Genetic diversity and epizootiology of Chlamydophila psittaci prevalent among the captive and feral avian species based on VD2 region of ompA gene

To study genetic diversity and occurrence of Chlamydophila psittaci, a total of 1,147 samples from 11 avian orders including 53 genera and 113 species of feral and captive birds were examined using ompA gene based nested PCR. Three types of chlamydiae: C. psittaci (94.12%), C. abortus (4.41%) and unknown Chlamydophila sp. (1.47%) were identified among 68 (5.93%) positive samples (Psittaciformes-59, Ciconiiformes-8 and Passeriformes-1). Based on nucleotide sequence variations in the VD2 region of ompA gene, all 64 detected C. psittaci strains were grouped into 4 genetic clusters. Clusters I, II, III and IV were detected from 57.35%, 19.12%, 10.29% and 7.35% samples respectively. A single strain of unknown Chlamydophila sp. was found phylogenetically intermediate between Chlamydophila species infecting avian and mammalian hosts. Among Psittaciformes, 28 out of 81 tested species including 10 species previously unreported were found to be chlamydiae positive. Chlamydiosis was detected among 8.97% sick and 48.39% dead birds as well 4.43% clinically normal birds. Therefore, it was observed that though various genetically diverse chlamydiae may cause avian chlamydiosis, only a few C. psittaci strains are highly prevalent and frequently associated with clinical/subclinical infections.     

Yersinia species in the dunnock (Prunella modularis) in sub-alpine habitats of the Western Carpathians

The study presents the prevalence of Yersinia species in dunnok Prunella modularis from the sub-alpine zone of the Western Carpathians. Bacteria were detected from cloacal and pharyngeal swabs from 97 specimens using PCR assay. Yersinia enterocolitica showed the highest prevalence (47.4%) from among the determined Yersinia species. Yersinia species (except Y frederiksenii) were detected more frequently in pharyngeal than cloacal samples. The highest prevalence of yersiniosis was detected in April (Yersinia spp. - 80%, Y. enterocolitica - 70%). No statistically differences were observed in the prevalence of Yersinia spp. between males and females and between juveniles and adult birds. Bacterial contamination did not affect body weight or tarsus length.     

Prevalence of Chlamydophila felis by PCR among healthy pet cats in Italy

Conjunctival swabs were taken from 60 healthy pet cats and tested for Chlamydophila felis by PCR assays to amplify the ompA, omp2 and groEL genes. Chlamydial DNA was detected in 2 (3.3%) cats, one of which had been vaccinated against C. felis eight months before sample collection. The nucleotide and predicted amino acid sequences of three genes from two cats showed 100% identity with the same regions amplified from conjunctival swabs of cats in the same geographic area.     

三种方法对禽鹦鹉热嗜性衣原体疑似病例病原的流行调查

目的利用免疫荧光抗体法、PCE-ELISA、PCR法分别检测北京市及周边地区禽鹦鹉热嗜性衣原体疑似病例的病原,以了解和评价该病原流行状况。方法收集临床疑似禽衣原体的父母代、商品代家禽、SPF鸡喉头拭子、气囊组织、子宫黏膜,组织标本固定后采用直接免疫荧光染色测定衣原体包含体;喉头拭子、子宫黏膜处理后以PCE-ELISA定量测定衣原体;以衣原体保守性高的序列设计CTU/CTL引物扩增病料组织的omp-1基因片段。结果直接荧光染色法显示家禽衣原体平均阳性率为38.7%,组织检出率依次为子宫黏膜、气囊、喉头拭子;PCE-ELISA检测显示平均阳性率为58.7%,其中SPF鸡阳性率为10.0%,健康肉鸡达到30.0%;PCR法检测家禽衣原体平均阳性率为71.6%,SPF鸡为10.0%。样本的目的基因与标准禽衣原体6BC同源性超过99%。结论种禽场和商品养殖场均发现疑似病例中禽鹦鹉热嗜性衣原体感染情况较为严重。荧光抗体染色、PCE-ELISA可用于临床实践中进行快速、准确检测。     

Prevalence of <Emphasis Type="Italic">Chlamydophila psittaci</Emphasis> in wild birds—potential risk for domestic poultry,pet birds,and public health?

