水貂出血性肺炎的实验室诊断方法研究进展

水貂出血性肺炎是由铜绿假单胞菌引起的严重威胁养貂业的重要疾病之一。文中概述了水貂出血性肺炎的实验室诊断方法研究进展,在病原学方面,主要依靠细菌分离培养、生化试验、特异性抗原及其抗体的检查等;在分子生物学方面,主要依靠国内外相继建立起的PCR技术和LAMP技术等。介绍和比较了各种方法的优缺点和实用性,为临床兽医科技工作者建立快速、简便、准确、实用的诊断提供参考。     

检测水貂出血性肺炎绿脓杆菌的比色LAMP法的建立与应用

为了快速有效检测水貂出血性肺炎病原绿脓杆菌,本研究结合金属指示剂HNB特性建立了快速检测绿脓杆菌比色LAMP法。根据绿脓杆菌外毒素A基因设计引物,建立了检测LAMP法;并且对该方法进行了特异性、灵敏性分析;同时进行了初步应用。结果显示,该方法特异性强,灵敏性好,可检测167CFU/mL的菌体,对病貂肺脏检出率高。结果表明,该比色LAMP法可以有效地检测水貂出血性肺炎绿脓杆菌。     

G＋B＋C三个血清型绿脓杆菌灭活疫苗大规模接种后的不良反应和保护效果观察

观察绿脓杆菌的G＋B＋C三个血清型制备的灭活疫苗大规模接种后的速发接种反应及保护性效果观察.用绿脓杆菌G＋B＋C三个血清型制备的疫苗以小鼠和水貂为实验室观察组,同时设定阴性对照,观察疫苗免疫后的速发接种反应;在养殖场,选择常年因绿脓杆菌引起的水貂出血性肺炎的养殖户,采用预防免疫和发病期紧急预防接种两种方式,并回访保护效果.在实验室对水貂和小鼠皮下接种疫苗,接种后7～21 d观察接种部位和解剖小鼠检查病理学变化,没有出现任何异常反应.从2012年和2013年共制备的5个批次14万头份的疫苗,在对临床推广应用的回访中,只有一个养殖户少量水貂出现接种部位化脓,经查是由于注射部位的肌肉吸收性降低而引起,对预防接种的10万头份中,接种后没有发生绿脓杆菌引起的出血性肺炎,在对4万头份的由于绿脓杆菌引起出血性肺炎的紧急接种中,在5～10 d达到控制本病的目的.结论：G＋B＋C三个血清型绿脓杆菌疫苗速发接种反应发生率低,预后良好,无论是预防接种还是发病后紧急接种,均具有较好的保护性.     

Field studies: pseudomonas pneumonia of mink

Epizootics of pneumonia in mink caused by Pseudomonas aeruginosa were investigated to characterize the serotype of organisms and to identify possible predisposing factors. Most epizootics were associated with P aeruginosa Fisher serotype 1, and a few were associated with 3 other serotypes. There were no predisposing factors identified that could be used to differentiate farms affected and those not affected with pseudomonas pneumonia. Cultural studies indicated that P aeruginosa was present in mink from affected and nonaffected herds. Organisms isolated included serotypes associated with naturally occurring disease. Serostudy results were similar among herds. A prospective field vaccination trial did not yield definitive results, since only slight losses occurred in both vaccinated and nonvaccinated mink. Significant levels of antibody were detected in mink 15 to 17 weeks after they were given a single dose of P aeruginosa lipopolysaccharide vaccine.     

An outbreak of fatal hemorrhagic pneumonia caused by Streptococcus equi subsp. zooepidemicus in shelter dogs

An outbreak of fatal hemorrhagic pneumonia with 70~90% morbidity and 50% mortality occurred in an animal shelter in Yangju, Gyeonggi Province, Korea. Clinically, the affected dogs showed severe respiratory distress within 48 h after arriving in the shelter. The dead were found mainly with nasal bleeding and hematemesis. At necropsy, hemothorax and hemorrhagic pneumonia along with severe pulmonary consolidation was observed, though histopathological analysis showed mainly hemorrhagic bronchopneumonia. Lymphoid depletion was inconsistently seen in the spleen, tonsil and bronchial lymph node. Gram-positive colonies were shown in blood vessels or parenchyma of cerebrum, lung, liver, spleen, and kidney. Also, Streptococcus (S.) equi subsp. zooepidemicus was isolated from the various organs in which the bacterium was microscopically and histologically detected. In addition, approximately 0.9 Kb specific amplicon, antiphagocytic factor H binding protein, was amplified in the bacterial isolates. In this study, we reported an outbreak of canine hemorrhagic bronchopneumonia caused by S. equi subsp. zooepidemicus in an animal shelter in Yangju, Korea.     

