HAEMATOLOGICAL AND BIOCHEMICAL PANELS IN TYRRHENIAN LOGGERHEAD SEA TURTLES (CARETTA CARETTA) RESCUED BY THE MTRC: A COMPARATIVE

ANALYSIS ACCORDING TO DIFFERENT CLINICAL FINDINGS AND SPECIFIC PATHOLOGIES

A. DONADIO1,2, A. AFFUSO1, P. SANTORO3, M. CIAMPA1, G. TREGLIA1, S. HOCHSCHEID1.1 MARINE TURTLE RESEARCH CENTER, STAZIONE ZOOLOGICA ANTON DOHRN, PORTICI, ITALY, 2 DEPARTMENT OF BIOLOGY, UNIVERSITY OF NAPLES FEDERICO II, NAPLES, ITALY, 3 DIAGNOSTICA DI LABORATORIO S.R.L., NAPOLI, ITALY

BLOOD WORK ON SEA TURTLES IS IMPORTANT BECAUSE IT ALLOWS THE ACHIEVEMENT OF A RELIABLE DIAGNOSIS AND PROGNOSIS AND A DEEPER KNOWLEDGE OF SEA TURTLE PHYSIOLOGY.

CLINICAL CONDITIONS, LIFE STAGE, PHYSIOLOGICAL STATE, METHODS USED INFLUENCE HEMATOLOGICAL AND BIOCHEMICAL PARAMETERS. THEREFORE, THE COLLECTION OF BASIC DATA FROM INDIVIDUALS ADMITTED AT SEA TURTLE REHABILITATION

FACILITIES IS IMPORTANT TO IMPROVE THE DATASET OF BASELINE HEMATOLOGICAL AND BIOCHEMICAL VALUES FOR THE MEDITERRANEAN LOGGERHEAD TURTLE.

THIS STUDY AIMS TO IDENTIFY POSSIBLE CORRELATIONS BETWEEN TURTLE STATUS AND BIOCHEMICAL AND HAEMATOLOGICAL VALUES AND TO ASSESS IF, DURING THE REHABILITATION PERIOD, THE CHANGES IN THE PATIENT’S HEALTH ARE MIRRORED BY

VARIATIONS IN BLOOD PARAMETERS.

31 LOGGERHEAD SEA TURTLES WERE CONSIDERED: THEY WERE RESCUED IN SOUTHERN TYRRHENIAN COASTAL WATERS AND ON BEACHES OF LAZIO AND CAMPANIA BETWEEN SEPTEMBER 2016 AND JULY 2018 AND HOSPITALIZED AT THE MARINE

TURTLE RESEARCH CENTRE (MTRC) OF THE STAZIONE ZOOLOGICA ANTON DOHRN (FIG 1).AT ARRIVAL, THE ANIMALS WERE WEIGHED AND THE STRAIGHT CARAPACE LENGTH MEASURED WITH A CALIPER (FIG 2, FIG 3). THE GENERIC HEALTH STATUS

WAS EVALUATED, THE BODY CONDITION INDEX (BCI) CALCULATED AND THE BLOOD WORK PERFORMED (FIG 4).

THE PARAMETERS CONSIDERED AND MEASUREMENT UNITS ARE REPORTED IN THE TABLES BELOW (TAB 3, TAB 4). HEMOGLOBIN CONCENTRATION WAS MEASURED BY A HEMOGLOBINOMETER (HEMATOLOGY ANALYZER 9000). HEMATOCRIT WAS

MEASURED USING THE MICROHEMATOCRIT METHOD. BLOOD CELLS WERE COUNTED IN BÜRKER'S HEMOCYTOMETER AFTER DILUITION. PLASMA BIOCHEMICAL ANALYTES WERE MEASURED USING GLOBAL 240 ANALYZER, ELECROLYTES WITH SFRI ISE 4500 ANALYZER AND THE GEL ELECTROPHORESIS WAS PERFORMED WITH INTERLAB MICROTECH 648ISO.