To determine the prevalence of Chlamydophila psittaci in wild birds, cloacal swabs from 527 songbirds, 442 waterfowl, 84 feral pigeons, and 38 cormorants were examined by Chlamydiaceae-specific real-time polymerase chain reaction (PCR) and ArrayTube microarray assays for chlamydial species determination and genotyping of C. psittaci. Inconclusive cases were further characterized by conventional PCR methods targeting the chlamydial outer membrane protein A, 16S, 23S, and intergenic spacer genes followed by sequencing of the PCR product. Swabs of 19 water birds (tufted ducks and pochards), 12 pigeons, and one songbird were tested positive by the Chlamydiaceae-specific real-time PCR. While C. psittaci genotypes B (n = 5) and E (n = 1) were identified in feral pigeons (n = 9), the genotype could not be identified in the remaining three cases. Sequence data of Chlamydiaceae-positive cases (n = 23; 19 waterfowl, three pigeons, one songbird) indicated the presence of nonclassified chlamydial agents (n = 20) and C. psittaci (n = 3) by 16S rRNA PCR and sequencing. In conclusion, C. psittaci was not detected in waterfowl and songbirds, but C. psittaci proved prevalent in urban feral pigeons, where it poses a significant risk for humans.     

Isolation of Vibrio cholerae from aquatic birds in Colorado and Utah

Vibrio cholerae was isolated from cloacal swabs and freshly voided feces collected from 20 species of aquatic birds in Colorado and Utah during 1986 and 1987. About 17% (198 of 1,131) fecal specimens collected from July 1986 through August 1987 contained the organism. Both O1 and non-O1 V. cholerae strains were isolated from the fecal specimens. Isolates from eight birds (representing five species) agglutinated in O group 1 antiserum. Supernatants of broth cultures from three isolates which typed as V. cholerae O1 serotype Ogawa gave reactions typical of cholera toxin when tested on Y-1 mouse adrenal cell cultures. Several serovars of non-O1 V. cholerae were isolated from the fecal specimens; serovar 22 was the most prevalent type. All non-O1 isolates were cytotoxic to Y-1 mouse adrenal cells. Only non-O1 V. cholerae was detected in water samples collected from the habitat of the birds. The results of this study suggest that aquatic birds serve as carriers and disseminate V. cholerae over a wide area.     

Isolation of Vibrio cholerae from aquatic birds in Colorado and Utah.

Vibrio cholerae was isolated from cloacal swabs and freshly voided feces collected from 20 species of aquatic birds in Colorado and Utah during 1986 and 1987. About 17% (198 of 1,131) fecal specimens collected from July 1986 through August 1987 contained the organism. Both O1 and non-O1 V. cholerae strains were isolated from the fecal specimens. Isolates from eight birds (representing five species) agglutinated in O group 1 antiserum. Supernatants of broth cultures from three isolates which typed as V. cholerae O1 serotype Ogawa gave reactions typical of cholera toxin when tested on Y-1 mouse adrenal cell cultures. Several serovars of non-O1 V. cholerae were isolated from the fecal specimens; serovar 22 was the most prevalent type. All non-O1 isolates were cytotoxic to Y-1 mouse adrenal cells. Only non-O1 V. cholerae was detected in water samples collected from the habitat of the birds. The results of this study suggest that aquatic birds serve as carriers and disseminate V. cholerae over a wide area.     

Occurrence of <Emphasis Type="Italic">Chlamydiaceae</Emphasis> in non-symptomatic free-living raptors in Spain

Few studies have investigated the role of raptors as natural reservoirs of Chlamydiaceae spp. and the preferred anatomical sites where these bacteria can be detected in non-symptomatic wild birds. We investigated the occurrence of Chlamydiaceae in 54 non-symptomatic adult free-living birds belonging to 14 species sampled upon reception in a raptor rehabilitation centre in Spain, and ten juvenile birds from five species born and reared in the centre for subsequent release into the wild. Swabs from conjunctivae, choanae and cloacae were taken to detect Chlamydiaceae DNA by a family-specific polymerase chain reaction (PCR) and a nested genus- and species-specific PCR. Chlamydiaceae DNA was detected in adult raptors belonging to 12 species (85.7%), mainly in conjunctival (40.6%) and, to a lesser extent, choanal (17.2%) swabs, but never in cloacal samples. Neither the genus nor the species of Chlamydiaceae could be confirmed by the nested PCR assay. Our results suggest that most of the raptor species investigated, especially the Common Kestrel (Falco tinnunculus) and Eurasian Griffon Vulture (Gyps fulvus), can be natural reservoirs of Chlamydiaceae spp. in the wild. Failure to identify the organisms at genus and species level might have been due to the poor quality and low concentration of DNA in the samples or to the presence of hitherto unclassified Chlamydiaceae species.     

