Indian Journal of Animal Research

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Indian Journal of Animal Research, volume 56 issue 12 (december 2022) : 1552-1556

Gross and Histopathological Findings of Naturally Occurring Anaplasma marginale Infection in Cattle

Dhruba Das1, Kalyan Sarma1,*, Parimal Roychoudhury1, G.E. Chethan1, R. Ravindran1, Sikdar Jabidur Islam1, H. Prasad1, J.B. Rajesh1, Biswadeep Behera1, Farhin Aktar Choudhury1
1College of Veterinary Sciences and Animal Husbandary, Central Agricultural University, Selesih, Aizawl-796 014, Mizoram, India.
Cite article:- Das Dhruba, Sarma Kalyan, Roychoudhury Parimal, Chethan G.E., Ravindran R., Islam Jabidur Sikdar, Prasad H., Rajesh J.B., Behera Biswadeep, Choudhury Aktar Farhin (2022). Gross and Histopathological Findings of Naturally Occurring Anaplasma marginale Infection in Cattle . Indian Journal of Animal Research. 56(12): 1552-1556. doi: 10.18805/IJAR.B-4283.
Background: Bovine anaplasmosis is a haemolytic disease of cattle caused by an obligate intra-erythrocytic bacterium Anaplasma marginale and is characterized by high fever, dyspnoea, anaemia, icterus, decreased milk production and death. The present study was undertaken to study the gross and histopathological changes caused by naturally occurring A. marginale infection in cattle. 

Methods: History and clinical examination of animals revealed inappetance/anorexia, presence of tick infestation, high fever, pale or icteric mucous membranes, tachycardia, dyspnoea and lymphadenomegaly. The diagnosis of A. marginale infection was done by blood smear examination and further confirmed by polymerase chain reaction and sequencing analysis. During the present study, two animals did not respond to treatment and were succumbed to A. marginale infection. A systematic post-mortem examination was performed on the animals and gross lesions were recorded. The tissue samples from various vital organs such as heart, lungs, liver, spleen and kidneys were collected in 10% neutral buffered formalin and processed for histopathological examination. 

Result: The post-mortem examination revealed emaciated carcass with yellowish discoloration of serosal surfaces of the abdominal organs, haemorrhagic heart, congestion of lungs, hepatomegaly, distension of the gallbladder and splenomegaly. The major histopathological changes noticed included myocardial degeneration and necrosis, interstitial pneumonic changes, degenerative changes in hepatocytes and renal tubular epithelial cells, enlargement of red pulp area of spleen with histiocytic proliferation and hemosiderosis. Histopathology also revealed inflammatory process characterized by infiltration of mononuclear cells in various organs. Very few reports are available on the pathological aspects of A. marginale infection and thus the outcome of the present study can yield valuable information to aid in the diagnosis of bovine anaplasmosis under field conditions.
Bovine anaplasmosis is a haemolytic disease of cattle caused by an obligate intra-erythrocytic bacterium Anaplasma marginale and is characterized by high fever, dyspnoea, anaemia, icterus, decreased milk production and death (Zivkovic et al., 2007; Spare et al., 2020). It is an economically important disease which is common throughout tropical and sub-tropical regions of the world including India (Kocan et al., 2010). A. marginale can be transmitted biologically by ticks and mechanically by blood-sucking arthropods or blood-contaminated fomites (Battilani et al., 2017). A. marginale is host-specific and cattle and water buffaloes are highly susceptible to infection (Yang et al., 2017). Purebred animals and dairy cows are very susceptible to per-acute anaplasmosis and death occurs within hours of the onset of clinical signs (Kocan et al., 2010). In acute infection, A. marginale invades red blood cells and cause destruction of red blood cells resulting in anaemia and even death in severe cases. Very few published reports are available regarding gross and histopathological changes caused by A. marginale infection. So, the present research was undertaken to study the gross and histopathological changes caused by naturally occurring A. marginale infection in cattle.
Study area
 
The study was conducted in the Aizawl district of Mizoram.
 
