Proposal of Chlamydia pecorum sp. nov. for Chlamydia strains derived from ruminants.

Chlamydia pecorum sp. nov. is proposed as the fourth species of the genus Chlamydia on the basis of the results of a genetic analysis of Chlamydia strains that were isolated from cattle and sheep which had various diseases, including sporadic encephalitis, infectious polyarthritis, pneumonia, and diarrhea. The levels of DNA-DNA homology between C. pecorum and strains of C. psittaci, Chlamydia pneumoniae, and Chlamydia trachomatis were less than 10%. Several DNA probes were used to identify C. pecorum. The C. pecorum strains were distinguished from C. psittaci strains by the results of immunological assays, including an immunofluorescence antibody assay performed with monoclonal antibodies and an immunoblot analysis of the immunological specificity of the major outer membrane protein. Species identification was based on results obtained from DNA analyses and serology. The type strain of C. pecorum is strain ATCC VR628.     

Host range of chlamydiaphages phiCPAR39 and Chp3

The host range of phiCPAR39 is limited to four Chlamydophila species: C. abortus, C. caviae, C. pecorum, and C. pneumoniae. Chp3 (a newly discovered bacteriophage isolated from C. pecorum) shares three of these hosts (C. abortus, C. caviae, and C. pecorum) but can additionally infect Chlamydophila felis. The ability to support replication was directly correlated with the binding properties of the respective bacteriophages with their host species. Binding studies also show that phiCPAR39 and Chp3 use different host receptors to infect the same host cells: cell binding is sensitive to proteinase K treatment, confirming that the chlamydiaphage receptors are proteinaceous in nature.     

两种侵袭宿主方式的优势种马胃蝇飞行能力研究

为了解两种不同侵染宿主方式马胃蝇的飞行行为,本研究利用飞行磨系统测定了黑腹胃蝇Gasterophilus pecorum（以牧草为产卵载体）和肠胃蝇G. intestinalis（以宿主体毛为产卵载体）的飞行能力。结果表明:（1）肠胃蝇总飞行时间和距离均显著高于黑腹胃蝇,分别为后者的5.52倍和7.65倍,但平均飞行速度无显著性差异（P>0.05）。（2）黑腹胃蝇雌虫的飞行时间、距离和速度均略高于雄虫,而肠胃蝇雌虫除平均飞行速度外的飞行参数均低于雄虫。（3）肠胃蝇吊飞期间的体重消耗（24.38%）显著高于黑腹胃蝇（14.07%）;黑腹胃蝇雌雄成虫甘油三酯含量均显著下降,但两者差异不显著（P>0.05）。飞行距离差异反映了两种不同侵染宿主方式马胃蝇的飞行能力发生了适应性的变化,而总飞行时间为两种马胃蝇飞行距离差异的主导因素。     

The aberrant parasitism of horse botflies (Diptera: Gasterophilidae)

Alongside with a high intensity of infection of horses with botfly larvae there was observed mass aberrant parasitism of horse botflies in farms of Astrakhan, Guryev and Uralsk Provinces, and in the Kalmyk ASSR in 1980-1981 and 1987. As a result of extremely high aggregation of horse botfly larvae in their usual localization places, Gasterophilus pecorum larvae remained, due to interspecific competition, in nonspecific places (oral cavity, pharynx), adapted to new habitats and normally developed. Their number varied from 260 to 750 specimens. Localization of G. pecorum larvae in the mentioned departments of the alimentary canal results in serious morbidity of horses.     

Detection of Chlamydia species-specific serum antibodies by prior adsorption of common genus-specific antibodies

To establish a method for the detection of Chlamydia species-specific antibodies to the three species of Chlamydia responsible for human disease, the author attempted to remove Chlamydia genus-specific antibodies by prior adsorption with heterologous Chlamydia antigen. The effects of adsorption with heterologous antigen were investigated by the microplate immunofluorescence antibody technique. The Chlamydia genus-specific antibodies in immune animal sera were significantly reduced by prior adsorption with heterologous Chlamydia antigen. Chlamydia pecorum which does not infect humans was found to be useful for the adsorption. A preliminary test using Chlamydia trachomatis-infected human sera showed that this adsorption method with C. pecorum is applicable to the serodiagnosis of human Chlamydia infections.     

