Haematology and blood chemistry references values

Haematology and blood chemistry references values for clinically healthy redwattled lapwing (Vanellus indicus) Sajid Umar, Kiran Aqil, Rizwan Qayyum, Muhammad Younus, Qamarun-Nisa, Shahzad Ali, Muhammad Ali Shah, Muhammad Irfan, Muhammad Usman, et al. European Journal of Wildlife Research ISSN 1612-4642 Eur J Wildl Res DOI 10.1007/s10344-016-1052-7

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Author's personal copy Eur J Wildl Res DOI 10.1007/s10344-016-1052-7

SHORT COMMUNICATION

Haematology and blood chemistry references values for clinically healthy red-wattled lapwing (Vanellus indicus) Sajid Umar 1,2 & Kiran Aqil 3 & Rizwan Qayyum 3 & Muhammad Younus 4 & Qamar-un-Nisa 5 & Shahzad Ali 6 & Muhammad Ali Shah 7 & Muhammad Irfan 8 & Muhammad Usman 9 & Asif Ali 10 & Akbar Ali 10 & Adnan Ayan 11 & Muhammad Yaqoob 12

Received: 7 December 2015 / Revised: 30 August 2016 / Accepted: 13 September 2016 # Springer-Verlag Berlin Heidelberg 2016

Abstract Red-wattled lapwings (Vanellus indicus) are medium-sized birds endemic to the wetlands of south and west Asia. Their population is decreasing due to loss of habitat, shrinkage of wetlands and poaching. Fifty-two red-wattled lapwings (RWL) were captured from wetlands of Punjab province during summer, 2014 (n = 52). All birds appeared to be in good body condition and no abnormalities were noted during physical examination. Haematological and plasma biochemical parameters of RWL of both sexes were analysed in

order to determine reference values, taking sex and age into account. No statistical differences in haematology and blood chemistry parameters were observed between genders within age groups except for CH and TG which were significantly higher in females. Differences between juveniles (J) and adults (A) were identified for TPP (J < A), MCH (J > A) and MCHC (J > A), urea (J < A), uric acid (J < A) and creatinine (J < A). These results provide reliable reference values for the clinical interpretation of haematologic results for the species.

* Sajid Umar [email protected] Kiran Aqil [email protected] Rizwan Qayyum [email protected] Qamar-un-Nisa [email protected] Shahzad Ali [email protected] Muhammad Ali Shah [email protected] Muhammad Irfan [email protected] Muhammad Usman [email protected] Akbar Ali [email protected] Adnan Ayan [email protected]

Muhammad Yaqoob [email protected] 1

Department of Pathobiology, National Veterinary School of Toulouse, Toulouse, France

2

Department of Pathobiology PMAS Arid Agriculture University, Rawalpindi, Pakistan

3

Veterinary Research Institute (VRI), Lahore, Pakistan

4

Department of Pathobiology, College of Veterinary and Animal Sciences, Jhang, Pakistan

5

Department of Pathology, University of Veterinary and Animal Sciences, Lahore, Pakistan

6

Department of Wildlife and Ecology, University of Veterinary and Animal Sciences, Lahore, Pakistan

7

Department of Pathobiology, PMAS Arid Agriculture University, Rawalpindi, Pakistan

8

Laboratory of Physiology and Cell Signalling, College of Veterinary Medicine, KNU, Daegu, South Korea

9

Poultry Research Institute (PRI), Rawalpindi, Pakistan

10

Department of Livestock and Dairy development, Punjab, Pakistan

11

Department of Parasitology, Faculty of Veterinary Medicine, Adnan Menderes University, Aydin, Turkey

12

Department of Clinical Sciences, PMAS Arid Agriculture University, Rawalpindi, Pakistan

Author's personal copy Eur J Wildl Res

Keywords Red-wattled lapwings . Vanellus indicus . Haematology . Plasma biochemistry . Reference values

blood chemistry references values for clinically healthy RWL. In present study, we collected blood samples from RWL in wetlands of Punjab province, Pakistan. Here, we established baseline values for haematologic and plasma chemistry parameters that will provide a baseline for comparisons allow researchers to monitor the health status of RWL in captivity and will provide a reference for comparison with diseased RWL.

