Surveys on Coxiella burnetii infections in Swedish cattle, sheep, goats and moose.

BACKGROUND: Q fever is a zoonotic disease caused by the bacterium Coxiella burnetii. Prevalence data in ruminant species are important to support risk assessments regarding public and animal health. The aim was to investigate the presence of or exposure to C. burnetii in cattle, sheep, goats and moose, and to compare two enzyme-linked immunosorbent assays (ELISAs). National surveys of antibodies against C. burnetii were performed for dairy cattle (n=1537), dairy goats (n=58) and sheep (n=518). Bovine samples consisted of bulk milk, caprine of pooled milk, and ovine of pooled serum. Antibodies were investigated in moose samples (n=99) from three regions. A one-year regional cattle bulk milk survey was performed on the Isle of Gotland (n=119, four occasions). Cattle, sheep and goat samples were analysed with indirect ELISA and moose samples with complement fixation test. For the sheep, goat, and parts of the cattle survey, samples were run in parallel by ELISAs based on antigens from infected ruminants and ticks. Bulk milk samples from the regional cattle survey and vaginal swabs from a subset of the sheep herds (n=80) were analysed for the agent by polymerase chain reaction. Spatial clustering was investigated in the national cattle survey. RESULTS: The prevalence of antibodies in dairy herds was 8.2% with large regional differences. High risk clusters were identified in the southern regions. The prevalence among dairy herds on the Isle of Gotland varied from 55.9% to 64.6% and 46.4% to 58.9.0% for antibodies and agent, respectively, overall agreement between agent and antibodies was 85.2%. The prevalence of antibodies in sheep was 0.6%, the agent was not detected the vaginal swabs. Antibodies were not detected in goats or moose, although parts of the moose samples were collected in an area with high prevalence in cattle. The overall agreement between the two ELISAs was 90.4%. CONCLUSIONS: The prevalence of antibodies against C. burnetii in dairy cattle in Sweden shows large regional differences. The results suggest that C. burnetii is a rare pathogen among Swedish moose, dairy goat and sheep. ELISAs based on ruminant and tick antigen performed in a similar manner under Swedish conditions.

Multilocus variable-number tandem-repeat analysis scheme for chlamydia felis genotyping: comparison with multilocus sequence typing.

Chlamydia felis is an important ocular pathogen in cats worldwide. A multilocus variable-number tandem-repeat analysis (MLVA) system for the detection of tandem repeats across the whole genome of C. felis strain Fe/C-56 was developed. Nine selected genetic loci were tested by MLVA in 17 C. felis isolates, including the C. felis Baker vaccine strain, and 122 clinical samples from different geographic origins. Analysis of the results identified 25 distinct C. felis MLVA patterns. In parallel, a recently described multilocus sequence typing scheme for the typing of Chlamydia was applied to 13 clinical samples with 12 different C. felis MLVA patterns. Rare sequence differences were observed. Thus, the newly developed MLVA system provides a highly sensitive high-resolution test for the differentiation of C. felis isolates from different origins that is suitable for molecular epidemiological studies.

Development and evaluation of a real-time polymerase chain reaction method for the detection of Mycoplasma felis.

Infection by Mycoplasma felis is associated with ocular and respiratory disease in cats and respiratory disease in horses. A correct diagnosis is beneficial since the use of specific antimycoplasmal treatment can lead to resolution. The objective of the present study was to develop a real-time polymerase chain reaction (PCR) method based on dual-labeled fluorogenic probe technology, targeting the gene encoding elongation factor Tu (tuf ), for the fast and specific detection of M. felis. Specificity was achieved by basing the assay design on partial sequencing of the tuf gene in strains and clinical isolates of M. felis as well as other mycoplasma species. The detection limit of the developed assay was in the order of 10 copies of target DNA, and no cross-reaction was observed with a panel of several mycoplasma species. Compared to a previously published conventional PCR protocol, the novel assay had equal or slightly improved performance in terms of sensitivity and specificity when analyzing 100 conjunctival swab samples from cats with clinical signs of infection.

