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1.
Nasal lavage fluid was collected from 155 tortoises, mostly Testudo spp., that were kept as companion animals and suffered from nasal discharge. Examination for chlamydial DNA by PCR assays targeting the ompA, ompB, and groESL genes, as well as the 16S rRNA signature region and the 16S-23S intergenic spacer, respectively, revealed 16 (10.3%) positive animals. Sequence analysis of PCR products indicated high homology to the family Chlamydiaceae. Phylogenetic trees constructed from partial sequences of the ompA and 16S rRNA genes showed that the present samples clustered outside the nine species of Chlamydia and Chlamydophila. Sequences of the nearest relative, Chlamydophila pecorum, were still clearly distinct from those of the positive tortoise samples. This suggests that the tortoises had been infected by Chlamydia-like agents, the taxonomic identity and pathogenic importance of which has yet to be established.  相似文献   

2.
Hemotrophic mycoplasmas (hemoplasmas) are bacteria that attach to red blood cells of mammals, leading to acute and/or subclinical disease in infected animals. It has been suggested that Mycoplasma ovis, a hemoplasma that infects sheep and goats worldwide, may also infect deer. The aim of this study was to evaluate whether South American deer are infected with M. ovis. EDTA-anticoagulated blood samples from a herd of 32 captive South American deer were collected. DNA extraction of blood samples was performed followed by PCR amplification of the 16S and 23S rRNA genes, and sequencing of products. Using M. ovis PCR, 27/31 (87%) were positive, including 21/22 Mazama nana; 2/3 Mazama americana and 4/6 Blastocerus dichotomus. Sequencing of the nearly entire 16S rRNA gene of 26/27 positive samples showed 98.2-98.8% identity to M. ovis of sheep (GenBank, AF338268) and 98.6-99.4% identity to M. ovis-like of a fawn (FJ824847); the 23S rRNA gene from one of these isolates and the fawn's had 97.6% identity. The remaining isolate had just 94.9% identity to the 16S rRNA gene of M. ovis and only 89.4% identity to the 23S rRNA gene of the fawn's M. ovis. This is the first report of M. ovis in captive South American deer, revealing a high prevalence of hemoplasma infection in these animals.  相似文献   

3.
A total of 210 frog samples originating either from a mass mortality (1991/1992) or from routine postmortem investigations of the years 1990 to 2004 were examined retrospectively for a possible involvement of Chlamydiae. For a prevalence study of Chlamydia in a selected Swiss amphibian population, 403 samples from free-ranging Rana temporaria were examined. Histopathology, immunohistochemistry using a monoclonal antibody against chlamydial lipopolysaccharide, and a 16S rRNA polymerase chain reaction (PCR) followed by DNA sequencing were performed on the formalin-fixed and paraffin-embedded tissues. Using PCR, 8 of 54 (14.8%) frog samples from the mass mortality (1991/1992) were positive for Chlamydia suis S45. A control group of healthy Xenopus laevis had 3 of 38 positive samples, sequenced as C suis S45 (2/3) and an endosymbiont of Acanthamoeba species UWE1 (1/3). Chlamydophila pneumoniae TW-183 was detected from exotic frogs kept in a zoo. Of the frogs collected for the prevalence study, 6 of 238 (2.5%) tested positive, 1 each for C suis S45, Cp pneumoniae TW-183, and uncultured Chlamydiales CRG22, and the remaining 3 revealed Chlamydophila abortus S26/3. In immunohistochemistry, there were 2 positive labeling reactions, 1 in intestine and the other in the epithelium coating the body cavity, both testing positive for Cp pneumoniae TW-183 in PCR. Histologically there were no lesions recorded being characteristic for Chlamydia. Although there is a prevalence of Chlamydia in Swiss frogs, no connection to a mass mortality (1991/1992) could be established. For the first time, C suis S45 and Cp abortus S26/3 were detected in frog material.  相似文献   