Introduction

Materials and methods

The red-wattled lapwing is a medium-sized plover in the Charadriidae family which is endemic to the wetlands of south and west Asia (India, Pakistan, Iran, Afghanistan, Iraq and Bangladesh) and extends into Turkey, the south-eastern most region of Europe. RWL resides in the open countryside, ploughed fields, grazing patches and margins and dry beds of water bodies with a preference of sites in close proximity to freshwater (Ali 1996; Ali and Ripley 2001; Pamela et al. 2005; Muralidhar and Barve 2013). Wetlands are fragile ecosystems which are fast deteriorating and shrinking due to manmade activities. The red-wattled lapwing has important ecological and cultural impact in these wetland systems. RWL can play a significant role in control of harmful insects. In Punjab, the local name is Btiteeri^ where it is believed that the laying of eggs by the lapwing on high ground is an indication of rains to come. The red-wattled lapwing is classified as a species of least concern on the IUCN red list. The European population is stable yet very small and therefore meets the threshold for classification as endangered in Europe. Given the large, apparently stable neighbouring population in Asia, there is significant potential for rescue from outside the region (Jokimäki et al. 2005; Kirwan et al. 2008; IUCN 2012; Wiersma and de Juana 2014; Patel and Dhandhukia 2015; Dhandhukia and Patel 2015). Haematology and plasma chemistry values are useful in assessing individual and population health and fitness for wildlife species (Deem et al. 2001). These parameters may provide a more-sensitive indication of population condition than morphologic data alone (Gallo et al. 2013). Furthermore, they are useful diagnostic tools in clinical practice (Campbell and Ellis 2007) and are especially important in birds, which frequently show few overt clinical signs of disease (Gallo et al. 2013). Accurate reference data are also essential for assessing population health; however, its determination can be difficult in wild species due to the inherent variability of natural systems (Newman et al. 1997; Plischke et al. 2010). There are no reports of haematology and blood chemistry parameters in wetland RWL. No data are available regarding haematology and blood chemistry references values for clinically healthy RWL. The lack of documentation in scientific journals is surprising, since RWL are precious birds in Asia and their number is declining every year due to shrinking wetlands and poaching. To the best of our knowledge, this is the first report on haematology and

Sample collection and storage

Haematology and blood chemistry may be important tools for population health investigations on wetland RWL populations and will also be essential to differentiate health and diseased status of birds in future disease surveillance programmes.

A total of 52 RWL were captured from wetlands of Punjab province Pakistan during summer, 2014 by using static nets as described previously (Seddon et al. 1999). Nets of nylon mesh 1–1.5 m high and 50–100-m long were set up across or perpendicular to RWL habitats, with the leading edge raised by fine cane poles and one edge trailing. In theory, RWL are herded slowly towards the nets by the trappers. When the RWL are close to the nets, they are pushed harder, and in trying to evade the trappers, will become entangled in the net or caught within the box. RWL were physically restrained and blood collection was performed from the right cutaneous ulnar vein within 5 min of restraint. Blood samples were immediately transferred to ethylene diamine tetra acetic acid (EDTA) and lithium heparin tubes (BDH Chemicals Limited, Poole, England) for haematology and blood chemistry analysis, respectively. Fifty-two individuals were sampled. Five individuals were excluded from the analysis because they had clinical signs of disease (e.g., dermatitis, dyspnoea and loose droppings), one blood samples discarded due to haemolysis, and three individuals were excluded because they had been subjected to stressful events (social group disruption or transference to new cages, or both) within the 12 h prior to the blood collection. Therefore, 43 RWL were included in the final analysis (15 male adult, 9 female adult, 10 male juveniles and 9 female juveniles). The study was performed in accordance with the ethics standards of the Committee on Animal Experimentation of Veterinary Research Institute (VRI) Lahore, Pakistan. The following protocol was followed for the sex and age of each bird: since the male and female RWL are similar in appearance, while the juvenile has duller plumage and has white throat dot. The sexual differentiation being made according to the voice, shriller in the case of the males and deeper in the females, expressed well by the words ‘did-he-do-it pity-to-do-it’ (Saxena and Saxena 2013). Sample processing and analysis Thin blood smears were prepared immediately after blood collection and fixed with 99 % methanol. Samples were transported to the laboratory and analysed within 2–6 h. Routine