Evolutionary relationships among members of the genus Chlamydia based on 16S ribosomal DNA analysis.

Nucleotide sequences from strains of the four species currently in the genus Chlamydia, C. pecorum, C. pneumoniae, C. psittaci, and C. trachomatis were investigated. In vitro-amplified RNA genes of the ribosomal small subunit from 30 strains of C. pneumoniae and C. pecorum were subjected to solid-phase DNA sequencing of both strands. The human isolates of C. pneumoniae differed in only one position in the 16S rRNA gene, indicating genetic homogeneity among these strains. Interestingly, horse isolate N16 of C. pneumoniae was found to be closely related to the human isolates of this species, with a 98.9% nucleotide similarity between their 16S rRNA sequences. The type strain and koala isolates of C. pecorum were also found to be very similar to each other, possessing two different 16S rRNA sequences with only one-nucleotide difference. Furthermore, the C. pecorum strains truncated the 16S rRNA molecule by one nucleotide compared to the molecules of the other chlamydial species. This truncation was found to result in loss of a unilaterally bulged nucleotide, an attribute present in all other eubacteria. The phylogenetic structure of the genus Chlamydia was determined by analysis of 16S rRNA sequences. All phylogenetic trees revealed a distinct line of descent of the family Chlamydiaceae built of two main clusters which we denote the C. pneumoniae cluster and the C. psittaci cluster. The clusters were verified by bootstrap analysis of the trees and signature nucleotide analysis. The former cluster contained the human isolates of C. pneumoniae and equine strain N16. The latter cluster consisted of C. psittaci, C. pecorum, and C. trachomatis. The members of the C. pneumoniae cluster showed tight clustering and strain N16 is likely to be a subspecies of C. pneumoniae since these strains also share some antigenic cross-reactivity and clustering of major outer membrane protein gene sequences. C. psittaci and strain N16 branched early out of the respective cluster, and interestingly, their inclusion bodies do not stain with iodine. Furthermore, they also share less reliable features like normal elementary body morphology and plasmid content. Therefore, the branching order presented here is very likely a true reflection of evolution, with strain N16 of the species C. pneumoniae and C. psittaci forming early branches of their respective cluster and with C. trachomatis being the more recently evolved species within the genus Chlamydia.     

Simultaneous differential detection of Chlamydophila abortus, Chlamydophila pecorum and Coxiella burnetii from aborted ruminant's clinical samples using multiplex PCR

Background  

Chlamydiosis and Q fever, two zoonosis, are important causes of ruminants' abortion around the world. They are caused respectively by strictly intracellular and Gram negative bacterium Chlamydophila abortus (Cp. abortus) and Coxiella burnetii (C. burnetii). Chlamydophila pecorum (Cp. pecorum) is commonly isolated from the digestive tract of clinically inconspicuous ruminants but the abortive and zoonotic impact of this bacterium is still unknown because Cp. pecorum is rarely suspected in abortion cases of small ruminants. We have developed a multiplex PCR (m-PCR) for rapid simultaneous differential detection of Cp. abortus, Cp. pecorum and C. burnetii in clinical samples taken from infected animals.     

Chlamydiosis: seroepidemiologic survey in a red deer (Cervus elaphus) population in Italy

Chlamydiae are obligate, intracellular, gram-negative bacteria that are responsible for important diseases in humans, other mammals, and birds. Studies have shown that chlamydiae could be present in wild ruminants, but the serodiagnostic method most commonly used did not allow identification of chlamydial species. We determined the prevalence of antibodies to Chlamydia pecorum, Chlamydia suis, Chlamydia abortus, and Chlamydia psittaci in 271 red deer (Cervus elaphus) of a central Italian population, by using the microimmunofluorescence test that shows antibody response against genus-specific and species-specific antigens. No sera had detectable antibodies to C. pecorum and C. abortus. Antibodies were detected against C. psittaci (9.6%) and C. suis (3.3%). Antibody response could be related to contact of the red deer with birds and wild boars (Sus scrofa), respectively, and confirm an extended host range of individual Chlamydia species. In view of the potential zoonotic risk related to exposition of C. psittaci, our findings suggest surveillance of wild ruminants as potential reservoirs for chlamydiae.     

