Molecular evidence for chlamydial infections in the eyes of sheep

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.     

Ocular chlamydiales infections of western barred bandicoots (Perameles bougainville) in Western Australia.

The western barred bandicoot (Perameles bougainville) is an endangered species, free ranging on only two islands off the coast of Western Australia (Dorre and Bernier Islands). Conservation efforts are currently directed at reintroducing these marsupials into predator-proof enclosures and habitats in historical distribution ranges on the mainland in the southwest of Western Australia and in South Australia. In September 2000, 19 western barred bandicoots were captured on Bernier Island for translocation, and 11 of these had evidence of at least one of the following eye conditions: corneal opacity, conjunctivitis, ocular discharge, and blepharitis. Five bandicoots were examined, and conjunctival and cloacal swabs were collected. Polymerase chain reaction for Chlamydiales was positive in four bandicoots. Four Chlamydiales types were identified by gene sequencing, including a strain of Chlamydia pecorum different from strains previously found in koalas and several new Chlamydiales genotypes. The bandicoots responded excellently to treatment with oxytetracyline weekly for 6 wk, and topical oxytetracycline and neomycin were administered topically to both eyes s.i.d. for 4 mo.     

Small mammal virology.

Most viral infections in small mammals are transient and rarely produce clinical signs. When clinical signs do appear, they are often of a multifactorial etiology such as respiratory infection with Sendai virus and the bacteria M. pulmonis in rodents. Diagnosis is generally made based on clinical signs, while therapy involves treatment for concurrent bacterial infections and supportive care. Small mammals may carry zoonotic viruses such as LCMV, but natural infections are uncommon. Viral diseases are rare (or largely unknown) for hedgehogs, chinchillas, and prairie dogs, while no known naturally occurring, clinically relevant viral diseases exist for gerbils and sugar gliders. This article is intended to aid the clinician in identifying viral infections in small mammals and to help determine the significance each virus has during clinical disease.     

不同病例动物源大肠杆菌的分离鉴定及16SrDNA同源性分析

为研究不同病例发病动物的发病原因,并对不同病例病料分离出的细菌进行16S rDNA同源性分析,本试验对10种不同病例发病动物病料进行细菌分离培养并对分离获得的细菌进行微生物学鉴定,设计1对16S rDNA基因引物,对分离出的10株细菌进行PCR扩增、测序及16S rDNA同源性分析。结果显示,分离获得的10株细菌经微生物学鉴定均为大肠杆菌,10株大肠杆菌中哺乳类动物病例犬乳房炎、犬子宫蓄脓、犬肺炎、犬皮肤化脓疮、奶牛乳房炎、犊牛腹泻6株大肠杆菌之间16S rDNA同源性为100.0%,家禽类动物病例肉鸽腹泻、肉鸡腹泻、野鸡腹泻和白孔雀腹泻4株大肠杆菌之间16S rDNA同源性也为100.0%,10株不同病例动物来源大肠杆菌之间16S rDNA同源性为97.5%~100.0%。本试验探明了10种不同病例发病动物的发病原因均为大肠杆菌感染引起,且10株大肠杆菌16S rDNA之间具有高度同源性。     

CATTLE AND THE PARALYSIS TICK IXODES HOLOCYCLUS

Paralysis of domestic stock by the paralysis tick Ixodes holocyclus is chiefly a disease of young animals (especially calves) and of non-habituated stock introduced into tick-infested country in spring. The tick has a wide host range, but its principal hosts are bandicoots. The tick has one generation per year and the adult female, which causes almost all paralysis, is abundant in spring and early summer and occurs most commonly in overgrown or regrowth country where bandicoots are abundant. The distribution and behavior of the long and the short-nosed bandicoots are reviewed. The number of ticks required to induce paralysis in cattle and the protection from paralysis afforded by prior experience of the tick are discussed.     