Detection of low pathogenic avian influenza viruses in wild ducks from Canada: comparison of two sampling methods

Surveillance for avian influenza viruses in wild birds was initiated in Canada in 2005. In 2006, in order to maximize detection of highly pathogenic avian influenza viruses, the sampling protocol used in Canada's Inter-agency Wild Bird Influenza Survey was changed. Instead of collecting a single cloacal swab, as previously done in 2005, cloacal and oropharyngeal swabs were combined in a single vial at collection. In order to compare the two sampling methods, duplicate samples were collected from 798 wild dabbling ducks (tribe Anatini) in Canada between 24 July and 7 September 2006. Low pathogenic avian influenza viruses were detected significantly more often (P<0.0001) in combined oropharyngeal and cloacal samples (261/798, 33%) than in cloacal swabs alone (205/798, 26%). Compared to traditional single cloacal samples, combined samples improved virus detection at minimal additional cost.     

Prevalence of thermotolerant Campylobacter in partridges (Perdix perdix)

Aim:  To estimate the prevalence of thermotolerant Campylobacter spp. in commercially reared partridges ( Perdix perdix ) in southern Italy.
Methods and Results:  Cloacal swabs of partridges ( n  =   240), equally distributed between male and female birds, from a game bird farm located in the Southern Italy were examined for the prevalence of thermotolerant Campylobacter spp. The samples were processed in order to detect thermotolerant Campylobacter spp. by culture methods. The positive samples were then confirmed by multiplex polymerase chain reaction. Thermotolerant Campylobacter spp. were isolated from 118 (49·2%) of the 240 cloacal swabs examined. As proved by PCR, 100% of the strains were identified as Campylobacter coli (118/118), and 15 (12·7%) out of the 118 positive samples were also positive for Campylobacter jejuni . In contrast, Campylobacter lari was not identified. Adult partridges showed a significantly higher prevalence ( P  <   0·05) than younger ones.
Conclusion:  These results reinforce the assumption that game birds may be considered as potential carriers of Campylobacter spp. for human being and other animal species.
Significance and Impact of the Study:  Although an earlier 1986 publication described the prevalence of Campylobacter coli in commercially reared partridges, this is the first report to confirm the species of Campylobacter using a PCR test.     

Oral and cloacal microflora of wild crocodiles Crocodylus acutus and C. moreletii in the Mexican Caribbean

Bacterial cultures and chemical analyses were performed from cloacal and oral swabs taken from 43 American crocodiles Crocodylus acutus and 28 Morelet's crocodiles C. moreletii captured in Quintana Roo State, Mexico. We recovered 47 bacterial species (28 genera and 14 families) from all samples with 51.1% of these belonging to the family Enterobacteriaceae. Fourteen species (29.8%) were detected in both crocodile species and 18 (38.3%) and 15 (31.9%) species were only detected in American and Morelet's crocodiles, respectively. We recovered 35 bacterial species from all oral samples, of which 9 (25.8%) were detected in both crocodile species. From all cloacal samples, we recovered 21 bacterial species, of which 8 (38.1%) were detected in both crocodile species. The most commonly isolated bacteria in cloacal samples were Aeromonas hydrophila and Escherichia coli, whereas in oral samples the most common bacteria were A. hydrophila and Arcanobacterium pyogenes. The bacteria isolated represent a potential threat to crocodile health during conditions of stress and a threat to human health through crocodile bites, crocodile meat consumption or carrying out activities in crocodile habitat. We especially warn about the presence of Salmonella arizonae and S. typhi, which cause enteritis and septicemia in crocodiles and salmonellosis and typhoid fever in humans. The risk of bacterial contamination from crocodiles to humans could increase in the future because of the accelerated destruction of crocodile habitat, which could lead to an augmentation of human-crocodile interactions. Information on bacterial diversity reported here could help in the choice of antibacterial products in case of infections that are of crocodile origin.     