Study animals
 
Six cattle suspected to be affected with haemoprotozoan or rickettsial infections were included in the present study. History and clinical examination of animals revealed inappetance/anorexia, presence of tick infestation, high fever, pale or icteric mucous membranes, tachycardia, dyspnoea and lymphadenomegaly.
 
Collection of blood samples
 
The blood samples (approximately 3.0 mL) were collected from each cattle by venipuncture of jugular vein into vials with dipotassium ethylenediaminetetraacetic acid (K2 EDTA).
 
Diagnosis
 
Blood smear examination
 
Blood smears (including peripheral) were prepared on clean grease free glass slides and fixed with methanol for 1 min. The smears were stained with Giemsa stain as per the standard procedure (Jain, 1986) and examined under oil immersion objective for the presence of any haemoprotozoan or rickettsial infection.
 
Polymerase chain reaction (PCR)
 
Around 0.5 mL of blood was used for deoxyribonucleic acid (DNA) extraction. Total genomic DNA from all the collected blood samples was extracted using commercially available DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) as per manufacturer’s instructions. The extracted DNA was eluted in 100 μL elution buffer and stored at -20°C until further analysis. The DNA amplification for the diagnosis of Anaplasma spp. infection was carried out by using oligonucleotide PCR primers [Ana-rpoBF (5'GCTGTTCCTA GGCTYTCTTACGCGA3') and Ana-rpoBR (5'AATCRAGCC AVGAGCCCCTRTAWGG3')] targeting 576 bp of the RNA polymerase beta subunit (rpoB) gene as per the method described earlier (Dahmani et al., 2017). The PCR-amplified products were resolved on 1.5% agarose gel in Tris-acetate-EDTA (TAE) buffer. The agarose gel was visualized under ultraviolet (UV) light in a gel documentation system (Molecular Imager® Gel Doc™ XR+System, Bio-Rad, USA).
 
Collection of tissue samples
 
During the present study, two animals were succumbed to infection. A systematic post-mortem examination was performed on the animals and important gross lesions were recorded. The tissue samples from various vital organs such as heart, lungs, liver, spleen and kidneys were collected in 10% neutral buffered formalin and processed for histopathological examination. The tissue samples were processed by paraffin sectioning method as described by Luna (1968). The paraffin sections of 6-8 micron thickness were cut and stained by routine Mayer’s haematoxylin and eosin as per the method described by Drury and Wallington (1980) and analyzed by using light microscope.
Diagnosis and treatment of A. marginale infection
 
The blood samples were found positive for Anaplasma spp. infection by using microscopic examination of blood smears (Fig 1A). It was again confirmed by PCR, as evident by the presence of expected 576 bp product in agarose gel under UV transilluminator (Fig 1B). Partial gene sequencing of rpoB gene revealed that the samples are identical (99.2-100%) to other sequences of A. marginale available at GenBank (GenBank accession number: MT755603 and MT755604). The blood samples were found negative for commonly occurring haemoprotozoan diseases such as babesiosis, theileriosis and trypanosomosis. All the animals were treated with intravenous oxytetracycline at the dose rate of 10 mg/kg, daily for 3 days along with supportive therapy comprising anti-pyretic, multivitamins and haematinics. Intravenous oxytetracycline can be used as an effective drug against bovine anaplasmosis (Radostits et al., 2007). Unfortunately, two animals did not respond to treatment and succumbed to infection after two days of initiation of therapy. The primary effect of A. marginale infection is the induction of extravascular hemolytic anaemia and the resultant anaemic anoxia is presumed to be the cause of death (Abba et al., 2016). The remaining four animals showed uneventful recovery.
 

