Hematological values of some Bothrops species (Ophidia - Crotalidae) in captivity

Hematological values of some Bothrops species (Ophidia - Crotalidae) in captivity

J.C. TROIANO

, J.C. VIDAL, E.F. GOULD, J. HEKER, J. GOULD, A. U. VOGT , C. SIMONCINI , E. AMANTINI , A. DE ROODT

1 Area de Iología, Museo Argentino de Ciencias Naturales "Bernardino Rivadavia", Av. Angel Gallardo 470 (1405) Buenos Aires, Argentina; 2 Fundación de Estudios Biológicos, Gral. Urquiza 1940/42 (1109) Buenos Aires, Argentina; 3 Instituto Nacional de Producción de Biológicos Carlos G. Malbran (ANLIS) Av. Velez Sarsfield 560 Buenos Aires- Argentina.

ABSTRACT. Blood samples of 50 healthy specimens from each of the following species: Bothrops alternatus, Bothrops jararacussu, Bothrops moojeni, and Bothrops neuwiedi diporus all kept in captivity were taken to determine the hematocrit (PCV) value, red blood cell count (RBC), total leukocyte (WBC) and differential leukocyte count, thrombocyte count, mean corpuscular volume (MCV), hemoglobin concentration (HbC), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). These hematological parameters were compared to those obtained from other Bothrops species. PCV values, RBC, hemoglobin, WBC count, and differential leukocyte count are within the range of values reported for other Bothrops species, while the thrombocyte count was significantly lower. All the hematological parameters obtained from the four studied Bothrops species were higher than those described for B. ammodytoides.

KEY WORDS: hematology, snakes, Bothrops, captivity.

INTRODUCTION

Snakes of the genus Bothrops are crotalids. Six species, namely Bothrops alternatus, B. ammodytoides, B, jararaca, B. jararacussu, B. moojeni, and B. neuwiedi diporus have been described in Argentina. They usually display an aggressive behavior, being responsible for most human and animal envenomings. Bothropic envenomings are characterized by hemorrhage, edema, and necrosis at the bite site, accompanied by generalized hemorrhaging and shock, which may lead to death or produce extensive tissue damage. Since the only specific treatment of bothropic envenomation is antivenom administration, these snakes must be kept in captivity and regularly milked to obtain venom for the preparation of specific antivenom. Venom yield depends on snake health, which can be difficult to ascertain due to the lack of reference values. Characterization of the hematological and clinical chemical parameters in snakes kept in captivity may be a good indication of health. Therefore, we started a systematic study of hematologic and clinical chemical parameters in crotalid species from Argentina (Bothrops and Crotalus).

In this paper, we analyze the hematological parameters of four Bothrops species native to Argentina in an attempt to establish their normal reference intervals.

MATERIALS AND METHODS

Fifty healthy specimens from each of the following species: Bothrops alternatus, Bothrops jararacussu, Bothrops moojeni, and Bothrops neuwiedi diporus of different ages and sexes, captured in Argentina (deparasitized upon arrival and quarantined for one month) were kept in captivity at one of the following: Fundación de Estudios Biológicos, and Instituto Nacional de Producción de Biológicos Serpentaria. The specimens were considered healthy when the following conditions were met: (1) absence of clinical signs of disease, wounds, abscesses, or parasites; (2) adequate nutritional condition without clinical signs of dehydration; (3) regular diet within the last six months; and (4) feces parasitological tests should be negative.

Each snake was kept in an individual box at 22-28ºC and 40-60% humidity with natural seasonally adjusted light/dark periods, as in the different seasons of the year. All the specimens fed spontaneously on living prey (one adult mouse 20-25g a week) and received filtered water ad libitum. All the samples were obtained during 1998. In order to take the blood samples, the snakes were removed from the boxes using a metallic hook, with the head and the body firmly restrained by two technicians. Blood (2.0 ml) was obtained by a third technician by venipuncture of the caudal vein (7), using disposable 21G-needles fitted to sterile 3.0 ml syringes. The use of anesthetics or sedative agents, which are known to induce significant alterations in the hematologic parameters (9), was avoided.

Although venipuncture of the caudal vein was usually successful, in a few cases it failed and a clear fluid, sometimes mixed with blood was obtained. These samples were discarded and venipuncture was repeated using clean needles and syringes.

Immediately after sample collection, three blood smears were prepared, air dried, and stained with a mixture of May-Grünwald & Giemsa stains in absolute methanol (19). The sample was transferred to a clean glass tube containing heparin (sodium salt, 20 µl/ml of blood) as anticoagulant and then used to determine the hematological parameters. Hematocrit (PCV) was determined by the microhematocrit method. Hemoglobin was measured by mixing 20µl of blood with 2.5 ml of Drabkin solution. Cyanmethemoglobin formation was determined by absorbance at 540 nm and compared to that of a hemoglobin standard (Wiener Laboratories, Argentina). Red blood cells, total leukocyte and thrombocyte counts were performed on a 1:200 dilution of blood in a solution as described by Otis (16), which allowed erythrocytes, leukocytes, and thrombocytes to be counted in the same standard Neubauer hemocytometer. Hematimetric indexes were calculated according to the original Wintrobe (28) formulae.