European hares in Chile: a different lagomorph reservoir for Mycobacterium avium subsp. paratuberculosis?

Ruminants are the principal host for infection by Mycobacterium avium subsp. paratuberculosis (Map), the cause of Johne's disease. Based on studies of a Map-infected population of European rabbits (Oryctolagus cuniculus) in Scotland, lagomorphs as a broad taxonomic order were proposed as potential nonruminant reservoirs for Map. To determine whether a different lagomorph species may serve as a wildlife reservoir, we investigated Map infection in European hares (Lepus europaeus) sharing habitat with known Map-infected dairy cattle in southern Chile. Fecal, mesenteric lymph node, and ileal samples were aseptically collected from 385 wild hares for liquid culture and real-time polymerase chain reaction identification of acid-fast isolates. All tissue samples were also acid-fast stained and examined microscopically. We isolated Map from at least one tissue from 48 hares (12.6%) and fecal samples from 16 hares (4.2%). No Map was found in tissues of eight of the fecal-culture-positive hares. Histologically, all tissues from all hares were within normal limits, and no acid-fast organisms were observed in any sample. Active infection, implying amplification of the organism secondary to resultant disease, was not evident. With this report Map isolations on a population versus incidental detection have now been made from two lagomorph species. However, although the rabbit population studied in Scotland appears to function as a Map reservoir, the hares studied in Chile appear to be a dead-end host, serving only as potential mechanical vectors for the organism.

Shedding of chlamydiae in relation to titers of serum chlamydiae-specific antibodies and serum concentrations of two acute-phase proteins in cats without conjunctivitis.

OBJECTIVE: To investigate shedding of chlamydiae from conjunctiva and genital tracts of cats without clinical signs of conjunctivitis or other infectious disease in relation to their titers of serum antibodies against chlamydiae and to serum amyloid A (SAA) and serum α(1)-acid glycoprotein (AGP) concentrations. ANIMALS: 62 healthy cats. PROCEDURES: Serum from each cat was analyzed for antibodies against chlamydiae and for SAA and AGP concentrations. Swab samples from the conjunctival sac and genital tract were analyzed with a real-time PCR assay for Chlamydiaceae. RESULTS: 4 of 8 of cats with high antibody titers (ie, 1,600) shed chlamydiae, but only from the conjunctiva. Chlamydiae could not be detected in samples from cats with lower antibody titers nor from any genital tract samples. In cats with antibody titers of 1,600, mean ± SD SAA concentration was significantly higher when chlamydiae were detected in conjunctival swab samples (3.9 ± 1.0 mg/L) than when no chlamydiae were detected (1.4 ± 1.0 mg/L). However, SAA concentration was greater than the limit for an acute-phase response in only one of those cats. There was no significant difference in serum AGP concentrations between cats with high titers that were or were not shedding chlamydiae. Nine of 30 (30%) cats (5 with and 4 without detectable serum antibodies against chlamydiae) that had been mated developed reproductive disorders. CONCLUSIONS AND CLINICAL RELEVANCE: Clinically normal cats with high chlamydiae-specific antibody titers can shed and thus transmit chlamydiae. Venereal spread from cats without clinical signs of infection is likely not common.

Survival of Mycobacterium tuberculosis and Mycobacterium bovis in human urine.