4.
OBJECTIVE: To detect and characterize the full range of chlamydial infections in cats with ocular disease by use of polymerase chain reaction (PCR) assays, cytologic examination, immunohistochemical analysis, and evaluation of clinical information including status for feline herpesvirus-1 (FeHV-1). SAMPLE POPULATION: DNA extracted from 226 conjunctival samples obtained from cats with clinically diagnosed keratitis or conjunctivitis and 30 conjunctival samples from healthy cats. PROCEDURE: PCR assays for the 16S rRNA gene specific for the order Chlamydiales and a new Chlamydophila felis (formerly Chlamydia psittaci) species-specific 23S rRNA gene were performed. Seventy-four conjunctival samples were prepared with Romanowsky-type stain, grouped on the basis of inflammatory pattern, and screened for chlamydial inclusions by use of immunohistochemical analysis. Clinical information and FeHV-1 status were recorded. RESULTS: 26 (12%) specimens had positive results for the only known feline chlamydial pathogen, C felis. Surprisingly, an additional 88 (39%) were positive for non-C felis chlamydial DNA. Identification of non-C felis chlamydial DNA by direct sequencing revealed 16S rRNA gene sequences that were 99% homologous to the sequence for Neochlamydia hartmannellae, an amebic endosymbiont. Chlamydial prevalence was significantly higher in cats with ocular disease. CONCLUSIONS AND CLINICAL RELEVANCE: Application of a broad-range detection method resulted in identification of a new agent associated with ocular disease in cats. Finding chlamydia-like agents such as N hartmannellae in coinfections with their obligate amebic host, Hartmannella vermiformis, raises questions about the potential role of these microorganisms in causation or exacerbation of ocular disease in cats.  相似文献   

5.
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.  相似文献   

6.
Objective To determine the presence of chlamydial species including recently described chlamydial agents as well as the human pathogen Chlamydophila pneumoniae in feline conjunctivitis. Animal studied Twenty five cats without and 49 cats with conjunctivitis were tested for chlamydia using a Chlamydiaceae real time (RT) PCR (targeting the 23S rRNA gene sequence), a Chlamydiales PCR (targeting the 16S rRNA gene sequence), and cell culture. The PCR products of all positive samples were sequenced and subsequently analyzed using a basic local alignment search tool search. Results Chlamydiaceae RT PCR and subsequent sequence analyses identified C. pneumoniae in five cats in the conjunctivitis group. The presence of Chlamydophila felis was shown in two cats with conjunctivitis. Chlamydiae related to uncultured members of Chlamydiales were detected in three conjunctivitis cases and in one cat without clinical symptoms. Conclusion This study detects for the first time, the known human pathogen C. pneumoniae in feline conjunctivitis cases using Chlamydiaceae RT PCR and sequence analyses.  相似文献   

7.
猪附红细胞体PCR检测方法的建立和初步应用   总被引：22，自引：1，他引：22  
基于猪附红细胞体广东株16S rRNA基因的序列特点，设计合成种特异性引物，建立了猪附红细胞体PCR检测方法。该方法能特异性扩增523bp的猪附红细胞体16SrRNA基因片段，而对猪丹毒杆菌G4T10株、猪链球菌STl71株、多杀性巴氏杆菌E0630株、猪胸膜肺炎放线杆菌、猪肺炎支原体、鸡毒支原体和猫血巴尔通氏体CA株的基因组DNA没有扩增带出现。对猪附红细胞体基因组DNA的最小检测量为160pg。通过对38份临床样品的检测，8份为猪附红细胞体感染阳性，其余为阴性。结果表明，建立的PCR检测方法具有极高的敏感性和特异性，可用于急性猪附红细胞体病和临床健康带菌猪的诊断。  相似文献   

8.
Ocular infections by chlamydiae are associated with ocular disease manifestations such as conjunctivitis and keratitis in humans and animals. Limited evidence exists that members of the order Chlamydiales can also cause ocular disease in sheep. In the current study, the prevalence of chlamydiae in the eyes of sheep was investigated by using PCR methods. Data obtained in sheep by broad-range 16S rRNA order Chlamydiales-specific PCR were compared to the prevalence of antibodies against chlamydiae detected by a competitive enzyme-linked immunosorbent assay (cELISA). Flocks tested included a clinically healthy flock and two flocks suffering from ocular disease and with histories of Ovine Enzootic Abortion (OEA). PCR detected DNA of Chlamydophila (Cp.) abortus and Cp. pecorum in the eyes of both healthy and sick animals but also identified Chlamydia (C.) suis and a variety of uncultured chlamydia-like organisms. Good correlation was found between the presence of Cp. abortus DNA in sheep conjunctival samples and seropositivity detected by cELISA. Despite these findings, no association was found between the presence of chlamydial DNA in the sheep conjunctival samples and the onset of clinical disease. These results suggest that the biodiversity of chlamydiae in the eyes of sheep is greater than that previously thought. Further investigations are needed to determine whether a causal relationship between infection by chlamydiae and ocular disease exists in these animals.  相似文献   