To obtain differential leukocyte counts, the stained blood smears were examined under an optical microscope using an oil-immersion lens. At least 200 cells were counted in each slide. Due to the existing inconsistency in the nomenclature, the morphological nomenclature proposed by Hawkey & Dennet (12) was employed. Thus, leukocyte types will be described as mononuclear cells (lymphocytes, monocytes, and azurophiles) or as granulocytes (heterophils, basophils, and eosinophils).

Data analysis - The results were analyzed by parametric descriptive statistics analysis and expressed as mean ± standard deviation (0 ± SD). Comparison between groups was performed by one-way ANOVA test and Tukey’s multiple comparison as post-ANOVA test. All statistical analyses were performed using Prism 2.1, Graph Pad Inc.

RESULTS

Hematological parameters of Bothrops alternatus, B. jararacussu, B. moojeni, and B. neuwiedii diporus are shown in Table 1. Comparison between data in Table 1 show that although PCV values do not display significant differences between species at the level of p < 0.05, RBC count in B. moojeni appears to be 20% lower than those in the other three species (p < 0.01). On the other hand, hemoglobin concentrations in B. moojeni and B. alternatus appear to be 15 % lower than the corresponding values for B. neuwiedi diporus and B. jararacussu (p < 0.01). Since RBC count and hemoglobin concentration vary in the same direction in B. moojeni and vary inversely in B. alternatus, the values of MCH and MCHC in B. moojeni were the highest and those in B. alternatus the lowest in the species studied.

WBC and thrombocyte counts in B .n. diporus were significant higher than the same values in B. altenatus, B. moojeni, and B. jararacussu (p<0.01). No statistically significant differences in differential leukocyte count were observed between the species (p>0.05).

DISCUSSION

This study was performed in an attempt to standardize normal reference intervals for hematological parameters in Argentine crotalids kept in captivity. In similar studies, where large numbers of samples have to be analyzed, extreme care should be taken to prevent sample contamination. Blood samples were obtained by venipuncture of the caudal vein, which is harmless for the specimen under study and usually successful. However, in a few cases it failed and a clear fluid sometimes mixed with some blood was obtained. A similar problem was reported in venipuncture of the jugular vein of the green sea turtle (Chelonia mydas) by Samour et al. (21). It has been suggested (27) that this fluid may be lymph from lymphatic vessels lying close to the veins, and this is supported by the observation that its chemical composition is similar to that of blood plasma (21). Mixed samples produced extreme variations in cell counts, thus they were discarded. Clean needles and syringes were employed to repeat the procedure.

PCV values in B. alternatus, B. jararacussu, B. moojeni, and B. n. diporus obtained in this study are within the range of the corresponding values described for Brazilian specimens of B. jararacussu, B. moojeni, B. leucurus, and B. jararaca (18,23), as well as those described for Agkistrodon piscivorus (13); Bitis arietans (11), and Crotalus durissus terrificus (24). These values are lower than those reported for: Cerastes cerastes (14); Crotalus horridus (8); the Australian elapids, Notechis scutatus, Pseudonaja nuchalis, Pseudechis porphyriacus, and Austerelaps superbus (5); the colubrids, Thammophis sirtalis (28), Pituophys sayii (21), and Natrix natrix (13); and the boids, Boa constrictor constrictor and Python regius (21).

PCV values observed in three of studied Bothrops species are 10-15% higher (p < 0.01) than those reported for B. ammodytoides (25), while that of B. alternatus is in the limit of statistical significance.

RBC counts in the four species studied are within the range described for: other Bothrops species, such as Bothrops jararaca and B. leucurus, (13,23,26); some crotalids, such as Agkistrodon piscivorus (13) and Crotalus cerastes (14); colubrids, Waglerophis merremii (23), Pituophis sayii (21), Natrix natrix (28), Coluber ventrimaculatus (4), and Lampropeltis gettulus (13). On the other hand, they are significantly lower than those described for other crotalid species, such as Crotalus durisssus terrificus (24), Crotalus horridus (8), Cerastes vipera, and Cerastes cerastes (1); Australian elapids, Pseudonaja nuchalis, Pseudechis porphyriacus, and Austerelaps superbus (2); opystoglyphous colubrid, Dispholidus typus (13); and boids, such as Boa constrictor constrictor and Python regius (20). RBC counts obtained with the four studied Bothrops species are 25-30 % higher than that previously reported for B. ammodytoides (25).