The prevalence of three species of feline haemoplasmas in samples submitted to a diagnostics service as determined by three novel real-time duplex PCR assays

Three distinct species of feline haemoplasmas are recognised: Mycoplasma haemofelis (Mhf), Candidatus Mycoplasma haemominutum (CMhm) and Candidatus Mycoplasma turicensis (CMt). These species differ in pathogenicity as Mhf and CMt can be associated with anaemia whereas CMhm usually results in few clinical signs. The purpose of this study was to develop quantitative real-time PCR assays for the detection of all three feline haemoplasma species combined with an endogenous internal control and to determine the prevalence of infection, using these assays, in 1592 EDTA blood samples submitted to Langford Veterinary Diagnostics, University of Bristol for haemoplasma testing. Primers and TaqMan probes were designed against published 16S rDNA sequences. These assays were combined with a feline 28S rDNA-specific assay to produce three duplex assays. The assays detected 1-10 copies of a sequence-specific plasmid per PCR. None of the assays showed cross-reactivity with 10(6) copies of a sequence-specific plasmid from the non-target haemoplasma species. Real-time PCR was performed on all samples using the three assays. Seven samples were negative for feline 28S rDNA and were excluded from the study. Of the remaining 1585 samples, 45 (2.8%), 177 (11.2%) and 27 (1.7%) samples were positive for Mhf, CMhm and CMt, respectively, including 11 Mhf/CMhm, 10 CMhm/CMt and two Mhf/CMt dual infections and two triple infections. The results of this study demonstrate the utility of these new duplex PCR assays for the detection of haemoplasma infections. CMhm was the most common infection and CMt infections were often associated with co-infection with other haemoplasma species, especially CMhm.     

Development of a PCR test for the identification of Staphylococcus intermedius based on the 16S rDNA sequence

The development of a PCR assay based on the 16S ribosomal RNA gene (rDNA) sequence was carried out for the identification of Staphylococcus intermedius. Sixty-six strains of S. intermedius, 70 of Staphylococcus aureus and 2 of Staphylococcus hyicus were examined for the assay. The 16S rDNA, of which the PCR target fragment makes up 901 bp corresponding to the sequence data of the gene, was detected in all strains of S. intermedius, but it was not detected in any strains of either S. aureus or S. hyicus. These results suggest that the PCR allows a simple and precise identification of S. intermedius.     

Detection of novel chlamydiae in cats with ocular disease

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.     

Novel gastric helicobacters and oral campylobacters are present in captive and wild cetaceans

The mammalian gastric and oral mucosa may be colonized by mixed Helicobacter and Campylobacter species, respectively, in individual animals. To better characterize the presence and distribution of Helicobacter and Campylobacter among marine mammals, we used PCR and 16S rDNA sequence analysis to examine gastric and oral samples from ten dolphins (Tursiops gephyreus), one killer whale (Orcinus orca), one false killer whale (Pseudorca crassidens), and three wild La Plata river dolphins (Pontoporia blainvillei). Helicobacter spp. DNA was widely distributed in gastric and oral samples from both captive and wild cetaceans. Phylogenetic analysis demonstrated two Helicobacter sequence clusters, one closely related to H. cetorum, a species isolated from dolphins and whales in North America. The second related cluster was to sequences obtained from dolphins in Australia and to gastric non-H. pylori helicobacters, and may represent a novel taxonomic group. Dental plaque sequences from four dolphins formed a third cluster within the Campylobacter genus that likely represents a novel species isolated from marine mammals. Identification of identical Helicobacter spp. DNA sequences from dental plaque, saliva and gastric fluids from the same hosts, suggests that the oral cavity may be involved in transmission. These results demonstrate that Helicobacter and Campylobacter species are commonly distributed in marine mammals, and identify taxonomic clusters that may represent novel species.     

