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Fatal Theileria orientalis N2 genotype infection among Asian water buffaloes (Bubalus bubalis) in a commercial dairy farm in Kerala, India KULANGARA VINODKUMAR*†, VARIKKOTTIL SHYMA, DAVIS KOLLANNUR JUSTIN, SIVASAILAM ASHOK, JOSEPH PARASSERY ANU, KATTILVEETIL MINI, VARIKKOTTIL MUHAMMEDKUTTY, SUCHITHRA SASIDHARAN and SUNANDA CHULLIPPARAMBIL Department of Veterinary Epidemiology and Preventive Medicine, College of Veterinary and Animal Sciences, Mannuthy, Trichur, Kerala, 680651, India (Received 14 June 2015; revised 5 October 2015; accepted 6 October 2015; first published online 2 November 2015) SUMMARY

Fifteen dairy buffaloes of a farm in the state of Kerala, India developed fatal oriental theileriosis within 2 months of their procurement. Typical piroplasms of Theileria orientalis were observed in the erythrocytes of all affected animals by Giemsa–Leishman staining of blood smears. Case fatality rate was 87·5% (seven out of eight) in the clinically progressed cases. Therapeutic management with anti-theilerial drugs buparvaquone and oxytetracycline led to recovery of seven other animals in less advanced stages of the disease. The aim of this study was to determine the reasons for increased virulence of this pathogen, hitherto considered to be benign. Acute haemolytic anaemia was the predominant haematological finding in the affected animals. Lymphocytic infiltration and degeneration of vital organs leading to functional derangement was the cause of the high mortality. Identification of T. orientalis was confirmed by polymerase chain reaction (PCR). DNA sequencing of the PCR products revealed close identity with already reported sequences of T. orientalis/buffeli N2 genotype. The sequences were deposited in GenBank with accession number KM609973 and KM043772. Rhipicephalus ticks, previously not reported as vectors for oriental theileriosis, were identified as the potential vectors. This is the first report of fatal oriental theileriosis in Asian water buffaloes. Key words: Theileria orientalis, Asian water buffaloes, fatal outbreak, pathology, genotype.

INTRODUCTION

Fatal outbreaks of theileriosis among cattle and buffaloes caused by Theileria parva (east coast fever) and Theileria annulata (tropical theileriosis) are well documented (Coetzer and Tustin, 2004). Oriental theileriosis, being considered to be a benign form, has not yet drawn similar attention. This disease, caused by a group of organisms variously designated as Theileria orientalis/Theileria sergenti/Theileria bufeli (Altangerel et al. 2011) has come under closer scrutiny in recent years owing to reports of severe outbreaks among cattle from many parts of the world (McFadden et al. 2011; Eamens et al. 2013). India is the largest producer of milk in the world and buffaloes contribute to more than 60% of the total milk production. The country is endemic for tropical theileriosis among cattle (Manuja et al. 2006) * Corresponding author: Department of Veterinary Epidemiology and Preventive Medicine, College of Veterinary and Animal Sciences, Mannuthy, Trichur, Kerala, 680651, India. E-mail: [email protected] † Current address: Department of Veterinary Epidemiology and Preventive Medicine, College of Veterinary and Animal Sciences, HARITHA, Mukkattukara, Nettissery(PO), Thrissur, Kerala, 680657, India.

while theileriosis among buffaloes remains unreported. There are reports of prevalence of oriental theileriosis among buffaloes in other Asian countries such as Sri Lanka, Vietnam, Thailand (Sarataphan et al. 2003) and China (Lan et al. 2010). These studies were conducted on blood samples collected from a number of apparently healthy animals from a wide geographic area. Detailed investigation of actual disease outbreaks among buffaloes has not been reported so far. This paper describes the development of clinical signs and haematopathological alterations during a fatal outbreak of oriental theileriosis among dairy buffaloes for the first time. The extent and progression of clinical disease was unusually severe in these cases. Since T. orientalis is still regarded as a pathogen of low virulence, the pathogenic processes that could be linked to the severity of this outbreak warranted a detailed study. The identification of the pathogen at the structural and molecular level established the presence of a genotype hitherto unreported in India. MATERIALS AND METHODS

Animals Fifteen adult dairy buffaloes belonging to breeds Murrah (n = 8) and Nili Ravi (n = 7) respectively

Parasitology (2016), 143, 69–74. © Cambridge University Press 2015 doi:10.1017/S0031182015001468