Local parasite lineage sharing in temperate grassland birds provides clues about potential origins of Galapagos avian Plasmodium

Oceanic archipelagos are vulnerable to natural introduction of parasites via migratory birds. Our aim was to characterize the geographic origins of two Plasmodium parasite lineages detected in the Galapagos Islands and in North American breeding bobolinks (Dolichonyx oryzivorus) that regularly stop in Galapagos during migration to their South American overwintering sites. We used samples from a grassland breeding bird assemblage in Nebraska, United States, and parasite DNA sequences from the Galapagos Islands, Ecuador, to compare to global data in a DNA sequence registry. Homologous DNA sequences from parasites detected in bobolinks and more sedentary birds (e.g., brown‐headed cowbirds Molothrus ater, and other co‐occurring bird species resident on the North American breeding grounds) were compared to those recovered in previous studies from global sites. One parasite lineage that matched between Galapagos birds and the migratory bobolink, Plasmodium lineage B, was the most common lineage detected in the global MalAvi database, matching 49 sequences from unique host/site combinations, 41 of which were of South American origin. We did not detect lineage B in brown‐headed cowbirds. The other Galapagos‐bobolink match, Plasmodium lineage C, was identical to two other sequences from birds sampled in California. We detected a close variant of lineage C in brown‐headed cowbirds. Taken together, this pattern suggests that bobolinks became infected with lineage B on the South American end of their migratory range, and with lineage C on the North American breeding grounds. Overall, we detected more parasite lineages in bobolinks than in cowbirds. Galapagos Plasmodium had similar host breadth compared to the non‐Galapagos haemosporidian lineages detected in bobolinks, brown‐headed cowbirds, and other grassland species. This study highlights the utility of global haemosporidian data in the context of migratory bird–parasite connectivity. It is possible that migratory bobolinks bring parasites to the Galapagos and that these parasites originate from different biogeographic regions representing both their breeding and overwintering sites.     

Succession in the intestinal microbiota of preadolescent turkeys

In the present study, automated ribosomal intergenic spacer analysis (ARISA), library sequence analysis, real-time PCR detection of Bacteroides uniformis and Campylobacter coli and dot-blot hybridizations of Clostridiaceae were used to identify trends in microbial colonization of the ceca of male turkeys. Two separate trials were performed with six and five birds, respectively. ARISA community profiles identified a period of community transition at week 12 of age in both trials. A significant increase of Ca. coli was also detected at week 12 in one trial, suggesting a possible correlation between microbiota destabilization and pathogen prevalence. Libraries of ribosomal small subunit 16S genes representing weeks 9, 11, 12 and 14 of both trials were sequenced. Whereas fingerprint and sequence analyses indicated significant differences in the species composition between the two trials, in general sequence library and dot-blot analyses indicated that Clostridia-like species decreased in prevalence over time. While B. uniformis prevalence in the two trials rose from 7% and 0% of the library clones at week 9 to 84% and 79% at week 11, real-time PCR did not support these results, with only approximately twofold and sixfold increases in internal transcribed spacer copy numbers observed.     

Intestinal spirochetes isolated from wild-living jackdaws, hooded crows and rooks (genus Corvus): provisionally designated "Brachyspira corvi" sp. nov

Intestinal spirochetes of genus Brachyspira are commonly isolated from mammalian and avian hosts, and several species have been reported to cause enteric disease in pigs and birds. Except for a previous publication on three isolates from corvid birds (order Passeriformes, family Corvidae, genus Corvus), of which two are further studied in this paper, no other reports exist on Brachyspira spp. of passerine birds. In this study, cloacal and intestinal swabs of small and large intestines were collected from 116 corvid birds of three species, i.e. jackdaws (Corvus monedula), hooded crows (Corvus corone cornix) and rooks (Corvus frugilegus), from four separate geographical locations in Sweden. Isolates were obtained by selective culture from 43 of 116 birds. All isolates were weakly hemolytic, indole-negative and lacked hippurate cleavage capacity. Examination by light microscopy did not indicate association with enteric disease in necropsied birds. Pure spirochete cultures were obtained by serial dilution and subculture, and selected isolates were analyzed by PCR (n=14), randomly amplified polymorphic DNA (RAPD) (n=14), and sequencing of the almost complete 16S rRNA (n=14), and partial nox genes (n=4). Positive reactions were noticed by PCR targeting a hexa-T segment of the 16S rRNA gene, which has been previously reported as a signature characteristic of Brachyspira pilosicoli. By 16S rRNA gene sequencing, the isolates formed a separate cluster related to genus Brachyspira, but not consistent with any presently recognized or proposed Brachyspira sp. The sequence similarity of the 16S rRNA gene among the isolates from corvid birds was 99.7-100%. Compared to 16S rRNA gene sequence data from all presently recognized and several proposed Brachyspira spp. the sequence similarity of the isolates from corvid birds varied between 94.1 and 96.5%. In a radial tree based on nox gene sequences, all four analyzed isolates from corvid birds formed a separate cluster. By RAPD analysis, the banding patterns of the isolates differed from all type strains of Brachyspira spp. Based on the results presented in this paper, we propose that the described isolates from corvid birds belong to a novel species within genus Brachyspira, with the provisional name "Brachyspira corvi" (cor'vi. L gen. n. corvi, of a crow).     

