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In this study, 20 P. aeruginosa strains were isolated from 112 farmed mink exhibiting hemorrhagic pneumonia in mideastern Shandong province, China. Serotype G (18/20) was the dominant serotype among the isolates with prevalence in mink, followed by serotype B (1/20), serotype C (1/20). The 9 virulence-associated genes of P. aeruginosa were tested using PCR. The prevalence of the virulence genes for the isolates were algD 95% (19/20), plcH 85% (17/20), exoY 80% (16/20), aprA 75% (15/20), lasB 70% (14/20), exoS 65% (13/20), exoT 60% (12/20) and toxA 60% (12/20), respectively. The 20 isolates were negative for exoU gene. The isolates exhibited multidrug resistance and cross resistance, using antimicrobial disc susceptibility assays. The animal experiments demonstrated that LD50% of the P.aeruginosa-CS-2 in the intratracheally challenged mink was 2.2 × 10 7.0  CFU, and 6.8 × 10 4.0  CFU in the intraperitoneally challenged mink. It implied that both the inoculation doses and the routes of inoculation could have influences on the pathogenicity of P. aeruginosa in mink. Therefore, the evolutionary and epidemiological surveillance of P. aeruginosa in mink should be further strengthened for public health., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Influenza A virus (IAV) and bacteria co-infection can influence the host clinical conditions. Both H9N2 IAV and Pseudomonas aeruginosa (P. aeruginosa) are potential pathogens of respiratory diseases in mink. In this study, to clarify the effects of H9N2 IAV and P. aeruginosa co-infections on hemorrhagic pneumonia in mink, we carried out to establish the mink models of the two-pathogen co-infections in different orders. Compared with the single infections with H9N2 IAV or P. aeruginosa, the mink co-infected with H9N2 IAV and P. aeruginosa showed severe respiratory diseases, and exacerbated histopathological lesions and more obvious apoptosis in the lung tissues. H9N2 IAV shedding and viral loads in the lungs of the mink co-infected with H9N2 IAV and P. aeruginosa were higher than those in the mink with single H9N2 IAV infection. Furthermore, the clearance of P. aeruginosa in the co-infected mink lungs was delayed. In addition, the anti-H9N2 antibody titers in mink with P. aeruginosa co-infection following H9N2 IAV infection were significantly higher than those of the other groups. This implied that H9N2 IAV and P. aeruginosa co-infection contributed to the development of hemorrhagic pneumonia in mink, and that P. aeruginosa should play a major role in the disease. The exact interaction mechanism among H9N2 IAV, P. aeruginosa and the host needs to be further investigated., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Hemorrhagic pneumonia in mink is a fatal disease caused by Pseudomonas aeruginosa. Very little is known about P. aeruginosa in relation to genotype and the mechanisms underlying antimicrobial resistance in mink. A total of 110 P. aeruginosa samples were collected from mink from Chinese mink farms between 2007 and 2015. Samples underwent molecular genotyping using pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST), antimicrobial susceptibility and its mechanism were investigated at the molecular level. The PFGE identified 73 unique types and 15 clusters, while MLST identified 43 (7 new) sequence types (ST) and 12 sequence type clonal complexes (STCC). Sequence types and PFGE showed persistence of endemic clones in cities Wendeng (Shandong, China) and Dalian (Liaoning, China), even in different timelines. The MLST also revealed the gene correlation of the mink P. aeruginosa across different time and place. The ST1058 ( n = 14), ST882 ( n = 11), and ST2442 ( n = 10) were the predominant types, among which ST1058 was the only one found both in Shandong province and Dalian (Liaoning, China). The MLST for P. aeruginosa infection in mink was highly associated with that in humans and other animals, implying possible transmission events. A small proportion of mink exhibited drug resistance to P. aeruginosa (9/69, 13%) with resistance predominantly to fluoroquinolone, aminoglycoside, and β-lactamase. Eight strains had mutations in the quinolone-resistance determining regions (QRDR). High proportions (65%; 72/110) of the fosA gene and 2 types of glpt deletion for fosmycin were detected. Furthermore, in the whole genome sequence of one multidrug resistant strain, we identified 27 genes that conferred resistance to 14 types of drugs.

Pseudomonas aeruginosa is an important animal pathogen and contributes to hemorrhagic pneumonia in mink. Between April 2011 and December 2016, samples of lung, liver, and spleen were collected from mink with this disease on 11 mink farms in 5 Chinese provinces. From these samples, we obtained 98 isolates of P. aeruginosa that belonged to 5 serotypes: G ( n = 58), I ( n = 15), C ( n = 8), M ( n = 5), and B ( n = 2); 10 isolates were not typeable (10/98). More than 90% of the isolates formed biofilms, and 85% produced slime. All 98 isolates were resistant to 10 antibiotics (oxacillin, ampicillin, penicillin G, amoxicillin, ceftriaxone, cefazolin, cefaclor, tilmicosin, tildipirosin, and sulfonamide). However, almost all were susceptible to gentamicin, polymyxin B, and amikacin. We identified 56 unique genotypes by pulsed-field gel electrophoresis. These findings have revealed genetic diversity and high antimicrobial resistance in P. aeruginosa isolated from mink with hemorrhagic pneumonia and will facilitate the prevention and control of this disease.

