Trop Anim Health Prod (2015) 47:933–937 DOI 10.1007/s11250-015-0811-x

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Serotyping of foot and mouth disease virus and Pasteurella multocida from Indian gaurs (Bos gaurus), concurrently infected with foot and mouth disease and haemorrhagic septicaemia Basavegowdanadoddi Marinaik Chandranaik 1 & Raveendra Hegde 1 & Beechagondahalli Papanna Shivashankar 1 & Papanna Giridhar 1 & Handenahally Kaverappa Muniyellappa 1 & Rajeshwar Kalge 1 & Benamanahalli Raju Sumathi 1 & Kumble Nithinprabhu 2 & Narasimhaiah Chandrashekara 1 & Venkataramanappa Manjunatha 1 & Nirupama Jaisingh 2 & Asha Mayanna 1 & Gowda Kallenahalli Chandrakala 1 & Sermaraja Kanaka 1 & Mudalagiri Dasappagupta Venkatesha 1

Received: 19 September 2014 / Accepted: 30 March 2015 / Published online: 19 April 2015 # Springer Science+Business Media Dordrecht 2015

Abstract We report the serotyping of foot-and-mouth disease virus (FMDV) and Pasteurella multocida from Indian gaurs which were concurrently infected with foot-and-mouth disease (FMD) and haemorrhagic septicaemia. Bannerghatta biological park (BBP), a national park located in the outskirts of Bengaluru city, Karnataka, India, is bordered by several villages. These villages witnessed massive outbreaks of FMD which spread rapidly to the herbivores at BBP. Post-mortem was conducted on carcasses of two Indian gaurs that died with symptoms of FMD. The salient gross findings included extensive vesicular lesions on the tongue, gums, cheeks, upper palate and hooves. Haemorrhagic tracheitis and ecchymotic haemorrhages on the heart were characteristic. The vesicular lesions of oral cavity were positive for ‘O’ type of FMD virus by sandwich enzyme-linked immuno sorbent assay (ELISA). The heart blood and spleen samples yielded growth of pure cultures of P. multocida. The isolates were typed as P. multocida type B using KTSP61 and KTT72 primers yielding specific amplicons of 620 bp. The phylogenetic analysis of the isolates was carried by sequencing of 1.4-Kbp nucleotides on the 16S ribosomal RNA (rRNA) gene of the isolates.

* Basavegowdanadoddi Marinaik Chandranaik [email protected] 1

Institute of Animal Health and Veterinary Biologicals, Hebbal, Bengaluru 560 024, Karnataka, India

2

Department of Veterinary Microbiology, Veterinary College, Bengaluru 560 024, India

Keywords Foot and mouth disease . Indian gaur . Pasteurella multocida . 16S rRNA gene sequencing

Introduction The Indian gaur (Bos gaurus) also called as Indian bison is the largest extant bovine and is native to South Asia and Southeast Asia. The species is listed as vulnerable on the IUCN red list since 1986 (Duckworth et al. 2008). Foot-and-mouth disease (FMD) is a transboundary, economically devastating and highly contagious viral disease of livestock, most importantly in cattle, buffalo, pigs, goats, and sheep. The causative foot-and-mouth disease virus (FMDV) is antigenically diverse having seven distinct serotypes and many variants within them. Being a single-stranded RNA virus, it confirms the quasi species nature with emergences and re-emergences of different genetic lineages with altered antigenicity within the serotypes, making vaccination-based control programme highly cost-effective, time-consuming and difficult to achieve. The disease is endemic and three serotypes of the virus, viz., O, A and Asia-1, are circulating in India (Patnaik et al. 2012). Though FMDV has been reported in more than 70 species of wildlife (Fiebre 2014), the detailed reports on FMD pathogenesis in wildlife are sparse; nevertheless, FMD can be destructive of wildlife (Thomson et al. 2003). The information available on FMD in Indian gaurs is limited and is mostly confined to news paper reports. There are a few reports of experimental induction of FMD in American bison (Rhyan et al. 2008); however, there is a scarcity of literature available on the systematic studies involving clinical findings, pathology,

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confirmation and molecular characterisation of the virus type involved in FMD outbreaks in Indian gaur (Indian bison). Generally, FMDV does not cause deaths in adult animals; it renders the ailing animal acutely immunosuppressed resulting in secondary bacterial complications, primarily by Pasteurella multocida (Venkataramanan et al. 2005). The state of Karnataka in India witnessed its worst outbreak of FMD during the year 2013–14, that took toll of more than 7000 domestic cattle (IAH&VB annual report 2013–14). Bannerghatta biological park (BBP), a national park located in the outskirts of Bengaluru city in Karnataka, is bordered by several villages. These bordering villages also witnessed massive FMD outbreaks with high morbidity in susceptible population. The disease rapidly spread to the herbivores at BBP. The outbreak took toll of several species of wild herbivores in the national park. In this paper, we report the cultural and molecular characterisation of P. multocida and serotyping of FMDV from Indian gaurs which were concurrently infected with FMD and haemorrhagic septicaemia (HS).