Clinical Sarcocystis neurona, Sarcocystis canis, Toxoplasma gondii, and Neospora caninum infections in dogs

Sarcocystis neurona, Sarcocystis canis, Toxoplasma gondii, and Neospora caninum are related apicomplexans that can cause systemic illness in many species of animals, including dogs. We investigated one breeder's 25 Basset Hounds for these infections. In addition, tissues from dogs and other non-canine hosts previously reported as S. canis infections were studied retrospectively. Schizonts resembling those of S. neurona, and recognized by polyclonal rabbit anti-S. neurona antibodies, were found in six of eight retrospective cases, as well as in two additional dogs (one Basset Hound, one Springer Spaniel) not previously reported. S. neurona schizonts were found in several tissues including the central nervous system, lungs, and kidneys. Fatal toxoplasmosis was diagnosed in an adult dog, and neosporosis was diagnosed in an adult and a pup related to the one diagnosed with S. neurona. No serological reactivity to S. neurona antibodies occurred when S. canis-like liver schizonts were retrospectively assayed from two dogs, a dolphin, a sea lion, a horse, a chinchilla, a black or either of two polar bears. Sequencing conserved (18S) and variable (ITS-1) portions of nuclear ribosomal DNA isolated from the schizont-laden liver of a polar bear distinguished it from all previously characterized species of Sarcocystis. We take this genetic signature as provisionally representative of S. canis, an assumption that should be tested with future sequencing of similar liver infections in other mammalian hosts. These findings further extend the uncharacteristically broad intermediate host range for S. neurona, which also causes a neurologic disease in cats, mink, raccoons, skunks, Pacific harbor seals, ponies, zebras, lynxes, and sea otters. Further work is necessary to delineate the causative agent(s) of other cases of canine sarcocystosis, and in particular to specify the attributes of S. canis, which corresponds morphologically to infections reported from wide range of terrestrial and marine mammals.     

Identification of a Mycoplasma ovis-like organism in a herd of farmed white-tailed deer (Odocoileus virginianus) in rural Indiana

Mycoplasma ovis is a hemoplasma parasite of sheep, goats, and reindeer; however, natural hemoplasma infection in white-tailed deer has not previously been reported. Subsequent to finding many coccoid, bacillary, and ring-shaped organisms, consistent with hemotropic mycoplasmas, on RBCs from a 72-day-old female white-tailed fawn, we sought to (1) identify the putative hemoplasma observed in blood from the fawn, (2) evaluate others in the herd for hemoplasma infection, and (3) identify clinicopathologic characteristics of hemoplasma-infected white-tailed deer. EDTA-anticoagulated whole blood was collected from the fawn and 8 apparently healthy does in the same herd. CBCs were performed on 7 nonclotted samples from the fawn and 6 does. DNA was extracted from all samples, followed by PCR amplification of bacterial (16S rDNA) and protozoal (18S rDNA) genes. The nearly complete 16S rDNA product from the fawn's sample was directly sequenced and compared with known sequences in the GenBank database. Samples from the fawn and 7 of 8 does were PCR-positive using hemoplasma-specific and M ovis-specific protocols. The fawn was PCR-negative for Anaplasma spp., Babesia spp., and Theileria spp. The 16S rDNA sequence from the fawn (GenBank accession number, FJ824847) was most closely related to M ovis (AF338268), having 98.5% sequence identity. The fawn had a mild nonregenerative anemia, a neutrophilic left-shift with toxic change, aspiration bronchopneumonia, and gastrointestinal disease. Hematologic values, including blood film evaluation, in infected does were unremarkable. The M ovis-like organism may have acted as either an opportunistic or primary pathogen in the fawn. The high occurrence of subclinical infections in the does suggests that white-tailed deer may act as wildlife reservoirs for M ovis.     

First Report on the Occurrence of ‘Helicobacter heilmannii’ in the Stomach of Rabbits

Gastric Helicobacter spp. have been described in a wide range of animal species, including dogs, cats, primates, swine, cattle and rodents. However, in lagomorphs--more specifically rabbits--gastric Helicobacter infections have never been reported. Biopsy specimens were collected from different stomach regions of 23 rabbits, including 10 pet rabbits, 10 industrial animals and 3 research animals. These were subjected to a PCR assay for the detection of Helicobacter DNA. Identification up to the species level was based on 16S rRNA sequence analysis and a recently developed multiplex PCR. Seven rabbits (four pet, one research animal and two industrial animals) tested positive in the Helicobacter genus-specific PCR in the stomach, with the corpus being predominantly positive. H. felis and H. salomonis, hitherto presumed to be naturally hosted by cats and dogs, were detected in three animals and one animal, respectively. One of these animals had been completely devoid of any form of contact with cats or dogs. A H. pullorum/H. rappini-like organism (96% 16S rDNA sequence similarity) was found in an industrially held rabbit. The helicobacters of the two remaining rabbits could not be identified up to the species level. To conclude, this is the first report on the occurrence of Helicobacter spp. in the stomach of rabbits. In view of the fact that H. felis and H. salomonis are put forward as having zoonotic potential, further research is necessary to investigate the implications of these findings not only for the rabbit but also for human health.     

