A. O. Vet. Fak. Derg.
37 (1) : 116-120, 1990
CRYOPRESERYATlON OF INFECTlYE LARVAE OF TRICHOSTRO:'-iGYLUS
YITRINUSı
Metin Alabay2 Zişan Emre' Harun Çerçi4
Trichostrongylus vitrinus cnfcktif lanalarının dondunılarak saklanması
Özet: Kil~fları çıkanınıış 3. dönem TrichostronRYlus vitdnus lar-valan fizyolojik su içinde sm azotta dondurulmuş ve 249 gün saklannıış-tır. Çözijldükten sonra canh kalan larva oram
%
29.2 olmuştur. Dahasonra larvalar, dondurmanııı enfektivite üzerindeki etkisini saptamak için 5 koyuna oralolarak verilmiştir. Larvalarm enfektivitesi zayif bulunmuştur.
Summary: Exsheathed third stage larvae of Trichostrongylus
vitrinus suspended in physiological sa/ine ıvere {rozen in liquid nitrogen and then stored for 249 days. Ajier thawing, the percentage of surviving larvae ıvas {ound to be 29.2
%.
Larvae were then orally trmısmitted to 5 sheep to determine the effect offreezing on ilifectivity. Tlıe infec-ti!'ity of tlıese larvae ıvas poor.Introduction
Successful cryopreservation of helminths comprısıng several
different cell types with differing volumes and permiability charac-teristics is intrinsically difficult to achieve. Those helminth species that can be cryopreserved are the free-livİng larvae of animal nema-todes (8). The infective larvae of many of these species can be succes-sfully cryopreserved simply by suspension in tap water and slow coling (i
oc /
min). Survival levels, as with Haemochus (3), are of tenextre-mely good. Those nematode species whose free-living third stage
ıThis study was sup!,orıed by Turkish Atomic Energy Ageney, Ankara. 2 Assoc. Prof. Lalahan Nuclear Reseaıch Institute of Animal Health, Parasitology Laboratory, Lalahan, Ankara.
3 DVM, PhD. Lalahan Nuclear Research Institute of Animal Health, Parasitology Labora!ory, Lalahan, Ankara.
4 BS,::. Lalahan Nuclcar Rescarch Institute of Anima! Health, Parasitology La-boratory, Lalahan, Ankara.
ENFEKTiı: LARVALARJ:-J DONDURULARAK SAKLANMASı 117
larvae retain the cuticle of the second stage as a protective sheath, invariably have to be exsheathed before they can be successfully cry-opreserved (2, 4, 9,
ıo,
i I). Artificial exsheathment is likely to fune-tion by removing a barrier to water movement allowing the larvae to dehydrate during cooling (8).Nematodes which remain enterily within a host throughout their life cycles, together with Schistosoma and Taenia, all appear to require the incorporation of crypoprotective additives (7, 8). Conversely, for most of the free-living larvae of nematodes of domestic animals, cryoprotectant addition leads to reduced survival (5, 9).
Several benefits could be gained from long term storage of cryop-reserved neınatode larvae. The considerable expenditure of time, labor and funds to continually maintain monospeeifie isolates in animals would be redueed and the risk of accidental contamination would be minimized.
Since Parfitt (Ii) reported that larvae of Nematodirus bat/us
survive freezing in liquid nitrogen and exsheathed Haemonclıus con-/or/us remain infective after four weeks cryopreservation (2), there has been increased interest in deep freeze storage of nematode larvae.
Ancylostoma ceylanicum (16), A. caninum (11), Dictyocaulus viviparus (9), Schis/osoma mansoni (7), N ippostrongylus hrasiliensis (I O), Coo-peria oncophora, Haemonchus con/or/us, Nema/odirus spathiger, Tric-hos/rongylus sp., Oesophagos/omunı sp. and Ostertagia sp. (4, 5, 6"
13) have all been suecessfully cropreserved. Van Wyk et aL. (14) examined the viability and the infectivity of 19 ruminant species af ter freezing and foun9 that generallyalı were infectiye after this treat-ment. Campbell et ai. (3) also reported that frozen H. con/or/us were as infective as normal larvae af ter 44 weeks of storage.