Materials and methods History of outbreak and collection of samples The Government of India has made stringent guidelines for compulsory FMD vaccinations of cattle living in 10-km vicinity of every national park in India. However, some villagers fail to get their animals vaccinated, owing to this non-compliance of guidelines, when a massive outbreak of FMD engrossed the entire state; unvaccinated susceptible cattle in many villages bordering BBP also witnessed FMD. The disease rapidly spread to unvaccinated, highly susceptible free-range wild herbivores at BBP. The disease was first seen in Indian gaurs which later spread to other wild herbivores, viz., nilgais (Boselaphus tragocamelus), chitals (Axis axis), blackbucks (Antilope cervicapra) and sambars (Cervus unicolor). The FMD outbreak took toll of several numbers of many herbivore species in BBP forest. We conducted post-mortem examination on two Indian gaur carcasses. Epithelial samples from vesicular oral lesions in transport medium (composed of equal amounts of glycerol and 0.04 M phosphate buffer), samples of all vital organs and heart blood were collected for laboratory examination. Serotype differentiating antigen detection enzyme-linked immuno sorbent assay The oral lesions and epithelial samples were subjected for sandwich enzyme-linked immuno sorbent assay (ELISA) for detection of FMD viral antigen and identification of viral serotype involved in the outbreak as per the procedures outlined by OIE (2012) using ELISA kits supplied by the Project Directorate on Foot and Mouth Disease (PDFMD), Muktheshwar, Uttaranchal state, India. For further characterisation of the virus involved in

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the outbreak, the tissue samples were submitted to PDFMD, Muktheshwar, which is a FAO reference laboratory for FMD for South Asian (SAARC) countries. Isolation of P. multocida The spleen and heart blood samples were subjected for microbiological culture and biochemical confirmation by catalase, indole, citrate and urease biochemical tests as per standard microbiological procedures of Quinn et al. (2011). Pathogenicity of P. multocida isolates was confirmed by mice inoculation test as per the procedures outlined in OIE (2008). Mice were maintained as per the standard institutional animal ethics committee guidelines. DNA extraction Five millilitres of nutrient broth inoculated with bacteria from a single colony of the isolate, and culture was incubated overnight with shaking. Bacterial cells were harvested by centrifugation at 1000×g for 15 min. Genomic DNA was extracted from the disrupted cells using DNA extraction kit procured from Amnion Biotech Pvt. Ltd., Bengaluru, Karnataka, India, following the protocols provided by the manufacturer. DNA extracted from vaccine strain of P. multocida obtained from vaccine production division of Institute of Animal Health and Veterinary Biologicals, Bengaluru, India, was used as positive control during PCR studies. 16S rRNA-based PCR We used a set of primers targeting the amplification of 16S ribosomal RNA (rRNA) gene of Pasteurella spp. The primers were forward primer 5′-CWG RCC TAN CAC ATG SAA GTC-3′ and reverse primer 5′-GRC GGW GTG TAC NAG GC-3′. The reaction mixture included 50 ng of genomic DNA, 100 ng of forward primer, 100 ng of reverse primer, 2 μl of 10 mM dNTPs mix, 5 μl of 10X Taq pol. buffer, 3 U of Taq polymerase enzyme and PCR grade water to make the volume to 50 μl. The PCR included initial denaturation at 94 °C for 5 min, and 35 repeated cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1.30 min and a final extension of 5 min at 72 °C. The PCR products were electrophoresed in 1 % agarose gel along with 500-bp DNA ladder. The PCR-amplified products were extracted from agarose gel and eluted in 25 μl of nuclease-free water using Quiagen® gel extraction kit as per manufacturer’s instructions and were then subjected to nucleotide sequencing at Amnion Biotech Pvt. Ltd, Bengaluru, India. Nucleotide sequences were aligned with published sequences in GenBank. Phylogenetic tree was constructed and sequence analysis was performed in MEGA version 6.2, using the neighbour-joining tree method with 1000 bootstrap replicates (Tamura et al. 2007).

