describe hygiene measures implemented to reduce the risk of human infection. Fecal samples from different animal species were collected from the floor of pens to determine the prevalence of Salmonella spp., Campylobacter spp., verocytotoxin producing E. coli/ VTEC and Francisella tularensis. A questionnaire on hygiene measures, number of animals per species, housing system, care procedures and feeding was administered to every petting zoo to estimate exposure of visitors to zoonotic microorganisms. In total, 423 fecal samples were examined. Of these samples, 41 were positive for Campylobacter spp., which were mainly isolates from pigs and poultry (35% positive samples from each species). In pigs, 50% of the positive samples (6 samples) were typed as C. jejuni. The others were typed as C. coli (3) and C lan' (3), respectively. Five poultry isolates were typed as C. jejuni, and two as C. coli. Two samples were positive for Salmonella spp. Salmonella typhimurium was isolated from a goat, the other isolate could not be identified by serotyping. Neither Francisella tularensis nor verocytotoxin producing E. coli/ VTEC were found. The low prevalence of zoonotic microorganisms in Swiss petting zoos could be attributed to the cleanness of enclosures and animals, low stocking rates and good animal care. However, there is room for improvement concerning visitors' information on hygiene and hand washing. Furthermore, a strict separation between picnic - areas and animals should be enforced.     

Clinical Leptospira interrogans serogroup Australis serovar lora infection in a stud farm in The Netherlands

A Leptospira interrogans serogroup australis serovar lora infection in a stud farm is reported. During three successive years (1984-1986) clinical leptospirosis with a severe often rapid, fatal course was seen in 12 foals. Clinical examination revealed severe respiratory distress, depression and pyrexia. Other symptoms were diarrhea (2), jaundice (1), and an unsteady gait (1). Morphological characteristics of the disease were massive pulmonary haemorrhage and haemorrhagic-thrombotic or extracapillary glomerulonephritis with tubulonephrosis and interstitial oedema. In most foals high or increasing MAT titres to serovar bratislava were found; from one foal Leptospira interrogans serovar lora was isolated. Serological examination of all 56 mares at the farm (August 1986) revealed antibodies to serovar bratislava in 64 per cent of the animals. These findings support the idea that Leptospira interrogans serovar bratislava and closely related strains (in this study serovar lora) may be adapted to and maintained by the horse population.     

Experimental inoculation of raccoons (Procyon lotor) with Spiroplasma mirum and transmissible mink encephalopathy (TME)

The primary objective of this study was to determine whether or not Spiroplasma mirum would be capable of producing lesions of transmissible spongiform encephalopathy (TSE) when inoculated in raccoons (Procyon lotor) and, if that was possible, to compare the clinicopathological findings with those of transmissible mink encephalopathy (TME) in the same experimental model. For this purpose, 5 groups (n = 5) of raccoon kits were inoculated intracerebrally with either S. mirum and/or TME. Two other groups (n = 5) of raccoon kits served as sham-inoculated controls. All animals inoculated with TME, either alone or in combination, showed clinical signs of neurologic disorder and were euthanized within 6 mo post-inoculation (MPI). None of the carcasses revealed gross lesions. Spongiform encephalopathy was observed by light microscopy and the presence of abnormal disease-causing prion protein (PrP(d)) was detected by immunohistochemistry (IHC) and Western blot (WB) techniques in only the raccoons administered TME. Raccoons inoculated with Spiroplasma, but not administered TME agent, were euthanized at 30 MPI. They did not show clinical neurologic signs, their brains did not have lesions of spongiform encephalopathy, and their tissues were negative for S. mirum by polymerase chain reaction (PCR) and for PrP(d) by IHC and WB techniques. The results of this study indicate that Spiroplasma mirum does not induce TSE-like disease in raccoons.     