haematologic methods were used to determine the following parameters (Thrall et al. 2007; Prioste et al. 2012; Gallo et al. 2013): packed cell volume (PCV), haemoglobin (Hb), red blood cell count, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), white blood cell count, thrombocyte count, heterophil count, lymphocyte count, monocyte count, eosinophil count, basophil count and heterophil to lymphocyte ratio. Erythrocytes, leukocytes and thrombocytes were counted manually using the Natt-Herrick staining solution and a Neubauer chamber (Natt and Herrick 1952). Hb was measured with the cyanmethemoglobin method (Drabkin and Austin 1935). Differential leukocyte counts were performed manually for 100 cells on Rosenfeld-stained blood smears (Rosenfeld 1947; Campbell and Ellis 2007). The samples were centrifuged at 3000g for 10 min and the resulting plasma was kept at −18 °C until analysis. Plasma chemistries and enzymes were analysed for samples which were processed on a wet automated analyser (Hitachi Model 902 Automatic Analyser, Hitachi Science Systems, Ibaraki, Japan) at a commercial veterinary laboratory (Veterinary Research Institute (VRI) Lahore, Pakistan). Parameters measured included glucose, uric acid (UA), urea (U), creatinine (CR), cholesterol (CH), triglycerides (TG), total plasma protein (TPP), albumin to globulin ratio, calcium (Ca) and phosphorus (P) concentrations and alkaline phosphatase (ALP), lactate dehydrogenase (LDH), creatine phosphokinase (CPK), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities (Gallo et al. 2013).

Statistical analyses Before statistical analysis, we excluded outlying values due to bad sample quality, for example as a result of haemolysis (Meyer and Harvey 1992; Hochleithner 1994). Animals were separated into four categories (juvenile males, juvenile females, adult males and adult females). A Shapiro-Wilk test was performed to evaluate the distribution of each parameter. Parameters following normal distribution were tested using ANOVA; post hoc comparisons were performed with Tukey’s tests, comparing genders within age groups and then comparing age groups with genders combined. Parameters not following normal distribution even after natural logarithm transformation were tested using the Kruskal-Wallis tests; post hoc comparisons were performed using the Bonferronicorrected Mann-Whitney tests (Kleinbaum et al. 1998). Significance level was p < 0.05 for all tests.

Results There were no significant statistical differences in parameters between the juvenile males and juvenile females within their

age group, nor between the adult males and adult females within their age group except for TG and CH (Tables 1 and 2). Differences between males and females were found in TG (p < 0.05) and CH (p < 0.05) levels (Table 2) since females had higher values for these parameters at all ages. The only significant differences observed were between juveniles and adults in the following six parameters: TPP (p < 0.05), MCH (p < 0.05), and MCHC (p < 0.05), U (p < 0.05), UA (p < 0.05) and CR (p < 0.05). Haematology and blood chemistry values of RWL are presented in Tables 1 and 2, respectively.