The present study on cryopreservation of third stage larvae of
T. vi/rinu.>was made to investigate the viability and infectivity of these larvae.
Materials and Mcthods
Infective larvae of Trichos/rongylus vitrinus were cultured from faeces passed by a sheep with a monospecific İnfection. The method of obtaining clean infectiye larvae of Trichostrongylus has been des-cribed before (I). Prior to cryopreservation, the larvae had been
sto-118 M. ALABAY-Z. EMRE-H. ÇERÇi
red in tap water in a refrigerator for about 6 weeks. Larvae were
exsheathed with O. 16
%
NaOCI. As soon as mass exsheathment hadstarted as viewed under dark baekground illumination (approx.
after 45 min.), NaOCl was removed by eentrifugation and the larvae
resuspended in 0.09
%
NaCl solution. Then, larvae were plaeed infive eppendorf tubes eaeh containing 15000 larvae in i mL. 0.09
%
NaCl solution. The tubes were frozen by plaeing them direetly into liquid nitrogen.
Frozen larvae were stored for 249 days to determine their survival
eapability. Larvae were thawed quiekly by plaeing the tubes into a
water bath at 37cC. immediately upon removing them from starage
in the liquid nitrogen. Af ter the larvae were thawed, the percentage of live larvae was determined on the basis of motility. Five doses
eon-taining approximately 4500 larvae were then prepared. These larvae
were then orally given to five sheep to determine theeffeet of freezing on infeetivity. Faeeal agg eounts were earried out using the MeMaster method 14 day s af ter inoeulation of larvae.
Results
The pereentages of T. vitrinus larvae survıvmg starage for 249
days in liquid nitrogen were 3 i .3, 34.4, 33.3, 23.9 and 23.1
%
(amean of 29.2 %) in the five samples. Larvae that survived freezİng
and thawing were not as motile as nonfrozen larvae.
lt this study, beeause it was not possible to slaughter the sheep
to make worm eounts, the infectivity of larvae could only be
asse-ssed by faecal egg eounts. Although all sheep beeame infeeted with
frozen larvae, faecal egg counts were very low. Egg eounts reaehed maximum values of 50 to 150 egg per gramme of faeees on day 38th of the infeetion.
Disscussion
At the present study, larvae of T. vitrinus were frozen by plaeing
them direetly into liquİd nitrogen. That means rapid eooling has been used and 29.2
%
survival of larvae obtained. Coles ct aL. (5) used bothrapid and slow eooling for eryopreservation of exsheathed T.
colub-riformis and T. axei larvae and obtained 35 and O
%
survival with rapid and 80 and 95%
with slow eooling rates respeetively. They also reported that oral ehallenge with frozen larvae was slightlysueeess-ENFEKTİF LARVALARıN DONDURULARAK SAKLANMASı 119
ful with intestinal nematodes and laparotmy was needed to
reestab-lish a successful culture of T. coluhrıformis. lsenstein and Herlich (6) stored ensheathed T. axei and T. collubriformis infective larvae in liquid nitrogen vapour by cooling at a rate of i
oc /
min. and found low percentage of larvae surviving cryopreservation. However, larvae that survived were as infectiye as nonfrozen larvae in rabbits. These results show better recovery of larvae and infection could be obtainedafter slow cooling of exsheated larvae and folIowing laparotomy.
Nevertheless, Campbell and Thomson (4) cropreserved
exshe-athed larvae of T. coluhriformis by rapid cooling and obtained 71-93
%
survivaI. Moreover, Van Wyk ct ai. (14,ı
5) reported that T.colub-riformis, T. axei and T. falculatus were viable af ter 2 years of
cryopre-servation by rapid cooling, a mean of more then 90 ~~ of the L3 being
alive when thawed after this period. They also found that sufficient
numbers of cryopreserved L3 of T. falculatus and T. colubriformis
developed when dosed per os in suspension without having to resort laparotomy.
Results reported in the present experiment demonstrated that
cryopreserved larvae of T. vitrinus were neither as viable nor infec-tive as reported for other TricllOstrongylus sp. (15). However, as this study \Vas of limited scopc, further more extensive experiments need
to be completcd to confirm these findings.