Feline ocular chlamydiosis: clinical and microbiological effects of topical and systemic therapy

Conjunctival swabs taken from a two-month-old kitten showing ocular discharge were found to be positive for Chlamydophila felis by PCR and isolation. The cat was treated with topical 1% tetracycline ophthalmic ointment twice a day for 60 days. At the end of the treatment, the cat showed no ocular signs and conjunctival swabs resulted PCR and isolation negative for C. felis. Forty days later, the ocular discharge recurred and C. felis was isolated from conjunctival swabs taken from both the cat's eyes. Twenty days of doxycycline systemic treatment at 10 mg/kg once daily was started. The treatment resulted in a complete clinical recovery after a few days. C. felis was not isolated or amplified on the 10th day after beginning the treatment. The cat's conjunctival swabs were also PCR and isolation negative on the 10th, 30th, 60th, 90th, 120th and 240th days after the end of therapy.     

Measuring the gut microbiome in birds: Comparison of faecal and cloacal sampling

The gut microbiomes of birds and other animals are increasingly being studied in ecological and evolutionary contexts. Numerous studies on birds and reptiles have made inferences about gut microbiota using cloacal sampling; however, it is not known whether the bacterial community of the cloaca provides an accurate representation of the gut microbiome. We examined the accuracy with which cloacal swabs and faecal samples measure the microbiota in three different parts of the gastrointestinal tract (ileum, caecum, and colon) using a case study on juvenile ostriches, Struthio camelus, and high‐throughput 16S rRNA sequencing. We found that faeces were significantly better than cloacal swabs in representing the bacterial community of the colon. Cloacal samples had a higher abundance of Gammaproteobacteria and fewer Clostridia relative to the gut and faecal samples. However, both faecal and cloacal samples were poor representatives of the microbial communities in the caecum and ileum. Furthermore, the accuracy of each sampling method in measuring the abundance of different bacterial taxa was highly variable: Bacteroidetes was the most highly correlated phylum between all three gut sections and both methods, whereas Actinobacteria, for example, was only strongly correlated between faecal and colon samples. Based on our results, we recommend sampling faeces, whenever possible, as this sample type provides the most accurate assessment of the colon microbiome. The fact that neither sampling technique accurately portrayed the bacterial community of the ileum nor the caecum illustrates the difficulty in noninvasively monitoring gut bacteria located further up in the gastrointestinal tract. These results have important implications for the interpretation of avian gut microbiome studies.     

Characteristics of Cefotaxime-Resistant Escherichia coli from Wild Birds in The Netherlands

Cloacal swabs from carcasses of Dutch wild birds obtained in 2010 and 2011 were selectively cultured on media with cefotaxime to screen for the presence of extended-spectrum β-lactamase (ESBL)/AmpC-producing Escherichia coli. Subsequently, all cefotaxime-resistant E. coli isolates were tested by broth microdilution and microarray. The presence of ESBL/AmpC and coexisting plasmid-mediated quinolone resistance (PMQR) genes was confirmed by PCR and sequencing. To determine the size of plasmids and the location of ESBL and PMQR genes, S1 pulsed-field gel electrophoresis (PFGE) was performed on transformants, followed by Southern blot hybridization. The study included 414 cloacal swabs originating from 55 different bird species. Cefotaxime-resistant E. coli isolates were identified in 65 birds (15.7%) from 21 different species. In all, 65 cefotaxime-resistant E. coli ESBL/AmpC genes were detected, mainly comprising variants of bla_CTX-M and bla_CMY-2. Furthermore, PMQR genes [aac(6′)-lb-cr, qnrB1, and qnrS1] coincided in seven cefotaxime-resistant E. coli isolates. Overall, replicon typing of the ESBL/AmpC-carrying plasmids demonstrated the predominant presence of IncI1 (n = 31) and variants of IncF (n = 18). Our results indicate a wide dissemination of ESBL and AmpC genes in wild birds from The Netherlands, especially among aquatic-associated species (waterfowl, gulls, and waders). The identified genes and plasmids reflect the genes found predominantly in livestock animals as well as in humans.