Study of hemato-biochemical changes in cases of anaplsmosis in cattle

Introduction

In Indian economy livestock plays an important role and contributes up to 4.11 per cent in Indian GDPA. Total of 20.5 million rural residents depend on cattle for their livelihood. (DAHDF, 19 th Livestock Census 2012 report, 2014). Many viral, metabolic, and parasitic diseases concern India due to climate differences in tropical and subtropical countries. The global loss due to Ticks and Tick-Borne Diseases (TTBDs) is estimated to be between US$ 13.90 and 18.70 billion annually (De Castro, 1997) [9] , while in India the cost of controlling TTBDs has been estimated as US$ 498.7 million/annum (Minjauw and McLeod, 2003) [16] . The most common haemoprotozoan infections in animals are trypanosomosis, theileriosis, babesiosis, and anaplasmosis. Bovine anaplasmosis is considered as one of the economically important rickettsial diseases affecting ruminants. In India, A. marginale, A. centrale, and A. bovis are the prevalent species in cattle (Nair et al., 2013) [17] and they are principally transmitted by a tick of Rhiphicephalus spp. (Constable et al., 2017) [7] . Bovine anaplasmosis, formerly known as gall sickness, is an infectious, non-contagious, tick-borne disease of domesticated and wild ruminants caused by Anaplasma species characterized by fever, icterus, anorexia, anaemia, constipation, weakness, dehydration, laboured breathing, depression, abortion, and often death (Richey and Palmer, 1990) [20] . Diagnosis can be done by Giemsa stained blood smear examination (Maharana et al. 2016) [14] . Diagnosis based on microscopic examination is not reliable for detecting pre-symptomatic or carrier animals. In these cases, the infection is usually detected by presence of antibodies on a serologic test with confirmation by molecular detection methods such as Complement Fixation Test (CFT), Capillary Agglutination Assay, Card Agglutination Test (CAT), Indirect Fluorescent Antibody (IFA) Enzyme Linked Immunosorbent Assays (ELISA) and PCR (Aubry and Geale, 2011) [5] , (Carelli et al., 2007) [6] .

Materials and Methods

The present investigation was carried out at the Department of Veterinary Medicine, College of Veterinary Science and Animal Husbandry, Anand with the objectives to study haemato-biochemical changes in cattle affected with anaplasmosis. Total 66 Giemsa's-stained thin blood smears were examined from cattle suspected for anaplasmosis. Out of them, 22 (33.34%) cattle were found positive by microscopic examination seen as compact spherical masses, inside the red blood cells near the periphery of RBCs wall. Out of 66 samples 34 (51.52%) samples were found positive for anaplasma species by PCR. Out of those 18 animals were selected for hematobiological examination.

Sample Collection and Storage

Whole blood samples were collected from the jugular veins of the suspected animals in the vacutainers containing an anticoagulant, tripotassium ethylene diamine tetra-acetic acid (K3EDTA) and serum clot activators on day '0' from selected cases. The similar samples were also collected from cattle under healthy group. These blood samples were kept in ice box, transported to the lab within 2-3 hours, and processed on the same day. Blood samples taken in serum clot activator vials were centrifuged at 3000 rpm for 10 minutes. The sera samples were separated/aspirated with sterile pipette and were stored at -20 º C until final use for estimation of various biochemical parameters.

Haematological Examination

Haematological analysis of samples was performed from the whole blood collected from the jugular vein of healthy and infected cattle in a K3EDTA vacutainers. Estimation of haematological parameters was done by using autohaematological analyzer machine (Abacus junior Vet-5) at Veterinary Clinical Complex, which included following parameters.

Serum Biochemical Examination

All the biochemical parameters were analyzed by using standard procedures and assay kits on clinical chemistry analyzer (CKK 300) at Veterinary Clinical Complex as per manufacturer's instructions. The serum samples obtained from blood collected on day 0 from cattle affected with anaplasmosis and stored deep frozen were used for the estimation of following biochemical parameters.

Result and Discussions 3.1 Hematological Examination

In present study, whole blood samples were collected from all 18 cattle affected with anaplasmosis and a healthy group (n=6) on day 0 to determine the haematological profile. The mean (±SE) values of various haematological parameters and indices of cattle affected with anaplasmosis are shown in Table 3.1 and Figure 3.2 and are discussed in detail as follows. In present study, the mean value of haemoglobin recorded in healthy cattle was 11.75±0.76 g/dl and in cattle with anaplasmosis it was 6.23±0.20 g/dl. The level of haemoglobin was highly significantly (P<0.001) decreased in anaplasmosis affected cattle (Table 3.1, Figure 3.1). Present findings were in accordance with Shaukat et al.

(2019) [23] . Similar observations were also reported by Coskun et al. (2012) [8] , Sharma et al. (2013) [22] , Singh et al.