水貂杯状病毒生物学特性研究进展

水貂杯状病毒（calicivirus）为杯状病毒科成员,能够引起水貂出血性肺炎和胃肠炎等,该病在中国河北、山东等水貂养殖大省暴发并流行,致死性较高,已有学者从血性肺炎病例中分离并鉴定病毒。作者从水貂杯状病毒的分类地位、生物学特性、致病机理和疫病防制等方面进行了综述。     

Multidrug-Resistant Pseudomonas aeruginosa Isolates From Captive Reptiles

Pseudomonas aeruginosa represents an opportunistic pathogen for animals and humans that is often associated with high disease morbidity and, at times, mortality. Captive reptiles have been shown to be reservoirs of P. aeruginosa strains that can be sources of exposure to humans that come in contact with these animals. In this study, the prevalence of P. aeruginosa among subclinical captive reptile species and the antimicrobial sensitivity of bacterial isolates were investigated. Sixty-five oral swabs were collected from captive reptiles belonging to 15 different species in which no overt signs of disease were evident. From this group of animals, 46 (70.8%) isolates were identified as P. aeruginosa. All of the P. aeruginosa strains were shown to have a wide range of antibiotic resistance. At present, there is a paucity of data regarding the prevalence of P. aeruginosa in various reptile species; therefore, continued scientific investigations are indicated to determine the significance of P. aeruginosa infection as it relates to captive reptile species.     

Comparison of Pseudomonas aeruginosa isolates from mink by serotyping and pulsed-field gel electrophoresis

Isolates of Pseudomonas aeruginosa from clinical infections in mink were subjected to serotyping and pulsed-field gel electrophoresis (PFGE) using SpeI. A total of 212 isolates of P. aeruginosa from the year 1998 to 2001 were included in this study: 168 isolates from mink obtained from 74 farm outbreaks of haemorrhagic pneumonia. Isolates from mink were separated into 34 distinct clones by PFGE subtyping. All isolates from mink infected during the same farm outbreak were identical, except in one case where two different strains were isolated from mink obtained from the same farm outbreak. P. aeruginosa of specific PFGE types were found to cause clusters of outbreaks on several farms within a few weeks of each other. However, PFGE types of strains causing clusters of farm outbreaks changed from year to year. These results suggest that some outbreaks of haemorrhagic pneumonia are caused by pathogenic strains of P. aeruginosa spread between farms and animals either mechanically, or through feed or water from a common source, rather than by random nosocomial infections with strains from the farm environment.     

Genotypic identification of Pseudomonas aeruginosa strains isolated from patients with urinary tract infection

BackgroundNumerous nosocomial infections including urinary tract infection (UTI) have been reported to be linked to Pseudomonas aeruginosa (P. aeruginosa). This bacterium is one of the most common pathogen colonized in the urinary tract. The main purpose of this study was to evaluated the presence of antibiotic resistance genes and also the most frequent genotype patterns of P. aeruginosa in the patients with UTI hospitalized in different wards of hospitals.Materials and methodsIn this study, 70 strains of P. aeruginosa isolated of urine samples from the patients with UTI were assessed. The isolated strains were genotyped using Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) method. We have also analyzed the presence of TEM and SHV resistant genes in the isolates.ResultsA total of 70 P. aeruginosa strains was isolated from the UTI patients. Based on MLVA method, 61 various genotypes of P. aeruginosa were identified which grouped into two main clusters and 4 sub-clusters. Moreover, approximately 80% and 70% of isolated strains carried the TEM and SHV resistance genes, respectively.ConclusionOur findings showed that the majority of patients hospitalized in different wards of hospitals have experienced the urinary tract infection caused by P. aeruginosa. According to the genotyping results, a high diversity of the P. aeruginosa population was observed in the patients with UTI. Our results can provide a better understanding of the P. aeruginosa genotype distribution and epidemiology of infection, which can be applied as basic data for future antibiotic therapies.     