Tuberculosis is a zoonotic disease that mainly causes respiratory infection. However, it can also infect other organs such as the kidneys and bladder, which can lead to high counts of the organisms in the urine. Introducing urine diversion systems and reuse of the urine in agriculture may introduce new transmission routes for infection, increasing the risk of spread. This study evaluated the inactivation rate of mycobacteria in human urine for ensuring safe reuse in agriculture and examined whether current World Health Organization recommendations on storage time are sufficient for inactivating Mycobacterium tuberculosis and Mycobacterium bovis. In this study, a decimal reduction in M. tuberculosis and M. bovis in human urine containing 7 and 3 g NH(3)-N L(-1), respectively, was obtained in just over 10 days at 4°C and below three days at 22°C. This is considerably faster than previously reported reduction rates of mycobacteria in animal slurry at similar temperatures. Based on the present results, a storage time of five weeks at temperatures below 20°C or of two weeks at temperatures above 20°C is sufficient to prevent transmission of mycobacteria when recycling human urine. These values lie within the WHO recommended storage period.

Interlaboratory comparison of real-time polymerase chain reaction methods to detect Coxiella burnetii, the causative agent of Q fever.

The bacterium Coxiella burnetii, which has a wide host range, causes Q fever. Infection with C. burnetii can cause abortions, stillbirth, and the delivery of weak offspring in ruminants. Coxiella burnetii infection is zoonotic, and in human beings it can cause chronic, potentially fatal disease. Real-time polymerase chain reaction (PCR) is increasingly being used to detect the organism and to aid in diagnosis both in human and animal cases. Many different real-time PCR methods, which target different genes, have been described. To assess the comparability of the C. burnetii real-time PCR assays in use in different European laboratories, a panel of nucleic acid extracts was dispatched to 7 separate testing centers. The testing centers included laboratories from both human and animal health agencies. Each laboratory tested the samples using their in-house real-time PCR methods. The results of this comparison show that the most common target gene for real-time PCR assays is the IS1111 repeat element that is present in multiple copies in the C. burnetii genome. Many laboratories also use additional real-time PCR tests that target single-copy genes. The results of the current study demonstrate that the assays in use in the different laboratories are comparable, with general agreement of results for the panel of samples.

An investigation on the presence of Chlamydiaceae in Swedish dogs.

BACKGROUND: Bacteria belonging to the family Chlamydiaceae cause a broad spectrum of diseases in a wide range of hosts, including man, other mammals, and birds. Upper respiratory and genital diseases are common clinical problems caused by Chlamydiaceae. Very little is known about chlamydial infections in dogs. Few clinical reports on natural disease in dogs describe mainly conjunctival and upper respiratory signs, and the role of Chlamydiaceae in genital disease is unclear. The present study aimed at studying the prevalence of Chlamydiaceae in healthy dogs and in dogs with genital or upper respiratory disease, including conjunctivitis. METHODS: A real-time polymerase chain reaction (PCR) for Chlamydiaceae was used to detect any chlamydial species within this family. Swab samples from the conjunctiva and the mucosal membranes of the oropharynx, rectum and genital tract were taken from 79 dogs: 27 clinically healthy dogs, 25 dogs with clinical signs from the genital tract and 28 dogs with conjunctivitis. There were 52 female and 27 male dogs. From 7 of the male dogs, additional semen samples were analysed. RESULTS: No Chlamydiaceae were detected from any dog. CONCLUSIONS: Although the number of dogs that was included is limited, the results suggest that cases of Chlamydiaceae in dogs probably are related to infection from other species, and that dogs in general do not harbour Chlamydiaceae. Bacteria belonging to the family Chlamydiaceae do not seem to be of major importance for genital or ocular disease in Swedish dogs.

Infectious causes for feline upper respiratory tract disease--a case-control study.

The aim of this case-control study was to investigate the prevalence of microorganisms in group-living cats with clinical signs of upper respiratory tract disease (URTD), in in-contact cats and in cats in groups without URTD problems. Samples were taken from the ventral conjunctival fornix for analysis of feline herpesvirus-1 (FHV), Mycoplasma felis and Chlamydiaceae using a real-time polymerase chain reaction technique. The oropharynx was sampled for bacteriological culture and viral isolation. Specific infectious agents were identified in 11/20 (55%) of the case households, in 7/20 (35%) of the cats with clinical signs and in 3/20 (15%) of the control households, in 3/40 (7.5%) of the cats. Chlamydiae and M felis were only detected from case households, both from cats with URTD and from in-contact cats. The difference in prevalence between case and control households was statistically significant for M felis (P=0.047). The presence of M felis in cat groups was thus associated with clinical signs of URTD.