Discussion This is the first study of baseline haematology and plasma chemistry information for RWL. All birds sampled in this study were in good physical condition. There were no significant differences between genders for any of the parameters herein evaluated except TG and CH, similar to previous studies in other birds (Prioste et al. 2012; Tell and Citino 1992; Polo et al. 1998; Gee et al. 1981). Significant haematologic differences occurred depending on age classes: TPP, U, UA and CR were higher in adults than in juveniles, while both MCH and MCHC were higher in juveniles than in adults. These differences are likely not clinically important and may be attributable to sample size, age variations in sampled birds, capture method and reproductive stage (Gallo et al. 2013; Balasch et al. 1974; Newman et al. 1997). Haematocrit values were similar to those reported for other captive and free-living birds reported previously (Samour et al. 1998; Travis et al. 2006; Lloyd and Gibson 2006) but higher than findings of Newman et al. (1997) in free-ranging pelagic cormorants (Phalacrocorax pelagicus), Puerta et al. (1995) in sparrows (Passer domesticus) and Chou et al. (2008) in black-faced Spoonbills (Platalea minor). These differences are likely to species difference. Moreover, slightly higher haematocrits may be due to dehydration (Campbell and Ellis 2007). Total RBC counts were higher than those values published for other captive and free-ranging bird species (Balasch et al. 1974; Melrose and Nicol 1992; Samour et al. 1998). The variability in RBC counts was not surprising as this parameter has been reported to be highly inconsistent (1.5 × 106 to 6.6 × 106 cells/ml) among different bird species (Amand 1986) and can also be affected by season, time of day and environmental temperature (Campbell and Ellis 2007). Total WBC counts were higher than values reported for other wild bird species, both captive (Melrose and Nicol 1992; Lloyd and Gibson 2006) and free-ranging (Puerta et al. 1995; Travis et al. 2006; Lloyd and Gibson 2006), although this may reflect species differences and inherent variability in the WBC-counting techniques (Meyer and Harvey 1992). Nevertheless, general causes of leukocytosis include inflammation due to infection, trauma, toxicities and

Author's personal copy Eur J Wildl Res Table 1 Haematologic results for clinically healthy red-wattled lapwings (Vanellus indicus; n = 43) in Punjab Pakistan during summer 2014

Parameters

Mean

SD

Range

Age/sex group differences

Erythrocytes (cells/μl × 106)

3.8 13.5

0.52 1.2

2.4–4.9 9.4–15.8

– –

47 134 38

3.2 14 3.2

38–59 90–202 23–58

– – Juveniles (39 ± 3.3), adults (37.2 ± 2.0)

Thrombocytes ((cells/μl × 10 ) Leukocytes (cells/μl × 103) Heterophils (cells/μl × 103) Lymphocytes (cells/μl × 103)a Monocytes (cells/μl × 103)a Eosinophils (cells/μl × 103)a Basophils (cells/μl × 103)a

30 23.6 11.03 5.84 4.23 0.36 0.80 0.005

2.6 7.5 3.45 1.23 1.45 0.26 0.56 0.03

25–34 12.0–48 4.32–20.21 4.2–9.5 2.80–7.60 0.02–1.00 0.00–3.10 0.00–0.15

Juveniles (31.3 ± 2.9), adults (29 ± 3.7) – – – – – – –

Heterophils (%) Lymphocytes (%)

61 42

5.6 3.7

33–72 32–62

– –

Monocytes (%) Eosinophils (%) Basophils (%) Heterophil:lymphocyte ratioa Total solids (g/dl)a

0.7 0.2 0.1 1.23 1.27

1.3 0.1 0.4 0.48 0.39

0–2 0–0.9 0–0.8 0.43–2.33 2.5–4.60

– – – – –

Haemoglobin (g/dl)a Haematocrit (%) MCV (fl)a MCH (pg)a MCHC (%) 3 a

SD standard deviation a

Assumption of normality was not met for these parameters (Kruskal-Wallis test p < 0.05)

haemorrhage (Campbell and Ellis 2007). Heterophils were found to be the most abundant circulating leukocyte in the RWL, as has been reported in golden conures (Prioste et al. 2012); there are, however, other bird species for which Table 2

lymphocytes are most common (Gee et al. 1981; Amand 1986; Dobado-Berrios et al. 1998). Because heterophilia is usually present in inflammation processes, and the heterophil counts in this study were similar to those reported for

Biochemical blood values for clinically healthy red-wattled lapwings (Vanellus indicus; n = 43) in Punjab, Pakistan, during summer 2014