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P. multocida-specific and P. multocida type B-specific PCR The primer sequences targeting SP6 promoter 5′-TATTTAGG TGACACTATAG-3 and T7 promoter primer 5′-TAATACGA CTCACTATAGG-3 amplifying a region of 460 bp were used for confirmation of isolate as P. multocida. Primer sequences KTSP61-5′-ATCCGCTAACACACTCTC-3′ and KTT72-5′AGGCTCGTTTGGATTATGAAG-3′ amplifying a region of 620 bp were used for confirming the isolate as P. multocida type B. The PCR testing was done following the procedures of Kirsty et al. (1998).

Results Bannerghatta biological park (BBP) is a natural habitat for several species of wild animals, including herbivores. Like in any other national parks in India, BBP is bordered by several villages, whose populace primarily depends on agriculture and animal husbandry activities for their livelihood. Foot and mouth disease virus is highly contagious and can spread by all transmission routes described for a virus. Further, the virus can conveniently spread across to a distance of about 200 km by wind. Several villages that were bordering the vast area of BBP witnessed foot and mouth disease in cattle with high morbidity. The affected villages were hardly 4–5 km from the borders of BBP. Since the wild herbivores in the forest were in free range, they were unvaccinated and thus highly susceptible for a virulent FMD virus. A week after the disease broke out in bordering villages, two Indian gaurs at herbivore safari of BBP showed malaise and diminished appetite and appeared pyrexic with painful gaits. The disease spread rapidly, and within 2 days, many animals showed lameness in one or more feet and excessive salivation. In most severe cases, chewing motions, protrusion of the tongue and severe salivation were observed. Treatments with analgesics and antibiotics were started using dart guns; however, it was extremely difficult in free-range situations; hence, in most cases, it went in vain and were inconsistent in keeping up the dosages and schedules of treatments. Post-mortem of the two adult Indian gaurs which failed to respond to higher antibiotics revealed extensive vesicular lesions on the gums, cheeks and upper palate, sloughing of tongue epithelium, submandibular oedema, haemorrhagic tracheitis, frothy exudates in trachea and bronchi, edematous lungs, hydrothorax, extensive ecchymotic haemorrhages on the heart, hydro-pericardium, haemorrhages on the liver, spleen and kidney, mucosal surfaces of intestines and moderate splenomegaly were noticed. The inter-digital spaces (hoof) had maggot-infested wounds. Oral lesions and epithelial samples collected at postmortem were positive for ‘O’ type of FMD virus by sandwich ELISA. The clinical samples were further confirmed as FMD O serotype at PDFMD, Muktheshwar, and were grouped

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under ‘O/ME-SA/Ind2001’ lineage and ‘Ind2001d’ sublineage (PDFMD annual report 2013–14). The heart blood and spleen samples collected during postmortem yielded growth of Gram-negative bipolar organisms with dewdrop-like colonies. Biochemically, the organisms were catalase positive, citrate negative, indole positive and urease negative and failed to grow on MacConkey’s media. The isolates were pathogenic to mice, and the heart blood collected from the dead mice yielded growth of pure cultures of P. multocida. The primer sequences targeting SP6 promoter site of P. multocida amplified the specific region of a 460 bp confirming the isolate as P. multocida, and primer sequences KTSP61 and KTT72 yielded specific amplicons of 620 bp confirming the P. multocida isolate as belonging to P. multocida type B. The isolates recovered in this study were named as ‘P. multocida IAHVB 2013 Gaur isolates 1 and 2’. PCR using the primers targeting 16S rRNA gene yielded amplicons of 1450 bp. The nucleotide sequencing and phylogenetic analysis showed that the isolates were genetically more than 98 % identical between themselves and were genetically nearest to the Indian P. multocida isolates of bovine and caprine origin and were clustered under the same group (Fig. 1). Since the herbivores were in free range in the forest, it was not possible to assess the morbidity and mortality patterns; however, it was evident that the current FMD outbreak had taken toll of at least 30 chitals, 12 sambars, five blackbucks, two Indian gaurs and seven nilgais. Representative samples of oral epithelial lesions were collected from each of these species, and all the carcasses were disposed by burning. The collected representative samples were tested by sandwich Pasteurella multocida IAHVB 2013 Gaur Isolate-2 62

Caprine India DQ288146 Bovine India DQ286927 Pasteurella multocida IAHVB 2013 Gaur Isolate-1

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Yak China JQ726501 Bovine Iran FJ231206 Bovine Scotland AY299312 Porcine India DQ286929 Bovine UK AY324032 Ovine Iran FJ231209 Duck China JX869945 Caprine South KoreaFJ405344 64

Bovine Scotland AY299304 Bovine China JQ404476

Fig. 1 16S rRNA gene-based phylogenetic analysis of Pasteurella multocida isolates from Indian gaurs and other host species

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ELISA as described earlier and were found positive for FMD O serotype.