Seroprevalence of bovine leptospirosis in Garanhuns Municipal District,Pernambuco State,Brazil

The prevalence of Leptospira interrogans serovars in dairy cattle was determined by analyzing 464 serum samples from cows on 15 properties in Garanhuns municipal district, Pernambuco State, Brazil. A microscopic seroagglutination test including 12 serovars of Leptospira interrogans as antigens was used. Samples with titres 100 were considered positive. Two hundred and twenty-one (47.63%) of the samples were positive to one or more serovars. The prevalence of the serovars was hardjo (21.98%), bratislava (15.73%), castellonis (11.64%), tarassovi (10.56%), pyrogenes (1.72%), icterohaemorrhagiae (1.08%), pomona (0.86%), wolffi (0.86%), grippotyphosa (0.86%), djasiman (0.43%), canicola (0.21 %), and copenhageni (0.21%).     

A serological study of bovine leptospirosis in the district of Taranaki

A cross-sectional serological survey of dairy cattle in Taranaki in 1979-80 indicated that 62% (551/891) of the animals had evidence of Leptospira interrogans serovar hardjo infection as disclosed by the microscopic agglutination test. Titres to Leptospira interrogans serovar pomona were demonstrated in only 4% (23/591) of the animals examined. The high prevalence of hardjo infection is suggestive of an endemic infection whilst the low level to pomona is indicative of sporadic infection. In a detailed examination of 10 herds, 9 revealed high (55%-91%) prevalence of serological reactions to hardjo and the herd profiles of titres, indicated that the animals had become infected at one to two years of age. A field strain of hardjo from cattle as well as the usual laboratory strain (hardjoprajitno) was incorporated in the test but there were no significant differences between the results given by the two antigens.     

Tularemia as a cause of fever in a squirrel monkey

CASE DESCRIPTION: A 3-year-old female squirrel monkey (Saimiri sciureus sciureus) was examined because of sudden onset of lethargy and fever. CLINICAL FINDINGS: On initial examination, the monkey was weak and febrile and had petechiae on both thoracic limbs. Following collection, blood samples were slow to clot. During the next week, the monkey developed anemia and thrombocytopenia; Francisella tularensis was isolated from blood samples. TREATMENT AND OUTCOME: Treatment with gentamicin resulted in the monkey's gradual return to health, but inguinal lymphadenopathy developed after drug administration was discontinued. Francisella tularensis was isolated from a fine-needle aspirate of an enlarged lymph node. Treatment with streptomycin resulted in resolution of infection. By use of biochemical and molecular tests, the microbial isolate was characterized as F tularensis subsp holarctica. Results of a microagglutination assay confirmed that the monkey had developed serum antibodies against F tularensis. CLINICAL RELEVANCE: With timely diagnosis, treatment of tularemia in the squirrel monkey was successful. Francisella tularensis is the cause of a highly infectious zoonotic disease, and infection with this microorganism is enzootic in wildlife throughout the Northern Hemisphere. Tularemia should be considered in the differential diagnosis of febrile disease in animals of any species. Even limited or indirect exposure of humans or other animals to outdoor environments in which reservoir hosts and arthropod vectors are present can lead to transmission of F tularensis. Francisella tularensis is a class A agent of bioterrorism, and all cases of tularemia (regardless of species) should be reported to public health officials.     

Immunohistochemical demonstration of Francisella tularensis in lesions of cats with tularemia.

An immunohistochemical test was developed and validated for detection of Francisella tularensis antigen in tissues of cats with fatal tularemia. Ten cases of naturally occurring tularemia in cats were positive both by isolation of F. tularensis and immunohistochemical identification of F. tularensis antigen. Nine additional cases with lesions typical of tularemia were positive for F. tularensis antigen, although bacterial cultures were not performed. Immunohistochemical identification of F. tularensis in formalin-fixed tissue is valuable for establishing a rapid etiologic diagnosis under circumstances where fresh tissues may not be available for isolation and identification of the organism.     

