The
PCR was standardized taking two sets of oligonucleotide primers; one targeted
at Large Subunit rRNA gene (LSU rRNA
gene) and the other targeted at Mitochondrial Cytochrome Oxidase subunit I gene
(Mt Cox 1 gene) employing conventional PCR and multiplex PCR respectively.
Upon agarose gel electrophoresis, amplified product size at 286 bp and 984 bp were
observed in case of conventional PCR and multiplex PCR respectively (fig. 11
& 12) for all the three infected pigs (N1,M1 and M2)
confirming the cysts to be Cysticercus
cellulosae.

Jardim et al. (2006) observed that the
primer pair TBR-3/TBR-6 targeting the LSU rRNA gene amplified specific
fragments of 286 bp from T. solium and did not show any
cross reaction with DNA obtained from Taenia hydatigena, Taenia
taeniaeformis, Hymenolepis
diminuta, Anoplocephala
magna, Paranoplocephala
mamillana, Moniezia
expansa, Homo sapiens,
Bos taurus nor Sus scrofa after selecting novel primers for the
species-specific identification of
Taenia saginata and
Taenia solium. The gene
is specific and conserved for taenid cestodes and the primer TBR-3 was selected
from the conserved region and TBR-6 from semi-conserved region of the LSU
rRNA gene which are being used in the present study.Emphasis is being given on the utility of
mitochondrial DNA (mtDNA) for molecular discrimination of taeniid taxa of closely
related organisms because of its relatively rapid rate of evolution, maternal
inheritance and it does not recombine. The advent of the polymerase chain
reaction (PCR) has provided a highly sensitive approach that is now widely used
to target mtDNA sequences for Echinococcus and Taenia identification
purposes, including discrimination of eggs (McManus, 2006). The availability of
complete or almost complete mtDNA sequences for several parasitic helminthes
provide a rich source of genetic markers for phylogenetic analysis and study of
genetic variability in helminth groups (Le et al., 2000).

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Sreedevi
et al. (2011) employed PCR test targeting
LSU rRNA gene and the Mt Cox 1 gene to detect Taenia solium
DNA in muscle lesions for validation of the meat inspection results of slaughtered
pigs which yielded products of 286 and 984 bp respectively in cysticercosis
positive cases. Yamasaki et al. (2004a)
established the multiplex PCR for
differential diagnosis of taeniasis and cysticercosis with Mt Cox 1 gene which yielded evident
differential products unique for Taenia saginata and Taenia asiatica and
for American/African and Asian genotypes of Taenia solium with molecular
sizes of 827, 269, 720, and 984 bp, respectively. Mt Cox 1 gene was also
targeted for the molecular diagnosis of neurocysticercosis in humans from formalin-fixed,
paraffin embedded specimen of brain (Yamasaki et al., 2004b), histological section of a subcutaneous
cysticercosis case (Shih et al.,
2010), ante-mortem diagnosis of cysticercosis in pigs (Ramahefarisoa et al., 2010)

Conclusion

In
the present study, the pigs slaughtered in the Mumbai and Nagpur region were reared
in semi-intensive system and free range system with every chance of open access
to the faeces of tapeworm carriers. The presence of various inflammatory cells
surrounding the cyst was evident in visceral organs. The eosinophils seemed to
be predominant. Neuronal degeneration, gliosis, neuronophagia were being
observed in the brain.

Conventional
post-mortem inspection may sometimes leads to misdiagnosis of other
morphological changes in affected muscles and organs. Looking at
histopathological sections, it is difficult to ascertain the exact etiology of
the disease. Therefore, molecular diagnostics methods have been considered for
validation of macroscopic lesions as these tests were found to be highly
specific and sensitive. Both the conventional and multiplex PCR employed in the
present study for the molecular confirmation of metacestodes produced amplified
product at 286 bp and 984 bp respectively, confirming the cysts to be those of Taenia solium.

Cysticercosis
and neurocysticercosis remain a serious neglected problem in marginalized
communities in many areas of India mainly due to poverty, ignorance, lack of
suitable diagnostic & management capacity and inappropriate prevention and
control strategies. This disease can be prevented if pigs are reared in
intensive farming with proper sanitary management practices. People should be
made aware of this disease both from zoonoses and food safety point of view and
proper control measures should be strictly followed in order to minimize the
risk of infection. 

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