Detection of all Chlamydophila and Chlamydia spp. of veterinary interest using species-specific real-time PCR assays

The aim of the present study was to analyse the occurrence of chlamydiae in several mammalian host species. Clinical samples that previously tested positive in a Chlamydiaceae-specific real-time PCR were retested using six species-specific real-time PCR assays to identify the chlamydial species involved. Chlamydophila (Cp.) abortus was the agent most frequently found in cattle, sheep, horses, goats, and pigs. Detection in cattle of Cp. psittaci (11% of samples) and Chlamydia (C.) suis (9%), as well as Cp. psittaci in a goat sample was somewhat unexpected. DNA of two different chlamydiae was identified in 56 (12.7%) of 440 samples tested. Cp. felis was the predominant species found in cats, while in guinea pigs and rabbits only Cp. caviae was detected. Interestingly, the latter two pathogens were also identified in samples from dogs. The data show that mixed chlamydial infections are not rare and suggest an extended host range of individual species.     

Detection of fluoroquinolone resistance level in clinical canine and feline Escherichia coli pathogens using rapid real-time PCR assay

Fluoroquinolones are used to treat infections caused by Escherichia coli in canine and feline veterinary patients, particularly those infecting the urinary tract. The gyrA gene is a primary target causing fluoroquinolone resistance in Gram negative coliforms, with mutations in codons 83 and 87 generally associated with high-level of resistance E. coli clinical isolates. We have developed a fluorescence resonance energy transfer (FRET) quantitative PCR to identify enrofloxacin-resistance in clinical E. coli isolates that carry mutations in codons 83 and 87 of gyrA. This real-time quantitative PCR assay is rapid, economical, and sensitive compared with cultured antimicrobial susceptibility testing. The assay identified as few as four genome copies per reaction from culture and 19 genome copies in urine. For the 70 isolates tested, the sensitivity was 87.5% (95% CI = 75–95.3%) (n = 42/48), specificity was 100% (95% CI = 87.3–100%) (n = 22/22), whereas accuracy was 91.4% (95% CI = 82.3–97%) (n = 64/70). Furthermore, we were able to accurately differentiate between the wild type and mutants E. coli directly from infected canine urine samples (n = 5) within 2 h. These results were confirmed by sequence alignments of the PCR products and comparison with the susceptibility testing. The FRET-PCR assay appears to have promising clinical application as an early diagnostic tool for rapid and sensitive detection and differentiation of the level of fluoroquinolone resistance among clinical E. coli isolates that may facilitate design of the dosing regimen.     

In vitro antagonistic activities of Lactobacillus spp. against Brachyspira hyodysenteriae and Brachyspira pilosicoli

The sensitivity of Brachyspira hyodysenteriae and Brachyspira pilosicoli, respectively the causative agents of Swine Dysentery and Porcine Intestinal Spirochaetosis to two probiotic Lactobacillus strains, L. rhamnosus CNCM-I-3698 and L. farciminis CNCM-I-3699 was studied through viability, motility and coaggregation assays. The cell-free supernatant of these lactobacilli contains lactic acid, that is stressful for Brachyspira (leading to the formation of spherical bodies), and lethal. It was demonstrated for the first time the in vitro coaggregation properties of two probiotic Lactobacillus strains (active or heat-treated) with two pathogenic strains of Brachyspira, leading to (1) trapping of spirochaetal cells in a physical network as demonstrated by SEM; (2) inhibition of the motility of Brachyspira. Such in vitro studies should encourage in vivo studies in animal model to evaluate the potential of the use of probiotic lactobacilli through a feeding strategy for the prevention of B. hyodysenteriae and B. pilosicoli.     

Seroprevalence of antibodies to Encephalitozoon cuniculi and Encephalitozoon intestinalis in humans and animals

