Clinico-pathological investigation of serum proteins in odontocetes

An initial assessment in stranded marine mammals, including evaluation of clinico-pathological variables, is a preliminary and critical step to define treatment and assessing the suitability of the animals for rehabilitation. Serum protein electrophoresis (SPE) is the most reliable method to determine the distribution of serum protein fractions and it is considered an essential step to evaluate the health status of animals, providing clinical useful information. The measurement of APPs in association to serum proteins’ fractions can supplement and extend the baseline information obtained from the complete blood cell count, fibrinogen, and standard serum chemistry panel. The thesis is divided in two main chapters, the first one focused on the serum protein electrophoresis (SPE) and the second one on the acute phase proteins evaluation In the first chapter, 38 under human care bottlenose dolphin serum samples were screened with agarose gel electrophoresis (AGE) to determine the reference intervals (RIs) of the serum proteins. Four main protein fractions were evident in all the animals tested: albumin, α-globulins, β-globulins, and γ-globulins. The RIs for the serum protein fractions were: albumin 45.0 ± 4.0 g/L, α-globulins 8.0 ± 1.0 g/L, β-globulins 5.0 ± 2.0 g/L, and γ-globulins 7.0 ± 2.0 g/L. Compared to previously published data in free ranging bottlenose dolphins, in our samples the concentration of total protein, α-globulins, and γ-globulins were slightly lower, while the concentration of albumin and the albumin/globulins ratio were slightly higher. The lower concentration of ‘’inflammatory’’ proteins associated to a higher concentration of albumin and the consequent higher albumin/globulins ratio reported in our study could reflect a lower antigenic stimuli in the animals housed in aquaria compared to the free-ranging populations. Moreover, in 8 electropherograms, we noticed that the base of the albumin peak was wider compared to the electropherograms of the other animals. For this reason, the same serum samples used for AGE were evaluated also with capillary zone electrophoresis (CZE), a more sensitive electrophoretic method. With CZE 9 out of 38 samples showed a double albumin peak. However, all these samples except 2 had an albumin peak wider than that observed with AGE in dolphins classified as non bisalbuminemic by CZE; furthermore a wider albumin peak was also noted with AGE in one sample with normal CZE profile. We report for the first time the presence of hereditary bisalbuminemia in two groups of related bottlenose dolphins identified by means of CZE and we confirm that AGE could fail in the identification of this alteration. To understand the genetic basis of bisalbuminemia, the albumin gene of 15 bottlenose dolphins belonging to two distinct families were reconstructed by direct comparison of its full length cDNAs with the provisional sequence of bottlenose dolphin albumin gene. Eighteen albumin gene variations were identified in the bottlenose dolphins studied (15 non-synonimous and 3 synonymous). In order to identify the non-synonimous variations able to cause bisalbuminemia, the genotype-phenotype correlations within the two families were studied. Two heterozygous non-synonymous variations that co-segregate with the ‘’bisalbuminemia’’ phenotype detected by SPE were identified: c.483C>G p.Phe146Leu in exon 4 and c.487T>C p.Tyr163His in exon 5. The genetic analysis of bottlenose dolphins’ albumin gene showed a significant polymorphism and two mutations associated with bisalbuminemia. Moreover, we were able to identify the autosomal codominant trait of this condition in dolphins, a similar pattern of inheritance to that in humans. The in silico analysis and the comparison between dolphin and human variations support the hypothesis that the variation p.Tyr163His could be more likely responsible for bisalbuminemia. In the second chapter double radial immunodiffusion (DRI), western blot (WB) analysis, and spectrophotometric measurement using immunologic or enzymatic assays were employed on serum samples of bottlenose dolphins and striped dolphins to validate, establish RIs, and evaluate the diagnostic accuracy of two positive acute phase proteins (C-reactive protein, CRP and serum amyloid-A, SAA) and one negative APP (serum paraoxonase-1, PON-1). With DRI none of the antibodies (Abs) against CRP and SAA cross-reacted with the serum samples of bottlenose dolphins and striped dolphins. Both the anti-SAA Abs tested were latex-conjugated, because produced for automatic immunoturbidimetric assays. The presence of latex associated to the Abs may have interfered to the migration of the Abs across the agarose gel. WB analysis for anti-CRP antibodies showed a weak positivity for striped dolphins and a pattern of positivity in the serum samples of bottlenose dolphins similar to those observed in dog, with multiple bands. However, we are not able to exclude the possibility that this pattern may represent an unspecific signal. The discouraging result obtained with the automated measurement of dolphins CRP (0.00 mg/L) seemed to confirm the hypothesis that the anti-human CRP Ab used does not recognize the cetaceans’ CRP, based also on the low homology of the amino acid sequence. On contrary, the SAA is highly conserved between different species. The automated measurement of SAA provided results with good precision; the SAA concentration in the whole set of bottlenose dolphins samples was 8.7 ± 11.8 mg/L. In addition, for the SAA concentration no differences were noted between different storage time, between the sex of the animals, and between pregnant and non-pregnant animals. The lack of differences in SAA concentration between males and females, and pregnant and non-pregnant animals allowed us to establish the SAA RIs using samples from the whole population instead of establish partitioned RIs .Moreover, a stability in SAA concentration in serum samples with long storage time was demonstrated. PON-1 activity was determined using 4 different substrates using enzymatic assays. The PON-1 activity using paraoxon as substrate provided results with good. The PON-1 activity in the whole set of bottlenose dolphins samples was 6.7 ± 4.6 U/L. As for the concentration of SAA, no differences in PON-1 activity were noted, based on sex, and between pregnant and non-pregnant animals. On contrary, the PON-1 activity for the long storage samples was significantly lower compared to the short storage samples. To evaluate the genetic influence of the single nucleotide polymorphisms (SNPs) in the PON-1 activity, we sequenced the two most studied SNPs of human PON-1 gene, the Q192R and the L55M. Based on the sequence analysis, all the dolphins were homozygous for methionine in L55M SNPs and for arginine in Q192R SNPs. Despite all the animals are homozygous for the phenotype associated to a higher paraoxonase activity in humans, the bottlenose dolphins’ PON-1 activity is low and it seems not useful to discriminate between healthy and diseased animals. The PON-1 activity using 4-nitrophenyl acetate (4-nPA) as substrate was higher compare to those obtained using paroxon, providing results with good precision and accuracy but no significant difference were noted between healthy dolphins and diseased dolphins. However, our results are based on a limited number of animals so we cannot exclude that, including a higher number of animals with different diseases, a more drastic change in PON activity will be evident.