9.
为筛选出检测猪支原体更为特异、敏感的PCR检测方法,本试验分别以16S rRNA、50S rRNA和膜蛋白OxaA为靶基因进行PCR检测,并从其敏感性、特异性和临床样本检出率等方面进行了比较。结果显示,以膜蛋白OxaA和16S rRNA为靶基因的PCR方法敏感性最高,最小检测DNA量为1.86 fg/μL,而以50S rRNA为靶基因的PCR方法最小检测DNA量为18.6 fg/μL;3种靶基因引物均扩增不出大肠杆菌、猪链球菌、猪肺炎支原体、牛附红细胞体等基因片段,具有较好的特异性;通过对临床60份血液样本的检测结果表明,以膜蛋白OxaA基因设计的引物检出率最高,为25%(15/60),明显高于16S rRNA基因的21.6%(13/60)和50S rRNA基因的18.3%(11/60)。本试验为猪支原体病的诊断及流行病学调查提供了更为敏感、特异的检测技术。  相似文献   

10.
The purpose of this study was to characterize, at the molecular level, the Ehrlichia canis strains involved in naturally occurring canine monocytic ehrlichiosis (CME) in Greece, and to investigate if any sequence diversity exists between the 16S rRNA genes of those involved in the mild non-myelosuppressive or the severe myelosuppressive form of CME. To this end, amplification of the ehrlichial 16S rRNA gene was attempted by nested polymerase chain reaction (PCR) assays in bone marrow (BM) aspirates from 20 dogs tentatively diagnosed as having non-myelosuppressive (n=10, group A) or myelosuppressive (n=10, group B) CME. PCR assay using E. canis-specific primers revealed that 15 BM samples, including all group A and 5 group B dogs, were positive. Using universal PCR primers, a nearly full-length 16S rRNA gene could be amplified from 13 BM samples, including 9 group A and 4 group B dogs. The 16S rDNA analysis based on secondary structure revealed that all sequences of the Greek strains were identical to each other and indicated 100% identity among some American (Venezuelan and Brazilian), European (Greek), Middle Eastern (Turkish) and Asiatic (Thailand) strains. The results of this study suggest that the E. canis strains involved in the non-myelosuppressive and myelosuppressive forms of CME in Greece share an identical 16S rRNA genotype.  相似文献   

11.
We examined the presence of hemoplasmas, hemotropic mycoplasmas, among 11 sheep (Ovis aries) with regenerative and hemolytic anemia and found six of them were positive by real-time PCR. The positive samples were then subjected to conventional PCR for direct sequencing of the 16S rRNA gene. Nucleotide sequences of all the positive samples were identified as the 16S rRNA gene of `Candidatus Mycoplasma haemovis' by phylogenetic analysis, demonstrating the infections with this particular hemoplasma species in Japan.  相似文献   

12.
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13.
Nineteen blood samples collected from free-ranging wild Japanese serows, Capricornis crispus, between 2006 and 2008 in Iwate prefecture were examined for the hemoplasma infection by real-time PCR targeting the 16S rRNA gene. Five (26.3%) out of the 19 samples were positive in real-time PCR with an average melting temperature at 85.18 °C. The positive samples in the real-time PCR were reconfirmed by conventional PCR, and one of them was successful for direct DNA sequencing. The nucleotide sequence of the 16S rRNA gene of the representative stain was identical to that of Mycoplasma ovis. This was the first demonstration of hemotropic mycoplasma infections among the free-living Japanese serows in Japan.  相似文献   