Thymidylate synthases of Mycoplasma mycoides subsp. mycoides SC and Ureaplasma parvum are flavin-dependent.

Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides subsp. mycoides SC) is the causative agent of contagious bovine pleuropneumonia (CBPP), one of the most serious bacterial diseases in cattle and buffalo. Ureaplasma parvum (U. parvum) colonizes the human urogenital tract, and has been associated with urethritis and premature birth. The de novo synthesis of thymidylate (dTMP) is essential and catalyzed by thymidylate synthase (TS), encoded by either the thyA or the thyX genes. No homologs to either thyA or thyX have been identified in the U. parvum and M. mycoides subsp. mycoides SC genomes. Here we report the identification, partial purification and characterization of M. mycoides subsp. mycoides and U. parvum TS. Our results showed that the M. mycoides subsp. mycoides SC and U. parvum TS apparently are flavin-dependent, having similar enzymatic activities but no sequence homology to other known ThyX proteins. Up to date there are 11 Mollicutes species lacking both thyA and thyX gene. Therefore, the finding described here most likely constitutes a new enzyme family specific for Mollicutes. These M. mycoides subsp. mycoides SC and U. parvum TS enzymes could be ideal targets for future development of agents against Myoplasma infections.

Multiplex screening of surface proteins from Mycoplasma mycoides subsp. mycoides small colony for an antigen cocktail enzyme-linked immunosorbent assay.

A recombinant antigen cocktail enzyme-linked immunosorbent assay (ELISA) for diagnosis of contagious bovine pleuropneumonia (CBPP) was developed after careful selection of antigens among one-third of the surface proteome proteins of the infectious agent Mycoplasma mycoides subsp. mycoides small colony (M. mycoides SC). First, a miniaturized and parallelized assay system employing antigen suspension bead array technology was used to screen 97 bovine sera for humoral immune responses toward 61 recombinant surface proteins from M. mycoides SC. Statistical analysis of the data resulted in selection of eight proteins that showed strong serologic responses in CBPP-affected sera and minimal reactivity in negative control sera, with P values of <10(-6). Only minor cross-reactivity to hyperimmune sera against other mycoplasmas was observed. When applied in an ELISA, the cocktail of eight recombinant antigens allowed a fivefold signal separation between 24 CBPP-affected and 23 CBPP-free sera from different geographical origins. No false-positive results and only two false-negative results were obtained. In conclusion, the selected recombinant mycoplasma antigens qualified as highly specific markers for CBPP and could be employed in both a suspension bead array platform and a cocktail ELISA setting. This set of proteins and technologies therefore offers a powerful combination to drive and further improve serological assays toward reliable, simple, and cost-effective diagnosis of CBPP.

Evaluation of different sampling methods and results of real-time PCR for detection of feline herpes virus-1, Chlamydophila felis and Mycoplasma felis in cats.

OBJECTIVE: To investigate how different sampling techniques affect detection of DNA from feline herpes virus Type 1 (FHV-1), Chlamydophila felis and Mycoplasma felis and to study the correlation between positive test results and clinical signs in cats. ANIMALS: Fifty-one cats; 24 with ocular signs and 27 healthy control cats. PROCEDURES: Samples were collected from all cats using cotton swabs, conjunctival and corneal biopsies, and corneal scrapings. Samples were analyzed for presence of FHV-1, C. felis, M. felis, and feline DNA, defined by 28S rDNA, by using real-time PCR. RESULTS: In affected cats, FHV-1 was detected in only one cat; C. felis and M. felis were not detected in any affected cats. None of the three organisms was detected in any control cats. Feline DNA was demonstrated in all conjunctival samples, in 82% of corneal swabs, 92% of corneal scrapings, and 100% of keratectomy samples. CONCLUSIONS: Because of the generally low detection rate for FHV-1, C. felis, and M. felis DNA in this study, differences regarding sampling technique could not be determined and correlation between positive test results and degree of clinical signs could not be made. Detection of feline DNA in most samples irrespective of sampling technique, suggests a low prevalence of FHV-1, C. felis and M. felis in this population of cats.

