Evidence of Clinicopathological Changes during Equine Leptospirosis

Muhammad Luqman Sohail1,*, Muhammad Sarwar Khan1, Muhammad Ijaz1, Muhammad Avais1, Muhammad Yasir Zahoor2, Omer Naseer1 and Muhammad Usman Saleem3

1Department of Clinical Medicine and Surgery, University of Veterinary and Animal Sciences, Lahore, Pakistan

2Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan

3Department of Biosciences, Faculty of Veterinary Sciences, Bahauddin Zakariya University, Multan, Pakistan

ABSTRACT

Leptospirosis is a globally spread zoonotic disease effecting a wide range of animal species and humans. 18 Leptospira positive horses based on ELISA and 9 healthy horses were included in the study. Blood profile, serum biochemistry and mineral profile were analyzed at day 0, 7 and 21 of infection. Results showed significant decrease (P<0.05) in red blood cells (RBC), packed cell volume (PCV), hemoglobin (Hb), mean corpuscular hemoglobin concentration (MCHC) and platelets while total leucocytes count (TLC), neutrophils, eosinophils, basophils, lymphocytes, monocytes, red cell distribution width (RDW) and mean corpuscular volume (MCV) were increased significantly (P < 0.05) which indicated hemolytic anemia. While, serum biochemistry showed significant elevation in alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN) and creatinine which indicate renal and liver damage. Concentration of Na, K, Ca, Mg, P, Cu and zinc in serum were significantly decreased (P< 0.05), indicating tubular damage in kidneys.

Article Information

Received 05 October 2015

Revised 13 October 2016

Accepted 26 November 2016

Available online 27 April 2017

Authors’ contribution

MSK, MA and MI designed the experiment. MYZ and MLS executed the experiment. MUS and ON analysed the data and wrote the manuscript.

Key words

Hemolytic anemia, Liver damage, Proximal tubular damage, Pulmonary hemorrhagic syndrome, Wet digestion.

DOI: http://dx.doi.org/10.17582/journal.pjz/2017.49.3.849.853

* Corresponding author: vet.luqman@gmail.com

0030-9923/2017/0003-0849 $ 9.00/0

Copyright 2017 Zoological Society of Pakistan

INTRODUCTION

Leptospirosis is globally spread infectious disease which infects a variety of domestic and wild animals such as sheep, horse, cattle, dog, raccoon, pig, skunk, and even humans are not the exception (Kojouri et al., 2009; Saleem et al., 2013). It is caused by spirochetes Leptospira interrogans along with its more than 260 serovars (Tonin et al., 2012a). Prevalence of leptospiral serovars varies in a particular geographic region and for every serovar there exists one or more than one maintenance hosts which serve as the reservoir of infection (Balamurugan et al., 2013). It occurs sporadically throughout the year in these areas, with the highest incidence in warmer months (Tilahun et al., 2013), marked by heavy rainfall (Himani et al., 2013; Hamond et al., 2014). In milder cases horses may have fever, anorexia and listlessness but severe infection involves jaundice (mostly in foals, rarely in adult horses), anemia, conjuctival suffusions, pathechial hemorrhage on the mucosa and hematuria (Verma et al., 2013; Bauverud et al., 2009). Leptospirosis provokes clinical abnormalities that can lead to significant changes in hematological and other laboratory findings. Decreased erythrocyte count, low Hb concentration, PCV and lymphocytes are common lab findings (Tonin et al., 2012b). Leucocytosis, neutrophilia, eosinopenia, and lymphopenia, along with high serum bilirubin concentration have also been reported in equine leptospirosis (Pinna et al., 2013). Liver function tests revealed elevated levels of serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST) (Tonin et al., 2012 a). A comprehensive understanding of hematological, biochemical and mineral alterations is necessary to study the systemic effects and clinical pathology of leptospiral infection in horses, so present study was conducted to mark changes in clinicopathological parameters during equine leptospirosis.

Material and Methods

For the current study 18 leptospirosis ELISA positive horses were selected, along with 9 healthy horses (n=27). Routine feed and fresh water were provided ad-libitum during the experiment. Formal permission from the Ethical Review Committee for the Use of Laboratory Animals, University of Veterinary and Animal Sciences, Lahore were taken.