The objective of this study was to investigate the potential of using phages as a therapy against hemorrhagic pneumonia in mink both in vitro and in vivo. Five Pseudomonas aeruginosa (P. aeruginosa) strains were isolated from lungs of mink with suspected hemorrhagic pneumonia and their identity was confirmed by morphological observation and 16S rDNA sequence analysis. Compared to P. aeruginosa strains isolated from mink with hemorrhagic pneumonia in 2002, these isolates were more resistant to antibiotics selected. A lytic phage vB_PaeP_PPA-ABTNL (PPA-ABTNL) of the Podoviridae family was isolated from hospital sewage using a P. aeruginosa isolate as host, showing broad host range against P. aeruginosa. A one-step growth curve analysis of PPA-ABTNL revealed eclipse and latent periods of 20 and 35 min, respectively, with a burst size of about 110 PFU per infected cell. Phage PPA-ABTNL significantly reduced the growth of P. aeruginosa isolates in vitro. The genome of PPA-ABTNL was 43,227 bp (62.4% G+C) containing 54 open reading frames and lacked regions encoding known virulence factors, integration-related proteins and antibiotic resistance determinants. Genome architecture analysis showed that PPA-ABTNL belonged to the "phiKMV-like Viruses" group. A repeated dose inhalational toxicity study using PPA-ABTNL crude preparation was conducted in mice and no significantly abnormal histological changes, morbidity or mortality were observed. There was no indication of any potential risk associated with using PPA-ABTNL as a therapeutic agent. The results of a curative treatment experiment demonstrated that atomization by ultrasonic treatment could efficiently deliver phage to the lungs of mink and a dose of 10 multiplicity of infection was optimal for treating mink hemorrhagic pneumonia. Our work demonstrated the potential for phage to fight P. aeruginosa involved in mink lung infections when administered by means of ultrasonic nebulization.

The biological characteristics and molecular epidemiology of Pseudomonas aeruginosa associated with mink hemorrhagic pneumonia from Shandong province of eastern China were determined in this study. From 2010 to 2011, 30 mink P. aeruginosa isolates were identified from lung, fecal and feed samples of clinical cases and subjected to serotyping, antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE) using SpeI. The P. aeruginosa isolates belonged to four serotypes-21 of type G, four of type I, three of type M, one of type B, and one non-typable strain. The strains were divided into four large groups as determined by PFGE. Isolates from the group 2 were highly homologous and were obtained from the same region as an epidemic. All of the isolates were sensitive to piperacillin, piperacillin/tazobactam, ceftazidime, cefepime, imipenem, amikacin, gentamicin and tobramycin and resistant to ampicillin, cefuroxime and cefuroxime axetil. A high frequency of resistance was found to ampicillin/sulbactam, cefazolin, cefotetan, ceftriaxone, nitrofurantoin, and trimethoprim/sulfamethoxazole (96.7%). Resistance to ticarcillin/clavulanic acid, ciprofloxacin and levofloxacin was less common (13.3%). There was no relationship between antibiotic resistance and serotype distribution of the isolates. The epidemic serotype of P. aeruginosa from the mink hemorrhagic pneumonia in Shandong province was type G, which was a clone of commonly found in this province. These findings reveal the genetic similarities and antimicrobial susceptibility profiles of P. aeruginosa from clinical cases of mink hemorrhagic pneumonia and will facilitate the prevention and control of the disease in Shandong province of China., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Hemorrhagic pneumonia can be a major cause of mortality in farmed mink in the fall. In its classic form, hemorrhagic pneumonia is caused by the bacterium Pseudomonas aeruginosa. In recent years, however, outbreaks of this type of pneumonia that are associated with hemolytic Escherichia coli have also occurred in farmed mink. The purpose of this study was to compare histological lesions of acute hemorrhagic pneumonia associated with both P. aeruginosa and E. coli in mink, including a description of tissue distribution of pathogens, in an attempt to differentiate between the 2 disease entities based on histopathology. The study included material submitted for diagnostic investigation to the National Veterinary Institute in Denmark from 2006 to 2009. Altogether, 19 cases of hemorrhagic pneumonia with a pure lung culture of P. aeruginosa and 18 cases of hemorrhagic pneumonia with a pure lung culture of E. coli were examined. Formalin-fixed paraffin-embedded lung tissue obtained from the mink was examined by histology and fluorescence in-situ hybridization (FISH). It was possible to detect a slight histological difference between hemorrhagic pneumonia caused by P. aeruginosa and by E. coli, as P. aeruginosa was most often found surrounding blood vessels and lining the alveoli, while E. coli showed a more diffuse distribution in the lung tissue. Furthermore, P. aeruginosa often elicited a very hemorrhagic response in the lung, while infection with E. coli was associated with a higher frequency of alveolar edema and mild lymphoid cuffing in the lungs.