Hemoglobin concentrations in B. alternatus, B. jararacussu, B. moojeni, and B. n. diporus are within the range described for other Bothrops species, such as B. jararaca and B. leucurus (18,23,26); and other crotalids, such as Crotalus horridus (8), Crotalus cerastes (14), and Crotalus durissus terrificus (24); colubrids, Pituophis sayii (22) and Waglerophys merremii (23); and elapids, Notechis scutatus, Pseudonaja nuchalis, Pseudechis porphyriacus, Austerelaps superbus (5), and Naja haje haje (2). Hemoglobin concentrations in the studied Bothrops species are about 35% higher than those reported for: Bothrops ammodytoides (25); and colubrids, Heterodon contortix, Natrix natrix, and Thammophis sirtalis (28), which were 15-20% lower than the values obtained in viperids, such as Cerastes vipera and Cerastes cerastes (1).

WBC counts (10-13 cells x 10 /l) obtained from the four studied Bothrops species are within the range reported for: B. ammodytoides (24), B. moojenii, B. leucurus, and B. jararaca (18,23); and Bitis arietans (15), Crotalus cerastes (5), Crotalus durissus terrificus (24), Boa constrictor constrictor, and Phyton regius (20). However, they are significantly lower than those reported for Cerastes vipera and Cerastes cerastes (1).

Thrombocyte counts obtained from the four studied Bothrops species are within the range reported for B. ammodytoides (25) and Crotalus durissus terrificus (24), but 200-300% lower than those reported by Rodrigues et al. (32-44000 cells per mm ) for Brazilian specimens of B. jararaca, B. leucurus, B. jararacussu, and B. moojeni.

Although it has been reported that the most abundant leukocyte type in the rat snake is the azurophil, whereas in other snakes it is the heterophil (6,10), the differential leukocyte counts in the four studied Bothrops species showed a high percentage (50-52%) of lymphocytes. This result is in agreement with previous reports on B. ammodytoides (25), B. jararaca, B. leucurus, B. jararacussu, B. mojenii (18), Bitis arietans (15), Crotalus cerastes (14), Crotalus durissus terrificus (24), Crotalus adamanteus (3), Boa constrictor constrictor, Python regius (20), Ablabophis rufulus, Crotaphopeltis hotamboeia (17), Coluber ventrimaculatus (4), and Naja haje haje (2), but is higher than that reported in Bothrops jararaca (23).

The low percentage of basophils in the differential leukocyte counts is in accordance with B. ammodytoides (25), B. jararaca (22), Bitis arietans (15), Crotalus durissus terrificus (24), and Crotalus adamanteus (3). High percentages of this type of leukocyte were reported in South African snakes, such as Ablabophis rufulus, Causus rombheatus, Crotaphopeltis hotamboeia, Psammophis subtaenitus subtaenitus, and Naja nigricollis, with a maximum of 22.3 % in Psammophis subtaenitus subtaenitus (17) and the colubrid Coluber ventrimaculatus, in which basophils represent 4-8% of the total leukocyte count (4).

Further studies confirm whether age, sex, and season of the year modify these hematological parameters are in progress.