Reference~
I. Alabay, M., Çerçi, H. and Aykol, F. (1985). An il1lproved ıııeıhod of obtainiııg deaıı
infeetive lG/Tae of Triehoslroııgylııs vitrinııs. Doğa Bilim Derg. DI, 9: 229-231.
2. Campbell, W.c., Blair, L.S. and Egerton, J.R. (1972). MotWty and iııfeetiviıy of Haeıııoııclıııs eoll/orlııs lanııe lIjier freeziııg. Vet. Ree., 91: 13.
3. Campbell, W.c., Blair, L.S. and Egerton, J.R. (1973). Uniıııpaired iııfeetivity of the
nematode Haemonehııs eolılorlUs afier Feeziııg for 44 weeks iıııhe preseııee of liquid ııitrogen. J. Parasit., 59: 425-427.
4. Campbell, W.c. and Thornson, B.M. (ı973). Surviral o/ııeıııatode larvae ajier Feeziıı/;
over liquid ııitrogeıı. Aust. vet. J., 49: iLO-II J.
5. Cole~, G.c., Sirnpkin, K.G. and Briscoe, M.G. (1980). Routine eryopreservatioıı o/
yuıııinatıt nellllltode larvae. Res. vet. Sei., 28: 391-392.
6. Isenstein, R.S. and Herlich, H. (I972). Cryopreserrııtion of iıı/eetil'e ıhird slage lıırvae
120 M. ALABAY-Z. EMP.E-l-I. ÇERÇi
7 . Jame~, E.n. (198 ı). Selıisıosom(/ mansoni: Cryopreserraıioıı of selıisıosomula by
11'.0-sırp addiıioıı of ('(haııediol aııd rapid eooling. ExpI. Parasit., 52: 105-116. 8. James, E.R. (J985). Cryopresermıion of he/miths. Parasit. Taday., ı:134-139.
'). James, E.R. and Peacock, R. (ı986). Stlldies 011ıhe cryopresenalioıı ol" Dieıyocaulus
l'iriparııs (Ilemaıoda) ıhird slage lanae. J. Helmith., 60: 65-73.
LO. Kelly, J.D. and Campbell, W.c. (ı974). Sıınh.al of Nipposırongylus brasiliensis larva;? ajier freezing over liqııid nilrogen. Int. J. ParasitaI., 4: 173-176.
i i. Kelly, J.D., Campbell, W.c. and Whitlock, H.V. (1976). fııfectiviı)' of /lııcylos"f"ma caniııulIIll/l"l'ae ajier ji-ecziııg ova liqııid niırogen. AlIs~. vet. J., 52: 14ı-ı4~. 12. Parfitt, J. W. (1971). Decp ji-eeze preserrali,m of Ilemaıade larvae. Res. vet. Sei., ı2:
488-489.
13. Ramıı, Th., Eckcrt, J.and Christen, C. (ı986). Eıfiil!rtmgen mil der Gejiierkonsf:rl'i-eruııg driller Larveıısledien I"OnTrichosırongylideıı d?r Wiederkaıırr. Sch •.vcizcr Arclı.
Ticrheilk., 128: 79-86.
14. Van Wyk, J.A., Gerber, II.M. and Van Aardt, W.P. (1977). Cryopreservalioıı o( ıh'! infeeıire larvae ol" ıhe commaıı nema/odes of /"IImiııalı/s. Onderstcpooıt J. vel. Res., 44: 173-194.
ı5. Van Wyk, J.A., Gerher H.M. am! Alves, M.n. (1984). M,'ıhods of iııfesling s!?eeD wiı/ı gasıro-iıııesıiııal nema/odes ajia cr)'opreserraıioıı: Dosing ol" larva,7iıı gelalin capsııles compared ıo dosing of larvae in waıer sııspensioıı. Onde! stepoor[ J. vet. Res., sı: 217-221.
16. Vatter, J.C.M. and Klaver-\'Vesseling, J.C.M. (1977). lJnimpaircd infecıivi!y of Aney.
