In this study, we report a comparison of mink from three farms, one of which had been living with AMDV for decades and had mostly asymptomatic mink, one that had been infected for a couple of years and had mink with clinical AD, and one that was AMDV-free. The results show correlations and differences between antibody response, viremia, virus copy numbers, pathology, and transcriptomes between the tree farms. We also conducted a 2.5-year follow-up in farm conditions and observed a high proportion of mink that remained ELISA negative but PCR positive throughout the follow-up.
Due to the lack of vaccine or treatment, AMDV control relies greatly on diagnostics with serological tests like CIEP and ELISA. Interestingly, up to 43% of ELISA negative mink were PCR positive from blood in our follow up. A high proportion of antibody-negative and PCR positive mink has also been noted earlier by Farid et al. who found that 16.5% and 40.0% of CIEP negative mink were PCR positive from spleen (Farid, 2020; Farid and Ferns, 2017). In China, 12.5% of CIEP negative mink from 5 farms were PCR positive from spleen (Wang et al., 2014) and in Sweden, 4.5% of free-ranging mink were ELISA negative but PCR positive from spleen (Persson et al., 2015). As the sensitivity of ELISA has been shown to be comparable to that of CIEP (Andersson and Wallgren, 2013; Knuuttila et al., 2009), differences in the protocol are unlikely to fully explain the high proportion in our study. One possible explanation for the ELISA negative results of AMDV-infected individuals would be fresh infections, as detectable antibody response may form later than detectable viremia (Farid et al., 2015). However, it is most likely not the only explanation, as many of the mink remained ELISA negative throughout the follow-up. It is also likely that decades of breeding have led to the selection of low antibody producers, as was also suggested by Farid et al. (Farid and Ferns, 2017). It should be noted that ELISA negative results does not necessarily mean no AMDV antibodies at all, but it means that they were below the detection limit that was determined using a commonly accepted guideline. It has also been shown that in some cases (e.g. low inoculum doses), antibody titer may decrease over time (Farid and Hussain, 2020), but as these mink are constantly re-exposed by diverse strains circulating in the farms, that is not the most likely explanation for the low antibody titers in this study. Mink from farm 1 showed some fluctuation in antibody titers and also occasional high titers despite being asymptomatic, which is not unheard of, as asymptomatic non-Aleutian mink may also have at least transiently high antibody titers (Bloom et al., 1975).
Due to decades of breeding, it is difficult to say how common consistently seronegative but PCR positive mink are in other farms, but false negative ELISA/CIEP results would explain the unsuccessful attempts to fully eradicate AMDV from infected farms. Further studies should be conducted on ELISA/CIEP negative mink in other AMDV infected farms with different disease status (freshly infected naïve animal population vs long-term infected farm with established pathogen-host relationship) to determine the extent of this phenomenon, as false negative results in diagnostics greatly hinder the eradication attempts. These results and the results from previous studies (Farid and Ferns, 2017) also bring up the option to co-exist with the virus by breeding tolerant mink as eradication has proven to be difficult and no fully effective vaccine exists. However, virus may cause problems when entering the naïve non-tolerant mink population, which is supported by the fact that farm 1 reported usually temporarily having more symptomatic mink when new mink were introduced into the farm.
Previous studies have shown that AMDV can persist in tissues even when viremia in blood, feces, and mouth is transient (Farid and Hussain, 2020; Jensen et al., 2014). After the first sampling, we consistently detected virus in blood samples, which is somewhat contradictory to that finding, but may be explained by the constant re-exposure to the virus in this study as compared to experimental infections. AMDV was, on the other hand, only transiently detected in feces which indicated that mink did not frequently shed the virus in feces. Phylogenetic analysis from blood showed the virus strains frequently changed between samplings. Different sequences in different samplings can result either from virus evolution within the host (Canuti et al., 2016; Virtanen et al., 2019) or from clearance of one virus strain and infection with another. In this case, the reason is probably a combination of both. In many cases, virus strains locate in totally different branches, with tMRCA being up to 21 years in different samplings, which speaks more for virus clearance and reinfection by another strain. Sanger sequence raw data also frequently showed overlapping AMDV sequences, most likely resulting either from coinfection or within-host evolution. Another interesting observation was different virus strains in tissues and blood, but this result is not uncalled for as the same phenomenon has been detected with other viruses like HIV (Haggerty and Stevenson, 1991) and can result from either coinfection or within-host evolution. As the farm has been infected for an exceptionally long time, great variation in virus strains is not a surprise, considering AMDV has been shown to have an exceptionally high substitution rate, most likely due to the intense farming practices (Virtanen et al., 2019). Multiple introductions into the farm are another possibility.