The presence of antibodies against Encephalitozoon cuniculi (E. cuniculi) and Encephalitozoon intestinalis (E. intestinalis) was examined in 215 samples from humans and in 488 samples from five different species of domestic and companion animals in Slovakia. The 215 human samples and samples from 90 swine, 123 non-infected cattle (cattle), 24 cattle infected with bovine leukosis virus (BLV-positive cattle), 140 sheep and 111 dogs were examined by the enzyme-linked immunosorbent assay (ELISA). Samples with serum titres 1:200 or higher were considered as positive. Specific anti-E. cuniculi antibodies were found in humans (0.9%), swine (52%), cattle (2%), sheep (9%) and dogs (15%) except for the BLV-positive cattle at the titre of 1:200. The titre of 1:400 was detected only in humans (0.5%). The presence of specific anti-E. intestinalis antibodies at the titre of 1:200 was confirmed in humans (6%), swine (51%), cattle (11%), BLV-positive cattle (13%) and dogs (6%) but not in sheep. The anti-E. intestinalis antibodies reached the 1:400 in humans (1%), swine (4%) and BLV-positive cattle (17%). The presence of specific anti-E. intestinalis antibodies at the titre of 1:600 was observed only in one swine (1%). Significant differences were observed in animals at titres 1:200 and 1:400 (chi-squared test: p < 0.0001) for both pathogens and in humans only for E. cuniculi at the titre of 1:400 (chi-squared test: p < 0.0075).     

Moprhological diversity of cultured canine gastric Helicobacter spp.

The cell morphology, the number of flagella, the occurrence of periplasmic fibrils and ultrastructural structures of five groups of cultured canine gastric Helicobacter spp. were compared. The study included four strains of Helicobacter felis, four strains of Helicobacter bizzozeronii, one strain of ‘Flexispira’, six strains of an unnamed spiral organism 2 and one strain of an unnamed spiral orgaism 3 which were isolated from gastric biopsies. Cultures were studied with negative staining, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Bacterial dimensions were measured from the negative staining samples and values were tested with ANOVA and Bonferroni tests. The organisms studied differed from each other morphologicall. H. felis was a slightly spiraled organism with periplasmic fibrils. ‘Flexispira’ was a thin and straight organism with periplasmic fibrils. H. bizzozeronii was a tightly spiraled organism. Spiral organism 2 was loosely spiraled and thicker than the other organisms. Spiral organism 3 was a short curved rod having a single bipolar flagellum. The other species had multiple flagella. As a conclusion the canine gastric Helicobacter spp. can be differentiated from each other morphologically with an electron microscope. The morphological differences were mainly found in the structures involved in motility. The importance of the differences may lie in their impact on the colonization in a gastric mucous environment.     

Rapid virulence typing of Pasteurella multocida by multiplex PCR

The gram-negative bacterium Pasteurella multocida constitutes a heterogeneous species associated with wide range of disease in many animals. Isolates are classified into five groups based on capsular antigen (capA, B, D, E and F). Recently, a new valuable PCR-based method was introduced to determine the epidemiological correlation between P. multocida infection and existence of virulence genes including tbpA, pfhA, toxA and hgbB. However, this method is tedious and laborious. Thus, in the current study, we designed a reliable multiplex PCR method for rapid detection of virulence genes in P. multocida. Eighty seven strains of P. multocida isolated from various clinically healthy and infected hosts were examined by uniplex PCR method for each virulence associated genes. Based on our improved and simplified multiplex PCR method, rapid detection of four virulence genes was accomplished. It is proposed that its implementation may benefit the epidemiological investigations.     

Allochronic seasonal peak activities of Dermacentor and Haemaphysalis spp. under continental climate in Hungary

Hard ticks (Acari: Ixodidae) were collected from the vegetation at monthly intervals in 2008 on six places of three different biotopes in Hungary. Except for Haemaphysalis concinna and H. punctata, predominance of females was observed in the questing population. From among the species with biphasic activity period, metastriate ticks (Dermacentor spp. and H. inermis) had higher prevalence of host-seeking males in the autumn than in the spring, as opposed to prostriate Ixodes ricinus. In comparison with the permanently mild weather experienced at the beginning of 2007, sharply rising temperatures during consecutive winter days in January and February of 2008 appeared to be more efficient in triggering a 1–2 month earlier spring peak activity of hard ticks, except for I. ricinus. Regarding seasonality, D. marginatus was most numerous in February and March, whereas D. reticulatus in September and October; H. punctata showed peak activity in March–April, H. concinna in May, and H. inermis in November–December. Within these genera such a temporal difference (allochrony) between seasonal peak activities of sympatric species under continental climate is described for the first time.     

