Anne Sofie Hammer

First introduction of highly pathogenic H5N1 avian influenza A viruses in wild and domestic birds in Denmark, Northern Europe

BackgroundSince 2005 highly pathogenic (HP) avian influenza A H5N1 viruses have spread from Asia to Africa and Europe infecting poultry, humans and wild birds. HP H5N1 virus was isolated in Denmark for the first time in March 2006. A total of 44 wild birds were found positive for the HP H5N1 infection. In addition, one case was reported in a backyard poultry flock.ResultsFull-genome characterisation of nine isolates revealed that the Danish H5N1 viruses were highly similar to German H5N1 isolates in all genes from the same time period. The haemagglutinin gene grouped phylogenetically in H5 clade 2 subclade 2 and closest relatives besides the German isolates were isolates from Croatia in 2005, Nigeria and Niger in 2006 and isolates from Astrakhan in Russia 2006. The German and Danish isolates shared unique substitutions in the NA, PB1 and NS2 proteins.ConclusionThe first case of HP H5N1 infection of wild and domestic birds in Denmark was experienced in March 2006. This is the first full genome characterisation of HP H5N1 avian influenza A virus in the Nordic countries. The Danish viruses from this time period have their origin from the wild bird strains from Qinghai in 2005. These viruses may have been introduced to the Northern Europe through unusual migration due to the cold weather in Eastern Europe at that time.

Identification and characterization of deer astroviruses

The threat of emerging infectious viruses in humans requires a more effective approach regarding virus surveillance. A thorough understanding of virus diversity in wildlife provides epidemiological baseline information about pathogens and may lead to the identification of newly emerging pathogens in the future. In this study, diarrhoea samples from an outbreak of gastrointestinal illness in a Danish population of European roe deer were gathered for which no aetiological agent could be identified. Large-scale molecular RNA virus screening, based on host nucleic acid depletion, sequence-independent amplification and sequencing of partially purified viral RNA, revealed the presence of novel astroviruses, CcAstV-1 and CcAstV-2, in two of ten diarrhoea samples. Whether these viruses were responsible for causing diarrhoea remains to be determined. Phylogenetic analyses on amplified sequences showed that these viruses were most closely related to each other, were a novel species in the genus Mamastrovirus and may represent two different serotypes.

Serovars of Salmonella from captive reptiles

The distribution on serovars of 60 Salmonella isolates from reptiles kept in captivity in Denmark during the period 1995–2006 was investigated. The isolates were all recovered from clinical specimens submitted to the National Veterinary Institute. A majority of the samples were from reptiles in zoological gardens or similar, while a minor number was from reptiles kept in private homes. A total of 43 serovars were detected, most of them being what is usually called exotic serotypes, and many not having a trivial name, while a few isolates belonged to well‐known human pathogenic serovars, such as S. Enteritidis, S. Typhimurium, S. Bovismorbificans. One isolate was rough and two were non‐typeable. Isolates from turtles belonged to the subspecies enterica, while many isolates from both sauria and snakes belonged to other subspecies. The findings underline the potential zoonotic risk by handling reptiles in zoological garden or other public settings, or keeping pet reptiles in private homes.

Implementation and validation of a sensitive PCR detection method in the eradication campaign against Aleutian mink disease virus

Aleutian mink disease virus (AMDV) is a severe progressive disease causing multiple different clinical syndromes in mink. In Denmark, the disease is notifiable and under official control. The control programme, based on serological screening, has confined successfully AMDV to the northern part of Denmark. However, re-infections and new introductions of virus into farms require a confirmatory virological test to verify the positive test results of single animals and ultimately to investigate disease transmission. A one step PCR amplifying a 374-base fragment of the NS1 gene of AMDV was compared to the counter-current immune electrophoresis (CIE) routinely used in the serological screening programme. Mink organs (n=299) obtained from 55 recently infected farms and 8 non-infected farms from 2008 to 2010 were tested by PCR, and the results were found to have a high correlation with the serological status of the mink. The relative diagnostic sensitivity of the PCR was 94.7%, and the relative diagnostic specificity was 97.9% when read in parallel with the CIE. PCR positive samples were sequenced and phylogenetic analysis revealed high similarity within the analysed AMDV strains and to AMDV strains described previously.

