Foot-and-mouth disease in elephants in Kerala state of India during 2013

Abstract

Foot-and-mouth disease (FMD) is a highly contagious acute vesicular disease of the cloven-hoofed animals including cattle, buffalo, sheep, goats, pigs along with more than 70 wildlife species. During the year 2013, FMD outbreaks were recorded in the southern peninsular India comprising the states of Karnataka, Tamil Nadu, Kerala and Andhra Pradesh. Besides domestic livestock, captive elephants in Kerala were also affected by FMD. The suspected FMD outbreak in six elephants occurred in Neendoor of Kottayam district, Guruvayoor and Thrissur of Thrissur district in Kerala during November - December 2013. The first clinical signs recorded in the elephants were loss of appetite and lameness with mild fever. Frank lesions were grossly evident on the tongue, palate and inner mucous membrane of trunk with exudates from nostrils. There was copious salivation often appeared to be drooling. Severe lameness led to recumbency. Erosive lesions were also noticed in foot-slipper. The foot with blisters turned to open sores making the animals difficult to stand and walk. Clinical samples (foot/oral/tongue/trunk/nasal epithelium) from the FMD-suspected elephants were collected in 50% phosphate buffered saline/glycerol medium (pH-7.5). Supernatants of the homogenized clinical samples were used in a serotype differentiating antigen detection ELISA and samples found negative were further subjected to multiplex PCR. All clinical samples were found positive for FMD virus (FMDV) serotype O in antigen detection ELISA and in mPCR. The VP1 region based phylogenetic analysis indicated the involvement of O/Middle East-South Asia/Ind2001d sub-lineage of FMDV serotype O, which was also responsible for severe disease in domestic livestock in southern states of India during 2013. © 2016, Indian Council of Agricultural Research. All rights reserved.

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Present address: 1Scientist (drmrout@gmail.com); 3Research
Associate; 4Scientist (SS); 5Senior Scientist; 6Project Director.
2Co-PI, FMD Collaborating Centre, Chief Disease Investigation
Office, Palode, Thiruvananthapuram.
Foot-and-mouth disease (FMD) is a highly contagious
acute vesicular disease of the cloven-hoofed animals
including cattle, buffalo, sheep, goat, pig along with more
than 70 wildlife species and considered as a menace to the
livestock industry (Pattnaik et al. 2012, Ding et al. 2013).
FMD in India is enzootic, where three serotypes of the virus
such as O, A and Asia 1 are prevalent and about 80% of the
outbreaks are attributed to serotype O (Subramaniam et al.
2013). The elephant (Elephas maximus) has been considered
as an integral part of India’s history, tradition, myth and
culture. During 2013, many FMD outbreaks due to serotype
O were recorded in India. Besides domestic animals,
outbreaks were also recorded in captive elephants in the
state of Kerala. The present study investigates the FMD
virus (FMDV) infection in elephants of Kerala with regard
to partial characterization and phylogenetic analysis of the
viruses involved in the disease outbreak.
MATERIALS AND METHODS
The suspected FMD outbreak in six elephants occurred
Indian Journal of Animal Sciences 86 (6): 627–631, June 2016/Article
https://doi.org/10.56093/ijans.v86i6.59128
Foot-and-mouth disease in elephants in Kerala state of India during 2013
MANORANJAN ROUT1, NANDAKUMAR S. NAIR2, BISWAJIT DAS3, SARAVANAN SUBRAMANIAM4,
JAJATI KESHARI MOHAPATRA5 and BRAMHADEV PATTNAIK6
ICAR-Project Directorate on Foot and Mouth Disease, IVRI Campus, Mukteshwar, Uttarakhand 263 138 India
Received: 4 September 2015; Accepted: 26 October 2015
ABSTRACT
Foot-and-mouth disease (FMD) is a highly contagious acute vesicular disease of the cloven-hoofed animals
including cattle, buffalo, sheep, goats, pigs along with more than 70 wildlife species. During the year 2013, FMD
outbreaks were recorded in the southern peninsular India comprising the states of Karnataka, Tamil Nadu, Kerala
and Andhra Pradesh. Besides domestic livestock, captive elephants in Kerala were also affected by FMD. The
suspected FMD outbreak in six elephants occurred in Neendoor of Kottayam district, Guruvayoor and Thrissur of
Thrissur district in Kerala during November - December 2013. The first clinical signs recorded in the elephants
were loss of appetite and lameness with mild fever. Frank lesions were grossly evident on the tongue, palate and
inner mucous membrane of trunk with exudates from nostrils. There was copious salivation often appeared to be
drooling. Severe lameness led to recumbency. Erosive lesions were also noticed in foot-slipper. The foot with
blisters turned to open sores making the animals difficult to stand and walk. Clinical samples (foot/oral/tongue/
trunk/nasal epithelium) from the FMD-suspected elephants were collected in 50% phosphate buffered saline/glycerol
medium (pH-7.5). Supernatants of the homogenized clinical samples were used in a serotype differentiating antigen
detection ELISA and samples found negative were further subjected to multiplex PCR. All clinical samples were
found positive for FMD virus (FMDV) serotype O in antigen detection ELISA and in mPCR. The VP1 region
based phylogenetic analysis indicated the involvement of O/Middle East-South Asia/Ind2001d sub-lineage of FMDV
serotype O, which was also responsible for severe disease in domestic livestock in southern states of India during
2013.
