Molecular assessment of visitor personal protective equipment contamination with the Aleutian mink disease virus and porcine circovirus-2 in mink and porcine farms.

Personal protective equipment (PPE) is an element of biosecurity intended to prevent the access or spread of diseases in farms. Nevertheless, to date no extensive reports exist about the effectiveness of different available PPE on farms. Thus, our aim was to estimate the degree of protection of PPE from viral contamination during farm visits. Two farms, infected with Aleutian mink disease virus and porcine circovirus-2 respectively, were visited by six visitors wearing different combinations of PPE: coveralls with hood and bootcovers, both with a certified barrier to infective agents (certified PPE group) and non-certified bootcover and coverall without hood (non-certified PPE group). Seventy-two swab samples from PPE and both hair and street clothes under PPE were taken after the visit and analysed by qPCR. Our results reveal viral exposure during visits, and the external protections of body and shoes were contaminated in all cases (24/24). In addition, protection from viral contamination varied noticeably according to the biosecurity elements used. A higher number of positives were detected in the non-certified PPE group than in the certified PPE group, both in elements under external protections (14/18 vs 3/18) and also in hair (4/6 vs 0/6). In fact, non-certified bootcovers broke during visits, resulting in viral contamination of the internal elements under them; these are consequently not suitable for using with wrinkled surfaces usually found in farm facilities. Thus, certified coveralls should be used in order to prevent contaminations, and workers and personnel of farms should be trained in their proper use. qPCR is a useful tool in the risk management of biosecurity programmes, and our results may serve as a model to evaluate different biosecurity measures.

Lesser housefly (Fannia canicularis) as possible mechanical vector for Aleutian mink disease virus.

Flies are known vectors for a variety of infectious diseases in animals. In fur mink farming, one of the most severe diseases is Aleutian disease, which is caused by the Aleutian mink disease virus (AMDV). The presence of large fly populations is a frequent issue in mink farms; however, no studies assessing their role as AMDV carrier vectors have been conducted to-date. In order to determine the presence of AMDV in aerial flies from an infected mink farm, flies (n = 254) and environmental swab samples (n = 4) were collected from two farm barns. Fannia canicularis (L.) (Diptera: Muscidae) represented more than 99% of the fly population. One hundred and fifty specimens of this species were divided into pools of ten flies and analysed by qPCR, and positive samples were further sequenced. All fly pools and environmental samples tested positive for AMDV, and sequence analysis revealed identical genotypes in both types of samples. This is the first report of AMDV contamination in flies from mink farms, suggesting that F. canicularis may act as an AMDV vector. These results may be of interest for epidemiological studies and also for the improvement of control measures against this virus in mink farms.

Evolutionary analysis of whole-genome sequences confirms inter-farm transmission of Aleutian mink disease virus.

Aleutian mink disease virus (AMDV) is a frequently encountered pathogen associated with mink farming. Previous phylogenetic analyses of AMDV have been based on shorter and more conserved parts of the genome, e.g. the partial NS1 gene. Such fragments are suitable for detection but are less useful for elucidating transmission pathways while sequencing entire viral genomes provides additional informative sites and often results in better-resolved phylogenies. We explore how whole-genome sequencing can benefit investigations of AMDV transmission by reconstructing the relationships between AMDV field samples from a Danish outbreak. We show that whole-genome phylogenies are much better resolved than those based on the partial NS1 gene sequences extracted from the same alignment. Well-resolved phylogenies contain more information about the underlying transmission trees and are useful for understanding the spread of a pathogen. In the main case investigated here, the transmission path suggested by the tree structure was supported by epidemiological data. The use of molecular clock models further improved tree resolution and provided time estimates for the viral ancestors consistent with the proposed direction of spread. It was however impossible to infer transmission pathways from the partial NS1 gene tree, since all samples from the case farms branched out from a single internal node. A sliding window analysis showed that there were no shorter genomic regions providing the same phylogenetic resolution as the entire genome. Altogether, these results suggest that phylogenetic analyses based on whole-genome sequencing taking into account sampling dates and epidemiological data is a promising set of tools for clarifying AMDV transmission.