Detection and identification of Toxocara canis DNA in bronchoalveolar lavage of infected mice using a novel real-time PCR

Toxocarosis is a zoonosis with worldwide distribution caused by Toxocara spp. of dogs and cats. In humans, diagnosis relies mainly on detection of parasite-specific antibodies. Although serological assays in current use have defined sensitivity and specificity, the problem of cross-reactivity still remains, particularly in areas of endemic polyparasitism. Microscopic detection of the parasite in tissue biopsies is not recommended for diagnosis because larvae can be difficult to locate, and finding the parasite eggs in faeces is not applicable since the larvae do not develop to the adult stage in the human host. In this study we describe a novel real-time PCR (‘Nemo-PCR’) that, in combination with DNA sequencing, allows the detection and identification of Toxocara canis and other nematodes in the Superfamily Ascaridoidea. Results indicate that this approach can detect Toxocara spp. DNA in bronchoalveolar lavage (BAL) of experimentally-infected mice. For diagnostic purposes further studies are necessary to evaluate this assay including testing human BAL fluid. The availability of such a direct assay would improve diagnosis of toxocarosis particularly for patients with pulmonary signs and symptoms.     

Preliminary findings of Salmonella spp. in captive green iguanas (Iguana iguana) and their environment

Captive reptiles are routinely identified as reservoirs of Salmonella spp. and reports of reptile-associated salmonellosis are increasing. Unfortunately, little is known about the epidemiology of Salmonella spp. and green iguanas. We did a limited survey of a green-iguana farm in El Salvador to identify sources of Salmonella spp. in green iguanas and their environment. A limited number of samples for microbiological culture were collected from iguanas (adult, hatchling, and embryos) and their environment (food, water, soil, shelter, insects, and wild-caught lizards). Salmonella spp. was isolated from the intestine of both adult (3/20) and hatchling iguanas (8/20). There was no evidence of Salmonella spp. in the reproductive tracts of female iguanas (0/10). Salmonella spp. was isolated from the surface of 40% (7/16) of the egg surfaces tested. Salmonella spp. was not identified from the externalized yolk-sac of the iguana embryos tested. Soil samples from a breeding pen and a nest were both positive for Salmonella spp. Eight different Salmonella spp. serotypes were identified in this survey. These results suggest that horizontal transmission of Salmonella spp. is a potential source of exposure to hatchling iguanas at this facility.     

小鼠细胞因子实时定量PCR检测方法的建立及应用

采用荧光SYBR Green I建立小鼠细胞因子mRNA实时定量PCR的检测方法;并利用建立的实时定量PCR的检测方法时感染H5N1禽流感病毒的BALB/c小鼠不同时间采集的肺脏组织中几种重要致炎细胞因子及趋化因子mRNA表达水平进行了检测.对HSN1禽流感病毒感染的BALB/c小鼠肺脏细胞因子的检测具有高度的特异性,检测的灵敏度为10~1～10~2拷贝数.H5N1禽流感病毒感染BALB/c小鼠后,小鼠肺脏中IL-1β、IL-6、TNFα、MIG、IP-10、RANTES、MIF和HMGB1细胞因子mRNA表达水平与时照组小鼠相比均有明显差异.建立的实时定量PCR能在基因转录水平敏感和特异地反应细胞因子的表达水平,该技术在基础和临床免疫研究中,具有良好的应用价值.     

Echinococcosis in pigs and intestinal infection with Echinococcus spp. in dogs in southwestern Lithuania

Cystic echinococcosis is a major emerging zoonosis in many Eastern European and Asian countries. Post slaughter examinations of 684 pig livers in Lithuania revealed significantly higher numbers of Echinococcus granulosus infections in animals from family farms (13.2%; 95% CI 10.7–16.2) as compared with those from industrial farms (4.1%; 95% CI 0.8–11.5). The prevalence was also significantly higher in pigs older than 1 year than in younger ones. In addition, in 0.5% of the pigs from the family farms, infertile and calcified E. multilocularis lesions were identified by PCR. Faecal samples from rural dogs (n = 240) originating from 177 family farms in 12 villages were investigated for taeniid eggs with two methods. Significantly more dogs excreting taeniid eggs were diagnosed with the flotation/sieving method (n = 34) as compared to the modified McMaster method (n = 12). Multiplex PCR performed with DNA from taeniid eggs isolated from faeces of 34 dogs revealed 26 infections with Taenia spp., 9 with E. granulosus and 2 with E. multilocularis (4 cases with concurrent Taenia spp. and E. granulosus or E. multilocularis infections). Genotyping of E. granulosus cyst tissues from 7 pigs, 1 head of cattle and from E. granulosus eggs from 8 dog faeces revealed the genotype G6/7 (‘pig/camel strain’) in all cases. The high infection pressure with Echinococcus spp. in family farms necessitates initiating control programs.     

