In vitro cultivation 2019 - In vitro cultivation of Setaria digitata worms in artificial media

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In vitro cultivation of Setaria digitata worms in artificial media
Shantaveer S. Biradar*
and Placid E. D’Souza
Department of Veterinary Parasitology, Hebbal, Veterinary College, Bengaluru (India)
E-mail: shantuvet-AT-gmail.com (
*Corresponding author)
Abstract: Setaria digitata is one of the common filarid worm found in cattle. The adult
worms found in the peritoneal cavity in male animals after slaughter. The objective of
this study was to investigate the survival, activity and release of excretory secretory
(ES) products from S. digitata, maintained in vitro in artificial media. The live adult S.
digitata worms (n = 12) comprising 2 males and 10 females were collected from the
peritoneal cavity of naturally infected male cattle slaughtered at KMPMCL abattoir,
Bangalore. The in vitro cultivation of adult S. digitata worms was done in 3 ml of
Dulbecco’s Modified Eagle’s Medium (DMEM) per worm (36ml for 12 worms) along
with antibiotics and antifungal agent incubated at 370
c in 5% carbon dioxide
atmosphere in the incubator. All the worms incubated in DMEM were alive and very
active for 48 hours. The activity was moderate and later they became sluggish and
worms start disintegrating from third day. Copious amounts of ova and microfilaria
were shed by the incubated worms after 24 hrs. The excretory secretory (ES) products
were obtained after centrifugation and the sediment contained microfilaria and ova.
Keywords: Setaria digitata, ova, microfilariae, DMEM, Antigenic ES products.
Introduction
The economic losses and debilitating effects caused by filariosis severely affect
man and animal power resources in developing countries. The common species of
Setaria causing microfilariosis in cattle was found to be S. digitata. The incidence of
56.8 and 12.5 per cent microfilariosis in cattle caused by S.digitata reported in
Karnataka and Orissa respectively (Sundar et al., 2005; Mohanty et al., 2000). The
adult worms are found in the peritoneal cavity and the larvae known as microfilariae
are found in the blood. Immature forms of S. digitata are causing a condition called as
‘‘cerebrospinal nematodiasis or kumri’’ in unnatural hosts. Excretory secretory (ES)
antigens play a major role in immunity to filarid nematodes of cattle and to extract
these antigens worms need to be maintained in artificial culture media (Sundar and
D’Souza, 2013). The present study was undertaken to investigate the survival, activity
and release of different developmental stages along with excretory secretory products
of S. digitata.
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Materials and Methods
The live adult S. digitata worms (n = 12) were collected from peritoneal cavity of
naturally infected male animals slaughtered at KMPMCL abattoir, Bangalore. The
worms were collected in sterile sample container with PBS (pH-7.3) and immediately
transported to the laboratory and washed thoroughly in 3 changes of phosphate buffered
saline (pH 7.3) every 15 min. The in vitro cultivation of adult S. digitata worms was
done as per method of Sundar and D’Souza (2013).
The in vitro cultivation of adult S. digitata worms was done in 3 ml of
reconstituted DMEM (Himedia, Mumbai) per worm (36ml for 12 worms) incubated at
370
c in 5 % carbon dioxide atmosphere in the incubator (Plate 1). The medium was
replenished every 24 h with fresh DMEM medium. The viability of the worms was
monitored daily for active motility. Mortality and other changes were also recorded. In
the early stages of incubation, incubating fluid transferred in to a small petridish to
observe release of different developmental stages of S. digitata and the process of
formation of sheathed microfilariae.
The spent media was collected every day and was finally pooled. The
membrane components were removed by centrifugation at 10,000 rpm for 60 min at 40
c
and the supernatant containing the antigen was dialysed against sucrose overnight
under refrigeration followed by reverse dialysis overnight against PBS. The
concentrated material was collected and considered as ES-antigen and it is aliquoted
and stored at -200
c until further use.
Result and Discussion
All the worms incubated in DMEM were alive and very active for 48 hours. The
activity was moderate and later they became sluggish and worms start disintegrating
from third day. Copious amounts of ova and microfilaria were shed by the incubated
worms after 24 hours. The sediment contained microfilaria and ova.
Development of microfilaria starting from the stage of egg to fully developed
microfilaria (L1) was observed. The eggs were oval in shape containing embryonic
mass inside (Plate 2) and observed larval development inside the egg (Plate 3).
Microfilariae having round anterior and pointed posterior ends with the egg shell
remained on the larvae forming a sheath around it were found in incubating medium
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once they show motility. The present observations on the development of egg stage to
L1 stage of S. digitata were in agreement with reports of Decruse and raj. 1990
The ES antigens responsible for pathogenicity of microfilariosis were released
during incubation during hatching of embryos. However, antigenic characterization of
ES antigens is in progress.
References
Decruse SW, Raj RK. Histological studies on female Setaria digitata (von Linstow
1906), a filaria of bovine, Bos indicus. Proceedings of the Indian Academy
of Sciences (Animal Sciences). 1990; 99:103-112.
Mohanty, M.C., Sahoo, P.K., Satapathy, A.K and Ravindran, B., 2000, Setaria digitata
infections in cattle: Parasite load, microfilaraemia status and relationship to
immune response. J. Helminthol 74: 343-347.
Sundar, S. T. B., D’Souza, P. E. and Jagannath, M. S. (2005). Prevalence of Setariosis
in Cattle and buffaloes in Karnataka. J. Parasitic Dis, 29: 147-149
Sundar, S.T.B and D’Souza, P.E., 2013. Survival, activity and release of antigenic
excretory secretory products and microfilariae of Setaria digitata
maintained in artificial media. Journal of Parasitic Diseases: Official
Organ of the Indian Society for Parasitology., 39 (1):107-109.
Plate 1. S. digitata in DMEM medium Plate 2: Embryonated egg of S. digitata
(10X).
Plate 3: Egg with coiled larvae (100X).