Conception rate,uterine infection and embryo quality after artificial insemination and natural breeding with a stallion carrier of Pseudomonas aeruginosa: a case report

Background

Pseudomonas aeruginosa may cause venereal disease and infertility in horses. A Pseudomonas aeruginosa - carrier stallion, often unresponsive to artificial vagina collection, was used to naturally breed mares. Semen collected from the same stallion was also used to perform artificial inseminations. Pregnancy rates, embryo quality and incidence of uterine infection were compared between inseminated or naturally-bred mares.

Methods

P. aeruginosa was isolated from swabbing of the penis, prepuce and distal urethra of the stallion. Before being bred or inseminated, clitoral/vestibular samples were collected from all mares, and cultured for isolation of P. aeruginosa. At the first observed estrus, endometrial swabs were also collected. All mares subjected to natural mating (NS) were re-evaluated for P.aeruginosa by culture of clitoral and endometrial swabs. Artificial inseminations (AI) were performed either with fresh-extended semen (11 AI/7 mares) or frozen semen (10 AI/7 mares). The stallion was also used to breed 3 mares (4 services). For embryo collection, 2 mares were inseminated with fresh-extended semen (1 AI/mare), and 2 additional mares were inseminated with frozen semen (2 AI/mare). Two mares were naturally-bred with a total of 9 services, for embryo collection. All mares were examined after AI or natural service (NS), for uterine pathologies. Embryo recoveries were attempted passing a catheter with inflatable cuff connected to a sterile flexible 2-way flushing catheter, through the cervix. Flushed media was recovered into an Em-Con filter, and embryos searched using a stereoscope. Embryos were graded from 1 (excellent) to 4 (degenerated/dead).

Results

Pregnancy rates obtained after NS was 50% per cycle. However, more than half of the NS resulted in uterine disease, while uterine pathology was seen only in 22% of the time following AI. Half of the mares bred by NS got positive to P. aeruginosa. Percentage of embryo recovery rates was identical after AI or NS (66.7%). The 4 embryos recovered after AI were classified as Grade 1, while after NS only 2 out of the 6 recovered embryos were Grade 1.

Conclusion

a) there was no evidence of reduced fertilization after AI or NS, b) a numerically higher incidence of uterine disease was noticed after NS, c) venereal transmission of P. aeruginosa after NS was confirmed, d) a lower percentage of G1 embryos may be obtained after NS. Overall, the data supports the indication for P. aeruginosa-carrier stallions to be bred by AI rather than by NS, and raises the possibility that P. aeruginosa may affect embryo quality.     

Post-antibiotic effect of orbifloxacin against Escherichia coli and Pseudomonas aeruginosa isolates from dogs

Orbifloxacin is a fluoroquinolone drug used widely in companion animal medicine. In this study, we firstly determined post-antibiotic effects (PAEs) and post-antibiotic sub-minimum inhibitory concentrations (MIC) effects (PA-SMEs) of orbifloxacin for two strains each of Escherichia coli and Pseudomonas aeruginosa from dogs, and these parameters were compared with those of enrofloxacin. At twice the MIC, the PAEs of orbifloxacin ranged from -0.28-0.93 h (mean, 0.29 h) for E. coli and -0.18-1.18 h (mean, 0.37 h) for P. aeruginosa. These parameters were not significantly different for E. coli and shorter for P. aeruginosa, compared to enrofloxacin (P < 0.05). Continued exposure to 0.1, 0.2, and 0.3 the MIC of orbifloxacin resulted in average PA-SMEs of 0.55, 1.11, and 2.03 h, respectively, for E. coli, and 1.04, 1.40, and 2.47 h, respectively, for P. aeruginosa. These PA-SMEs, which had no significant differences with those of enrofloxacin, were significantly longer than the corresponding PAEs (P < 0.05). These results suggest that the PA-SME of orbifloxacin for E. coli and P. aeruginosa can be meaningfully prolonged by increase of sub-MICs.     