Characterization of the koala biovar of Chlamydia pneumoniae at four gene loci--ompAVD4, ompB, 16S rRNA, groESL spacer region

Koalas are infected with two species of Chlamydia, C. pecorum and C. pneumoniae. While it is known that significant genetic diversity occurs in the C. pecorum strains infecting koalas, very little is known about the C. pneumoniae strains that infect this host. In the current study, 10 isolates of koala C. pneumoniae were analysed at four gene loci and found to be different to both the human and horse C. pneumoniae strains at all loci (biovar differences ranging from 0.3% at groESL up to 9.0% at ompAVD4). All koala biovar isolates studied were found to be 100% identical at ompAVD4 (all 10 isolates) and at ompB (all three isolates) gene. This lack of allelic polymorphisms at ompAVD4 has now been observed for koala C. pneumoniae, human C. pneumoniae, guinea pig inclusion conjuctivitis C. psittaci and feline conjuctivitis C. psittaci and may be correlated to a lack of antibody response to the chlamydial major outer membrane protein (MOMP) in these same strain/host combinations. This study also provides the first documented case of natural C. pneumoniae infection causing a severe and extended respiratory episode in a captive koala population. This captive episode is in contrast to most free-range observations in which koala C. pneumoniae is rarely documented as causing respiratory, ocular or urogenital tract disease.     

Genomic Relatedness of Chlamydia Isolates Determined by Amplified Fragment Length Polymorphism Analysis

The genomic relatedness of 19 Chlamydia pneumoniae isolates (17 from respiratory origin and 2 from atherosclerotic origin), 21 Chlamydia trachomatis isolates (all serovars from the human biovar, an isolate from the mouse biovar, and a porcine isolate), 6 Chlamydia psittaci isolates (5 avian isolates and 1 feline isolate), and 1 Chlamydia pecorum isolate was studied by analyzing genomic amplified fragment length polymorphism (AFLP) fingerprints. The AFLP procedure was adapted from a previously developed method for characterization of clinical C. trachomatis isolates. The fingerprints of all C. pneumoniae isolates were nearly identical, clustering together at a Dice similarity of 92.6% (+/- 1.6% standard deviation). The fingerprints of the C. trachomatis isolates of human, mouse, and swine origin were clearly distinct from each other. The fingerprints of the isolates from the human biovar could be divided into at least 12 different types when the presence or absence of specific bands was taken into account. The C. psittaci fingerprints could be divided into a parakeet, a pigeon, and a feline type. The fingerprint of C. pecorum was clearly distinct from all others. Cluster analysis of selected isolates from all species revealed groups other than those based on sequence data from single genes (in particular, omp1 and rRNA genes) but was in agreement with available DNA-DNA hybridization data. In conclusion, cluster analysis of AFLP fingerprints of representatives of all species provided suggestions for a grouping of chlamydiae based on the analysis of the whole genome. Furthermore, genomic AFLP analysis showed that the genome of C. pneumoniae is highly conserved and that no differences exist between isolates of respiratory and atherosclerotic origins.     

Comparative scanning electron microscopy of Gasterophilus third instars

Comparison of cuticular features, including spine distribution and shape, structure of the maxillae and mandibles, the cephalic sensillae and the terminal abdominal segments of third instar Gasterophilus haemorrhoidalis, Gasterophilus inermis, Gasterophilus intestinalis, Gasterophilus meridionalis, Gasterophilus nasalis and Gasterophilus pecorum was conducted using scanning electron microscopy. One or more features distinguished among the species, with the exception of G. haemorrhoidalis and G. intestinalis, which shared all morphological characteristics. The features presented in this study complement and extend those presented in Zumpt's compendium. The function of some features described by Zumpt (e.g. the 'warts' on the rim of the respiratory chamber) is clarified and the presence of previously described sensory structures in the grooves on the maxillae of G. intestinalis is called into question.     