Aleutian mink disease virus in striped skunks (Mephitis mephitis): evidence for cross-species spillover.

Aleutian mink disease virus (AMDV) causes a parvovirus infection, initially characterized in American mink (Neovison vison), that may have harmful effects on wild populations of susceptible animals. In North America, where American mink are native, the origin, host range, and prevalence of AMDV in wild species is not clear. We studied striped skunks (Mephitis mephitis) and raccoons (Procyon lotor) to determine whether species sympatric with mink are potential reservoirs in the transmission of AMDV to wild mink and mink farms. Antibodies to AMDV were detected in 41% of skunk serum samples (143/347) and AMDV nucleic acids were detected in 32% (14/40) of skunk spleen samples by PCR, indicating that AMDV exposure and infection were frequent in skunks. We detected no AMDV antibodies in 144 raccoon blood samples. Phylogenetic analysis revealed a newly identified AMDV haplogroup consisting of isolates from Ontario skunks and a free-ranging domestic mink from Ontario. Our findings of frequent AMDV infection in skunks, close genetic similarity between skunk and mink AMDV isolates, and evidence of AMDV transmission from skunks to mink support the hypothesis that skunks may be acting as alternative hosts and reservoirs of AMDV to wild mink through cross-species virus spillover.

Inactivation of Aleutian mink disease virus through high temperature exposure in vitro and under field-based composting conditions.

Disposal of manure contaminated with Aleutian mink disease virus (AMDV) is a significant concern to the mink industry. Inactivation of AMDV under field conditions has received limited attention in the scientific literature. We evaluated the thermal inactivation of AMDV in vitro and during composting of mink manure. Spleen homogenate containing AMDV was heated under controlled conditions at 45°C, 55°C, and 65°C for 3 days. Results of the in vitro study identified complete absence of viral replication in mink at 65°C only. Next, manure-mixed AMDV packed in polyester pouches was inserted in different layers of three replicate mink manure compost piles. The virus was retrieved after the compost piles had undergone a heating period and subsequently returned to ambient temperatures. Temperature regimes in the compost piles were categorized as ≥65°C, ≥60-64°C, and ≥55-59°C. Initially, layer-wise composite virus samples were assayed for virus replication in mink. Twenty-one-day post-inoculation (p.i.) plasma tested for AMDV and antibodies indicated infection in 40%, 80%, and 100% of mink inoculated from samples originating from the top, center and bottom layers of the piles, respectively. Subsequently, the virus was extracted from individual pouches in compost layers achieving thermal activity ≥65°C and was tested in mink. No antibodies or virus was detected in plasma taken weekly up to day 21 p.i. PCR data of bone marrow and lymph nodes collected on day 21 p.i. also showed no AMDV. However, mink that received virus from positive control manure indicated infection in their plasma as early as 1 week p.i.

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.

Aleutian mink disease virus and humans.

Reports of a possible relationship between Aleutian mink disease parvovirus (AMDV) and human infection are rare. However, 2 mink farmers with vascular disease and microangiopathy similar to that in mink with Aleutian disease were found to have AMDV-specific antibodies and AMDV DNA. These findings raise the suspicion that AMDV may play a role in human disease.

Infection with Aleutian disease virus-like virus in a captive striped skunk.