水貂绿脓杆菌分离株的血清型分析和Eric-PCR指纹图谱多样性研究

为了掌握山东地区水貂出血性肺炎流行情况,从2012年山东省内多个养貂社区进行水貂出血性肺炎流行病学调查。从病貂中分离获得48株绿脓杆菌,分别测定了分离株的生化特性、血清型以及部分菌株对小鼠与水貂的半数致死量（LD50）,同时运用Eric-PCR方法对分离进行了进行分型。结果显示,48株分离菌株与标准菌株生化特性基本一致,血清型G型为36株,血清型Ⅰ型为7株,血清型C型为4株;48株分离株大多数对恩诺沙星最敏感;动物试验显示,48株分离株对小鼠与水貂均能致病,所选4株分离株对小鼠与水貂的LD50有差异,且水貂对所选菌株显著敏感于小鼠;分子分型分为13个基因型。结果表明,该地区水貂出血性肺炎病原呈现遗传多样性。本试验为水貂出血性肺炎的防治提供了理论依据。     

ASYMMETRIC NASAL MUCOSAL THICKENING IN HEALTHY DOGS CONSISTENT WITH THE NASAL CYCLE AS DEMONSTRATED BY MRI AND CT

The nasal cycle is a physiological phenomenon that causes regular cyclical congestion and decongestion of the venous sinusoids lining the nasal mucosa. The purpose of this prospective study was to describe magnetic resonance imaging (MRI) and computed tomographic (CT) features of the normal nasal cycle in a group of dogs. Five dogs were recruited that met the following criteria: 8 to 15 months old, nonbrachiocephalic breed, no clinical signs or history of nasal disease, and undergoing anesthesia for problems unrelated to the nasal cavity. Nasal MRI (n = 5) and CT scans (pre‐ and postcontrast, n = 5) were acquired. Images were evaluated subjectively by two board‐certified radiologists and objectively by a diagnostic imaging intern using regions of interest placed on each side of the nasal cavity. Findings were compared using Cohen's kappa coefficient and Students t‐test on log‐transformed data. All dogs showed diffuse unilateral mucosal thickening of the rostral part of the nasal cavity in both MRI and CT studies. This mucosal thickening shifted sides between examinations in three dogs. Changes appeared most marked on T2‐weighted scans. No asymmetric mucosal changes were seen in the mucosa of the ethmoturbinates, vomer–nasal septum, hard palate or the frontal sinuses in any patient on MRI or CT. Computed tomographic contrast enhancement of the thickened mucosa was not statistically significant (P‐value < 0.08). In conclusion, the normal nasal cycle may cause asymmetrical mucosal changes in the rostral part of the nasal cavity that mimic MRI and CT characteristics previously reported for inflammatory disease in dogs.     

Pseudomonas pneumonia of mink: pathogenesis, vaccination, and serologic studies

Fulminating pneumonia was produced in mink by the intratracheal administration of Pseudomonas aeruginosa. The sequence of pulmonary lesions was focal inflammation, focal necrosis, and widespread inflammation and necrosis. Secondary lesions of peracute hemorrhage and necrosis were the result of bacterial spread via the airways. Invasion of vessel walls by P aeruginosa was a terminal event and was secondary to bacillary invasion and necrosis of adjacent tissues. Regional (lymphatic) and systemic spread of bacteria followed the development of pulmonary lesions, but there was little morphologic evidence of tissue damage in other organs. Immunofluorescence studies showed that P aeruginosa antigen was dispersed within pulmonary cells and was free in the lung parenchyma. Mink surviving beyond postinfection hour 60 had a macrophage infiltration into limited pulmonary lesions. A vaccine trial was conducted with P aeruginosa lipopolysaccharides (LPS) used as antigen, and an enzyme-linked immunosorbent assay was used to detect antibody. Antibody was detected in mink after vaccination with LPS or natural exposure. Mink with antibody to LPS, from vaccination or naturally acquired, were resistant to experimental infection.     

The association between pneumonia and atrophic rhinitis in slaughter weight Swine

Four hundred and sixty-two pigs from 37 herds were examined at slaughter for the presence and extent of turbinate atrophy and pneumonia. Turbinate atrophy was scored by measuring the mm space between the turbinate bone and the floor of the nasal cavity on both sides of the nasal septum. The total percentage of pneumonic involvement for each lung was evaluated by scoring the percentage of each lobe that was consolidated.

There was a low, positive correlation between individual scores of turbinate atrophy and the associated percentage of lung involved with pneumonia (r = + 0.177; p < 0.001). There was a postive correlation between the herd mean turbinate atrophy score and the herd mean percentage pneumonia score (r = + 0.515; p = 0.001).