Phylogentic analysis of Anaplasma ovis strains isolated from sheep and goats using groEL and mps4 genes

Evidence of Anaplasma spp. in goats and sheep in Cyprus has been demonstrated by previous research. Herein, further research was performed for the identification of the exact Anaplasma spp. resulting in the identification of Anaplasma ovis strains in all samples examined. We used a bioinformatics as well as a molecular approach (study of groEl and mps4 genes) in order to verify the validity of the results. All samples depicted the presence of A. ovis regardless of the host (goat or sheep).     

Quantitative real-time PCR and culture examination of Mycobacterium avium subsp. paratuberculosis at farm level

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease in ruminants and may contribute to Crohn's disease in humans. The aim of this study was to determine the occurrence and quantity of MAP in cattle feces and milk in the Iranian context. In addition, we evaluated the effect of cattle age as well as farming system as risk factors contributing to MAP load. For this, a total sample of 373 consisting of 150 cattle feces (CF), 150 individual cow's milk (ICM), as well as 73 bulk-tank milk (BTM) was collected randomly and regardless of the cattle health status. The samples were assayed using F57 quantitative real-time PCR (qPCR) and culture method. According to the results of qPCR which was found ∼10 times more sensitive than culture assay, MAP was detected in 68.66% (103/150) of the CF, 12% (18/150) of the ICM and 52.05% (38/73) of the BTM samples. In contrast to the previous reports, the quantity of MAP in the BTM (2.03–5.97 log cfu/50 ml) was statistically (p < 0.01) higher than the ICM (0.90–1.97 log cfu/50 ml). Data suggested a direct relation (p < 0.01) between the cattle age and the quantity of MAP in the CF samples, while the relation was not statistically significant (p > 0.05) for the ICM. In addition, MAP load in the BTM samples obtained from traditional farms was significantly (p < 0.01) higher than that of the industrial ones, while the differences in CF and ICM was not significant (p > 0.05).     

Detection of Fusobacterium necrophorum and Dichelobacter nodosus in lame cattle on dairy farms in New Zealand

Lameness in the dairy industry in New Zealand causes a problem in lost production, animal welfare and associated costs. To understand what bacteria may be present on the hooves of lame dairy cattle in this grass-fed system, samples were scraped from lame dairy cows and examined for the presence of Fusobacterium necrophorum (F. necrophorum) and Dichelobacter nodosus (D. nodosus) using the polymerase chain reaction (PCR). The PCR primers were designed to detect the presence of the lktA gene, which encodes a leukotoxin unique to F. necrophorum, and the fimA gene of D. nodosus. A total of 148 hoof scrapings were collected by farm staff over the period September 2005 to May 2006. F. necrophorum was detected in 79/148 of the samples, while D. nodosus was detected in 7/148 of the samples. The frequent finding of F. necrophorum within dairy herds in New Zealand is noteworthy and the occasional finding of D. nodosus on some dairy cattle suggests a possible role in both ovine and bovine hoof pathology.     

Microsporidiosis: Enterocytozoon bieneusi in domesticated and wild animals

Microsporidia are a ubiquitous group of obligate intracellular parasites that infect all major animal groups. Enterocytozoon bieneusi is the most commonly identified Microsporidia in humans and has also been reported worldwide in animals with importance in veterinary medicine (e.g. cats, dogs, horses, cattle and pigs). The identification of E. bieneusi in animals has raised the question of the importance of animal reservoirs in the epidemiology of this pathogen, and the implications of the infection with this pathogen in infected animals. Considerable genetic diversity within E. bieneusi has been found with over 90 genotypes identified based on the ITS nucleotide sequence of E. bieneusi spores recovered from the feces of infected humans and animals. Both host-adapted E. bieneusi genotypes with narrow host ranges and potentially zoonotic genotypes with wide host specificity have been identified. The information presented in this review should be useful in understanding the taxonomy, epidemiology, zoonotic potential, and importance in public health of E. bieneusi.     