鸡咽部混合菌群16S rDNA序列分析方法的建立

通过优化鸡咽拭子预处理方法、储存条件、提取咽拭子标本中的细菌总DNA条件、细菌16SrDNA保守区通用引物设计及PCR扩增条件,成功建立了鸡咽部菌群16SrDNA序列分析方法,并初步分析了健康鸡的咽部菌群种类和组成,同时发现鸡咽部菌群中未知细菌和未可培养细菌所占的比例很大,需要深入研究其对鸡呼吸道疾病的影响。     

Modest genetic differentiation among North American populations of Sarcocystis neurona may reflect expansion in its geographic range

Sarcocystis neurona is an important cause of neurological disease in horses (equine protozoal myeloencephalitis, EPM) and sea otters in the United States. In addition, EPM-like disease has been diagnosed in several other land and marine mammals. Opossums are its only definitive hosts. Little genetic diversity among isolates of S. neurona from different hosts has been reported. Here, we used 11 microsatellites to characterize S. neurona DNA isolated from natural infections in 22 sea otters (Enhydra lutris) from California and Washington and in 11 raccoons (Procyon lotor) and 1 striped skunk (Mephitis mephitis) from Wisconsin. By jointly analyzing these 34 isolates with 26 isolates previously reported, we determined that geographic barriers may limit S. neurona dispersal and that only a limited subset of possible parasite genotypes may have been introduced to recently established opossum populations. Moreover, our study confirms that diverse intermediate hosts share a common infection source, the opossum (Didelphis virginiana).     

Rapid and direct detection of clostridium chauvoei by PCR of the 16S-23S rDNA spacer region and partial 23S rDNA sequences

Clostridium chauvoei causes blackleg, which is difficult to distinguish from the causative clostridia of malignant edema. Therefore, a single-step PCR system was developed for specific detection of C. chauvoei DNA using primers derived from the 16S-23S rDNA spacer region and partial 23S rDNA sequences. The specificity of the single-step PCR system was demonstrated by testing 37 strains of clostridia and 3 strains of other genera. A 509 bp PCR product, which is a C. choauvoei-specific PCR product, could be amplified from all of the C. chauvoei strains tested, but not from the other strains. Moreover, this single-step PCR system specifically detected C. chauvoei DNA in samples of muscle from mice 24 hr after inoculation with 100 spores of C. chauvoei, and in clinical materials from a cow affected with blackleg. These results suggest that our single-step PCR system may be useful for direct detection of C. chauvoei in culture and in clinical materials from animals affected with blackleg.     

Use of a polymerase chain reaction for detection of Mycoplasma dispar in the nasal mucus of calves.

A polymerase chain reaction (PCR) assay was developed for the detection of Mycoplasma dispar in nasal mucus samples collected from calves. The target DNA sequence was the 16S rRNA gene, and the fragment was selected within a region of high polymorphism. The specificity and detection limit of the method were determined. This method was then used for the detection of M. dispar in nasal swabs collected from 301 calves, including 155 clinical samples from animals showing signs of respiratory disease and 146 samples from healthy animals. PCR with generic primers was applied to the detection of Mollicutes, followed by the detection of M. dispar. Mollicutes were detected in 52.05% of clinical samples from healthy animals and in 90.96% of samples from sick animals. Mycoplasma dispar was detected in 6.16% of healthy animals and in 34.84% of sick animals. The PCR assay was useful in verifying the presence of M. dispar in calves and may be a useful tool in monitoring this mycoplasma in cattle herds.     

