Acute Phase Proteins in Marine Mammals: State of Art, Perspectives and Challenges

The term “acute phase response” (APR) is referred to a nonspecific and complex reaction of an organism that occurs shortly after any tissue damage, such as infection, trauma, neoplasia, inflammation, and stress. The APR can be identified and monitored with some laboratory tests, such as the concentration of several plasma proteins, the acute phase proteins (APPs). The APPs are components of the non-specific innate immune response, and their plasma concentration is proportional to the severity and/or the extent of tissue damage. The evaluation of health status of marine mammals is difficult because the classical clinical signs of illness used for human and domestic animals are difficult to recognize and understand. For this reason, in the past years, several efforts were done to identify laboratory markers of disease in these animals. The APPs have demonstrated their role as early markers of inflammation in veterinary medicine, thus several APPs were tested in marine mammals, such as C-reactive protein (CRP), serum amyloid-A (SAA), and Haptoglobin (Hp). However, the difficulty to extrapolate the knowledge about APPs in one species to another, the lack of specie-specific reagents, the absence of data about negative APPs have hampered their extent use in marine mammals. Herein, the state of art of APPs in marine mammals is reviewed, with particular attention to pre-analytical and analytical factors that should be taken into account in validation and interpretation of APPs assays. Moreover, the current application, potential utility and the future developments of APPs in marine mammals is highlighted and discussed

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

Relationship of diagnostic accuracy of renal cortical echogenicity with renal histopathology in dogs and cats, a quantitative study

Renal cortical echogenicity is routinely evaluated during ultrasonographic investigation of the kidneys. Both in dog and cat previous ex-vivo studies have revealed a poor correlation between renal echogenicity and corresponding lesions. The aim of this study was to establish the in-vivo relationship between renal cortical echogenicity and renal histopathology

correlation between renal histopathology and renal ultrasound in dogs

Abstract Fifty-three privately owned dogs were included in the study. Ultrasonography of the kidneys was performed ante mortem. All the dogs died or were euthanized for reasons unrelated to this study. Histopathology of both kidneys was performed, and a degeneration and an inflammation score ranging from zero to two was assigned by consensus between two pathologists. A numerical score based on a three level semi-quantitative scale (0, 0.5, 1) was assigned by consensus between two of the authors to the following ultrasonographic abnormalities: cortico-medullary definition, echogenicity of the renal cortex, echogenicity of the medulla, renal shape, cysts, scars, mineralizations, subcapsular perirenal fluid accumulation, pyelectasia. The scores deriving from the consensus were summed to create a summary index called renal ultrasound score (RUS). Statistically significant differences in cortico-medullary definition, echogenicity of the renal cortex, echogenicity of the medulla, renal shape, scars and pyelectasia were evident between the degeneration score groups. There were significantly different distributions of cortico-medullary definition, renal shape and scars between the inflammatory score groups. There were statistically significant differences in the RUS between the degenerative score groups (F = 24.154, p-valu

Immunohistochemical Markers of Apoptotic and Hypoxic Damage Facilitate Evidence-Based Assessment in Pups with Neurological Disorders

Seizures in puppies often present a diagnostic challenge in terms of identifying and treating the underlying cause. Dog breeds with mutations of the MDR1-gene are known to show adverse reactions to certain drugs, yet metabolic imbalance exacerbated by physiologically immature organs and other contributing pathologies require consideration before arriving at a diagnosis. This study analysed the brains of two male, 5-week-old Australian Shepherd siblings that died after displaying severe neurological symptoms upon administration of MilproVet(®) to treat severe intestinal helminth infection. Despite the initial symptoms being similar, their case histories varied in terms of the symptom duration, access to supportive therapy and post-mortem interval. Histopathology and immunohistochemistry were used to obtain more information about the phase of the pathological processes in the brain, employing protein markers associated with acute hypoxic damage (β-amyloid precursor protein/APP) and apoptosis (diacylglycerolkinase-ζ/DGK-ζ, apoptotic protease activating factor 1/Apaf1, and B-cell lymphoma related protein 2/Bcl-2). The results seem to reflect the course of the animals’ clinical deterioration, implicating that the hypoxic damage to the brains was incompatible with life, and suggesting the usefulness of the mentioned immunohistochemical markers in clarifying the cause of death in animals with acute neurological deficits

Cytologic scoring of equine exercise-induced pulmonary hemorrhage: Performance of human experts and a deep learning-based algorithm