The age at slaughter was known for 95 pigs from four herds and was not significantly correlated with the mm of turbinate atrophy, or the percentage of pneumonia.

     

Investigation of repeated vaccination as a possible cause of glomerular disease in mink

The search for antigens capable of causing immune-complex-mediated glomerulonephritis continues. Modified live-virus vaccines commercially available for veterinary use are a possible source. In this study, repeated vaccination of mink with live-virus vaccines was investigated as a model for vaccine-induced glomerular injury. Three groups of 10-wk-old mink, 15 per group, were vaccinated once with 4-way vaccine against distemper, Pseudomonas aeruginosa infection, botulism and mink viral enteritis. Subsequently, all mink in each group each were vaccinated either with the 4-way vaccine, a monovalent canine distemper vaccine, or saline. Glomerular function was assessed at 2-wk intervals by determining the urinary protein:creatinine (P:C) ratio. Kidney sections taken at necropsy, 20 wk after the 1st vaccination, were examined by light and immunofluorescent microscopy for deposition of immunoglobulin and complement. There was no statistically significant difference between the treated and control groups based on average urinary P:C ratio medians. Light microscopic changes were detected in glomeruli, but Fisher's exact test showed no significant differences between any of the treatment groups. Deposition of immunoglobulin but not complement was significantly more frequent (P < 0.05) in the glomeruli of animals that received multiple injections of the 4-way vaccine than in the glomeruli of those given only the monovalent canine distemper vaccine or saline. These findings suggest that repeated vaccination may increase the glomerular deposition of immunoglobulin. Further studies are required to determine if the increased deposition of immunoglobulin contributes to the development of glomerular damage and to identify the antigens driving production of the deposited immunoglobulin.     

Mycoplasma bovis infections in Swiss dairy cattle: a clinical investigation

Mycoplasma bovis causes mastitis in dairy cows and is associated with pneumonia and polyarthritis in cattle. The present investigation included a retrospective case–control study to identify potential herd-level risk factors for M. bovis associated disease, and a prospective cohort study to evaluate the course of clinical disease in M. bovis infected dairy cattle herds in Switzerland. Eighteen herds with confirmed M. bovis cases were visited twice within an average interval of 75 d. One control herd with no history of clinical mycoplasmosis, matched for herd size, was randomly selected within a 10 km range for each case herd. Animal health data, production data, information on milking and feeding-management, housing and presence of potential stress- factors were collected. Composite quarter milk samples were aseptically collected from all lactating cows and 5% of all animals within each herd were sampled by nasal swabs. Organ samples of culled diseased cows were collected when logistically possible. All samples were analyzed by real-time polymerase chain reaction (PCR). In case herds, incidence risk of pneumonia, arthritis and clinical mastitis prior to the first visit and incidence rates of clinical mastitis and clinical pneumonia between the two visits was estimated. Logistic regression was used to identify potential herd-level risk factors for M. bovis infection. In case herds, incidence risk of M. bovis mastitis prior to the first visit ranged from 2 to 15%, whereas 2 to 35% of the cows suffered from clinical pneumonia within the 12 months prior to the first herd visit. The incidence rates of mycoplasmal mastitis and clinical pneumonia between the two herd visits were low in case herds (0–0.1 per animal year at risk and 0.1-0.6 per animal year at risk, respectively). In the retrospective-case-control study high mean milk production, appropriate stimulation until milk-let-down, fore-stripping, animal movements (cattle shows and trade), presence of stress-factors, and use of a specific brand of milking equipment, were identified as potential herd-level risk factors. The prospective cohort study revealed a decreased incidence of clinical disease within three months and prolonged colonization of the nasal cavity by M. bovis in young stock.     

A survey of Aleutian mink disease virus infection of feral American mink in Nova Scotia

Spleen samples from 14 mink that were trapped in 4 counties of Nova Scotia were tested for the presence of the Aleutian mink disease virus (AMDV) by polymerase chain reaction. Viral DNA was not detected in samples from Kings County (n = 2), but was detected in all the mink sampled from Colchester (n = 2) and Halifax (n = 6) counties, and 3 of 4 mink from Yarmouth County. The high level of AMDV-infected mink in Colchester and Halifax counties may pose a serious threat to the captive mink and wild animal populations. Because treatment of infected free-ranging mink is not an option, AMDV control strategies for the captive mink should be primarily focused on bio-security to protect clean ranches.