Hemorrhagic pneumonia is an acute and fatal disease of farmed mink caused by Pseudomonas aeruginosa. The pathogenesis of this disease has not yet been resolved. Mink are the only animals known to be susceptible to acute, contagious, and fatal lung infections caused by P. aeruginosa. The purpose of this study was to investigate the correlation between dose-response and season of infection and to clarify whether Danish mink are carriers of P. aeruginosa on their nasal mucosa during the season for hemorrhagic pneumonia. To elucidate the pathogenesis of the disease, an infectious dose-response trial was carried out on adult mink and mink kits, both in the season for hemorrhagic pneumonia (November) as well as out of season (July). It proved difficult to infect mink via the intra-nasal route. Only 4 out of 60 infected mink developed clinical disease and were euthanized, all of them in November, illustrating that predisposing factors in the mink itself and not infectious dose might be crucial for disease development. We were able to culture P. aeruginosa from the nasal cavity of the clinically healthy experimental mink 8 d after inoculation. This indicated that the mink can carry P. aeruginosa on their nasal mucosa without developing the disease. It was not possible, however, to culture P. aeruginosa from the nasal cavity of clinically healthy mink obtained from farms in November, which indicates that the organism is not a normal part of the nasal mucosal flora of mink.

Hemorrhagic pneumonia in mink (Neovison vison) is caused by Pseudomonas aeruginosa and is an acute and fatal disease in farmed mink. Earlier work has demonstrated that some outbreaks of hemorrhagic pneumonia are caused by pathogenic strains while most outbreaks are caused by local strains. The objective of this study was to determine the genetic and geographical relationship among outbreaks of hemorrhagic pneumonia by pulsed-field gel electrophoresis typing of P. aeruginosa isolates. Furthermore, chosen isolates were typed by a commercial genotyping method based on single nucleotide polymorphisms (SNPs) and compared to a larger dataset of human and environmental origin. The bacterial isolates were obtained from diagnostic samples from 2002 to 2009 and contained 164 isolates from 95 outbreaks on 90 farms. Our results show that most outbreaks of hemorrhagic pneumonia in mink are caused by distinct strains of P. aeruginosa. We also identified related P. aeruginosa strains which, together with two prevalent but unrelated clones, caused one third of the outbreaks of hemorrhagic pneumonia supporting the sparse literature on this subject. None of the SNP typed strains were identified in a large dataset of human and environmental origin., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

Investigation of a total of 354 Ps. aeruginosa strains by "active" pyocine typing, using Govan & Gillies' indicator strains (21, 24) by which it may be possible to demonstrate the sources and routes of pseudomonas infection revealed that: (1) of the isolated pyocine-producing types 85.5% could be fitted into Govan & Gillies' classification, (2) 11.1% of all strains tested did not produce pyocine, (3) p3 was the only pyocine type isolated from an enzootic of fatal, haemorrhagic pneumonia in experimental minks debilitated by change of environment and inoculation with plasmacytosis (Aleutian disease), (4) types 1--5, 8, 10, 17, 29, and 37 could be isolated from the blue foxes and their farm environment, and (5) types 1--3, 5, 6, 9, 10, 15, 17, 28, 31, and 37 were identified in hospitalized dogs and their hospital environment. Pyocine type p1 was predominant in all groups except that of experimental minks and samples from the room in which they were kept. This type, plus type p3, made up more than half the typed strains of Ps. aeruginosa. The sources of infection comprised: (1) Carriers among the blue foxes and their winter watering troughs. During periods without frost the troughs were not cleaned, but were left containing more or less water contaminated with urine, faeces, hairs, and dust--a well-suited habitat for Ps. aeruginosa. (2) In the case of the experimental minks the infection may have been derived from a dog with pseudomoniasis in the hospital, brought to the experimental room in the course of feeding and cleaning. (3) The infection in the Small Animal Clinic seems to be maintained by frequent admission of patients for treatment, especially of chronic otitis externa/media caused by widely resistant strains of Ps. aeruginosa.

We have characterized 20 Pseudomonas aeruginosa strains isolated from pneumonic mink lungs with regard to elastase production and serotype. P. aeruginosa PAO1, a well-characterized elastase-producing strain, and two elastase-deficient mutants of PAO1 were used for comparative purposes. Elastase activity was assayed on elastin agar and by using 14C-elastin coated microtiter plates. Elastase antigen was measured using a double antibody sandwich ELISA (enzyme-linked immunosorbent assay). Total proteolytic activity was determined on skim milk agar plates. The results from ELISA showed that all strains produced antigenically similar elastase, although the amounts produced varied considerably between strains. There was a good correlation regarding elastase assays between ELISA and 14C-elastin, elastin agar and total proteolytic activity on skim milk agar. No correlation was found between serotype and elastase activity. The results showed that the 14C-elastin assay is a simple and sensitive method of determining elastolytic activity of P. aeruginosa strains.