胸腺嘧啶依赖型金黄色葡萄球菌小菌落突变株的分离鉴定

本试验对由患慢性奶牛乳房炎奶样中分离出的1株疑似金黄色葡萄球菌小菌落突变株(SCVs)进行形态观察、金黄色葡萄球菌相关保守基因片段(nuc、nucA、16S rDNA) 多重PCR扩增鉴定、药敏试验、生理生化特性研究及补偿试验。结果显示分离出1株金黄色葡萄球菌SCVs,该菌含有金黄色葡萄球菌菌种特异性基因nuc和nucA;与金黄色葡萄球菌质控菌株ATCC 25923的抑菌圈大小明显不同;菌落形态主要表现为菌落细小、生长缓慢、溶血能力下降;凝固酶活性下降;耐盐能力降低;革兰氏染色为革兰氏阳性球菌,呈葡萄状排列;补偿试验鉴定该金黄色葡萄球菌SCVs为胸腺嘧啶依赖型。结果表明成功分离鉴定出1株胸腺嘧啶依赖型金黄色葡萄球菌SCVs,为由金黄色葡萄球菌SCVs引起的奶牛慢性乳房炎的预防和控制及其致病机制的研究奠定前期基础。     

Waddlia, Parachlamydia and Chlamydiaceae in bovine abortion

The etiology remains unknown in many cases of bovine abortion in Switzerland. Bacteria of the Chlamydiales order are known abortive agents, therefore cases of bovine abortion from three representative regions of Switzerland were investigated in this study. Particularly Chlamydiaceae as well as the Chlamydia-like organisms Waddlia and Parachlamydia were of interest, especially because of their possible zoonotic potential. Placenta samples (n=343) were tested for these bacteria by different PCR-methods, immunohistochemistry and serology for Chlamydia abortus. Additionally an attempt for the isolation of Waddlia and Parachlamydia was made by co-cultivation in amoebae. In 67.3% of the 343 cases a necrotizing and/or purulent placentitis was found histologically. By real-time PCR, 0.9% (3/343) of the cases were positive for Waddlia, 13.4% (46/343) positive for Parachlamydia and 14.6% (50/343) positive or questionable positive for Chlamydiaceae. Of these samples, confirmation by immunohistochemistry was possible in 2/3 cases for Waddlia, 25/46 for Parachlamydia and 4/50 for Chlamydiaceae. Of the 50 cases positive or questionable positive for Chlamydiaceae, species-identification by ArrayTube Microarray or 16S rRNA PCR resulted in 41 cases positive for C. abortus whereas the presence of Chlamydia suis was confirmed in four and Chlamydia pecorum in one case. This study brought evidence for the importance of different members of Chlamydiales in different regions of Switzerland although Waddlia is not occurring in a high prevalence. On the other hand mixed infections with different Chlamydiales as well as with other abortigenic agents could be found.     

A survey for piroplasmids in horses and Bactrian camels in North-Eastern Mongolia

Equine piroplasmosis caused by Babesia caballi and Theileria equi is widespread in Asia. The presence of these haemozoans in Mongolia was previously confirmed in domestic as well as in reintroduced Przewalski horses in which they cause significant pathology. The data on occurrence of piroplasms from Bactrian camels in Asia is lacking. A total of 192 horses, 70 Bactrian camels, and additional 16 shepherd dogs from the Hentiy province were included in our study. No clinical signs typical for piroplasmid infection were observed during the field survey. Microscopic examination revealed the presence of T. equi in blood smears from 67% of examined horses, with camels and dogs being negative. A two step PCR approach was used to detect piroplasms in peripheral blood. In the first "catch all" PCR reaction, amplification of the 496 bp-long fragment of the SSU rRNA gene enabled the detection of Babesia and Theileria spp. Second round multiplex PCR reaction used for species discrimination allowed the amplification of T. equi- and B. caballi-specific 340 bp and 650 bp-long regions of the SSU rRNA, respectively. This assay detected T. equi in 92.7% of horses, while the infections with B. caballi and dual infections were rare. In both PCR setups, camels and dogs were negative indicating that in the studied region, these hosts do not share piroplasms with horses.