Discussion In India, a systematic FMD control programme (FMDCP) was initiated in the year 2003, with the objective of creating FMDfree zones. By 2010, out of the 640 districts in the country, only 221 districts were covered under FMDCP, wherein about 120 million cattle, buffaloes and pigs in these 221 districts areas are vaccinated twice a year to build up strong herd immunity (Annual report of Govt. of India 2013–14). During this period, the vaccination of cattle, buffaloes and pigs in nonFMDCP areas (419 districts) was done under other vaccination programmes which were not as stringent as in FMDCP programme. Further, reluctance of the farmers to get their cattle vaccinated against FMD (due to the fear of drop in milk yield after vaccination) is an important epidemiological factor in poor herd immunity against FMD. A combination of all these factors resulted in a massive FMD outbreak in Karnataka state during 2013. The lesions noticed in the animals were pathognomonic of a severe and acute FMD in wildlife in general and bison in particular (Thomson et al. 2003; Rhyan et al. 2008). The vesicular lesions noticed in the animals and detection of O type of FMD virus are in accordance with Grubman and Baxt (2004) and Chandranaik et al. (2014) who have reported involvement of O type of FMD virus in over 80 % of FMD outbreaks, worldwide. Clustering of the current virus type into O/ME-SA/Ind2001 lineage and further into sub-lineage Ind2001d is in accordance with the previous reports of Rout et al. (2014), who have recorded the O/ME-SA/Ind2001 lineage FMD virus as the cause of outbreaks in Karnataka state in 2012. Further, PDFMD, Muktheshwar, has established that the sub-lineage Ind2001d virus was responsible for the massive outbreak of FMD in Karnataka state during 2013 (PDFMD annual report 2013–14). Growth of pathogenic P. multocida from the heart blood samples collected from animals showing symptoms of submandibular oedema, haemorrhagic tracheitis and haemorrhages on all vital organs is in accordance with the descriptions of haemorrhagic septicaemia in cattle (Quinn et al. 2011). This concurrent infection of FMD with HS is in agreement with the findings of Venkataramanan et al. (2005) who have reported P. multocida as the most common secondary bacterial pathogen responsible for deaths during FMD outbreaks. Identification of different parameters involved in the immune response elicited against FMD virus remains unclear; however, it is proven that FMD virus induces great degree of imunosuppression during acute infections (Grubman and Baxt 2004; Diaz et al. 2009; James and Edwards 2011). These factors combined with host factors of painful lesions which

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reduce feed intake in the ailing animals create an environment for microorganisms that generally remain as carriers/latent in hosts to take the upper hand and produce disease as pathogens. FMD-induced immunosupression might have resulted in uncontrolled multiplication of P. multocida causing septicaemia and death. The primer sequences targeting SP6 and T7 promoter regions amplifying a region of 460 bp for confirmation of isolate as P. multocida and the primer sequences KTSP6 and KTT72 amplifying a region of 620 bp for typing the isolates as P. multocida type B described by Kirsty et al. (1998) have been the most widely used primers for confirmation of P. multocida worldwide (Anina et al. 2009; OIE 2008), and the results confirmed the isolates recovered during this study as P. multocida type B. 16S rRNA genome-based PCR and nucleotide sequencing showed the isolates to have the highest nucleotide sequence identity with P. multocida isolates of Indian cattle and goats. This may be attributed to fact that the villagers surrounding the national park generally leave their cattle and small ruminants for grazing near to the national park resulting in possible cross contaminations of bacterial pathogens through contaminated grass, water or air. Further, it is important to note that there were no genomic sequences of P. multocida isolates originating from Indian gaur deposited in the GenBank for comparison, and possibly, this is the first report on the molecular study of P. multocida from Indian gaur. As it is difficult to vaccinate the wild herbivores that are susceptible for the FMD virus, continuous vaccination to all susceptible domestic animals, twice a year, covering all 640 districts of the country is the only way to control this devastating disease. Previous experiences in countries where FMD has been eradicated, viz., Australia, New Zealand, USA and European countries, have shown that eradication of FMD in domestic animals has consequently resulted in eradication of the disease in wild susceptible animals also, possibly through population immunity (Thomson et al. 2003). Acknowledgments The authors are thankful to the Project Directorate on Foot and Mouth Disease, Muktheshwar, India, for confirming the FMD samples. Conflict of interest The authors declare that they have no conflict of interest.

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Serotyping of foot and mouth disease virus and Pasteurella multocida from Indian gaurs (Bos gaurus), concurrently infected with foot and mouth disease and haemorrhagic septicaemia.

We report the serotyping of foot-and-mouth disease virus (FMDV) and Pasteurella multocida from Indian gaurs which were concurrently infected with foot...
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