The development of a real-time PCR to detect pathogenic Leptospira species in kidney tissue

A LightCycler real-time PCR hybridization probe-based assay that detects a conserved region of the16S rRNA gene of pathogenic but not saprophytic Leptospira species was developed for the rapid detection of pathogenic leptospires directly from processed tissue samples. In addition, a differential PCR specific for saprophytic leptospires and a control PCR targeting the porcine beta-actin gene were developed. To assess the suitability of these PCR methods for diagnosis, a trial was performed on kidneys taken from adult pigs with evidence of leptospiral infection, primarily a history of reproductive disease and serological evidence of exposure to pathogenic leptospires (n=180) and aborted pig foetuses (n=24). Leptospire DNA was detected by the 'pathogenic' specific PCR in 25 tissues (14%) and the control beta-actin PCR was positive in all 204 samples confirming DNA was extracted from all samples. No leptospires were isolated from these samples by culture and no positives were detected with the 'saprophytic' PCR. In a subsidiary experiment, the 'pathogenic' PCR was used to analyse kidney samples from rodents (n=7) collected as part of vermin control in a zoo, with show animals with high microagglutination titres to Leptospira species, and five were positive. Fifteen PCR amplicons from 1 mouse, 2 rat and 14 pig kidney samples, were selected at random from positive PCRs (n=30) and sequenced. Sequence data indicated L. interrogans DNA in the pig and rat samples and L. inadai DNA, which is considered of intermediate pathogenicity, in the mouse sample. The only successful culture was from this mouse kidney and the isolate was confirmed to be L. inadai by classical serology. These data suggest this suite of PCRs is suitable for testing for the presence of pathogenic leptospires in pig herds where abortions and infertility occur and potentially in other animals such as rodents.     

Rapid diagnosis and quantification of Francisella tularensis in organs of naturally infected common squirrel monkeys (Saimiri sciureus)

Francisella tularensis, a small Gram-negative facultative intracellular bacterium, is the causative agent of tularaemia, a severe zoonotic disease transmitted to humans mostly by vectors such as ticks, flies and mosquitoes. The disease is endemic in many parts of the northern hemisphere. Among animals, the most affected species belong to rodents and lagomorphs, in particular hares. However, in the recent years, many cases of tularaemia among small monkeys in zoos were reported. We have developed a real-time PCR that allows to quantify F. tularensis in tissue samples. Using this method, we identified the spleen and the kidney as the most heavily infected organ containing up to 400 F. tularensis bacteria per simian host cell in two common squirrel monkeys (Saimiri sciureus) from a zoo that died of tularaemia. In other organs such as the brain, F. tularensis was detected at much lower titres. The strain that caused the infection was identified as F. tularensis subsp. holarctica biovar I, which is susceptible to erythromycin. The high number of F. tularensis present in soft organs such as spleen, liver and kidney represents a high risk for persons handling such carcasses and explains the transmission of the disease to a pathologist during post-mortem analysis. Herein, we show that real-time PCR allows a reliable and rapid diagnosis of F. tularensis directly from tissue samples of infected animals, which is crucial in order to attempt accurate prophylactic measures, especially in cases where humans or other animals have been exposed to this highly contagious pathogen.     

Prevalence of Coxiella burnetii infections in aborted fetuses and stillborn calves

Coxiella burnetii infections are mostly subclinical in cattle, but can occasionally be associated with abortion. In the present study, 100 aborted fetuses or stillborn calves that were submitted for postmortem examination between September 2007 and March 2008 were examined for infection with C burnetii. Samples of both pooled fetal tissues and placental cotyledon were tested using a real-time PCR assay. In addition, the sections of placental cotyledon were examined using immunohistochemistry (IHC). The IHC of four placentas was positive. The PCR results of the IHC-positive placentas were high positive (HP); the PCR results of the organs of these four fetuses and calves varied from low positive (LP) to HP. The four IHC-positive fetuses had a gestation length of seven to nine months. All four placentas had histological signs of inflammation, but only one of four placentas had gross pathological signs of inflammation possibly due to a concomitant infection with Bacillus licheniformis. Five other IHC-negative placentas had (high) positive PCR results; the PCR results of the organs of these fetuses were LP or negative. The present study indicates that C burnetii infections are detected in a limited percentage of aborted fetuses and stillborn calves by IHC. To assess the importance of placentas with PCR-positive and IHC-negative test results, more research is needed.     