A robust method for bacterial lysis and DNA purification to be used with real-time PCR for detection of Mycobacterium avium subsp. paratuberculosis in milk.

A possible mode of transmission for the ruminant pathogen Mycobacterium avium subsp. paratuberculosis (MAP) from cattle to humans is via milk and dairy products. Although controversially, MAP has been suggested as the causative agent of Crohn's disease and its presence in consumers' milk might be of concern. A method to detect MAP in milk with real-time PCR was developed for screening of bulk tank milk. Pellet and cream fractions of milk were pooled and subjected to enzymatic digestion and mechanical disruption and the DNA was extracted by automated magnetic bead separation. The analytical sensitivity was assessed to 100 organisms per ml milk (corresponding to 1-10 CFU per ml) for samples of 10 ml. The method was applied in a study of 56 dairy herds to compare PCR of farm bulk tank milk to culture of environmental faecal samples for detection of MAP in the herds. In this study, 68% of the herds were positive by environmental culture, while 30% were positive by milk PCR. Results indicate that although MAP may be shed into milk or transferred to milk by faecal contamination, it will probably occur in low numbers in the bulk tank milk due to dilution as well as general milking hygiene measures. The concentration of MAP can therefore be assumed to often fall below the detection limit. Thus, PCR detection of MAP in milk would be more useful for control of MAP presence in milk, in order to avoid transfer to humans, than for herd prevalence testing. It could also be of value in assessing human exposure to MAP via milk consumption. Quantification results also suggest that the level of MAP in the bulk tank milk of the studied Danish dairy herds was low, despite environmental isolation of MAP from the herds.

Expression and immunogenicity of six putative variable surface proteins in Mycoplasma mycoides subsp. mycoides SC.

Variable surface protein Vmm and five Vmm-type proteins from Mycoplasma mycoides subsp. mycoides SC were analysed to determine whether these proteins are expressed in vivo in animals affected by contagious bovine pleuropneumonia (CBPP) and in vitro. Recombinant versions of these proteins were constructed and expressed in Escherichia coli after mutation of the TGA Trp codons to TGG. These proteins were then analysed by dot and Western blotting with sera from CBPP-affected cattle. Furthermore, affinity-purified polyclonal antibodies to the recombinant proteins were used in Western and colony blotting to look for expression of the putative Vmm-type proteins in cultured M. mycoides SC. This study demonstrates that immunoglobulins in CBPP sera recognize all putative Vmm-type proteins tested, indicating that these proteins or their homologues are expressed by mycoplasmas during natural infections. Vmm and one of the putative Vmm-type proteins showed variable expression in vitro.

New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis.

BACKGROUND: Johne's disease, a serious chronic form of enteritis in ruminants, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). As the organism is very slow-growing and fastidious, several PCR-based methods for detection have been developed, based mainly on the MAP-specific gene IS900. However, because this gene is similar to genes in other mycobacteria, there is a need for sensitive and reliable methods to confirm the presence of MAP. As described here, two new real-time PCR systems on the IS900 gene and one on the F57 gene were developed and carefully validated on 267 strains and 56 positive clinical faecal samples. RESULTS: Our confirmatory PCR systems on IS900 were found sensitive and specific, only yielding weak false positive reactions in one strain for each system. The PCR system on F57 did not elicit any false positives and was only slightly less sensitive than our primary IS900-system. DNA from both naturally infected and spiked faeces that tested positive with our primary system could be confirmed with all new systems, except one low-level infected sample that tested negative with the F57 system. CONCLUSION: We recommend using the newly constructed DH3 PCR system on the F57 gene as the primary confirmatory test for PCR positives, but should it fail due to its lower sensitivity, the DH1 and DH2 PCR systems should be used.