Key words: Elephant, Foot-and-mouth disease, India, Kerala
in Neendoor of Kottayam district, Guruvayoor and Thrissur
of Thrissur district in Kerala during November - December
2013. Clinical samples (foot/oral/tongue/trunk/nasal
epithelium) from those elephants were collected in 50%
phosphate buffered saline/glycerol medium (pH-7.5).
Supernatants of the homogenized clinical tissue samples
were used in a serotype differentiating antigen detection
ELISA as per the method described by Bhattacharya et al.
(1996) for confirmation of the serotype of the virus involved
in the outbreak. Samples found negative in ELISA were
further subjected to serotype differentiating multiplex
polymerase chain reaction (mPCR). Total RNA was
extracted from the tissue samples using RNeasy Mini Kit.
Reverse transcription was performed using M-MLV reverse
transcriptase and reverse primer NK61 (Knowles and
Samuel 1995). The serotype differentiating mPCR was
performed using Hotstar Kit essentially as described
previously (Giridharan et al. 2005). The mPCR products
were visualized on ethidium bromide stained 2% agarose
gel.
PCR amplification of VP1 region was performed using
Pfu DNA polymerase and the primer combination of ARS4
and NK61 (Knowles and Samuel 1995) following thermal
conditions described earlier (Hemadri et al. 2002). Cycle

628 ROUT ET AL. [Indian Journal of Animal Sciences 86 (6)
12
sequencing reactions of gel purified PCR products were
carried out using BigdyeV3.1 terminator kit and sequences
were resolved on ABI 3130 genetic analyzer. The nucleotide
sequences were aligned using clustal W algorithm
(Thompson et al. 1994). Phylogenetic analysis was
conducted using MEGA 6.06 software (Tamura et al. 2013)
employing the best fit nucleotide substitution model,
TN93+G+I. Phylogenetic tree was reconstructed using
Neighbor joining (NJ) method available and the robustness
of the tree topology was evaluated with 1000 bootstrap
replicates.
RESULTS AND DISCUSSION
The first clinical signs recorded in the suspected
elephants were loss of appetite and lameness with mild
fever. Frank lesions were grossly evident on the tongue,
palate and inner mucous membrane of trunk with exudates
from nostrils. There was copious salivation often appeared
to be drooling. Severe lameness led to recumbency. Erosive
lesions were also noticed in foot-slipper. The foot with
blisters turned to open sores making the animals difficult
to stand and walk. The lesions in mouth and feet of elephants
can be severe, which may be attributed to the invasiveness
of the virus strains and highly susceptible nature of the
elephant to the virus, but mechanical stress to which the
affected tissues are subjected might also be an important
factor contributing to the severity of the disease. In
elephants, the excessive stress on the feet due to heavy
weight of the animal may result in severity of the foot
lesions. Moreover, in Kerala, since elephants are mostly
rented out for ‘ezhunnellippu’ (parading with statues of
deities mounted on the elephant’s back) during temple
festivals, they need to work continuously for several hours
that may aggravate the foot lesions in the affected elephants.
However, the affected elephants should not be used for this
purpose; otherwise the infectious virus may rapidly spread
to other healthy animals depending upon its quantum and
virulence. Investigation of an outbreak of FMD in elephants
used for ceremonial purposes in Nepal found that the titre
of the virus recovered from tongue epithelium was of the
order of 106 to 108 ID50 per gram of tissue (Mahy 2005).
Hence, due care must be taken to keep the affected animals
segregated from the healthy ones.