High-throughput Immunophenotyping of 43 Ferret Lymphomas Using Tissue Microarray Technology

To validate the use of the tissue microarray (TMA) method for immunophenotyping of ferret lymphomas, a TMA was constructed containing duplicate 1-mm cores sampled from 112 paraffinembedded lymphoma tissue specimens obtained from 43 ferret lymphoma cases. Immunohistochemical (IHC) expression of CD3, CD79α, and Ki-67 (MIB-1) was determined by TMA and whole mount (WM) staining of each individual case for result comparison. There was a high correlation between CD79α and CD3 results comparing ferret TMA and WM sections (kappa statistic 0.71–0.73 for single-core TMA and 0.79–0.95 for duplicate-core TMA) and between continuous data from Ki-67 staining of ferret TMA sections and WM sections (concordance correlation coefficients 0.77 for single cores and 0.87 for duplicate cores). Subsequently, a panel of commercially available antibodies was applied to the TMA for the analysis of expression in ferret lymphomas. The results of this study confirmed previously published results suggesting specific cross-reactivity of the applied IHC markers (CD3, CD79α, Ki67) with ferret lymphoma tissue. Other IHC markers (CD45Ro, bcl2, bcl10, MUM1, CD30, vimentin) were also expressed in subsets of the included ferret lymphomas. Further studies are necessary to determine the usefulness of these markers for diagnostic and prognostic evaluation of ferret lymphomas. In conclusion, the TMA technology was useful for rapid and accurate analysis of protein expression in large archival cohorts of ferret lymphoma cases.

Usage of antimicrobials and occurrence of antimicrobial resistance among bacteria from mink

The usage of antimicrobials for treatment of mink and the occurrence of antimicrobial resistance among the most important bacterial pathogens in mink was investigated. The aim of the study was to provide data, which may serve as a basis for the formulation of recommendations for prudent use of antimicrobials for mink. A total of 164 haemolytic staphylococci, 49 haemolytic streptococci, 39 Pseudomonas aeruginosa, 13 Pasteurella multocida, and 1093 Escherichia coli isolates from Danish mink were included in the study. A high frequency of resistance among S. intermedius was found for tetracyclines (54.7%), followed by penicillin (21.7%), lincosamides (20.4%), macrolides (19.1%), and spectinomycin (18.5%). Very low frequencies of resistance were recorded for other antimicrobials. The highest frequency among the E. coli isolates was recorded for ampicillin, streptomycin, sulphonamides, and tetracyclines, whereas resistance to other antimicrobials was rare. All P. aeruginosa were sensitive to gentamicin and colistin and sensitive or intermediate to enrofloxacin, whereas most isolates were resistant to all other antimicrobials. All P. multocida and haemolytic streptococci were sensitive to penicillin. There was a steady increase in the use of antimicrobials during the period 2001-2006, the majority of the prescribed amount being extended spectrum penicillins followed by aminoglycosides, sulphonamides with trimethoprim, and macrolides.

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.

High prevalence of Aleutian mink disease virus in free-ranging mink on a remote Danish island.

Aleutian mink disease virus (AMDV) causes severe disease in farmed mink (Neovison vison) worldwide. In Denmark, AMDV in farmed mink has been confined to the northern part of the mainland since 2002. From 1998 to 2009, samples from 396 free-ranging mink were collected from mainland Denmark, and a low AMDV antibody prevalence (3% of 296) was found using countercurrent immune electrophoresis. However, on the island of Bornholm in the Baltic Sea, a high prevalence (45% of 142 mink) was detected in the free-ranging mink. Aleutian mink disease virus was detected by polymerase chain reaction in 32 of 49 antibody-positive free-ranging mink on Bornholm, but not in mink collected from other parts of Denmark. Sequence analysis of 370 base pairs of the nonstructural gene of the AMDV of 17 samples revealed two clusters with closest similarity to Swedish AMDV strains.

Monitoring chronic infection with a field strain of Aleutian mink disease virus.

Aleutian mink disease virus (AMDV) readily spread within farmed mink and causes chronic infections with significant impacts for welfare and economy. In the present study a currently circulating Danish AMDV strain was used to induce chronic experimental infection of farmed mink. PCR was used to detect viral DNA in full blood, organs, faeces and oro-nasal swabs weekly for the first 8 weeks and then biweekly for another 16 weeks after AMDV challenge inoculation of wild type mink. The mink (n=29) was infected and seroconverted 2-3 weeks after AMDV inoculation and AMDV antibodies persisted during the maximum experimental period of 24 weeks. Viraemia and faecal excretion of viral DNA was detected in the mink (n=29) at various and intermittent time intervals. Excretion of viral DNA in oro-nasal swabs was detected for 1-8 weeks in 21 mink. This highlights the risk of transmitting AMDV between infected farms. PCR was successfully used to detect viral DNA in organs 8, 16 and 24 weeks after AMDV inoculation with only minor differences between these weeks which is of diagnostic interest. This AMDV challenge model was also used to mimic natural infection of susceptible sapphire mink. Four of 6 sapphire mink were infected indirectly via the AMDV inoculated wild type mink whereas the other 2 sapphire mink remained uninfected.

Typing of Pseudomonas aeruginosa from hemorrhagic pneumonia in mink (Neovison vison)

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.