Kulangara Vinodkumar and others

were procured from the State of Punjab in the northern part of India which is known for its high yielding buffaloes. They were immediately transported to a commercial dairy farm in Palakkad District of the State of Kerala in the southern tip of the country across a distance of 3500 km. This constituted the initial stock for the owners. The animals were housed in a single shed in the farm. Clinical signs started to manifest by the eighth week of their arrival. All 15 animals were in various stages of progression of the disease when examined by our team for the first time after 10 weeks of their arrival in the farm. Clinical evaluation Affected animals were subjected to detailed clinical examination every day. Peripheral blood smears and lymph node aspirate were prepared and examined by Leishman–Giemsa staining from each buffalo. Approximately 20 microscopic fields were checked per smear for estimating the parasitaemic status. An automatic haematology counter (Goodhealth Inc., New Delhi) was used for the complete haematological examination of heparinized blood samples. Serum biochemistry assessment was done with a semi-automatic biochemical analyser (Transasia, India). Post-mortem examinations followed by a detailed evaluation of histopathological changes were done for all the seven dead animals. Concurrent T. annulata infection was ruled out by polymerase chain reaction (PCR) (d’Oliveira et al. 1995) for species-specific Tams 1 (MSA) gene in the 721 bp region. Alternative causes of haemolytic anaemia such as Anaplasma, Babesia and Trypanosoma were considered and eliminated by negative results of repeated microscopical examination of peripheral blood smears. Specific PCR targeting the merozoite surface glycoprotein 45 (gp45) for Babesia bigemina and rhoptry-associated protein 1 (rap-1) specific for Babesia bovis (Mtshali et al. 2014) were also employed to rule out babesiosis. Dung samples were screened to check for trematode parasite infestations. Fodder lots were scrutinized for the presence of toxic plants. Adult ticks collected from buffaloes were morphologically examined and identified using the guide to identification of species (Wall and Shaerer, 2001). Molecular investigations Confirmation of identity of theileria organisms was done by amplification of 18SrRNA gene as described by Allsopp et al. (1993). Further discrimination of T. orientalis was done by amplifying and comparing the major piroplasm surface protein (MPSP) gene (Tanaka et al. 1993). Five samples were selected at random and duplicate PCR products from them were purified using a commercial kit

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(GeneJET*) employing spin column technology. Every PCR was done in duplicate and we used products from both PCR for sequencing to reduce amplification errors and increase accuracy. The products were subjected to nucleotide sequencing at SciGenome, Kochi. The sequences obtained were applied to Basic Local Alignment Search Tool in NCBI for homology analysis. A phylogenetic tree was constructed using the neighbour-joining algorithm with molecular distance estimation. Clustal V method with MegAlign programme (LaserGene/ DNAStar) software was employed for this purpose. The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al. 2004). All positions containing gaps and missing data were eliminated from the dataset. Translation of the sequences was done to detect significant amino acid changes in the structure of the genotype under study. Therapeutic management A treatment regime of Buparvaquone (2·5 mg kg−1 body wt. i/m twice 24 h apart) followed by oxytetracycline hydrochloride (10 mg kg−1 body wt. i/v daily for 5 days) was followed for all affected animals. Supportive therapy using fluids, parenteral supplements and haematinics was employed according to requirement.

RESULTS

Clinical evaluation Typical piroplasms of T. orientalis, appearing as thin or thick rods with light staining trailing cytoplasm (Aparna et al. 2011) were detected in the erythrocytes of all animals during microscopical examination of blood smears on the first day itself. Progression of clinical signs. The animals could be categorized into three based on the progression of clinical signs (Table 1). The first category (C1) included three animals with early clinical signs such as lethargy, reduced feed intake, hypogalactia and generalized depigmentation of skin. Four animals in which the clinical signs had progressed to peripheral lymphadenopathy, haemorrhagic conjunctivitis, lacrimation, anaemia, anorexia, agalactia and sternal recumbency were included in the second category (C2). Oedema of the dependant parts, especially the metacarpal region of one or more limbs was observed in all animals of this category. Animals in the terminal stages of the disease with severe anaemia, ventral oedema, jugular pulse, purulent nasal discharge, pneumonia, ataxia, dyspnoea and lateral recumbancy belonged to the third category (C3). Seven out of eight animals in this group died within 2 weeks from the beginning of therapy.

Fatal oriental theileriosis among Asian water buffaloes

Table 1. Classification of 15 buffaloes based on parasitaemia, clinical signs and end result of infection Parasitaemia status Clinical status

P1

P2

P3

Death/total

C1 C2 C3 Death/total

3 3 0 0/6

0 1 7 6/8

0 0 1 1/1

0/3 0/4 7/8 7/15

P1 – Stray organisms detected (10% of erythrocytes affected). C1 – Lethargy, reduced feed intake, hypogalactia and generalized depigmentation of skin. C2 – Peripheral lymphadenopathy, haemorrhagic conjunctivitis, lacrimation, anaemia, anorexia, agalactia and sternal recumbency. C3 – Ventral oedema, jugular pulse, anaemia, purulent nasal discharge, ataxia, dyspnoea and lateral recumbency.