14.
In 2001, the first case of bovine chlamydial abortion was reported in canton Graubunden, Switzerland. In this region, Chlamydophila (Cp.) abortus is endemic in small ruminants. Hence, we aimed to investigate the incidence of chlamydia-related abortions in cattle from Graubunden. During breeding seasons of 2003-2004, formalin-fixed and paraffin-embedded placenta specimens (n = 235) from late-term abortions in cattle were analyzed by histopathology, immunohistochemistry with a Chlamydiaceae-specific monoclonal antibody against chlamydial lipopolysaccharide (LPS), and 2 different polymerase chain reaction (PCR) methods (16 S ribosomal ribonucleic acid [rRNA] PCR, intergenic spacer [IGS-S] PCR), followed by PCR product sequencing. In 149 of 235 cases (63.4%), histopathologic lesions such as purulent and/or necrotizing placentitis were observed. Chlamydial antigen was clearly demonstrated in immunohistochemistry in only 1 of 235 cases (0.4%). Cp. abortus or Cp. psittaci was found in 12 of 235 (5.1%) and 10 of 235 cases (4.2%) by 16 S rRNA PCR and IGS-S PCR, respectively. However, we detected, by 16 S rRNA PCR, 43 of 235 cases (18.3%) to be positive for chlamydia-like organisms. In contrast to the situation in small ruminants in the canton Graubunden, bovine abortion from Cp. abortus seems not to play an important role. Nevertheless, zoonotic potential should be taken into account when handling abortion material from cattle. The significance of chlamydia-like isolates other than Waddlia chondrophila remains an open question in abortion and needs further investigation.  相似文献   

15.
The aim was to detect and characterize chlamydial infections in guinea-pigs (GP) with ocular disease, study their pathogenicity and zoonotic potential and to test for the presence of Acanthamoebae spp. in GP eyes and to investigate whether they could act as vectors for Chlamydia-like organisms. Overall 126 GP, of which 77 were symptomatic, were screened by clinical examination, cytology, gross pathology, histology, immunohistochemistry, polymerase chain reaction (PCR) and bacteriology. A new Chlamydiaceae-specific intergenic spacer rRNA gene PCR, designed to amplify this segment linking the 16S and 23S regions, was performed. DNA samples were also received from one owner including samples of his cat and rabbit. Guinea-pigs: 48 of 75 symptomatic, but only 11 of 48 asymptomatic GP were positive by PCR for Chlamydophila caviae guinea-pig inclusion conjunctivitis (GPIC) (P < 0.0001). Eighteen of 75 or 15/48, respectively, were positive for DNA from Chlamydia-like organisms. Acanthamoebae-DNA could be found in two GP, of which one was symptomatic. Owner, cat and rabbit: Samples of all three species were positive by PCR for C. caviae GPIC and the owner's one-day disposable contact lenses showed a positive PCR result for the Chlamydia-like organism Parachlamydia acanthamoebae. No Acanthamoebae-DNA could be detected. This study is the first to describe Chlamydia-like organisms in GP and to detect C. caviae GPIC in human, cat and rabbit. Therefore, C. caviae GPIC could pose a zoonotic potential. We believe that the finding of C. caviae GPIC in species other than GP is probably not unique.  相似文献   

16.
OBJECTIVE: To describe the detection of Ehrlichia platys in free-roaming dogs in Central Australia. PROCEDURE: Blood samples were collected from four dogs and examined for bacterial 16S ribosomal DNA using Polymerase Chain Reaction (PCR)-based assays. The three positive samples obtained were then sequenced and identification of the PCR product carried out. As a result of all three samples being identical to or closely related to part of the 16S rRNA gene of E. platys, blood samples were subsequently obtained from a further 24 dogs. These samples were screened using a PCR-assay to determine the presence of Ehrlichia DNA using genus-specific primers. The positive samples obtained from the screening process were then subjected to a further PCR-assay using E. platys specific primers. RESULTS: Of 28 dogs sampled, Ehrlichia DNA was detected in the blood of 13 dogs. Sequencing of the amplicons obtained indicated a high homology with the 16S rRNA gene for E. platys. When the E. platys-specific PCR was performed for 10 of those dogs, the 678 bp product obtained from the PCR amplification confirmed the identification as part of the 16S rRNA gene of E. platys in all 10 dogs. CONCLUSION: This study reports for the first time Ehrlichia carriage by dogs in Australia. It also indicates the usefulness of the PCR technique in rapidly and accurately identifying diseases that are otherwise difficult to detect. By using universal primers directed against bacterial 16S ribosomal DNA and sequencing analysis, the detection of potentially pathogenic Ehrlichia organisms that had not previously been found in Australia has been made possible.  相似文献   