Three ml blood was drawn from jugular venepuncture in each of plain (red top) and anticoagulant coated (purple top) vacutainers, by 20 gauge needle after disinfecting the collection site with alcohol swab. Blood collected in anticoagulant coated vacutainer was stored for hematological analysis and in plain vacutainers for biochemical and mineral analyses. Serum was immediately separated and refrigerated at -20°C till analyzed. Clinical pathological parameters including hematology, liver and renal function tests and serum mineral profile were analyzed at day 0, 7 and 21. Hematological parameters; Hemoglobin concentration (Hb), Hematocrit (PCV), Erythrocyte count, Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Red Cell Distribution Width (RDW), Total Leukocyte Count (TLC), Differential Leukocytic Count (DLC), and platelet count were measured by automated hematological analyser (Abacus junior vet 5, Diatron, Austria). Serum biochemical parameters, alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TB), blood urea nitrogen (BUN), and creatinine were analyzed using Pictus 700 chemistry analyzer (Diatron, Hungary).

Serum quantity of minerals (Cu, Zn, Na, K, Mg, Ca and P) was also determined at day 0 7, 21 and 35. Serum levels of zinc and copper were estimated by the method described by (Ali et al., 2013). Briefly, for wet digestion, a quantity of 0.5 mL serum and 5 mL nitric acid were taken in a digestion flask and was placed on hot plate at 90°C for 15 minutes until 2 ml material was left over. After cooling, 2.5 ml perchloric acid was added and heated until the volume was reduced to 2 ml, which was then stored in plastic bottles. These digested samples were used for the estimation of copper by atomic absorption spectrophotometer (Perkins Elmer Analyst 800, USA). Serum zinc level was estimated by using commercial kits in chemistry analyzer.

Determination of serum levels of sodium, potassium, magnesium, calcium, and phosphorus was done by automated blood chemistry analyzer hitachi 705 (Hitachi, Japan), using Thermo Fisher Scientific Inc. (USA) reagents.

Statistical analysis was done by using statistical package for social sciences (SPSS 16.0, SPSS, Chicago, USA). by one way ANOVA. The differences between diseased and healthy horses at different days were analyzed by Tukey’s test (P value< 0.05).

Results and Discussion

Haemolytic parameters

A comprehensive analysis of hematological parameters, liver and renal function indicators and mineral profile was performed at day 0, 7 and 21 of diseased and healthy horses to mark the changes in them. All the studied parameters were altered significantly at day 0, 7 and 21 (P<0.05). Table I shows changes in hematological parameters in diseased horses.RBC, Hb, PCV, MCHC and platelets showed significant decrease (P<0.05), while MCV, RDW, TLC, DLC increased significantly (P<0.05) during the course of disease.

Table I.- Hematological changes in diseased and healthy horses.

Parameters | Unit | Week 1 | Week 3 | Week 5
Diseased | Healthy | Diseased | Healthy | Diseased | Healthy
RBC | 10 12 | 3.26 ±03* | 5.89 ±0.04 | 3.28 ±0.03* | 5.88 ±0.03 | 3.27 ±03* | 5.84 ±0.03
Hb | g/dl | 7.24 ±0.02* | 11.87 ±0.03 | 7.21 ±0.02* | 11.89 ±0.39 | 7.22 ±0.02* | 11.93 ±0.04
PCV | % | 24.62 ±0.03* | 37.72 ±0.03 | 24.59 ±0.02* | 37.75 ±0.04 | 24.60 ±0.03* | 37.84 ±0.05
MCV | Fl | 58.47 ±0.03* | 41.42 ±0.04 | 58.41 ±0.03* | 41.44 ±0.03 | 58.48 ±0.03* | 41.53 ±0.04
MCH | Pg | 23.74 ±0.03* | 15.15 ±0.3 | 23.78 ±0.03* | 15.17 ±0.03 | 23.76 ±0.03* | 15.20 ±0.04
MCHC | g/l | 25.12 ±0.03* | 36.21 ±0.03 | 25.16 ±0.04* | 36.41 ±0.03 | 25.20 ±0.03* | 36.54 ±0.03
RDW | %cv | 27.04 ±0.03* | 19.35 ±0.04 | 27.11 ±0.03* | 19.37 ±0.03 | 26.94 ±0.03* | 19.45 ±0.04
TLC | 10 9 | 14.42 ±0.04* | 7.23 ±0.03 | 14.35 ±0.03* | 7.25 ±0.04 | 14.52 ±0.04* | 7.27 ±0.03
Neutrophils | % | 57.32 ±0.04* | 41.77 ±0.04 | 57.23 ±0.04* | 41.72 ±0.03 | 57.39 ±0.04* | 41.79 ±0.03
Basophils | % | 1.12 ±0.03* | 0.39 ±0.03 | 1.11 ±0.02* | 0.38 ±0.03 | 1.11 ±0.03* | 0.39 ±0.03
Eosinophils | % | 21.57 ±0.04* | 11.21 ±0.08 | 21.44 ±0.03* | 11.19 ±0.07 | 21.61 ±0.04* | 11.27 ±0.02
Lymphocyte | % | 64.81 ±0.03* | 45.61 ±0.03 | 64.88 ±0.04* | 45.63 ±0.03 | 64.74 ±0.03* | 45.75 ±0.03
Monocytes | % | 4.37 ±0.03* | 1.23 ±0.03 | 4.35 ±0.04* | 1.25 ±0.02 | 4.31 ±0.03* | 1.27 ±0.03
Platelets | 10 3 | 145.21 ±3.85* | 185.95 ±6.8 | 145.15 ±3.86* | 184.84 ±7.06 | 145.33 ±3.8*5 | 185.78 ±6.51

