First Report on Molecular Identification of Babesia bigemina in a Nervous Signs Evident Naturally Infected Holstein-Friesian Cattle from Central India

Bovine babesiosis is worldwide distributed and responsible for serious burden on livestock production and human livelihood and is attaining interest due its emerging zoonotic nature (Bock et al., 2004).  Bovine babesiosis is inadequately controlled diseases in various part of the globe. Ixodid ticks are responsible for transmission of babesial parasite to vertebrate host. Agrawal et al., 2017 and Sharma et al., 2013 reported only 2.17% and 2.43% prevalence of babesiosis in cattle in central part and Punjab state of India, respectively.  Blood protozoans have been found to produce nervous disorders in a variety of host viz. cerebral malaria in human caused by Plasmodium falciparum (Cecil and Loeb, 1959), turning sickness in cattle of East Africa, caused by Theileria parva (Mettam and Carmichael, 1936) and cerebral babesiosis in cattle caused by B.bovis (Bock et al., 2004). Economic losses caused by babesiosis in livestock of India are 57.2 million USD annually (Mcleod and Kristjanson, 1999). Depending on the size of merozoites, Babesia species have been grouped in to large (3-5µm) and small form (0.5-2.5 µm). There are different species of Babesia examined from bovine namely B. bigemina, B.bovis, B.divergens and B.major.  In India, B.bigemina is the major species reported from cattle although there are very few reports of B.bovis from India (Shastri et al., 1991 Gautam and Chhabra, 1983 Muraleedharan et al., 1984 Maharana et al., 2016). Intravascular haemolysis and haemoglobinuria is the main pathogenesis caused by B.bigemina while excessive production of cytokinin particularly IL-6, tumor necrosis factor -6 resulting in B.bovis caused cerebral babesiosis (CB). Cerebral babesiosis has been reported mainly by B.bovis and few cases of CB have been reported due to B.argentina. Although, B.argentina is considered as synonym of B.bovis (Mahoney and Mirre, 1977).  Due to the evidence of broader diversity of Babesia species by molecular studies, it is imperative to identify the Babesia species by molecular methods. The thin blood smear is a tradition method but gold standard for diagnosis of babesiosis and other blood protozoans but have limited sensitivity (Kaur et al., 2016). The molecular method using specific primer for PCR based amplification of genomic DNA overcome not only sensitivity issue but also gives confirmatory diagnosis (Singh et al., 2013). This paper highlights the first case of molecular based evidence of cerebral babesiosis caused by Babesia bigemina in naturally infected Holstein Friesian cattle. 

In the year 2015, a Holstein Friesian cattle aged 3 years and weighing 300 kg, owned by a dairy owner was showing high rise of temperature (104°F), lateral recumbency, paddling of legs at frequent interval, convulsion, severe dyspnea, peculiar sound during open mouth breathing, pale color of eye conjunctiva  and mucous membrane of vagina, sever anemic (Fig 1). The previous case history revealed that animal was showing the said clinical signs from last four days along with passing of coffee color urine. But at the time of examination the urine was transparent.



Aseptically 3 ml blood sample were collected from ear vein of suffering cow in blood collection vial containing EDTA anticoagulants. The reason to collect the blood sample from ear vein was that as capillary blood may contain up to 20 times higher B.bovis than blood of general circulation. While there is no distribution discrimination of B. bigemina parasitized cell in the blood circulation of host (Callow et al., 1993). Peripheral thin blood smear was prepared from animal at the site of collection and immediately fixed with methanol. Blood sample brought to Department of Veterinary Parasitology, College of Veterinary Science and A.H, Jabalpur and blood smear stained by standard protocol for Giemsa staining and examined under oil immersion of microscope.

Genomic DNA was isolated from the collected blood sample using QIAamp® DNA blood mini kit following the manufacturer’s recommendations. In brief, approximately 200 µl of the blood sample was mixed with 20 µl of proteinase-K and 200µl of lysis buffer in a 2.0 ml microcentrifuge tube. The homogenous suspension was thoroughly vortexed and incubated at 56°C for 10 min. Subsequently, 200 µl of ethanol was added to the lysate and again vortexed. The mixture was then applied to QIAamp spin column and centrifuged at 8000 rpm for 1 min. Thereafter, 2 washings were given with wash buffers and DNA was eluted in 200 µl of elution buffer and stored at -20°C till further use. Genomic DNA of B. bigemina was isolated from infected blood showing parasitaemia in Giemsa stained blood smear examination and utilized as positive control. Genomic DNA was also isolated from the whole blood of infection-free, 3-day-old bovine calf and used as a negative control.
 