Parameters Total plasma protein (g/dl)a Uric acid (mg/dl)a Urea (mg/dl)a Creatinine (mmol/l)a Glucose (mg/dl)a Albumin: globulin ratioa Alkaline phosphatase (IU/L) Lactate dehydrogenase (IU/L)a Creatine phosphokinase (IU/L) Aspartate aminotransferase (IU/L) Alanine aminotransferase (IU/L)a Calcium (mg/dl) Phosphorus (mg/dl) Triglyceride (mg/dl)a Cholesterol (mg/dl)

Mean 3.61 4.6 7.8 0.33 195.55 1.30 2216.28 687.25 777.33 395.18 76.92 7.22 4.55 101.63 338.69

SD 0.54 1.05 3.15 0.009 24.58 0.24 468.39 222.51 316.44 37.13 10.12 0.66 0.77 28.24 44.34

Range

Age/sex group differences

2.5–5.2 2.46–11.2

Adults (3.89 ± 3.0), juveniles (3.5 ± 4.2) Adults (4.9 ± 2.0), juveniles (4.2 ± 1.4)

2.00–21.00 0.2–0.6 120.00–295.00 0.60–1.80 136.00–4900.00 322.00–1322.00 376.00–1623.00 138.00–603.00 36.00–136.00 5.30–12.90 2.46–7.5 38.00–200.00 165.00–599.00

Adults (8.12 ± 4.0), juveniles (7.5 ± 2.2) Adults (0.38 ± .006), juveniles (0.29 ± 0.004) – – – –

SD standard deviation a

Assumption of normality was not met for these parameters (Kruskal-Wallis test p < 0.05)

– – – – Male (100.12 ± 2.5), female (103 ± 4.0) Male (340.22 ± 1.4), female (337 ± 3.0)


cormorants (Travis et al. 2006), it is possible that the increased leukocyte counts observed were of non-infectious origin and may be due to species difference. The differential WBC counts of RWL were comparable to published ranges for similar species, except for eosinophils in flightless cormorants and monocytes in black-faced cormorants, which showed higher counts (Melrose and Nicol 1992; Travis et al. 2006). Heterophils and lymphocytes make up the majority (i.e., >80 % combined) of WBCs in RWL irrespective of age and sex. These results agree with those reported for other birds (Newman et al. 1997; Travis et al. 2006; Rubio et al. 2012). Haematological values also depend on methodology differences such as sample size, restraining (physical or chemical) and use of anticoagulant (heparin or EDTA). RWL had lower levels of UA and CR than those reported for free-ranging flightless cormorants (Travis et al. 2006) and pelagic cormorants (Newman et al. 1997) but were similar to those reported for captive double-crested cormorants, common coots (Rubio et al. 2012) and adult stone curlews (Samour et al. 1998). However, the higher values of UA and CR are more difficult to interpret. RWL commence ovulation in summer, which could affect their protein metabolism. This has been reported in the house sparrow (Puerta et al. 1995). The diet of the lapwing usually includes a range of insects, snails and other invertebrates, mostly picked from the ground. They may also feed on some grains. Factors such as the amount of proteins ingested with the feed, the individual’s nutritional status at the moment of sampling or metabolic differences may be influencing the plasma UA and CR concentration of the birds (Newman et al. 1997; Jenni-Eiermann and Jenni 1998; Travis et al. 2006; Campbell and Ellis 2007). Plasma uric acid levels are good indicators of nutritional status in species of birds with few fat reserves, such as RWL, since protein catabolism is very rapidly activated during periods of food shortage, and leads to a continuous linear increase in the products of nitrogen excretion in the blood (Dobado-Berrios et al. 1998). In birds, CH and TG concentrations are affected by the qualitative composition of their diet and are regulated by lipid metabolism (Dobado-Berrios et al. 1998). RWL had lower levels of triglycerides than those reported for freeranging pelagic cormorants (Newman et al. 1997), redlegged partridges (Rodríguez et al. 2004) and sparrows (Puerta et al. 1995) but similar to those reported for captive double-crested cormorant, quails (Scholtz et al. 2009) and black-faced spoonbills (Chou et al. 2008). On the contrary, cholesterol levels were slightly higher than values reported for free-ranging flightless cormorants (Travis et al. 2006), red-legged partridges (Rodríguez et al. 2004) and pelagic cormorants (Newman et al. 1997) but similar to those reported for captive double-crested cormorants and quails (Scholtz et al. 2009). Increases in cholesterol have been associated with dehydration during fasting, high levels of dietary fat or age (Campbell and Ellis, 2007). Therefore,

