The observation that there was no difference among treatments for the measurements recorded over time (incidences of positive CIEP, PCR and IAT, the results of qELISA, total protein measured by a refractometer and IAT scores) and the traits recorded at the termination of the study (the proportion of spleen, lymph node and bone marrow samples harboring viral DNA, antibody titer, total serum protein, albumin, globulins and albumin/globulin ratio) suggest that kelp supplementation had no effect on viral replication, viral sequestration or immune response of mink to AMDV infection. To our knowledge, this is the first study on the effect of seaweed supplementation on a non-enveloped DNA virus, and the results contradict the antiviral effects of seaweed supplementation on enveloped viruses in vitro [11,12,13,14,15,16] and in vivo [7, 17, 18].
The most striking finding of the current study was the significant decrease of creatine and BUN in the mink supplemented with 1.5% kelp compared with the control group, implying that kelp supplementation significantly improved kidney function. The amounts of BUN in mink supplemented with both levels of kelp in the current study were higher than the estimates for healthy brown (16.2 mg dl-1, 5.79 mmol L-1) [22] and healthy dark mink (4.24 mmol L-1) [23], and agrees with a previous report that AMDV-infection significantly elevated its level [24]. Similarly, the amount of BUN in Royal pastel mink which did not show AD symptoms after inoculation with the low pathogenic Pullman strain and sampled from 8 to 126 dpi (12 to 37 mg dl-1, 4.3 to 13.2 mmol L-1) were close to the estimates in the current experiment, but six mink which showed AD symptoms in that study had greater values (23 to 144 mg dl-1, 8.2 to 51.4 mmol L-1) [25]. The current and the above reports clearly suggest that AMDV infection elevates the level of BUN, which is likely the result of trapped immune complexes in the glomeruli [26], which subsequently causes interstitial nephritis and renal dysfunction [1]. The kidneys also showed the greatest severity of AD lesions among organs of naturally infected [27] and experimentally inoculated mink [28]. The enhancement of health, reproduction and survival of the same groups of mink supplemented with 1.5% kelp [29] could have been the result of improved kidney function. The only published report on the effects of AMDV infection on liver function showed that infection did not have a significant effect on ALKP activity, whereas some other measures of liver damage (thymol turbidity, activities of glutamic oxaloacetic transaminase, glutamic pyruvic transaminase) significantly increased [24]. There is not enough information to assess the effects of AMDV infection on liver function with any degree of certainty, and provide a reason for the absence of any effect of kelp supplementation on the markers of liver health in the current study (ALKP and GGT activities).
Reports on the effects of dietary seaweed supplementation on the kidney and liver functions of different animal species are rare. Supplementation of a high-salt diet with a seaweed extract fed to salt-sensitive rats for 7 weeks decreased kidney damage indicated by decreased urinary protein excretion, increased creatinine clearance rate, reduced hypertensive glomerular sclerosis and decreased arterial injury in the kidney [30]. Feeding moderate amounts of a brown seaweed (Sargassum polycystum) extract significantly reduced the percentages of necrotic and degenerative liver and kidney cells in experimentally induced diabetic rats, which were attributed to antioxidant pigments or sulphated polysaccharides in the seaweed [31]. Replacing 1.0 and 3.0% of corn with green seaweed (Ulva lactuca) meal in a broiler diet in a 21-day study did not have any effect on total serum protein, albumin, globulins, or markers of liver function (AST, CK and GGT), but both seaweed supplements significantly reduced levels of ALT and uric acid compared with the control group [32]. On the contrary, dietary supplementation of ram lambs for 74 days with a green seaweed (Ulva lactuca) meal at 3 and 5% of dry matter did not have any effect on the levels of some blood parameters, including total protein, albumin, globulins, AST and ALT, and it was concluded that seaweed had no effect on liver or kidney functions [33]. The same green seaweed meal supplementation at 1 and 2% of dry matter did not have any effect on markers of kidney function (BUN, creatinine, total plasma protein) or liver enzymes (ALKP, AST, ALT) after 56 days of feeding male rabbits and 38 days of feeding pregnant female rabbits [34]. Dietary supplementation of layer hens with 0.5, 1.0% or 2.0% of two species of cultivated red seaweed did not have any effect on the concentration of some blood parameters, including total protein, CK, AST and uric acid during a 30-day feeding trial [35]. The above reports indicate that the effects of seaweed supplementation on kidney and liver functions in unchallenged animals of different species are inconclusive, and the positive effects of kelp on kidney function in the current study was likely because of the role that kidneys play in AMDV pathogenesis in an unknown manner, and deserves further investigation.