CASE DESCRIPTION: A 5-month-old captive female striped skunk (Mephitis mephitis) was evaluated because of lethargy, signs of depression, azotemia, and erythema of the skin around the eyes. CLINICAL FINDINGS: Antemortem diagnostic tests revealed renal disease but failed to identify an etiologic agent. A diagnosis of severe nonsuppurative interstitial nephritis was made on the basis of results of histologic examination of renal biopsy specimens. TREATMENT AND OUTCOME: The skunk was administered isotonic fluids SC daily and later every other day because of the handling-related stress. Because of the skunk's deteriorating condition, it was euthanized after 24 days of supportive care. Aleutian disease was diagnosed on the basis of positive results of a PCR assay that targeted the DNA from Aleutian disease virus (ADV); positive results for ADV were also obtained by use of plasma counterimmunoelectrophoresis and an ELISA. Genetic sequencing of the 365-base pair PCR product revealed 90% sequence identity with mink ADV. CLINICAL RELEVANCE: In the skunk of this report, infection with a skunk-specific parvovirus resulted in clinical signs and pathologic changes similar to those associated with ADV infection in mink. For skunks with signs of renal failure, differential diagnoses should include parvovirus infection. In confirmed cases of infection with this ADV-like virus, appropriate quarantine and biosecurity measures should be in place to prevent spread to other susceptible animals within a zoological collection.

Detection of Aleutian disease antibodies in feral American mink in southern England.

Fourteen of 27 American mink (Mustela vison) trapped in the upper Thames region were positive for anti-Aleutian disease antibodies. This demonstration of the occurrence of this viral disease in a feral American mink population suggests that it could threaten populations of at least two protected mustelids, the otter (Lutra lutra) and the polecat (Mustela putorius), and may also play a role in the apparent decline of local mink populations in Britain.

Investigation of an outbreak of Aleutian disease on a commercial mink ranch.

OBJECTIVE: To determine the modes of transmission of Aleutian mink disease in a natural outbreak. ANIMALS: 5,580 black and 9,087 brown mink from a ranch with an outbreak of Aleutian mink disease. PROCEDURE: Each mink had serum tested by counter-electrophoresis for Aleutian disease antibody. If a mink was seropositive for Aleutian disease virus by counter-electrophoresis, it was considered to be infected. Correlation of prevalence of the disease in kits and parents was determined. Spatial arrangement of infected and un-infected mink also was studied. RESULTS: Infected black dams were more likely to produce infected kits than were uninfected dams. In contrast, infected black sires were less likely to produce infected kits than were uninfected sires. In brown mink, in which prevalence of Aleutian disease was lower, transmission from infected dams and sires to kits was apparent. Infected black mink appeared to be more efficient in transmitting the disease horizontally than were infected brown mink. Although the spatial arrangement of infected mink indicated that mechanical transmission of the disease may be the most efficient mode of horizontal transmission, airborne transmission also occurred. CONCLUSIONS AND CLINICAL RELEVANCE: Infected sires with nonprogressive Aleutian disease may confer protection to their kits in the face of a severe outbreak. Brown mink may be less able to transmit the virus horizontally than are black mink. Airborne transmission is substantial, but may not be as efficient as mechanical transmission.

Investigation of the pathogenesis of transplacental transmission of Aleutian mink disease parvovirus in experimentally infected mink.

The transplacental transmission of Aleutian mink disease parvovirus (ADV) was studied in experimental infection of 1-year-old female non-Aleutian mink. The ADV-seronegative female mink were inoculated with ADV prior to mating or after the expected implantation of the embryos during pregnancy. A group of uninfected females served as a control group. Animals from each group were killed prior to or shortly after parturition. The in situ hybridization technique with radiolabeled strand-specific RNA probes was used to determine target cells of virus infection and virus replication. In both infected groups, ADV crossed the endotheliochorial placental barrier, although animals infected before mating already had high antibody titers against ADV at the time of implantation. The percentage of dead and resorbed fetuses was much higher in dams infected before mating. In the placentae of these mink, virus DNA and viral mRNA were detected in cells in the mesenchymal stroma of the placental labyrinth and hematoma but only occasionally in the cytotrophoblast of the placental hematoma. Placentae of animals infected during pregnancy showed in addition very high levels of virus and also viral replication in a large number of cytotrophoblast cells in the placental hematoma, which exhibited distinct inclusion bodies. In both groups, neither virus nor virus replication could be detected in maternal endothelial cells or fetal syncytiotrophoblast of the placental labyrinth. Fetuses were positive for virus and viral replication at high levels in a wide range of tissues. Possible routes of transplacental transmission of ADV and the role of trophoblast cells as targets for viral replication are discussed.