Fig 1


 
 
Gross lesions
 
A varying degree of changes were noticed in different organs of the body. Gross pathological observation revealed emaciated carcass with yellowish discoloration of serosal surfaces of the abdominal organs (Fig 2A). Icteric condition observed in the present study could be due to excessive destruction of red blood cells and similar findings were also reported by previous reports (Jaswal et al., 2015; Abba et al., 2016). Paintbrush haemorrhages and severely congested blood vessels on epicardial surface (Fig 2B) and presence of endocardial haemorrhages (Fig 2C) were the major gross findings noticed in heart. These findings are in consistent with the reports published earlier (Coetzee et al., 2005). The post mortem examination also revealed presence of severely congested lungs (Fig 2D), hepatomegaly with blunt edges and necrosis (Fig 2E), splenomegaly with discoloration (Fig 2F) and distended gall bladder with dark colored bile (Fig 2G). Pulmonary congestion, distension of gallbladder, enlargement of liver and spleen has been documented in both natural and experimental bovine anaplasmosis (Jaswal et al., 2015; Abba et al., 2016; Lima et al., 2019). Splenomegaly with congestion and discoloration reported in the present study may be due to erythrophagocytosis that results in excessive destruction of defective erythrocytes by macrophages within the spleen (Devos et al., 2006). No gross lesions were observed in kidney (Fig 2H). A dead foetus was found in the uterus of one animal (Fig 2I), whereas the other animal had a recent history of abortion. Deprivation of oxygen due to excess haemolysis may cause foetal death and abortion in pregnant cows (Whittier et al., 2009).
 

Fig 2


 
Histopathological observations
 
Histological examination of heart revealed presence of haemorrhage in the myocardium along with degeneration and necrosis (Fig 3A). These findings may be secondary to severe anaemia induced by A. marginale infection and similar findings were also documented in previous reports (Jaswal et al., 2015). Examination of lung tissues showed interstitial pneumonic and oedematous changes, thickening of interalveolar septum and infiltration of mononuclear cells (Fig 3B). Degeneration and necrosis of hepatocytes, congestion and haemorrhage, mild infiltration with mononuclear cells and enlargement of sinusoids were the major findings noticed in liver tissues (Fig 3C). Hepatic necrosis was more prominent around the central vein. Hepatic necrosis is also an important feature of bovine anaplasmosis and is generally more severe than in babesiosis (Devos et al., 2006). Histological examination of spleen revealed enlargement of red pulp area, histiocytic proliferation with hemosiderosis, serofibrinous exudation in the red pulp area and lymphoid proliferation in white pulp area (Fig 4A). Accumulation of hemosiderin pigment in the cells of reticuloendothelial system has already been reported in bovine anaplsmosis (DeVos et al., 2006). Centrilobular hypoxic hepatic necrosis and splenic hemosiderosis may be due to enhanced destruction of red blood cells (Anderson and Hurtado, 1989). Splenic changes are similar to those occurring in babesiosis except that there is generally more extensive accumulation of plasma cells in the red pulp in case of anaplasmosis (DeVos et al., 2006). Tissue section of kidneys obtained from A. marginale infected cattle showed degeneration and necrosis of tubular epithelial cells and infiltration of mononuclear cells in the interstitial space of renal parenchyma (Fig 4B). Congestion and haemorrhages were also observed. In overall, the histopathological changes observed in the present study are in agreement with previous reports (Jaswal et al., 2015; Abba et al., 2016; Lima et al., 2019). It is well known that anoxia causes degenerative changes and necrosis in parenchymatous organs. During A. marginale infection, the oxygen content of the blood is lowered due to less haemoglobin content which could lead to degenerative changes in liver, spleen and kidney.
 

fig3


 

fig4

A study on the pathological changes caused by A. marginale infection in cattle was undertaken. The major gross changes included hepatomegaly, distension of the gallbladder, pulmonary congestion, splenomegaly and haemorrhagic heart. The histopathological alterations included myocardial degeneration and necrosis, interstitial pneumonic changes, degenerative changes in hepatocytes and renal tubular epithelial cells, enlargement of red pulp area of spleen with histiocytic proliferation and hemosiderosis. Very few reports are available on the pathological aspects of A. marginale infection. Therefore, the outcome of the present study can yield valuable information to aid in the diagnosis of bovine anaplasmosis under field conditions.

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