Detection of Francisella tularensis subsp. holarctica in a European brown hare (Lepus europaeus) in Thuringia, Germany

The isolation of Francisella tularensis subsp. holarctica biovar II (strain 06T0001) from a European brown hare (Lepus europaeus) from Thuringia, Germany, is described for the first time. Identification of the microorganism was carried out by phenotypic characterisation, partial sequencing of the 16S rRNA gene and specific PCR using the primers TUL4-435/TUL4-863 and FtC1/FtC4. The epidemiology of tularemia in Germany is discussed and a risk assessment for humans is made.     

基因芯片技术在钩端螺旋体病传染源监测中的应用

为了将合成的核酸探针点样到玻片 上制备成基因芯片,先用常规PCR扩增动物 组织中钩端螺旋体特异片段,再用Cy3标记 引物,通过不对称PCR获取荧光素标记的靶 序列,然后与制备的芯片杂交,扫描仪记录结 果。同时,以PCR技术为对照,进一步观察 基因芯片技术检测动物传染源中钩端螺旋体 的特异性、敏感性及可行性。常规PCR扩增 问号钩端螺旋体阳性标本,出现单一482bp 的扩增产物,而双曲钩端螺旋体及其他螺旋 体、病原、正常动物组织均无任何DNA扩增 条带,芯片杂交结果与常规PCR方法结果一 致。用芯片和PCR对动物组织中不同浓度 钩端螺旋体的检测,最低检测浓度分别为10 条和100条钩端螺旋体,芯片检测的敏感性 高于常规PCR法。用基因芯片与PCR分别 对标本进行检测,5只人工感染的豚鼠肾、 肝、心、血等组织的检测结果均为阳性;28份 疫区野鼠肾标本阳性结果分别为8份和4 份,10份可疑病猪肾标本阳性结果分别7份 和5份;5份非疫区野鼠肾、5份正常猪肾标 本以及其他微生物的检测结果均为阴性。结 果表明,基因芯片技术可快速、灵敏、特异地 检测动物传染源中问号钩端螺旋体。     

Serological survey for evidence of Leptospira interrogans in free-living platypuses (Ornithorhynchus anatinus)

Objective   To survey free-living platypuses for evidence of Leptospira interrogans .
Methods   A serological study of Leptospira antibodies was carried out on 21 platypuses captured between May and October 2001 in the Wollondilly River 200 km south of Sydney, New South Wales.
Results   Positive reactions, all to the L Hardjo serovar, were seen in 14 (66%) of the captured animals, with adult males showing a higher prevalence of antibodies than adult females. Several individual platypuses showed a high titre of L Hardjo antibodies, and some animals demonstrated cross-reactions to the serovars L Medanensis and L Kremastos.
Conclusion   The serological findings demonstrate that these animals are constantly exposed to infection with Leptospira in their environment, but it is not known if platypuses suffer from clinical leptospirosis or if they mount an immune response, but are unaffected by the bacteria. The prevalence of Leptospira infection among the platypus population could not be precisely estimated because of the unknown number of individuals inhabiting the Wollondilly River inside the survey property. Domestic livestock, mostly cattle, may be the major source of Leptospira infection. The effects of this disease on population dynamics and on reproduction in wild platypuses are not well understood. The role of other wildlife in the transmission and maintenance of Leptospira in the environment is unknown.     

Seroepidemiological studies of zoonotic infections in hunters in southeastern Austria--prevalences,risk factors,and preventive methods

The aim of this study was to investigate the seroprevalences to zoonotic pathogens in hunters, to propose preventive measures and to obtain more information about the occurrence of zoonotic pathogens in local wild animal populations. From 146 male and 3 female hunters originating from the south-eastern Austrian federal states of Styria and Burgenland blood samples were taken and anamnestic data were obtained using a questionnaire. The serological investigations included the following viral, bacterial and parasitic zoonotic agents or zoonoses, respectively (antibody-seroprevalences in brackets): encephalomyocarditis virus (EMCV, 15%), Puumala-Hantavirus (10%), Newcastle Disease virus (NDV, 4%), borreliosis (IgG 42%, IgM 7%), brucellosis (1%), chlamydiosis (3%), ehrlichiosis (IgG 15%, IgM 3%), leptospirosis (10%), tularaemia (3%), Q fever (0%), Echinococcus multilocularis/E. granulosus (5%/11%), toxocariasis (17%). Out of a control group of 50 persons (urban population, no hunters) only one person was found to be seropositive for Toxocara canis and NDV and four for EMCV, all other results were negative in the control group. The high seroprevalences especially to Borrelia burgdorferi s.l., Ehrlichia spp., Leptospira interrogans, E. granulosus, E. multilocularis, encephalomyocarditis virus and Puumala virus demonstrate that hunters are particularly exposed to zoonotic pathogens. It should also be noted that one hunter was seropositive for Brucella abortus and five exhibited antibodies to Francisella tularensis. In these cases, as well as in the cases of the 15 seropositives for Leptospira interrogans, the suspected source of infection may--besides rodents--also include wild boars and brown hares. The infections with NDV and Chlamydophila psittaci may be traced back to contact with certain species of birds (potential risk: aviaries). For Hantaviruses, rodents are considered to be the main source of human infections.     