Exercise-induced pulmonary hemorrhage (EIPH) is a relevant respiratory disease in sport horses, which can be diagnosed by examination of bronchoalveolar lavage fluid (BALF) cells using the total hemosiderin score (THS). The aim of this study was to evaluate the diagnostic accuracy and reproducibility of annotators and to validate a deep learning-based algorithm for the THS. Digitized cytological specimens stained for iron were prepared from 52 equine BALF samples. Ten annotators produced a THS for each slide according to published methods. The reference methods for comparing annotator’s and algorithmic performance included a ground truth dataset, the mean annotators’ THSs, and chemical iron measurements. Results of the study showed that annotators had marked interobserver variability of the THS, which was mostly due to a systematic error between annotators in grading the intracytoplasmatic hemosiderin content of individual macrophages. Regarding overall measurement error between the annotators, 87.7% of the variance could be reduced by using standardized grades based on the ground truth. The algorithm was highly consistent with the ground truth in assigning hemosiderin grades. Compared with the ground truth THS, annotators had an accuracy of diagnosing EIPH (THS of < or ≥ 75) of 75.7%, whereas, the algorithm had an accuracy of 92.3% with no relevant differences in correlation with chemical iron measurements. The results show that deep learning-based algorithms are useful for improving reproducibility and routine applicability of the THS. For THS by experts, a diagnostic uncertainty interval of 40 to 110 is proposed. THSs within this interval have insufficient reproducibility regarding the EIPH diagnosis

Screening of candidate G-quadruplex ligands for the human c-KIT promotorial region and their effects in multiple in-vitro models

Stabilization of G-quadruplex (G4) structures in promoters is a novel promising strategy to regulate gene expression at transcriptional and translational levels. c-KIT proto-oncogene encodes for a tyrosine kinase receptor. It is involved in several physiological processes, but it is also dysregulated in many diseases, including cancer. Two G-rich sequences able to fold into G4, have been identified in c-KIT proximal promoter, thus representing suitable targets for anticancer intervention. Herein, we screened an \u201cin house\u201d library of compounds for the recognition of these G4 elements and we identified three promising ligands. Their G4-binding properties were analyzed and related to their antiproliferative, transcriptional and post-transcriptional effects in MCF7 and HGC27 cell lines. Besides c-KIT, the transcriptional analysis covered a panel of oncogenes known to possess G4 in their promoters. From these studies, an anthraquinone derivative (AQ1) was found to efficiently downregulate c-KIT mRNA and protein in both cell lines. The targeted activity of AQ1 was confirmed using c-KIT\u2013dependent cell lines that present either c-KIT mutations or promoter engineered (i.e., \u3b1155, HMC1.2 and ROSA cells). Present results indicate AQ1 as a promising compound for the target therapy of c-KIT-dependent tumors, worth of further and in depth molecular investigations

Cytologic scoring of equine exercise-induced pulmonary hemorrhage : performance of human experts and a deep learning-based algorithm

Exercise-induced pulmonary hemorrhage (EIPH) is a relevant respiratory disease in sport horses, which can be diagnosed by examination of bronchoalveolar lavage fluid (BALF) cells using the total hemosiderin score (THS). The aim of this study was to evaluate the diagnostic accuracy and reproducibility of annotators and to validate a deep learning-based algorithm for the THS. Digitized cytological specimens stained for iron were prepared from 52 equine BALF samples. Ten annotators produced a THS for each slide according to published methods. The reference methods for comparing annotator’s and algorithmic performance included a ground truth dataset, the mean annotators’ THSs, and chemical iron measurements. Results of the study showed that annotators had marked interobserver variability of the THS, which was mostly due to a systematic error between annotators in grading the intracytoplasmatic hemosiderin content of individual macrophages. Regarding overall measurement error between the annotators, 87.7% of the variance could be reduced by using standardized grades based on the ground truth. The algorithm was highly consistent with the ground truth in assigning hemosiderin grades. Compared with the ground truth THS, annotators had an accuracy of diagnosing EIPH (THS of < or ≥ 75) of 75.7%, whereas, the algorithm had an accuracy of 92.3% with no relevant differences in correlation with chemical iron measurements. The results show that deep learning-based algorithms are useful for improving reproducibility and routine applicability of the THS. For THS by experts, a diagnostic uncertainty interval of 40 to 110 is proposed. THSs within this interval have insufficient reproducibility regarding the EIPH diagnosis.The Dres. Jutta and Georg Bruns-Stifung für innovative Veterinärmedizin.https://journals.sagepub.com/home/vetCompanion Animal Clinical Studie

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.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