Occurrence and characterization of gastric Helicobacter spp. in naturally infected dogs

Helicobacter-like organisms are frequently observed in the stomach of dogs but the relationship between these microorganisms and gastric pathology has not been clearly established. Different species of helicobacters are known to be present in the canine stomach but their specific prevalence in naturally infected dogs is unknown. The aims of this study were to isolate and characterize helicobacters in canine gastric biopsies, to compare the commonly used tests for the identification of Helicobacter spp. and to determine the occurrence of these species in dogs. Twenty-three out of 25 dogs (92%) were positive for Helicobacter-like organisms in cytological screening. Culture was successful from biopsies of 5/25 dogs. The isolates were analyzed by electron microscopy, biochemical and physiological tests, whole protein analysis and 16S rDNA sequencing. Helicobacter felis was identified in four samples and Helicobacter bizzozeronii in one sample. Only the whole protein analysis in combination with electron microscopy was able to clearly discriminate the two species. Compared to the high prevalence of Helicobacter-like organisms, the occurrence of H. felis and H. bizzozeronii, was low (17 and 4%, respectively). No Flexispira rappini-like organisms or H. salomonis were detected. Electron microscopy revealed that H. bizzozeronii-like microorganisms were present in three additional biopsies where we were unable to culture any Helicobacter-like organisms. These observations indicate that in the stomach of dogs not all helicobacters are culturable. The unculturable bacteria appeared to be the prevalent ones and may represent different spiral organisms. The presence of distinct helicobacters with different characteristics can reflect different roles in the pathogenesis of canine gastric disease.     

Mycobacterium avium subsp. paratuberculosis in cow bulk tank milk in Cyprus detected by culture and quantitative IS900 and F57 real-time PCR

The possibility that Mycobacterium avium subsp. paratuberculosis (MAP) plays some role in the development of Crohn's disease in humans is attracting attention to milk and milk products originating from infected animals. In this study, we focused on the detection of MAP in 220 bulk tank milk (BTM) samples from all dairy cattle herds in Cyprus. In total, 63 (28.6%) BTM milk samples were found to be positive for MAP using quantitative real-time PCR assays for IS900 and F57. The presence of MAP in BTM was low, and was assessed to be several tens of MAP cells per one ml of BTM. Milk samples examined by cultivation were found to be negative for MAP in all 220 BTM. In two BTM samples cultivation and subsequent sequencing of 16S rRNA revealed two isolates of M. fortuitum.     

Bartonella spp. in cats from Buenos Aires,Argentina

In Argentina, data on the presence of members of the genus Bartonella is scarce. To increase knowledge about these zoonotic pathogens in this country, the presence and variability of Bartonella spp. was investigated in cats and dogs from Buenos Aires. Bartonella spp. was detected in 17.8% of cats, while all dogs tested negative by PCR and Reverse Line Blot. B. henselae was the most frequent species, being detected in 11.9% (14/101), while B. clarridgeiae was found in only 5.9% (6/101) of the cats. Afterwards, B. henselae isolates and positive blood samples were characterized by Multiple Locus Sequence Typing (MLST) and Multiple Locus Variable Number Tandem Repeats Analysis (MLVA). As result, four different MLST sequence types (ST) and eight MLVA profiles were identified. ST 1 was the most frequent variant found in cats, followed by ST 8. Interestingly, some of the MLVA profiles that were detected in this study have been previously associated with human disease, and represents a potential risk of infection. Veterinarians and physicians should consider the presence of these emerging pathogens in their diagnostic routine.     

Evaluation of PCR inhibitory effect of enrichment broths and comparison of DNA extraction methods for detection of Salmonella Enteritidis using real-time PCR assay

The best enrichment broth and DNA extraction scheme was determined for rapid and sensitive detection of Salmonella Enteritidis in steamed pork using real-time PCR. The inhibitory effect of commonly used Salmonella enrichment broths, Rappaport-Vassiliadis (RV) and Muller-Kauffmann tetrathionate with novobiocin (MKTTn), on real-time PCR was confirmed. The inhibition of PCR was statistically significant (p < 0.05) in RV and MKTTn, as compared with buffered peptone water (BPW) or phosphate-buffered saline. The inhibitory effect of the selective enrichment media was successfully removed by using a modified DNA extraction, PrepMan Ultra Reagent with an additional washing step or the DNeasy Tissue Kit. In three experiments, when applied to detection of Salmonella Enteritidis in steamed pork, the real-time PCR coupled with single 24 h enrichment with BPW performed better than double 48 h enrichment with BPW plus RV or MKTTn. The simple real-time PCR assay using BPW proved to be a rapid and sensitive test for detection of low concentrations of Salmonella Enteritidis in steamed pork samples as compared with the conventional culture method.