the slight increase in cholesterol levels observed in our study is likely related to dehydration from short-term fasting at the time of sampling. An increased level of CH could also be linked to ovulation and breeding season in females since CH is the precursor of steroid hormones (Rodríguez et al. 2004; Gallo et al. 2013). Previous studies have shown an increase in LDH, CPK and AST from capture stress (Travis et al. 2006). The low values found in our study might reflect tolerance to stress during handling. RWL had higher values of ALP than those reported by Newman et al. (1997) for free-ranging pelagic cormorants and lower than those reported for red-legged partridge (Rodríguez et al. 2004) and yellow-legged gulls (AlonsoAlvarez, 2005). Plasma concentrations of ALP, Ca and P tend to a maximum at the time of maximum mineralization and osteoblastic activity, reflecting the bird’s need to have a well-ossified skeleton just before flight, given their relative fragility and the stresses that they will have to withstand. The mobilisation of Ca and P needed for this process means that their concentrations rise in parallel with that of the enzyme (Gallo et al. 2013). In the present study, we found no significant correlation between the age and the concentration of any of these metabolites, unlike those workers, who reported significantly higher concentrations of ALP in young birds (Dobado-Berrios et al. 1998; Polo et al. 1998). The AP concentration is subject to hormonal control and may therefore function as a key mechanism in the regulation of growth in birds. Its plasma levels are influenced by the nutritional status of the birds and can vary rapidly in response to the availability of food. After a period of fast, there is a delay in the re-establishment of the normal levels of this enzyme. Our sample included only active and physically healthy birds, and no abnormalities or evidence of disease were noted during physical examination; thus, differences are likely not clinically important and may be attributable to age variations in sampled birds. RWL had lower values of ALT than those reported for other bird species (Rodríguez et al. 2004; Puerta et al. 1995; Rubio et al. 2012). This enzyme has limited clinical value in birds because it can be increased by pathologic changes in almost all tissues (Gallo et al. 2013), thus hindering result interpretation. Ca and P are essential for the formation and maintenance of the skeleton and together constitute most of the mineral content of the avian body (Blair 2008). Ca levels in RWL were similar to those reported for captive double-crested cormorant (Gallo et al. 2013), quails (Scholtz et al. 2009), sparrows (Puerta et al. 1995) and lower than those found in redlegged partridges (Rodríguez et al. 2004). Previous studies in free-living birds report lower Ca levels in adults than in fledglings (Gallo et al. 2013). However, such age differences are not seen in all avian species (Rodríguez et al. 2004; Scholtz et al. 2009). Levels of P were similar to those reported for common coots (Rubio et al. 2012) but lower than those described for free-ranging flightless cormorant (Travis et al. 2006) and


sparrows (Puerta et al. 1995). Phosphorus levels are known to vary widely and are also commonly elevated in young, growing animals (Gallo et al. 2013; Rubio et al. 2012). Differences in Ca and P levels may be attributable to age variations in sampled birds (Travis et al. 2006; Newman et al. 1997), to dietary differences or the capture and restraint methods used, or any combination thereof. In summary, our results provide reliable haematologic reference values for this species. Such reference values will be important to evaluate the health status of RWL and other related species thus allowing for better veterinary care and management of their population. Moreover, haematologic exams will be important tools in the case of potential release candidates for reintroduction programmes in order to avoid the release of sick birds which could carry infectious agents to immunologically naive wild populations. Acknowledgments We gratefully acknowledge Tamoor Azeem and Salman Ahmed Abid for their contributions in the field. We also thank the Department of Wildlife and Ecology, University of Veterinary and Animal Sciences, Lahore, for technical support. The authors gratefully acknowledge the fellowships to Sajid Umar from the Higher Education Commission (HEC), Pakistan.

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