Detection of chlamydiae in boar semen and genital tracts

Chlamydiae cause abortion and reproductive disorders in sows. Although organisms can infect the male genital tract, little is known about the disease situation in boars. Hence, we examined the prevalence of chlamydial infection in semen and genital tracts of boars. Samples collected from Swiss boars (group A: n=42), and boars from Germany (group B: n=39) were examined by bacteriology, LPS-ELISA, immunohistochemistry (IHC) and polymerase chain reaction (PCR). The latter methodology involved use of three PCR assays including 16Sig rDNA, IGS-S (intergenic spacer 16S/23S-Short) and IGS-L (intergenic spacer 16S/23S-Long) PCR for comparison methods. PCR sensitivity and the presence of potential PCR inhibitors were determined by spiking semen with Chlamydophila (Cp.) abortus DNA. Detection limits of the 16Sig and IGS-S PCR were 10 templates, while the IGS-L PCR was less sensitive (100 templates). Of 25 semen samples that were collected from group A, one semen sample was positive for Cp. psittaci and two were positive for Chlamydia-like organisms by 16Sig PCR. Screening of sera from Swiss boars revealed three animals with positive reactions in the LPS-ELISA, although we failed to detect chlamydiae within organs of these or sera-negative animals by IHC or IGS-S PCR. In group B, 10 ejaculates were positive for Chlamydia (C.) suis and two were positive for Chlamydia-like organisms by 16S PCR. The identification of DNA from Chlamydia-like organisms in semen from both groups of boars was surprising and a role for these bacteria in reproductive diseases requires further assessment. In conclusion, the prevalence of chlamydial infection was low in group A animals indicating that venereal transmission may not be significant for Chlamydia-associated reproductive diseases in pigs, although rare cases may occur.     

A multiplex PCR for the detection of Brucella spp. and Leptospira spp. DNA from aborted bovine fetuses

Bovine brucellosis and leptospirosis are important causes of bovine abortion around the world. Both diseases can be serologically diagnosed, but many factors may cause false positive and negative results. Direct methods based on bacteriological isolation are usually employed, but they are difficult, time consuming and dangerous. Monoplex polymerase chain reaction (PCR) have been successfully described for the detection of Brucella spp. and Leptospira spp. Aiming at improvement in the direct diagnosis, a multiplex PCR (mPCR) for the detection of these agents in aborted bovine fetuses is described. The detection threshold of the mPCR was evaluated in experimentally contaminated bovine clinical samples using a conventional proteinase K/SDS or a boiling-based extraction protocols. The mPCR was applied to two groups of clinical samples: 63 episodes of bovine abortion and eight hamsters experimentally infected with Leptospira interrogans serovar pomona. Adopting microbiological isolation as reference, the test showed a sensitivity of 100% in both groups of clinical samples. Seven samples collected from bovine fetuses were Brucella spp. culture negative but showed positive results in mPCR. Regarding Leptospira spp. detection, similar results were observed in three bovine clinical samples. All hamsters infected with Leptospira were positive in both microbiological culture and mPCR. The boiling extraction protocol showed better results in some clinical samples, probably by the removal of PCR inhibitors by heat treatment. The high sensitivity, simplicity and the possibility of detection of both bacteria in a single tube reaction support the use of the mPCR described in the routine diagnosis.