FIG 1. RED LINE INDICATES THE COAST MONITORED BY THE SEA TURTLE STRANDING AND

SALVE NETWORK COORDINATED BY THE SZNFIG 4. BLOOD SAMPLING AT THE MOMENT OF

ARRIVAL

FIG 5. PARAMETERS THAT EXHIBITED STATISTICALLY SIGNIFICANT DIFFERENCES AMONG GROUPS BASED ON BCI

TAB 1. PATIENT GROUPS BASED ON THE BCI

FIG 6. CHLOLESTEROL WAS THE ONLY PARAMETER THAT

EXHIBITED STATISTICALLY SIGNIFICANT DIFFERENCE AMONG

GROUPS BASED ON HEATH ASSESSMENT. RESULTS ARE

SHOWN IN LOGARITHMIC SCALE (LOG10)

TAB 2. PATIENT GROUPS BASED ON THE GENERAL

HEALTH ASSESSMENT

OUR HAEMATOLOGY VALUES GENERALLY FALL WITHIN THE REPORTED RANGES (TAB 3) (BASILE ET AL. 2011). DIFFERENCES IN THE BIOCHEMISTRY RESULTS MAY BE INFLUENCED BY THE

DIFFERENT ANALYSIS METHOD USED (TAB 4).

CONSIDERING BCI, THE TURTLES WERE DIVIDED INTO 4 GROUPS (TAB 1); THE DATA WERE TESTED WITH THE NON-PARAMETRIC KRUSKAL-WALLIS TEST AND THE ONES WITH P-VALUE <0,05 ARE DISPLAYED IN BOX-PLOT CHARTS (FIG 5).

TAB 3. SUMMARY OF BLOOD WORK PERFOMED IN THE PRESENT STUDY

10 ANIMALS WERE RELEASED DURING THE STUDY PERIOD. IN THESE CASES, BLOOD WORK WAS REPEATED JUST BEFORE THE RELEASE AND THE VALUES WERE COMPARED WITH THE ONES AT THE

ARRIVAL USING THE NON- PARAMETRIC WILCOXON RANK SUM TEST AND THE ONES STATISTICALLY DIFFERENT ARE DISPLAYED IN BOX-PLOT CHARTS (FIG 7).

DIFFERENCES IN HEMATOLOGICAL PARAMETERS WERE INVESTIGATED ALSO BY GROUPING THE PATIENTS ON THE BASIS OF HEALTH CONDITIONS (TAB 2, FIG. 6).

FIG 2. PATIENT WEIGHING FIG 3. TAKING MEASUREMENTS

FIG. 7 . COMPARISON OF BLOOD WORK RESULTS AT ARRIVAL AND BEFORE THE RELEASE, ONLY THOSE PARAMETERS FOR WHICH A GENERAL TREND WAS OBSERVED ARE SHOWN. A) ALBUMIN GENERAL INCREASE. B) BILIRUBIN GENERAL DECREASE, C) LDH GENERAL DECREASE, D) CPK GENERAL DECREASE

ACKNOWLEDGEMENT

The study was conducted under permit no. 24471/PNM issued by the Ministry of Environment.Financial support was provided by the Campania Region - UOD Gestione delle risorse naturali protette - Tutela e salvaguardia dell'habitat marino e costiero – Parchi e riserve naturali.We wish to thank Nicola Campomorto and all the volunteers belonging to the National Body for Animal Protection (ENPA – section of Salerno) for the precious work of rescue and recovery of sea turtles found in difficulty.AD wishes to thank UNEP's RAC/SPA, the Bern Convention, IUCN AND the 6th Mediterranean Conference on Marine Turtles for the assignment of the travel grant.