*Significant difference between diseased and healthy horses (P < 0.05). Hb, hemoglobin; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; PCV, packed cell volume; RBC, red blood cells; RDW, red cell distribution width; TLC, total leukocyte count.

These findings were in agreement with the studies of Pinna et al. (2013) and Oliviera et al. (2013), who reported a decrease in all mentioned parameters, because of hemolytic anemia caused by equine leptospiral infection. Same results were reported in dogs (Goldstein et al., 2006) and rats (Tonin et al., 2012). According to literature (Tonin et al., 2012), leptospirosis causes hemolytic anemia and capillary damage because of the hemolysin expressed by the bacteria. This caused a decrease in RBC, Hb, PCV, MCHC and increase in MCV and RDW. Cottle and Hughes (2010) reported same hematological alterations for hemolytic anemia, which is a frequent finding for equine leptospirosis.

Hematological analysis further revealed a significant increase in TLC and DLC, which is similar to the studies reported by (Oliviera et al., 2013) who reported same findings in equine leptospirosis, because leptospirosis is a bacterial disease and cause leucocytosis to combat bacterial infection (Goldstein et al., 2006; Lather et al., 2009; Pinna et al., 2010). The platelets were lesser in infected horses as compared to healthy ones which might be because of hemorrhages caused by the organism (Adler and Moctezuma, 2010) as pulmonary hemorrhage syndrome has been reported in Leptospira infected animals (Levett, 2011), humans (Amilasan et al., 2012) and dogs (Birnbaum et al., 1998).

Table II shows that liver enzymes; ALT, AST, ALP and TB increased significantly (P<0.05) at day 0, 7 and 21 in infected horses. Renal function parameters i.e. BUN and creatinine also showed significant increase (P<0.05) in diseased horses.

Same findings were reported by Pinna et al. (2010) and Adler and Moctezuma (2010) because of hepatocellular damage, caused by the leptospiral toxins during leptospiremia (Birnbaum et al., 1998). Levette (2001) reported elevated bilirubin and liver enzyme levels during leptospirosis which required many weeks for normalization which is in line with our findings.

BUN and creatinine were significantly increased because of renal damage caused by Leptospira (Sitprija, 2008). Adler and Moctezuma (2010) declared kidney as the colonization site during leptospirosis which may have caused renal damage, leading to increased BUN and creatinine in our study. Same abnormal changes in renal functions were reported by (Zala et al., 2014) during human leptospirosis.

Serum mineral profile was also analyzed during leptospiral infection and is presented in Table III. Sodium, potassium, calcium, magnesium, phosphorus, copper and zinc were decreased significantly (P<0.05) in horses with equine leptospirosis as compared to healthy horses.

Table II.- Serum biochemistry of diseased and healthy horses.

Parameters | Unit | Day 0 | Day 7 | Day 21
Diseased | Healthy | Diseased | Healthy | Diseased | Healthy
ALT | U/L | 33.67 ±0.92* | 25.37 ±0.52 | 33.62 ±0.75* | 25.31 ±0.55 | 33.59 ±0.92* | 25.39 ±0.50
AST | U/L | 263.65 ±4.94* | 227.95 ±1.99 | 263.81 ±4.8* | 227.89 ±2.02 | 263.57 ±4.94* | 227.85 ±1.94
ALP | U/L | 552.31 ±4.39* | 492.31 ±2.44 | 551.91 ±4.15* | 492.24 ±2.35 | 552.34 ±4.39* | 492.39 ±2.36
TB | mg/dl | 1.32 ±0.04* | 0.87 ±0.04 | 1.35 ±0.03* | 0.85 ±0.03 | 1.39 ±0.04* | 0.88 ±0.04
BUN | mg/dl | 37.41 ±0.54* | 29.19 ±0.90 | 37.35 ±0.48* | 29.27 ±1.10 | 37.42 ±0.54* | 29.41 ±0.67
Creatinine | mg/dl | 1.98 ±0.04* | 1.21 ±0.03 | 1.95 ±0.03* | 1.19 ±0.03 | 1.94 ±0.04* | 1.18 ±0.03

*Significant changes between diseased and healthy horses (P<0.05). ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; TB, total bilirubin; BUN, blood urea nitrogen.