(2014) [24] , Meenakshisundaram et al. (2014) [15] , Maharana et al. (2016) [14] , Ganguly et al. (2018) [11] , Subramanian et al. (2019) [25] , and Ola fadunsin et al. (2021) [19] . A significant reduction in the haematological parameters (Hb, PCV, RBC, and MCHC) might be due to the intravascular haemolysis of erythrocytes, increased erythrocyte phagocytosis by the reticuloendothelial system, and restricted erythropoietic activity in the bone marrow, which is a result of A. marginale infection (Sharma et al., 2013) [22] .

In [24,14,15,25,10,11] . The decreased value of PCV might be due to intravascular haemolysis of erythrocytes by Anaplasma spp. which invade and cause erythrophagocytosis. (2019) [23] . who also reported that there were significantly (P<0.05) decreased level of total erythrocyte count in anaplasmosis affected cattle as compared to healthy animals. Present findings were also in accordance with Coskun et al.

Total erythrocyte count (TEC; x10

(2012) [8] , Arunkumar and Nagarajan (2013) [4] , Sharma et al. (2013) [22] , Meenakshisundarm et al. (2014), Singh et al.

(2014) [24] , Maharana et al. (2016) [14] , Szabara et al. [10,11,25] . The decrease level of total erythrocyte might be due to erythrophagocytosis by reticuloendothelial system because of intravascular haemolysis of erythrocyte which was initiated by parasitic damage to erythrocytes and that leads to decrease in total erythrocyte count (Sharma et al., 2013) [22] .

The mean±SE value of mean corpuscular haemoglobin concentration (MCHC) recorded in healthy cattle was 34.37±0.74 g/dl and in cattle with anaplasmosis it was 31.08±0.55 g/dl. The level of mean corpuscular haemoglobin concentration was decreased highly significantly (P<0.004) in anaplasmosis affected cattle (Table 3.1, Figure 3.1). The present findings agreed with Ola-Fadunsin et al. (2021) [19] , who reported significantly decreased mean value of MCHC. Present findings were also in accordance with Arunkumar and Nagrajan (2013), Sharma et al. (2013) [22] , Maharana et al. (2016), and Shaukat et al. (2019) [14,23] . The significant decrease in MCHC might be due to intravascular haemolysis of erythrocytes, increased erythrophagocytosis by reticuloendothelial system and subsequently rapid release of immature erythrocytes into circulation (Ola-fadunsin et al., 2021) [19] .

Mean corpuscular volume (MCV; fl)

The mean±SE value of mean corpuscular volume (MCV) recorded in healthy cattle was 43.07±1.62 fl and in cattle with anaplasmosis it was 49.09±0.97 fl. The level of mean corpuscular volume was increased significantly (P<0.011) in anaplasmosis affected cattle (Table 3.1, Figure 3.1). The present findings agreed with Shaukat et al. (2019) [23] , who reported significant (P<0.05) increase in value of MCV in anaplasmosis affected cattle as compared to healthy cattle. Similar observations were also reported by Arunkumar and Nagarajan (2013) [4] , Sharma et al. (2013) [22] , Maharana et al. (2016) [14] , Ola-Fadunsin et al. (2021) [19] . Whereas Ganguly et al. (2018) [11] reported a nonsignificant decrease (MCV of cattle affected with anaplasmosis.

The increase in MCV might be due to rapid haemolysis of erythrocytes which leads to release of immature macrocytic erythrocytes into the circulation.

The mean ±SE value of eosinophils recorded in healthy cattle was 1.43±0.20% and in cattle with anaplasmosis it was 2.03±0.16%. The level of eosinophils was significantly (P<0.041) increased in anaplasmosis affected cattle as compared to healthy cattle (Table 3.1, Figure 3.1). Ganguly et al. (2018) [11] reported that the mean value of eosinophils was increased non-significantly in anaplasmosis affected cattle as compared to healthy cattle, while Hussain et al. (2017) [12] observed a significantly increased mean value of eosinophils in anaplasmosis affected cattle as compared to healthy cattle. However, lymphocytes and platelets count were decreased non-significantly. Furthermore, total leucocytes count (TLC), mean corpuscular haemoglobin (MCH), neutrophils, monocytes and eosinophils increased non-significantly in anaplasmosis affected cattle.

Serum Biochemical Profile

The mean ±SE value of serum albumin recorded in healthy cattle was 3.73±0.19 g/dl and in cattle with anaplasmosis 2.56±0.10 g/dl. It was highly significantly (P<0.001) decreased in anaplasmosis affected cattle (Table 3.2 and Figure 3.2). The present findings were in accordance with Ganguly et al. (2018) [11] and Ola-Fadunsin et al. (2021) [19] who observed significantly decreased albumin level of cattle affected with anaplasmosis. However, Szabara et al. (2016) observed mild hypoalbuminemia in cattle affected with anaplasmosis as compared to healthy group. Furthermore, Hussain et al. (2017) [12] reported no changes in biochemistry in cattle affected with anaplasmosis.