17.
In a previous study we described the use of a new set of PCR primers (CpsiA/CpsiB) specific of the conserved pmp-family genes of Chlamydophila abortus as an efficient tool for the detection of these bacteria in ruminants including also preliminary results on avian strains. In this work, the use of this set of primers was extended to representative strains of the six major avian serovars (serovars A-F) and to field isolates of C. psittaci. For all the studied representative strains, using purified genomic DNA as a template, CpsiA/CpsiB primers allowed, as observed for C. abortus, a minimal 10-fold PCR signal increase compared to the one observed with ompA specific primers. In comparison to primers targeting the 16S-23S rRNA intergenic spacer, similar or increased sensitivity was observed depending on the strain. All the field isolates were amplified with CpsiA/CpsiB primers. On clinical samples, our primers are the best among those tested for detection of C. psittaci by simple conventional PCR. RFLP experiments performed using PCR fragments amplified with the CpsiA/CpsiB primers gave promising results demonstrating that these primers may provide an interesting tool for molecular typing when the bacterium cannot be grown from pathological samples.  相似文献   

18.
Antimicrobial resistance (AMR), encoded by plasmid‐mediated AMR genes (ARGs), is an increasing global public health threat. Wildlife play a fundamental role as sentinels, reservoirs and potential vectors of ARGs. For the first time in Galapagos, we have identified and quantified the presence of ARGs in free‐living giant tortoises (Chelonoidis porteri). We performed ARG analyses by quantitative PCR of faeces collected from the cloaca of 30 tortoises widely distributed across Santa Cruz Island. Validated samples (n = 28) were analysed by a panel of up to 21 different ARGs and all 28 tortoise samples were positive to one or more genes encoding resistance. Thirteen of 21 tested ARGs were present in at least one sample, and 10 tortoises (35.7%) had a multi‐resistant pattern. We recommend additional research so we may more fully understand resistance patterns across taxa and geographical locations throughout the Galapagos archipelago, and the implications of ARGs for the health of wildlife, domestic animals, and humans. In this study, we found 100% of sampled giant tortoises had ARGs present in their faeces, suggesting a large‐scale distribution of these genes within the archipelago.  相似文献   

19.
Although hemoplasma infection in domestic animals has been well documented, little is known about the prevalence and genetic diversity of these bacteria in wild rodents. The present work aimed to investigate the occurrence of hemotrophic mycoplasmas in wild rodents from five Brazilian biomes, assessing the 16S rRNA phylogenetic position of hemoplasma species by molecular approach. Spleen tissues were obtained from 500 rodents, comprising 52 different rodent species trapped between 2000 and 2011. DNA samples were submitted to previously described PCR protocols for amplifying Mycoplasma spp. based on 16S rRNA, followed by sequencing and phylogenetic inferences. Among 457 rodent spleen samples showing absence of inhibitors, 100 (21.9%) were PCR positive to Mycoplasma spp. The occurrence of hemotropic mycoplasmas among all sampled rodents was demonstrated in all five biomes and ranged from 9.3% (7/75) to 26.2% (38/145). The Blastn analysis showed that amplified sequences had a percentage of identity ranging from 86 to 99% with other murine hemoplasmas. The ML phylogenetic analysis of 16S rRNA gene of 24 positive randomly selected samples showed the presence of ten distinct groups, all clustering within the Mycoplasma haemofelis. The phylogenetic assessment suggests the circulation of novel hemoplasma species in rodents from different biomes in Brazil.  相似文献   

20.
Strangles is a contagious equine disease caused by Streptococcus equi subsp. equi. In this study, clinical strains of S. equi (n=24) and Streptococcus equi subsp. zooepidemicus (n=24) were genetically characterized by sequencing of the 16S rRNA and sodA genes in order to devise a real-time PCR system that can detect S. equi and S. zooepidemicus and distinguish between them. Sequencing demonstrated that all S. equi strains had the same 16S rRNA sequence, whereas S. zooepidemicus strains could be divided into subgroups. One of these (n=12 strains) had 16S rRNA sequences almost identical with the S. equi strains. Interestingly, four of the strains biochemically identified as S. zooepidemicus were found by sequencing of the 16S rRNA gene to have a sequence homologous with Streptococcus equi subsp. ruminatorum. However, they did not have the colony appearance or the biochemical characteristics of the type strain of S. ruminatorum. Classification of S. ruminatorum may thus not be determined solely by 16S rRNA sequencing. Sequencing of the sodA gene demonstrated that all S. equi strains had an identical sequence. For the S. zooepidemicus strains minor differences were found between the sodA sequences. The developed real-time PCR, based on the sodA and seeI genes was compared with conventional culturing on 103 cultured samples from horses with suspected strangles or other upper respiratory disease. The real-time PCR system was found to be more sensitive than conventional cultivation as two additional field isolates of S. equi and four of S. zooepidemicus were detected.  相似文献   

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