REFERENCES

Received 14 June 1999

Accepted 01 September 1999

References

• 01 AL-BADRY KS., NUZHY, S. Haematological and biochemical parameters in active and hibernating sand viper. Comp. Biochem. Physiol. A Comp. Physiol., 1983, 74, 137-41.
• 02 AL-BADRY KS., EL DEIEB S., NUZHY S. Haematological changes correlated with the hibernation cycle in the Egyptian cobra (Naja haje haje). J. Therm. Biol., 1992, 17, 107-9.
• 03 ALLEMAN RA, JACOBSON ER, RASKIN RE. Morphologic, cytochemical staining and ultrastructural characteristics of blood cells from eastern diamondback rattlesnakes (Crotalus adamanteus). Am. J. Vet. Res., 1999, 60, 507-14.
• 04 BANERJE V., SHARON V. Hematological investigation on reptiles. IV. Coluber ventrimaculatus Proc. Zool. Soc. Calcuta, 1982, 33, 9-15.
• 05 BOARD PG., ROBERTS N., SHINE R. Studies on the blood of Australian elapid snakes-1. Morphology and composition. Comp. Biochem. Physiol. B Comp. Biochem., 1977, 56, 353-6.
• 06 BOUNOUS DI., DOTSON TK., BROOKS RL., RAMSAYC. Cytochemical staining and ultrastructural characteristic of peripheral blood leucocytes from the yellow rat snake (Elaphe obsoleta quadrivitatta). Comp. Haemathol. Int., 1996, 6, 86-91.
• 07 BUSH M., SMELLER J. Blood collection and injection techniques in snakes. Vet. Med. Small. Anim. Clin., 1978, 73, 211-4.
• 08 CARMICHAEL EB., PRETCHER PW. Constituents of the blood of the hibernating and normal rattlesnakes Crotalus horridus. J. Biol. Chem, 1945, 161, 693-6.
• 09 CUSTER R., BUSH M. Physiologic and acid base measures of gopher snakes during ketamine or halothane-nitrous oxyde anesthesia. J. Am.Vet. Med. Assoc., 1980, 177, 870-4.
• ¹⁰ DOTSON T.K., RAMSAY E. A color atlas of the blood cells of the yellow rat snake. Compend. Cont. Educ. Pract. Vet., 1994, 17, 1013-7.
• ¹¹ HATTINGS J., WILLEMSE GT. Hematological observations on the puff adder, Bitis arietans (Ophidia - Viperidae). Herpetologica, 1976, 32, 245-6.
• ¹² HAWKEY C.M., DENNET T.B. A colour atlas of comparative haematology Ipswich: Wolfe Medical, 1994: 1-129.
• ¹³ HUTTON K The blood chemistry of terrestrial and aquatic snakes. J. Cell. Comp. Physiol., 1958, 52, 319-28.
• ¹⁴ MAC MAHON JA., HAMMER AH. Hematology of the sidewinder (Crotalus cerastes). Comp. Biochem. Physiol. A Comp. Physiol., 1975 , 51, 53-8.
• ¹⁵ OTIS V.S. Hemocytological and serum chemistry parameters of the African puff adder Bitis arietans. Herpetologica, 1973, 29, 110-6.
• ¹⁶ OTIS V.S. Leucocyte and erythrocyte diluent for reptilian blood cells count. Copeia, 1974, 1, 253 - 5.
• ¹⁷ PIENAAR U. DE V. Haematology of some South African reptiles Johannesburg: University of Witwatersrand, 1962: 1-299.
• ¹⁸ RODRIGUES LM., MONTEIRO AO., MELGAREJO AR., ALMOSNY NRP., AGUIAR AS. Hematological evaluation of snakes of the genera Bothrops (Wagner, 1824). In: Congreso Mundial de la Asociación Mundial de Medicina Veterinaria de Pequeños Animales, 13, Buenos Aires, 1998. Resumenes... Buenos Aires: WSAVA, 1998, 2: 45.
• ¹⁹ ROSENFELD G. Corante pancromático para hematologia e citologia humana. Nova combinação dos componentes do May-Grünwald e do Giemsa num só corante de emprego rápido. Mem. Inst. Butantan, 1947, 20, 328-34.
• ²⁰ ROSSKOPF W., WOERPEL R., YANOFF S. Normal hemogram and blood chemistry values for Boa constrictor and pythons. Vet. Med. Small. Anim. Clin., 1982, 77, 822-3.
• ²¹ RYERSON DLA. Preliminary survey of reptilian blood. J. Entomol. Zool., 1949, 41, 49-55.
• ²² SAMOUR J., RILEY J., MARCH P., SAVAGE D., NIEVA A., JHONES O. Blood sampling techniques in reptiles. Vet. Rec., 1984, 114, 472-6.
• ²³ SANO-MARTINS IS. Hematologia comparada de serpentes Bothrops jararaca e Waglerophis merremii São Paulo., 1978 150p. (Disertação - Mestrado) - Universidade de São Paulo, Departamento de Biologia do Instituto de Biociências
• ²⁴ TROIANO JC., VIDAL JC., GOULD E., GOULD J. Hematological reference intervals of the South American rattlesnake (Crotalus durissus terrificus Laurenti, 1768) in captivity. Comp. Haemathol. Int., 1997, 1, 109-12.
• ²⁵ TROIANO JC., VIDAL JC., GOULD EF., MALINSKAS G., GOULD J., SCAGLIONE M., SCAGLIONE L., HEKER JJ., SIMONCINI C. Hematological and blood chemical values from Bothrops ammodytoides (Ophidia - Crotalidae) in captivity. Comp. Haemathol. Int., 1999, 9, 31-5.
• ²⁶ VALLE JR, LEAL-PRADO J. Nota sobre a hematologia dos ofidios. Indices da Wintrobe da Bothrops jararaca. Mem. Inst. Butantan, 1947, 17, 241-5.
• ²⁷ WILLETE-FRAHM M. Blood collection techniques in amphibians and reptiles. In: KIRK R. Ed. Kirk’s currents in veterinary therapy Philadelphia: Saunders, 1995: 344-8.
• ²⁸ WINTROBE R. Variations in size and hemoglobin concentration of erythrocytes in the blood of various vertebrates. Folia Haematol. Leipz., 1933, 51, 32-6.

Publication Dates

• Publication in this collection
  22 Sept 2000
• Date of issue
  2000

History

• Accepted
  01 Sept 1999
• Received
  14 June 1999