In present study, the mean ±SE value of serum globulin recorded in healthy cattle was 3.57±0.15 g/dl and in cattle with anaplasmosis it was 4.65±0.14 g/dl. The level of globulin was highly significantly increased (P<0.000) in anaplasmosis affected cattle as compared to healthy ones (Table 3.2 and Figure 3.2).

The present findings were in accordance with Nasreldin et al. (2020) [18] , who observed significant increase in the level of globulin in cattle affected with anaplasmosis. Similar finding was also reported by Sharma et al. (2013) [22] . The elevated serum globulin level observed in the A. marginale infected animals could be due to activation of a defensive immune response that leads to an increase in circulating immunoglobulin in the serum (Sharma et al., 2013) [22] .

The mean ±SE value of serum A/G ratio recorded in healthy cattle was 1.05±0.085 and in cattle with anaplasmosis 0.56±0.03 It was highly significantly decreased (P<0.001) in anaplasmosis affected cattle (Table 3.2 and Figure 3.2). The present findings are in accordance with Nasreldin et al. (2020) [18] who observed significantly decreased level of A/G ratio in female cattle affected with anaplasmosis.

In present study, the mean ±SE value of serum bilirubin recorded in healthy cattle was 0.89±0.04 mg/dl and in cattle with anaplasmosis it was 1.84 ±0.08 mg/dl. The level of bilirubin was highly significantly (P<0.000) increased in anaplasmosis affected cattle than in healthy cattle (Table 3.2 and Figure 3.2). The present findings are in consistent with Nasreldin et al. (2020) [18] who reported significant increase in the level of total bilirubin in the anaplasmosis affected cattle in comparison to healthy group. Similar observations were also reported by Coskun et al. (2012) [8] , Sharma et al. (2013) [22] , Jaseem et al. (2018) [11] , and Ola-Fadunsin et al. (2021) [19] . The elevated serum bilirubin levels could be due to haemolysis of parasitized erythrocytes. Additionally, higher serum bilirubin levels have been linked to haemolytic anaemia and hepatic dysfunction (Coskun et al., 2012) [8] .

Aspartate aminotransferase (AST; U/L)

In present study, the mean ±SE value of serum aspartate aminotransferase activity recorded in healthy cattle was 91.31±3.38 U/L and in cattle with anaplasmosis it was 122.72±2.76 U/L. The level of aspartate aminotransferase was highly significantly (P<0.000) increased in anaplasmosis affected cattle (Table 3 [8] , Jaseem et al. [10,11,18] . The increase in AST is related to a hepatic damage or muscular trauma induced by prolonged clinical recumbency in animals. Massive haemolysis may also occur, which when combined with hypoxia, can result in hepatic cell degeneration and glomerular dysfunction, resulting in a rise in AST and ALT enzymes.

Alanine Aminotransferase (ALT; U/L)

The mean±SE value of serum alanine aminotransferase activity recorded in healthy cattle was 35.94±4.24 U/L and in cattle with anaplasmosis it was 51.92±2.15 U/L. The level of alanine aminotransferase was significantly (P<0.010) increased in anaplasmosis affected cattle (Table 3 [10] and Ganguly et al. (2018) [11] and Nasreldin et al. (2020) [18] , who reported significant (P<0.05) increase in the serum alanine aminotransferase in cattle affected with anaplasmosis. However, Alekish and Iamail (2019) observed nonsignificantly increased alanine aminotransferase in anaplasmosis affected cattle. The present finding is conceivable since it is related to a hepatic damage or muscular trauma induced by prolonged clinical recumbency. These tissues might contain a large amount of enzymes that could be released into the bloodstream. (Coskun et al. 2012) [8] . According to the researchers, the rise in enzyme activity might be due to severe anaemia, which causes hypoxia and liver damage. Massive haemolysis may also occur, which, when combined with hypoxia, can result in hepatic cell degeneration and glomerular dysfunction, resulting in a rise in AST and ALT enzymes (Sharma et al. (2013) [22] . However non-significant changes were observed in the level of total protein and creatinine.

Conclusion

The haematological examinations revealed that the Mean±SE values of haemoglobin, packed cell volume, and total erythrocytes count were decreased highly significantly (p<0.01) and mean corpuscular haemoglobin concentration (MCHC) was decreased significantly (p<0.05) in anaplasmosis affected cattle as compared to healthy cattle. The serum biochemical examinations revealed that the Mean±SE values of total protein were decreased nonsignificantly, while albumin and A/G ratio were decreased highly significantly (p<0.01) in anaplasmosis affected cattle as compared to healthy control group. Globulin and alanine aminotransferase were increased significantly (p<0.05), whereas aspartate aminotransferase and bilirubin were increased highly significantly (p<0.01).

Acknowledgement

AcknowledgementThe authors are grateful to the Dean of Collage of Veterinary Science and Animal Husbundary, and authorities of Kamdhenu Universirty for the facilities and encouragement provided for this work.

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