Evidence of a conserved role for Chlamydia HtrA in the replication phase of the chlamydial developmental cycle

Identification of the HtrA inhibitor JO146 previously enabled us to demonstrate an essential function for HtrA during the mid-replicative phase of the Chlamydia trachomatis developmental cycle. Here we extend our investigations to other members of the Chlamydia genus. C. trachomatis isolates with distinct replicative phase growth kinetics showed significant loss of viable infectious progeny after HtrA was inhibited during the replicative phase. Mid-replicative phase addition of JO146 was also significantly detrimental to Chlamydia pecorum, Chlamydia suis and Chlamydia cavie. These data combined indicate that HtrA has a conserved critical role during the replicative phase of the chlamydial developmental cycle.     

In Vitro Susceptibility of Chlamydia pecorum to Macrolides,Tetracyclines, Quinolones and β-Lactam

The in vitro susceptibility of Chlamydia pecorum to two macrolides (clarithromycin and erythromycin), two tetracyclines (doxycycline and minocycline), two quinolones (ofloxacin and ciprofloxacin) and one β-lactam (ampicillin) was determined. The MICs were 0.004 to 0.008 μg/ml for clarithromycin, 0.008 to 0.031 μg/ml for doxycycline and minocycline, 0.063 to 0.125 μg/ml for erythromycin, 0.25 to 0.5 μg/ml for ofloxacin and 0.25 to 1.0 μg/ml for ciprofloxacin. The MIC for ampicillin was greater than 1,024 μg/ml. The results show clarithromycin and doxycycline are the two most effective drugs against C. pecorum.     

Diagnosis of ovine enzootic abortion using an indirect ELISA (rOMP91B iELISA) based on a recombinant protein fragment of the polymorphic outer membrane protein POMP91B of Chlamydophila abortus

Chlamydophila abortus is of major economic importance worldwide as one of the principal causes of abortion in sheep. Serological diagnosis of infection by the complement fixation test (CFT) is complicated by false positive reactions resulting from cross-reactive antibodies to Chlamydophila pecorum. To improve diagnosis an indirect enzyme-linked immunosorbent assay (iELISA) based on a recombinant protein fragment of the C. abortus polymorphic outer membrane protein POMP91B (rOMP91B iELISA) was assessed using a panel of 281 sera from experimentally and naturally infected sheep. The iELISA performed well, being more sensitive (84.2%) and specific (98.5%) than the CFT. Furthermore, the iELISA was better at differentiating C. abortus- from C. pecorum-infected animals. The new rOMP91B iELISA test will prove a valuable tool for the routine serodiagnosis of C. abortus infection.     

Application of a Mycoplasma group-specific PCR for monitoring decontamination of Mycoplasma-infected Chlamydia sp. strains.

Mycoplasma contamination of biological materials remains a major problem. Most contaminations are caused by the use of Mycoplasma-contaminated cell lines. We adapted a Mycoplasma group-specific PCR to detect Mycoplasma contamination in cell lines and demonstrate its use in monitoring decontamination procedures with Mycoplasma-contaminated suspensions of Chlamydia spp. Three different methods were investigated: the use of Mycoplasma-specific antiserum in cell culture, physical separation by the combined use of enzymatic treatment and differential centrifugation, and the use of detergents. With these methods only incubation with Triton X-100 resulted in decontamination of Mycoplasma-contaminated suspensions of several laboratory strains of Chlamydia pneumoniae, C. pecorum, and C. trachomatis. Only one C. pneumoniae strain, UZG-1, was sensitive to Triton X-100 treatment. Since 39 of 40 throat swabs from patients with symptoms of an upper respiratory tract infection had positive reactions in the Mycoplasma group-specific PCR, this procedure could also have clinical significance in attempts to propagate C. pneumoniae strains from clinical specimens.     

Seroepidemiologic survey for Chlamydia suis in wild boar (Sus scrofa) populations in Italy

We used serology to estimate the prevalence of exposure to chlamydiae in Italian populations of wild boars (Sus scrofa). Sera from 173 hunter-killed wild boars harvested during the 2006-2009 hunting seasons in three Italian regions were tested for antibodies to Chlamydia suis, Chlamydophila pecorum, Chlamydophila abortus, and Chlamydophila psittaci by the microimmunofluorescence test. Antibody titers to chlamydiae ≥ 1:32 were detected in 110 of the 173 samples tested (63.6%). Specific reactivity could be assessed only in 44 sera with antibody titers to C. suis that were two- to threefold higher than antibody titers against the other chlamydial species; the other 66 sera had similar reactivity against all the chlamydia species tested. Antibody to C. suis was detected in sera from wild boar populations with rare or no known contact with domestic pigs. These results suggest that the wild boar could be a chlamydia reservoir and may acquire chlamydiae independent of contacts with the domestic pig.     