Molecular characterization of thymidine kinase from Ureaplasma urealyticum: nucleoside analogues as potent inhibitors of mycoplasma growth.

Ureaplasma urealyticum (U. urealyticum), belonging to the class Mollicutes, is a human pathogen colonizing the urogenital tract and causes among other things respiratory diseases in premature infants. We have studied the salvage of pyrimidine deoxynucleosides in U. urealyticum and cloned a key salvage enzyme, thymidine kinase (TK) from U. urealyticum. Recombinant Uu-TK was expressed in E. coli, purified and characterized with regards to substrate specificity and feedback inhibition. Uu-TK efficiently phosphorylated thymidine (dThd) and deoxyuridine (dUrd) as well as a number of pyrimidine nucleoside analogues. All natural ribonucleoside/deoxyribonucleoside triphosphates, except dTTP, served as phosphate donors, while dTTP was a feedback inhibitor. The level of Uu-TK activity in U. urealyticum extracts increased upon addition of dUrd to the growth medium. Fluoropyrimidine nucleosides inhibited U. urealyticum and M. pneumoniae growth and this inhibitory effect could be reversed by addition of dThd, dUrd or deoxytetrahydrouridine to the growth medium. Thus, the mechanism of inhibition was most likely the depletion of dTTP, either via a blocked thymidine kinase reaction and/or thymidylate synthesis step and these metabolic reactions should be suitable targets for antimycoplasma chemotherapy.

An IS900-like sequence found in a Mycobacterium sp. other than Mycobacterium avium subsp. paratuberculosis.

The insertion sequence IS900 has been considered specific for Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) and has, therefore, been used as the target gene for diagnostic PCR of M. paratuberculosis. From a healthy dairy cow we have isolated and characterised a mycobacterium harbouring one copy of a sequence with 94% identity to IS900 at the nucleic acid level. The isolate was shown to be related to Mycobacterium cookii, as assessed by 16S rRNA sequencing. Strong amplifications were obtained with several PCR primers described for detection of IS900. This finding shows the need of alternative PCR systems based on other genes than IS900 to confirm the presence of M. paratuberculosis.

Intraspecific variation in the 16S rRNA gene sequences of Mycoplasma agalactiae and Mycoplasma bovis strains.

Intraspecific variation in the 16S rRNA genes of 17 Mycoplasma agalactiae and eight Mycoplasma bovis isolates was investigated to determine the degree of sequence variation in these two species and to determine whether the polymorphisms in the 16S rRNA genes could be used for the construction of an evolutionary tree and as epidemiological markers. A high degree of variation was found within isolates (between operons) and between isolates of both species. In contrast to M. capripneumoniae no distinct evolutionary pattern could be seen, probably because there are functional systems for gene conversion in M. agalactiae and M. bovis. However, the non-European isolates of M. agalactiae shared three characteristic nucleotides and European isolates from the same or neighbouring countries were very similar. Differences within isolates included both polymorphic positions and sequence length differences between operons. The amount of variation within isolates of the respective species ranged from zero to seven polymorphisms for M. agalactiae and from zero to four polymorphisms for M. bovis. The high degree of variation suggests the potential for misdiagnosis of species in diagnostic PCR assays based on the 16S rRNA gene sequences. All isolates of both species had a thymidine in position 912 (E. coli numbering) that causes streptomycin resistance in several bacterial species and which is characteristic for the members of the hominis group. As expected, when five M. agalactiae and three M. bovis isolates were tested for streptomycin susceptibility, they all demonstrated streptomycin resistance. M. agalactiae and M. bovis were found to have high intraspecific variation in their 16S rRNA gene and the polymorphisms patterns indicate that gene conversion takes place.