The current selection strategy for the establishment of AMDV-tolerant mink herds is based on low antibody titer [36] or low serum gamma globulin [37]. The genetic and biological causes of differences amongst AMDV-infected individuals for antibody titer and serum gamma globulin levels are not clearly understood. If low antibody titer and low gamma globulin level are the result of slow immune response of mink to infection, then long-term selection for low levels of these parameters may weaken the immune system of the mink with some unexpected consequences. If so, it is tempting to hypothesize that improving kidney function, using BUN or creatine levels, could be an alternative approach in selecting tolerant mink.
In agreement with previous reports [28, 38,39,40,41], AMDV DNA was detected in the blood by PCR in advance of seroconversion by CIEP, and viremia was short-lived, revealed by the rapid decline in PCR positive cases after 99 dpi. The observation that high percentage of the spleen and lymph node samples harbored the virus, along with a high percentage of seropositive mink at 451 dpi, but much fewer viremic mink at this time, agrees with the previous reports [27, 40,41,42,43,44] and implies that testing blood samples by PCR is not reliable for detecting AMDV infection in chronically infected mink herds. Smaller numbers of PCR positive bone marrow than spleen and lymph node samples agree with a previous report [38] and could have been caused by the method of sampling this tissue.
The observation that antibody production persisted throughout the experiment, even after the termination of viremia, agrees with previous reports for experimentally inoculated mink [25, 28, 38, 40, 41, 45]. Five mink (9.4%) in the current study remained CIEP negative for 451 days, which is a condition observed previously for experimentally inoculated [41] and chronically infected mink [27, 37, 46, 47]. AMDV DNA was detected in the blood and organs of one of these mink (1.9%), indicating that this individual was chronically infected but had low antibody titer throughout its life which was not detectable by CIEP, a finding which agrees with previous reports [27, 37, 42, 48]. The other four seronegative mink were viremic during the early period of the experiment but virus was not detected in their blood at the later dates or in their blood or organs at the termination of the experiment, implying that these animals likely cleared the virus, which was also observed previously [42, 49, 50]. These findings demonstrated that the long-term response of mink to AMDV infection with respect to virus replication and antibody production is complex and combining them in two categories (low and high antibody titers) may decrease the effectiveness of genetic selection for tolerance.
Although IAT has been used for detecting AMDV-infected mink for many years [51], it is not a specific test for AMDV infection because the serum gamma globulin level elevates in response to infection by all pathogens [52]. The incidences of IAT-positive mink in the present study were rather high for all treatments throughout the study (73.7 to 100%), and in agreement with previous studies, fluctuated over time [37, 50, 53]. The fluctuations could be, at least partly, the result of reduced bacterial contamination in feed and the environment during the cold seasons [37, 50]. In the current study, the decline in the incidence of IAT-positive cases from 31 to 56 dpi, which corresponded to October to November, and again from 366 to 451 dpi, corresponded to September to December (Fig. 3), could support this assumption.
Changes in antibody titer measured by qELISA, gamma globulin level measured by IAT score and total serum protein measured by a refractometer were parallel, i.e., increased until 56 or 99 dpi and then declined. The decline in antibody titer and serum gamma globulin level following initial increases were previously observed when animals were monitored for a long time [45, 54]. The declining trends of these parameters could have been the result of reduced viral replication [1] or death of animals with high levels of gamma globulin [25, 49, 55, 56] or high antibody titers [28, 45, 56].
The parallel changes in qELISA, IAT score and serum protein over time were manifested in the positive correlation coefficients among these measurements at different sampling occasions (Table 6). The greatest correlation coefficients were between serum protein measured by a refractometer and IAT scores (0.53 to 0.79) and the lowest were between serum protein and qELISA (0.36 to 0.48), possibly because gamma globulin constitutes a higher proportion of total serum protein than anti-AMDV antibodies. This assumption is supported by the greater correlation coefficients between total serum protein and globulin level (0.82 and 0.88) than between total serum protein and antibody titer (0.48 and 0.51) on 451 dpi (Table 9). The steady decline in the magnitude of correlation coefficients between IAT scores and qELISA over time (0.61 to 0.28) could be caused by the non-linear association between globulins and antibody titer [57], which is the result of declined antibody titer over time.