Seroepidemiology of Feline Chlamydiosis by Microimmunofluorescence Assay with Multiple Strains as Antigens

The prevalence of anti-chlamydia antibodies was examined in 232 cat sera collected in 1985 and from 1993 to 1995 from laboratories and veterinary hospitals located in 11 prefectures of Japan. The antibodies were determined by an indirect microimmunofluorescence test using six strains of feline Chlamydia: one strain each of avian- and guinea pig-derived C. psittaci and one strain each of C. pecorum, C. pneumoniae and C. trachomatis. Positive rates of IgG antibodies to chlamydiae were 34.4% in 1985 and 16.5–21.4% from 1993 to 1995. Positive rates of IgM antibodies to chlamydiae were 8.2% in 1985 and 6.6–14.3% from 1993 to 1995. Variations in antibody reactivity to the different feline strains were observed. The results suggest the wide prevalence of chlamydial infection in cats in Japan, and antigenic diversity in the feline strains of C. psittaci.     

Utility of mitochondrial and ribosomal genes for differentiation and phylogenesis of species of gastrointestinal bot flies

Larvae of Gasterophilus spp. (Diptera: Oestridae) cause gastrointestinal myiasis of equids. However, their identification may be problematic due to morphological similarities between species infesting identical regions of the digestive tract. In this study, genes encoding for mitochondrial cytochrome oxidase I (COI) and for the 16S and 28S ribosomal subunits of the most commonly encountered Gasterophilinae subfamily species [i.e., Gasterophilus haemorrhoidalis (L.), Gasterophilus inermis (Brauer), Gasterophilus intestinalis (De Geer), Gasterophilus nasalis (L.), and Gasterophilus pecorum (F.)] were studied, together with Gyrostigma pavesii (Corti), a rhinoceros parasite, and Hypoderma lineatum (De Villers), as outgroup taxa. Analysis identified interspecific differences that allowed their unequivocal identification. The high genetic homology among the sequences of G. haemorrhoidalis and G. intestinalis (i.e., 100, 99.86, and 99.46% in the 28S, COI, and 16S genes, respectively) strongly support the hypothesis that they are morphotypes of the same species. Phylogenetic analyses (maximum-likelihood and parsimony) were performed using PAUP; all analyses supported monophyly of subfamily Gasterophilinae. This study confirms the utility of the COI and 16S and 28S rRNA genes to address diagnostic and phylogenetic questions in Gasterophilus species.     

Biological properties and cell tropism of Chp2, a bacteriophage of the obligate intracellular bacterium Chlamydophila abortus

A number of bacteriophages belonging to the Microviridae have been described infecting chlamydiae. Phylogenetic studies divide the Chlamydiaceae into two distinct genera, Chlamydia and Chlamydophila, containing three and six different species, respectively. In this work we investigated the biological properties and host range of the recently described bacteriophage Chp2 that was originally discovered in Chlamydophila abortus. The obligate intracellular development cycle of chlamydiae has precluded the development of quantitative approaches to assay bacteriophage infectivity. Thus, we prepared hybridomas secreting monoclonal antibodies (monoclonal antibodies 40 and 55) that were specific for Chp2. We demonstrated that Chp2 binds both C. abortus elementary bodies and reticulate bodies in an enzyme-linked immunosorbent assay. Monoclonal antibodies 40 and 55 also detected bacteriophage Chp2 antigens in chlamydia-infected eukaryotic cells. We used these monoclonal antibodies to monitor the ability of Chp2 to infect all nine species of chlamydiae. Chp2 does not infect members of the genus Chlamydia (C. trachomatis, C. suis, or C. muridarum). Chp2 can infect C. abortus, C. felis, and C. pecorum but is unable to infect other members of this genus, including C. caviae and C. pneumoniae, despite the fact that these chlamydial species support the replication of very closely related bacteriophages.