During 2013–2014, a total of fifty outbreaks of FMD
due to serotype O were recorded in Kerala. The incidences
were reported in the districts of Thiruvananthapuram (04),
Kollam (07), Alappuzha (03), Pathanamthitta (02),
Kottayam (01), Idukki (04), Ernakulam (01), Thrissur (05),
Palakkad (04), Malappuram (04), Kozhikkode (05),
Wayanad (05), Kannur (02) and Kasaragod (03) (Annual
Report PDFMD 2013–2014). During the same period
several FMD incidences were also recorded in other three
southern states of the country. In Kerala, the apparent non-
structural protein (NSP) antibody seroprevalence against
FMDV in cattle and buffalo population stands at 6.87%
during 2014–2015 (Annual Report PDFMD 2014–2015).
All clinical samples collected from the suspected
elephants were found positive for FMDV serotype O in
antigen detection ELISA and in mPCR. The NJ tree
depicting phylogenetic relationships of serotypes O elephant
isolate is presented (Fig. 1). The recovered isolate grouped
within the lineage Ind2001 of Middle-East South Asia (ME-
SA) topotype, precisely in the sub-lineage Ind2001d, which
re-emerged in the year 2008 and has been dominating
serotype O outbreaks in the country since then
(Subramaniam et al. 2015). The elephant isolate grouped
closely with the contemporary isolates collected from
domestic animals in nearby areas, which indicates the
probable transmission of FMDV from domestic livestock.
Thus, tracing the origin of field outbreak through
characterization/sequencing of the virus genome followed
by phylogenetic analysis plays a pivotal role in
understanding the track of virus movement that has an
immense significance in the epizootiology of FMD.
India is enzootic for FMD with a complex disease
supporting environment. It must be remembered that most
of the reports of FMD in wildlife from Southeast Asia have
been derived from India and in almost every case, the
evidence of initiation of the outbreak was observed from
nearby livestock (Weaver et al. 2013). Similarly, in the
present case, the intermixing of infected domestic animals
with the elephants was suspected to have been the probable
source of infection. As mentioned earlier, in Kottayam and
Thrissur districts, several outbreaks occurred in the nearby
areas in cattle. At the same time, the elephants were also
found to have an unrestricted access to the grazing area
meant for the local cattle, which might have been a
presumable cause of transmission of virus either through
aerosol exposure or direct contact. Again, the elephants were
looked after by mahouts residing in FMD affected areas,
which might have probably facilitated mechanical
transmission of the virus causing the disease in the
elephants. Moreover, these elephants are not usually
vaccinated against FMD, for which the protective titre of
antibody remains at poor level. Strict movement restriction
pertaining to human and animals is also not followed in
any part of the country. Hence, intermingling of affected
animals with apparently healthy domestic cattle and other
susceptible livestock also play a vital role in transmission
of infectious virus. The symptoms of FMD being very mild
or inapparent in small ruminants, the disease very often go
unnoticed in these species, which also silently play an
important role in the epizootiology of the disease (Pay
1988). The virus also spreads through direct contact with
infected animals, farm tools, clothes, humans and tends to
affect other healthy animals. Sometimes, the same person
taking care of infected animals are also involved in the
maintenance of healthy stock in the farms, which can also
act as a contributing factor behind the spread of infection.
It is also noteworthy to mention that in Tanzania, FMD
outbreaks from 2001 to 2006 appeared to be the result of
uncontrolled human activity (Picado et al. 2011). The same
factor was also exemplified in Uganda, where human and
livestock movements were the predominant reason behind

June 2016] FMD IN ELEPHANTS IN KERALA 629
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FMD outbreaks (Ayebazibwe et al. 2010). The facts and
circumstances set forth above indicate that human and
livestock movements should be judiciously controlled
especially during the face of an outbreak. Thus, the
controlled movements of people and livestock as well as
proper vaccination before and during outbreaks can be
considered to be the effective means of handling FMD
outbreaks (Thomson 2011).
FMD in Indian elephant was previously reported by
Hedger and Brooksby (1976). Pyakural et al. (1976) also
reported and isolated FMDV serotype O from a natural
outbreak in Indian elephant. Strains of FMDV isolated from
elephants so far in India are type O, A, A22 and Asia 1
(Pattnaik and Venkataramanan 1989). Serotype Asia 1 was
Fig. 1. Neighbour-joining phylogenetic tree estimated at VP1 coding region (639 bp) depicting relationships of FMDV serotype O
isolates collected from elephants with contemporary isolates from domestic animals. Bootstrap values (>70%, out of 1000 replicates)
is shown near the nodes. The elephant isolate sequenced in this study is indicated by filled triangle.