The positive and rather large correlation coefficients between IAT score and serum globulins (0.80), total serum protein (0.75) and antibody titer (0.63) on 451 dpi (Table 9) point to the efficacy of this low-cost on-farm method for assessing serum globulin level and the state of animal health. The Spearman’s rank correlation coefficients between serum protein levels measured by a refractometer with globulins and serum protein measured by the Chemistry Analyzer (0.82 and 0.85) on 451 dpi were slightly greater than those for IAT scores, but its correlation coefficient with antibody titer was smaller (0.48), suggesting that the refractometer may be less accurate than IAT for assessing animal health. The superiority of IAT over the refractometer was also shown by the higher Spearman’s rank correlation coefficients between successive measures of IAT than serum protein measurements (Table 4).
The greatest Spearman’s rank correlation coefficients between successive measures were for qELISA results compared with IAT and serum protein measured by a refractometer (Tables 4 and 5), suggesting that antibody titer was more stable over time compared with total serum protein or gamma globulin levels. This is contradictory to a previous report that total serum protein was less variable over time compared with antibody titer and gamma globulin level [26]. Means of qELISA readings prior to inoculation (day 0) were numerically larger than those on 366 dpi for all treatments, which could be caused by the lack of adjustment for the background noise in different batches of samples tested by this private laboratory, signifying that this method requires some refinement as previously suggested [58]. Changes in antibody titer over time have previously been reported [26, 45, 54]. In another experiment, antibody titer was stable within 15 days but not when measured in October and the next February [59]. The declining trends in the magnitudes of qELISA, IAT and total serum protein over time, and intermediate correlation coefficients between successive measures of each parameter, suggest that a single measure is not an accurate determinant of the long-term level of these measurements, a point which needs to be taken into consideration when evaluating animals for tolerance to AMDV infection.
The mean and range of albumin in the current study were comparable to the previous estimates for AMDV-infected black mink [57] and slightly lower than the mean of healthy brown female mink (30 g L-1) [22]. Elevated levels of antibody titer and gamma globulin in AMDV-infected mink are associated with a decrease in albumin [24, 60, 61], through reduced albumin synthesis by the liver in order to regulate blood osmotic pressure [62, 63]. The observation that the correlation coefficient between albumin and globulins was negative and significant whereas there was no correlation between albumin and total serum protein, was the manifestation of the homeostatic effect of albumin. It may be concluded that the reduction in the level of albumin is an indication of the severity of hypergammaglobulinemia, regardless of the source of infection, and thus has low diagnostic specificity [63]. The lack of a correlation between albumin and antibody titer in the current and a previous study [57] confirms that albumin level is not an accurate diagnostic tool for AMDV infection.
The level of globulins in the present study was the non-albumin segment of the serum protein, of which gamma globulin is the largest fraction in AMDV-infected mink [60]. The level of globulins at 451 dpi was within the range of the previous estimates for AMDV-infected black mink [57], and also fell within the range of suggested values reported for gamma globulin for AMDV-infected mink, namely between 15 and 50% (30 mg/mL) [1, 61] or between 20 and 63% [55]. Globulin level had a moderate correlation coefficient with antibody titer in the current study (0.57), which was smaller than 0.81 [56] and 0.75 [54] previously reported for the correlation between antibody titer and gamma globulin level. The mean and range of A/G ratio in the current study were close to the previous estimates for AMDV-infected mink [24, 57]. The association between A/G and antibody titer in the current and a previous study [57] was negative and moderate, suggesting that A/G does not have a stronger prognostic advantage than globulin level or antibody titer.
Positive and significant correlation coefficients between the markers of kidney function (BUN and creatine, r = 0.85) and between markers of liver function (ALKP and GGT, r = 0.33) indicate that the two independent tests for each organ were confirmatory and the results were thus reliable. The levels of BUN, creatine, ALKP and GGT were not associated with any other blood parameters, antibody titer, total serum protein measured by a refractometer or IAT score, implying that the effects of AMDV infection on kidney and liver functions were independent of the immune response of mink to infection. The correlations between ALKP with albumin, BUN with A/G, creatine with total serum protein, globulins and A/G tended to be significant (P < 0.10) but difficult to interpret.