Haematological changes. Animals were also divided into three categories based on the parasitaemic status as determined by the number of erythrocytes infected with piroplasms (Table 2). Haematobiochemical alterations observed for each category are shown in Table 2. Six animals in the first category (P1) were in the early stages of the disease with only mild (10%) parasitaemia, which had haematological alterations similar to category P2. But the gamma glutamyl transferase (GGT) level was also severely elevated, indicating hepatic impairment and further progression of the disease. Differential diagnosis Microscopic examination of blood smears did not reveal any other aetiologic agent for haemolytic anaemia like Anaplasma, Babesia or Trypanosoma. Amplification of species-specific Tams 1 (MSA) gene at the 721 bp region for T. annulata was not

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observed in PCR of any of the samples. PCR targeting merozoite surface gp45 for B. bigemina and rap-1 specific for B. bovis also did not yield any positive results. The possibility of plant toxins was ruled out as the animals were stall fed with commercial concentrates and hybrid Napier fodder cultivated in the farm premises. Therapeutic management All seven animals in the first two clinical categories recovered upon treatment. But the health status and production performance of the recovered animals remained sub-optimal despite continuing supportive intervention for the next 3 months. Seven out of eight animals in the third clinical category died within 14 days despite intensive management. Post-mortem examination and histopathology Petechiae and echymosis of the vital organs and subcutaneous musculature were the primary lesions noticed. Thoracic and peritoneal effusions, cardiac myopathy, hydro-pericardium, pulmonary emphysema, multi-lobular congestion and consolidation of lungs were observed in five animals. Multiple white nodular foci were evident predominantly in the kidneys. Diffuse lymphocytic infiltration of vital organs, lymphoid depletion and oedema in lymphnodes were observed in sections of tissues from all the seven animals. Moderate hepatocytolysis and haemosiderosis coupled with lymphangiectasis were evident in the liver. Extensive involvement of the kidneys was also manifested as tubular necrosis, hyalinization and glomerulitis in all these cases. Molecular identification Amplification of 18SrRNA gene was evident in the PCR of samples from 15 animals with typical bands in the 1098 bp region. Amplification of species specific MPSP gene (770 bp) for T. orientalis was observed in all the 15 samples. The nucleotide sequences of 1098 bp amplicons obtained in this study had close homology (99%) with other sequences of T. orientalis/buffeli with accession numbers HM538215, HM538206, HQ840965 (from China), AB520955 (Japan), AF236094 (The Netherlands), AB000272 (Australia) and DQ287959 (Spain). They were deposited with GenBank accession number KM609973. The MPSP gene sequences determined in this study aligned closely in the phylogenetic tree (Fig. 1) with Genotype N2 sequences reported by Sivakumar et al. 2014 (accession nos. AB560829– AB560832). These were deposited in the GenBank with accession number KM043772. Translation of the sequences revealed differences in only two amino acids from other N2 genotypes.

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Table 2. Comparison of selected haematological and biochemical parameters in relation to parasitaemia among 15 buffaloes with Theileria orientalis infection

Haematological/ biochemical/ parameters PCV(%) TEC (106 µL−1) Platelets (104 µL−1) Hb (g dL−1) Protein (g dL−1) Globulin (g dL−1) GGT (μ L−1) Creatinine (mg dL−1)

Parasitaemia (15)

Normal (Hagawane et al. 2009)

P1 (6)

Mean

Mean ± S.D.

35 6·53 23·4 9·94 8·3 2·3 14 1·2

35·00 ± 2·00 6·82 ± 0·60 28·83 ± 2·87 10·13 ± 1·12 8·02 ± 1·00 4·75 ± 0·60 17·00 ± 2·19 1·20 ± 0·18

P2 (8) t-value# ns

0·00 1·174ns 4·639** 0·421ns 0·691ns 10·016** 3·354* 0·000ns

P3 (1)

Comparison of P1 and P2

Mean ± S.D.

t-value#

Mean

t-value

21·88 ± 2·167 4·03 ± 0·68 14·80 ± 1·45 8·69 ± 0·60 7·69 ± 0·91 5·66 ± 0·64 16·00 ± 3·67 2·50 ± 0·74

17·13** 10·358** 16·78** 5·92** 1·91ns 14·88** 1·544ns 4·991**

20·00 4·90 12·19 8·01 8·91 7·20 66·00 2·4

11·56** 7·96** 12·04** 3·12** 0·641ns 2·71* 0·590ns 4·19**

PCV, packed cell volume; TEC, total erythrocytic count; Hb, haemoglobin; GGT, Gamma glutamyl transpeptidase. P1 – Stray organisms detected (