Table III.- Serum mineral profile in diseased and healthy horses.

Parameters | Unit | Day 0 | Day 7 | Day 21
Diseased | Healthy | Diseased | Healthy | Diseased | Healthy
Sodium | mmol/l | 99.20 ±0.61* | 137.78 ±0.72 | 98.21 ±0.78* | 136.35 ±0.81 | 99.28 ±0.61* | 137.91 ±0.61
Potassium | mmol/l | 2.12 ±0.04* | 4.11 ±0.06 | 2.41 ±0.05* | 4.07 ±0.06 | 2.13 ±0.04* | 4.12 ±0.05
Calcium | mmol/l | 2.51 ±0.02* | 3.12 ±0.01 | 2.54 ±0.02* | 3.15 ±0.02 | 2.56 ±0.02* | 3.14 ±0.02
Magnesium | mmol/l | 0.41 ±0.03* | 0.780 ±0.05 | 0.39 ±0.03* | 0.74 ±0.03 | 0.44 ±0.03* | 0.77 ±0.03
Phosphorus | mmol/l | 0.62 ±0.02* | 1.12 ±0.04 | 0.67 ±0.03* | 1.09 ±0.04 | 0.59 ±0.02* | 1.15 ±0.04
Copper | mmol/l | 11.25 ±0.55* | 18.45 ±0.44 | 11.42 ±0.60* | 18.43 ±0.42 | 11.29 ±0.55* | 18.41 ±1.10
Zinc | mmol/l | 7.21 ±0.25* | 14.08 ±0.26 | 7.31 ±0.19* | 14.15 ±0.25 | 7.17 ±0.25* | 14.14 ±0.22

* Significant changes between diseased and healthy horses (P<0.05).

These clinico-pathological changes were notable during the course of disease and were consistent till the 21st day of analysis.

This is in agreement with the findings of Sitprija (2008) who reported low level of Ca, Mg, and P because of their abnormal excretion due to renal damage during leptospirosis. Low levels of Na and K were observed in our study, which favors the findings of Cerqueira et al. (2008) because of tubular defects in acute renal failure during leptospirosis, which may require recovery time of up to 6 months. Andrade et al. (2007) also reported Leptospira induced dysregulation of sodium transporters in kidneys. Tubular defects have been considered to be occurred because of inhibition of Na+/K+ ATPase. Na+/K+ pump is the driving force for reabsorption for potassium, which is inhibited by glycolipoproteins present in Leptospira (Ibrahim et al., 1997). This inhibitory effect causes potassium loss leading to lower potassium levels during leptospirosis. Sitprija (2008) described low levels of sodium as a sequel of increased levels of ADH during leptospiral invasion. Spichler et al. (2008) reported low levels of magnesium, same as found in our study, because of direct effect of leptospiral biochemical contents on proximal tubule and loop of Henle function. Same findings of low Na, K and Mg were reported in human leptospirosis by Araujo et al. (2010) because of proximal tubular injury, as this portion of nephron is main target for leptospiral colonisation. Proximal tubular injury decreases sodium and water reabsorption and increases sodium and water transport to the distal nephron, which may increase potassium excretion causing hypokalemia (Russell, 2000). Sitprija (2008) further reported decreased copper and zinc levels during tropical diseases as malaria and leptospirosis which is in agreement with the findings of our study. Low copper levels, indicating compromised immunity was reported in bovine leptospirosis by Mohammadi and Sakhaee (2015).

CONCLUSION

It is concluded that hemolytic anemia, liver and renal tubular damage impart significant clinico-pathological changes in hematology, serum biochemistry and mineral profile during equine leptospirosis.

ACKNOWLEDGMENTS

This work was completed in partial fulfillment of the requirements for the PhD degree in Clinical Medicine and Surgery at University of Veterinary and Animal Sciences Lahore, Pakistan. We are thankful to Higher Education Commission (HEC), Islamabad for the financial support to conduct this study (PIN no. 112-26696-2AV1-030).

Statement of conflict of interest

The authors declare that there is no conflict of interests regarding the publication of this article.

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