The relationship between capsid protein (VP2) sequence and pathogenicity of Aleutian mink disease parvovirus (ADV): a possible role for raccoons in the transmission of ADV infections.

Aleutian mink disease parvovirus (ADV) DNA was identified by PCR in samples from mink and raccoons on commercial ranches during an outbreak of Aleutian disease (AD). Comparison of DNA sequences of the hypervariable portion of VP2, the major capsid protein of ADV, indicated that both mink and raccoons were infected by a new isolate of ADV, designated ADV-TR. Because the capsid proteins of other parvoviruses play a prominent role in the determination of viral pathogenicity and host range, we decided to examine the relationship between the capsid protein sequences and pathogenicity of ADV. Comparison of the ADV-TR hypervariable region sequence with sequences of other isolates of ADV revealed that ADV-TR was 94 to 100% related to the nonpathogenic type 1 ADV-G at both the DNA and amino acid levels but less than 90% related to other pathogenic ADVs like the type 2 ADV-Utah, the type 3 ADV-ZK8, or ADV-Pullman. This finding indicated that a virus with a type 1 hypervariable region could be pathogenic. To perform a more comprehensive analysis, the complete VP2 sequence of ADV-TR was obtained and compared with that of the 647-amino-acid VP2 of ADV-G and the corresponding VP2 sequences of the pathogenic ADV-Utah, ADV-Pullman, and ADV-ZK8. Although the hypervariable region amino acid sequence of ADV-TR was identical to that of ADV-G, there were 12 amino acid differences between ADV-G and ADV-TR. Each of these differences was at a position where other pathogenic isolates also differed from ADV-G. Thus, although ADV-TR had the hypervariable sequence of the nonpathogenic type 1 ADV-G, the remainder of the VP2 sequence resembled sequences of other pathogenic ADVs. Under experimental conditions, ADV-TR and ADV-Utah were highly pathogenic and induced typical AD in trios of both Aleutian and non-Aleutian mink, whereas ADV-Pullman was pathogenic only for Aleutian mink and ADV-G was noninfectious. Trios of raccoons experimentally inoculated with ADV-TR and ADV-Utah all became infected with ADV, but only a single ADV-Pullman-inoculated raccoon showed evidence of infection. Furthermore, none of the ADV isolates induced pathological findings of AD in raccoons. Finally, when a preparation of ADV-TR prepared from infected raccoon lymph nodes was inoculated into mink and raccoons, typical AD was induced in Aleutian and non-Aleutian mink, but raccoons failed to show serological or pathological evidence of infection. These results indicated that raccoons can become infected with ADV and may have a role in the transmission of virus to mink but that raccoon-to-raccoon transmission of ADV is unlikely.

Aleutian disease in domestic ferrets: diagnostic findings and survey results.

Aleutian disease was diagnosed as the cause of posterior ataxia and paresis in domestic ferrets. Six serologically positive animals (four clinically affected and two unaffected) were investigated in detail and seven other clinically affected ferrets were also identified. The diagnostic findings included hypergammaglobulinaemia, histological lesions in the central nervous system and parvovirus-like particles in mesenteric lymph nodes. A wider serological survey of 446 animals owned by members of a ferret club revealed an incidence of 8.5 per cent seropositive animals.

Experimental transmission of Aleutian disease virus (ADV) to different animal species.