PCR assay
 
B.bigemina specific primers as per the protocol of (Figueroa et al., 1992) were used during the PCR assay.
The sequences of the primers were as follows:

Bbig278 Forward: 5′-CAT CTA ATT TCT CTC CAT ACC CCT CC-3′
Bbig278 Reverse:5′-CCT CGG CTT CAA CTC TGA TGC CAA AG-3′

For the PCR following components were used; master mix consisted of 2.5 µl of 10X PCR buffer, 0.5 µl of 10 mM dNTP mix, 2.0µl of 25 mM MgCl2, 1.0 U of recombinant TaqDNA polymerase, 1 µl each (15pmol) of Bbig278Forward and Bbig278Reverse primers and 5 µl of template DNA isolated from field and the volume was made up to 25µl with nuclease-free water. The cycling conditions were: initial denaturation at 94°C for 5 min, 37 cycles of denaturation at 94°C for 1 min, annealing at 59°C for 1 min and extension at 72°C for 1 min and the final extension was done at 72°C for 10 min. The PCR product was checked for amplification by electrophoresis on a 1.2% agarose gel and visualized using gel documentation system (Syngene, UK).

Haematological values namely haemoglobin, PCV, TLC, Neutrophils, Lymphocytes and Eosinophils of suffered animal were found 3.0g/dl, 11%, 4000/µl, 36%, 62% and 2%, respectively which indicated that cow was suffering from severe anemia, sever leucopenia, neutrophilia and eosinophilia at the last stage of disease.  The clinical picture of microscopic examination of blood smears showed anisocytosis with  Babesia organism approximately  in 30% RBCs. Morphologically these were visualized as pear shaped lied in pair forming an acute angle with size more than 3µm average length (Fig 2). The same sample in PCR based amplification gave the confirmed the presence of B.bigemina (Fig 3). 





The high rise of temperature as response to effect of unspecific toxic substances produced during the metabolism of Babesia. Subsequently, the heart rate was increased, marked dyspnea was developed. Similar Plus minus findings were also reported by several workers in cattle suffering from cerebral babesiosis. In case of B.bigemina, mild form of anemia has been reported by various workers (Vikrant et al., 2013) but in present study sever form of anemia has been reported might be due to high level of parasitaemia which leads to oxidative damage to the erythrocytes (Saleh, 2009) or suppression of erythropoiesis and phagocytosis of non infected erythrocyte due to changing in its antigenic surface (Esmaeilnejad et al., 2020). Haemoglobinuria was present in early stage of infection but urine was transparent in latter stage of infection which is in corroborated with findings of Bock, 2004.

The morphology of parasite in blood smear examination revealed that the present case is of B. bigemina and this was again confirmed by molecular method. In the previous studies it is stated that the cerebral form of babesiosis is caused by B. bovis. This might be the first report of cerebral babesiosis by B. bigemina.  In the present study B. bigemina was confirmed by traditional as well as molecular methods, although further investigations are required to prove B. bigemina as cause of cerebral babesiosis.  The relation of cerebral babesiosis by B. bigemina with other factors like breed of bovine, age, sex, prevalence of vectors should also be investigated.

Many species of Babesia have different characteristic with relation to severity of disease and drug susceptibility hence, accurate detection of Babesia species is required. Although there are number of reports of B.bovis caused cerebral babesiosis in cattle. B.bovis has the ability to markedly alter erythrocyte structure and function, causing parasitized RBCs to cytoadhesion or accumulate in the microvasculature and giving rise to fatal cerebral babesiosis, respiratory distress and multiorgan failure, contrary to B.bovis, in case of B.bigemina the pathogenesis of cerebral babesiosis is not studied (Wright and Goodger., 1988). There is lot of difference between biology of B.bovis and B.bigemina as sporozoites of B.bigemina are mainly transmitted by nymph while sporozoites of B.bovis are mainly transmitted by larvae of ticks.  Intravascular sequestrations of infected erythrocytes are very common in B.bovis and rarely occur with B. bigemina infections. B.bovis is more pathogenic than B. bigemina and only 1% level of parasitaemia is sufficient to produce acute form of disease while in case of B.bigemina parasitaemia level exceeds 10%. Hence, exact pathogenesis behind cerebral babesiosis in case of B.bigemina is required. Cerebral babesiosis caused by B.argentina has been reported (Callow and Mcgavin, 1963) but latter on it was confirmed that B.argentina is not a separate species but it is B.bovis. In a study conducted by Ajayi, 1978  of cerebral pathology in cattle, only 8% of the animals had babesiosis, of which only 10% exhibited heavily parasitized capillaries However, because of this low incidence and the fact that few studies have analysed CB, one should be careful in concluding that other Babesia species do not cause CB.

Haemoglobinuria is not a consistent feature of babesiosis in cattle hence, it is imperative if cattle is showing nervous symptoms along with anemia then same should be diagnosed to  rule out the possibility of B.bigemina infection. Presently there is no report of occurrence of molecular evidence of B.bigemina caused cerebral babesiosis in Holstein Friesian cattle.  This is a first report of its kind and hence placed on record.

This work was supported by Dean, College of Veterinary Science and Animal Husbandry, Jabalpur, India.