After years of selecting low antibody producers for breeding, farm 1 appeared to have been able to breed an AMDV-tolerant herd and their mink had litter sizes and pelt quality comparable to the average in Finland. Mink often produced low number of antibodies and had lower copies of virus in their tissues as compared to the mink from farm 2. In histopathological evaluation, mink from farm 1 had mild lesions similar to the lesions seen in AMDV infections in kidneys, spleen and liver, indicating that virus may have caused some tissue damage despite the lack of visible symptoms. Also, one of the mink that remained healthy most of the follow-up developed severe AD with typical histopathological lesions by the last sampling despite the fact that the farmers reported rarely having mink with clinical AD. The virus strain of that mink was the same as in some of the asymptomatic mink so the change of virus strain to a more pathogenic one is not the most likely explanation for the sudden onset of symptoms. More likely, this might be connected to the fact that mink were kept alive longer than they normally would have been. Possibly the tolerant mink were not completely unaffected by the virus but had a very slowly progressing form of the disease and some of the other mink in the follow up may have also developed AD if the follow-up had been continued. Kidneys have a good reserve capacity, and clinical signs of kidney failure may not be detected until kidney function declines to 25% or less (Cianciolo and Mohr, 2015).
With regard to other differences between color types, some of the previous studies have detected more antibodies in Aleutian type mink (Porter et al., 1984). We did not detect higher ELISA absorbances in sapphire mink and, on the contrary, white mink had the highest mean ELISA values in ¾ samplings even though the difference was statistically significant only in one sampling. Breeding might play a role in low numbers of antibodies, even in sapphire mink. Also, we did not observe differences in AMDV genome copy numbers in asymptomatic, AMDV-positive mink of different color types. However, our results are influenced by the fact that we focused on mainly asymptomatic mink (excluding one sick mink in the last sampling) and the results might be different in a naïve mink population that has not been previously exposed to the virus. The only difference detected between color types was that sapphire mink, which are considered more susceptible to the disease, had larger spleens in relation to their body weight. This is logical considering that AMDV infection is known to cause enlarged spleen both in farmed and free-ranging mink (Eklund et al., 1968; Zalewski et al., 2021), but a better comparison would require the inclusion of uninfected controls to take natural differences between color types into account. Another logical observation was that spleen size correlated positively with ELISA absorbances. ELISA absorbance also showed positive correlation with AMDV genome copy number in spleen (as copies/ng of DNA) and possible but not statistically significant correlation with copy number in kidneys. This is similar to the earlier findings that genome copy number in blood was greater in the farm with a clinical course of infection as compared to the farm with subclinical infection (Kowalczyk et al., 2018). One mink with the clinical form of AD from farm 1 had clearly higher copy numbers in both tissues as compared to clinically healthy mink. Mean copy number in kidney was greater than mean copy number in spleen in the farm with clinically sick mink, but the small sample size prevents any strong conclusions.
Transcriptome analysis revealed several up- or downregulated genes in infected mink as compared to non-infected mink and symptomatic mink as compared to non-symptomatic mink. These genes were involved not only directly in immune response but also other cellular processes. Many of the genes that were highly upregulated in symptomatic mink as compared to asymptomatic mink were related to innate immunity, which may partly be explained by the fact that mink from farm 1 had been infected for a long time but mink from farm 2 may have had an acute infection. The most significantly upregulated gene in mink from farm 2 (compared to farm 1) was ONECUT2 (log2FC=7.21), which activates the transcription of several liver genes. Interestingly, Karimi et al. detected several genes involved in liver development to be strongly selected between groups of different disease severity (Karimi et al., 2021). Bloom et al (Bloom et al., 1994), on the other hand, suggested a predominance of Th1 response (macrophage activation) over Th2 response (B cell activation and antibody production) in mink lacking the progressive disease. In addition to some proinflammatory genes, several genes involved in suppression of inflammatory response and activation of Th1 response were upregulated in farm 2. Also, considering the fact that farm 2 reported that the number of symptomatic mink was slowly decreasing, it appears that mink from farm 2 were also starting to tolerate the virus by suppressing inflammation and antibody production. It should be noted that some differences may have been caused by other factors than AMDV, including age, other infections, and possible differences in environmental conditions, but to minimize their effect, mink of same sex and color type were chosen, and the samples were collected at the same time of the year. These results give information about gene-level differences in mink with different AMDV status and help understand the mechanisms behind varying symptoms and immune response. Further studies, e.g., genome-wide association analysis, are needed to better understand the roles of environmental and genetic factors in AD severity.
One limitation to this study is the small sample size and the small number of farms. However, all the mink represented the same gender, excluding one male mink from farm 2. Limited sample size should also be taken into account when interpreting statistical tests. Especially with the non-significant correlations, results might have been different with a bigger sample size and non-significant differences should also be considered when planning further studies. Causes of death for the mink that died during the follow-up were also not reported, so it is not known if some of them died of AD. However, it is expected that some breeding females are lost during a long follow-up like this one for example due to nursing sickness. Even though decades of breeding and living with the virus is the most likely explanation for the different numbers of symptomatic mink in the farms, the effect of virus strain cannot be excluded either, as the two infected farms had different virus strains. More thorough analysis of virus sequences or complete genome sequencing was not conducted because frequent co-infections in farm 1 would affect the reliability of the sequencing results. However, the very diverse virus strains in farm 1 indicate that the dominance of the host effect on different disease severity in farms.