Two animals from each of 8 different species (mink, Finn raccoon, cat, dog, ferret, blue fox, mouse and rabbit) were inoculated with the highly virulent Utah I strain of ADV. Only the mink developed hypergammaglobulinemia. All animals produced antibodies to ADV antigens, but with different titres. Mink sera had much higher antibody titres than the other animal sera. Antibodies to the ADV-coded, non-structural polypeptide (p71) were found in mink, Finn raccoons and dogs only. Presence of this kind of antibodies was taken as evidence of ADV replication, because p71 was not present in the ADV inoculum. The animals were killed 4 weeks after virus inoculation. Homogenates of different organs from mink, Finn raccoons, ferrets, dogs, mice and the cat were shown to infect ADV-negative mink, which developed antibodies to ADV following inoculation. We conclude from the present studies that mink and Finn raccoons are potential threats as ADV transmitters. Cats, ferrets, dogs and mice may be considered potential ADV reservoirs, because they possibly harbour ADV for 4 weeks or longer. Blue foxes and rabbits did not seem to be a risk factor for ADV transmission.

Aleutian disease in ferrets.

When 32 antibody-free ferrets were inoculated with the highly mink-virulent Utah-1 strain of Aleutian disease virus (ADV), most developed ADV antibody starting 15 days after infection, but the antibody titers were much lower than those seen in mink. Relatively small amounts of ADV were demonstrated in CRFK cell culture, using ferret spleen and lymph node homogenates only 4 to 10 days after experimental infection, but low-level viral persistence for 180 days was shown by mink inoculation. The ferrets inoculated with the Utah-1 strain of ADV did not develop elevated gamma globulin levels, but did have mild tissue lesions. Forty-two percent of a group of 214, approximately 1-year-old, recently pregnant, female ferrets were found to have antibody to ADV. An analysis of the serum proteins of the ferrets with ADV antibody showed that they had a significant, but mild, elevation of their serum gamma globulin. Serial ferret-to-ferret transmission of a ferret strain of ADV by inoculation of spleen homogenates was demonstrated, and some of these ferrets developed liver lesions. Mink inoculated with ferret ADV made antibody, but did not develop hypergammaglobulinemia or tissue lesions. Although both ferret and mink strains of ADV replicate and persist in the ferret, they fail to cause severe disease of the type usually seen in the closely related mink. Mink and ferret ADV strains appear to be biologically distinct.

Transmission of Aleutian disease from mink with inapparent infections.

In apparent or nonprogressive Aleutian disease virus infection was considered a subclinical but persistent viral infection in which infected mink did not develop tissue lesions, hypergammaglobulinemia, or high antibody titers. Transmission of Aleutian disease virus from mink with this type of infection was measured. Mink with inapparent Aleutian disease appeared healthy and had normal gamma-globulin values, but were capable of transmitting the disease by direct and indirect horizontal contact. The risk of direct or indirect horizontal transmission from mink with inapparent infection was less than from mink with progressive Aleutian disease. Infection also was directly transmitted from the dam to the kits, but again the risk of infection from dams with inapparent infection was less than from dams with progressive Aleutian disease. Mink infected from their dams before weaning developed the disease more slowly than mink which became infected after weaning.

The nature of aleutian disease in mink. I. Two forms of hypergammaglobulinemia as related to method of disease transmission and type of lesion.

Aleutian mink disease is generally considered to precipitate spontaneously in ranch mink, with a lethal outcome. A two-year field study of a herd of susceptible mutant mink (sapphires and violets), however, has shown that all individual mink were affected from birth; the well state consisted of periodic low-level hypergammaglobulinemia accompanied by minute vascular occlusions. The spontaneous lethal change in an individual appeared to arise during one of these hypergammaglobulinemic episodes and thus represented a failure of the immune system to control an inherent virus-induced mononucleosis. The fact that the entire herd was affected by the periodic form from birth is considered strong evidence for vertical transmission at a rate of 100%. The incidence of spontaneous precipitation was found to be dependent on the level of hypergammaglobulinemia in the mother during pregnancy.