630 ROUT ET AL. [Indian Journal of Animal Sciences 86 (6)
14
earlier recorded from Indian elephant by Rahman et al.
(1988) and the probable source of infection could be traced
back to an outbreak of FMD due to serotype Asia 1 in cattle
and buffaloes. When FMD-affected cattle enter a reserve
forest or any wildlife premises, the chances of the disease
spreading among susceptible wildlife species including
elephants were reported to be very high. Mahouts are
sometimes also responsible for transmission of the virus
from the affected cattle in their house or locality as
mentioned in this case.
A vaccination-based FMD Control Programme
(FMDCP) was launched by the Department of Animal
Husbandry, Dairying and Fisheries (DAHD&F),
Government of India since August 2003–2004 covering 54
specified districts in the country, which involved biannual
vaccination of cattle and buffaloes with trivalent (O, A and
Asia 1) FMD vaccine. Under the same programme in 2010,
additional 167 districts were further included. So, currently,
this programme includes 221 districts of the country
covering all the states of southern peninsula including
Kerala. Three districts of Kerala namely, Trivandrum,
Kollam and Pathanamthitta were covered under FMDCP
during 2003–2004 and later in 2010–2011, eleven districts
were further added including the districts of Kottayam and
Thrissur, from where the clinical samples from FMDV
infected elephants were collected. In Kottayam and Thrissur
districts, biannual vaccination is being followed in bovine
population as in other districts under FMDCP. Mostly, cattle
and buffaloes are included under the vaccination programme
ignoring most of the small ruminants, pigs, wild and captive
animals. Thus, the elephants from the sampled districts were
also refrained from FMD vaccination drive. Moreover, there
are no commercially available FMD vaccines currently
approved for use in susceptible wild/captive animals
(Weaver et al. 2013). Still, increasing the vaccination
frequency to every 4 months with an altered dose may be
imperative with the presently available vaccines, as the in-
use vaccines are labile and hence must be kept at specified
temperatures from the time of manufacture to the point of
use through the maintenance of an unbroken cold chain with
proper handling that is exceptionally critical (Weaver et al.
2013). Concomitant post-vaccination monitoring is also
essential to determine if the current vaccination-based
control programmes are effective (OIE 2012).
There is no resident population of elephants reared in
the reported area. Elephants are usually brought to the area
for temple rituals and are taken out once the festival is over.
During this temporary period, routine vaccination and other
preventive measures were not adopted in these animals.
However, the elephant keepers in the nearby areas of
sampling were suggested to routinely follow FMD
vaccination along with other biosecurity measures in order
to prevent FMD in elephants. After the incidence of FMD,
biosecurity measures were strengthened during the elephant
camp held at Guruvayoor. Along with this, the state animal
husbandry department should also take initiatives to bring
these species under the umbrella of routine vaccination
campaign along with domestic cattle and buffaloes. The
pre- and post-vaccinated serum samples should be collected
and routinely tested in liquid phase blocking ELISA, which
will help to keep vigil over the kinetics of protective
antibody titre in the vaccinated elephants. To the best of
our knowledge, this appears to be the first confirmed report
of FMD in elephants based on partial characterization and
phylogenetic analysis of the virus genome. However, after
this incidence of FMD in 2013, the disease has not been
reported in these species in the particular region.
Hegde et al. (2010) having screened 37 elephant serum
samples from various zoological gardens and National Parks
in Karnataka for FMDV antibodies, suggested the necessity
of vaccination of these animals to maintain immunity
against the disease. Few reports state that free-ranging
elephants are rarely affected when outbreaks occur in
ruminants in their territory (Howell et al. 1973, Kalanidhi
et al. 1992); hence, clinical FMD has been supposed to be
a disease of elephants in captivity (Chakraborty and
Majumder 1990). Clinical cases of FMD are numerous for
Indian elephants, which may be linked to their higher
susceptibility to infection (Pyakural et al. 1976, Hedger and
Brooksby 1976). Still the possibility of virus transmission
between elephants cannot be underemphasized. Regular
surveillance thus becomes the prime need of the hour to
keep track on FMD incidences along with the circulating
serotypes and evolving strains of the virus. Each and every
outbreak in any susceptible species in an enzootic region
should assure rapid diagnosis followed by detailed scientific
investigation, so that the virus dynamics could be
understood paving the way for formulation of effective
control strategies.
ACKNOWLEDGEMENT
The authors are highly grateful to the Indian Council of
Agricultural Research (ICAR) for granting funds and
facilities for carrying out the work.
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