REFERENCE

Basile F., Di Santi A., Ferretti L., Bentivegna F., Pica A. (2011) Hematology of the Mediterranean population of sea turtle (Caretta caretta): comparison of blood values in wild and captive, juvenile and adult animals. Comp Clin Pathol DOI 10.1007/s00580-011-1306-4Bjorndal, K. A., Bolten, A. B., & Chaloupka, M. Y. (2000). GREEN TURTLE SOMATIC GROWTH MODEL: EVIDENCE FOR DENSITY DEPENDENCE. Ecological Applications, 10(1), 269–282. doi:10.1890/1051-0761(2000)010[0269:gtsgme]2.0.co;2

0

10

20

30

40

50

60

VERY GOOD GOOD LIGHT EMACIATED

Hae

mat

ocr

it (

%)

Haematocrit

Condition BCI* N

Very good > 1.75 7

Good 1.60<>1.75 11

Light 1.45<>1.60 10

Emaciated <1.45 3

*BCI calculated as (Mb/SCL3) X 1000

Condition N

Good/Acute stress 14

Subacute trauma 8

Chronic trauma 6

Fatal trauma 3

Reference Arrival

Juvenile Adult

WBC x mmc 14552 (2000-38000) 23824 (12000–45500) 22800 (13500–39000)

RBC x mmc 515968 (53000-1000000) 488362 (280000–760000) 411236 (338500–555600)

Hb 10.4 (1.3-16.9) 8 (2.3-12.5) 10.6 (7.1-13.2)

MCH 2.139 (1.259-3.2) 1.70 (0.61-2.84) 2.61 (2.10-3.30)

Ht % 32.7 (4-50) 23 (13-30) 29 (21-33)

TBC x mmc 16242 (3000-42000) 1965 (400–5500) 4920 (1000–9000)

MCV (fL) 669.9 (407.4-950) 487 (323–682) 723 (567–828)

MCHC (g/dl) 32 (29.8-36.4) 34 (18–47) 36 (28-40)

Heterophils (%) 85 (79-92) 62.4 (41-75) 52.6 (9.9-78)

Lymphocytes (%) 11.3 (5-20) 32.4 (23-55) 42.3 (20-85.4)

Monocytes (%) 1.2 (0-3) 2.6 (1-6) 1.8 (0.2-5)

Eosinophils (%) 2.3 (0-5) 2.6 (1-6) 2.3 (1-4.5)

Basile et al. 2011 Reference Arrival

Glucose (mg/dl) 144.3 (38-305)

BUN (mg/dl) 198.5 (60-541)

Creatinine (mg/dl) 0.4 (0.14-0.7)

Uric Acid (mg/dl) 0.6 (0.1-4.1)

Bilirubin (mg/dl) 0.14 (0.01-0.3)

Cholesterol (mg/dl) 328 (22-5280)

Triglycerides (mg/dl) 148.8 (10-894)

GOT (AST) (U.I.) 187.5 (80-410)

GPT (ALT) (U.I.) 15.6 (3-43)

LDH (U.I.) 532.2 (85-2105)

CPK (U.I.) 2839.6 (54-8247)

GGT 2.45 (1-4)

ALP (U.I.) 43.8 (16-78)

Sodium (mEq/L) 146.4 (142-156)

Potassium (mEq/L) 3.76 (2.5-8.1)

Chloride (mEq/L) 130.4 (92-967)

Calcium (mg/dl) 5.97 (4-10.1)

Phosphorus (mg/dl) (mg/dl) 8.37 (4.1-15.3)

Magnesium (mg/dl) 4.33 (1.2-10.5)

Albumin (gr/dl) 1.31 (0.2-1.9)

TAB 4. SUMMARY OF BIOCHEMISTRY RESULTS FROM

THE PRESENT STUDY

IN CONCLUSION

• OUR DATA CONTRIBUTE TO A DATABASE OF

REFERENCE VALUES FOR BLOOD WORK ON

MEDITERRANEAN SEA TURTLES

• SOME PARAMETERS HAVE BEEN IDENTIFIED THAT

ARE RELIABLE INDICATORS FOR A TURTLES HEALTH

STATUS.

• FURTHER DATA COLLECTION, ESPECIALLY FROM

LOCAL HEALTHY TURTLES, IS NECESSARY TO

BETTER UNDERSTAND THE VARIABILITY OF BLOOD

VALUES AND HOW IT CORRELATES WITH THE

TURTLES’ STATUS.