RESEARCH ARTICLE

Histopathological Studies Targeting Comparative Efficacyof Herbal and Standard Allopathic Immunomodulators AgainstVisceral Larva Migrans

Aman Dev Moudgil • Susamoy Mittra •

Rajesh Kumar Asrani • Virendra Singh

Received: 19 April 2014 / Revised: 23 June 2014 / Accepted: 21 August 2014

� The National Academy of Sciences, India 2014

Abstract Various anthelmintics are effective against the

adult stages of Toxocara canis, but possess a doubtful

efficacy against the tissue encysted larval stages because of

their low solubility and approximately no bioavailability

for the tissues. Hence, repeated administration of these

drugs may lead to development of resistance. The aim of

the present study was to evaluate the effect of three plant

extracts (methanolic extracts of Hippophae rhamnoides,

Hippophae salicifolia and Piper longum), in comparison to

an allopathic drug (levamisole) on the migratory behavior

of Toxocara canis larvae in tissues of Swiss albino mice

(paratenic hosts). Fourteen days after medication, the mice

were experimentally infected with embryonated eggs of

Toxocara canis. Histopathological changes in various

organs caused by migration of larvae were scored, based on

severity of tissue/cellular damage and were categorized

into four levels: normal (0), mild (?), moderate (??) and

severe (???) in various organs of mice belonging to

different groups, at days 14 and 28 post infection. The

severity of tissue damage was considered inversely pro-

portional to immunomodulatory efficacy. Hence, the find-

ings of this study revealed Hippophae salicifolia to be a

superior immunomodulator followed by Hippophae

rhamnoides, levamisole and Piper longum in Swiss albino

mice model.

Keywords Toxocara canis � Hippophae rhamnoides �Hippophae salicifolia � Piper longum � Levamisole

Introduction

Toxocara canis [1] is one of the most harmful helminth

parasites of dogs, possessing significant zoonotic potential.

The infection and propagation of parasite in the definitive

host depends on many factors such as age, geographical

area and climatic conditions [2]. The larval stages of T.

canis have obligatory tissue migratory phases with

remarkable longevity, resulting in generation of a drug

resistant reservoir in adult dogs [3, 4] and visceral larva

migrans in infected human beings [5, 6].

A widespread development in resistance and various

untoward effects caused by synthetic and allopathic drugs

have prompted many workers to look for herbal immuno-

modulators in the management of infectious diseases [7],

especially parasitic diseases. The current therapeutic

management strategies for the treatment of adult definitive

hosts (dogs) and paratenic hosts including human beings

become difficult due to uncertain efficacy of drugs as well

as covert nature of T. canis infection [8].

Keeping these facts in mind, present study was planned

to observe the effects of herbal immunomodulators on

migratory behavior of T. canis larvae in tissues of

A. D. Moudgil � S. Mittra

Department of Veterinary Parasitology, DGCN College of

Veterinary and Animal Sciences, CSK HP Krishi

Vishwavidyalaya, Palampur 176062, Himachal Pradesh, India

A. D. Moudgil (&)

Department of Veterinary Parasitology, College of Veterinary

Sciences (COVS), Guru Angad Dev Veterinary and Animal

Sciences University (GADVASU), Ludhiana, Punjab, India

e-mail: moudgil.aman@gmail.com

R. K. Asrani

Department of Veterinary Pathology, DGCN College of

Veterinary and Animal Sciences, CSK HP Krishi

Vishwavidyalaya, Palampur 176062, Himachal Pradesh, India

V. Singh

Seabuck Thorn, College of Basic Sciences, CSK HP Krishi

Vishwavidyalaya, Palampur 176062, Himachal Pradesh, India

123

Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.

DOI 10.1007/s40011-014-0425-5

experimentally infected mice. The study may prove helpful

for management of toxocarosis in pups and also to manage

the untoward effects of zoonosis to paratenic hosts, espe-

cially human beings.

Material and Methods

Collection of Debris Free Eggs

The eggs of T. canis were collected from the faeces of

donor pups. These eggs were harvested by floatation

technique using Sheather’s sugar solution [9] and cultured

for embryonation. After embryonation was complete, these

infective eggs were used to infest the experimental animals

as per Institutional Animal Ethics Committee’s guidelines

[10].

Preparation of Immunomodulators

Fresh leaves of sea buckthorn (Hippophae rhamnoides and

H. salicifolia) were collected and completely shade dried

and grounded separately with the help of pestle-mortar

initially followed by grinder (Cyclotec, 1093 sample mill,

Foss Tecator) in order to get a fine powder. Fifty grams of

powdered material for either species was mixed with

400 ml of absolute methanol separately and left for 24 h in

a 500 ml conical flask. The content was then filtered using

fine muslin cloth followed by Whatman filter paper no. 41.

The filtrate was concentrated into semisolid form using

vacuum evaporation (Rota Vap, Bucchi). The contents

were then freeze dried using lyophilizer (Lyoplus) in a

round bottom flask for 4–5 h until dark brown crystalline

form was achieved which was stored at 4 �C until used.

Good quality whole fruit of Piper longum were procured

from local market and dried completely in hot air oven at

60 �C for half an hour. These dried fruits were then ground

with the help of grinder (Cyclotec, 1093 sample mill, Foss

Tecator) to obtain fine powder. Then, methanolic extract of

P. longum was prepared as mentioned earlier.

Levamisole tablet (150 mg), (DICARIS�, Johnson and

Johnson, Jansen Cilag Pharmaceuticals, Mumbai) was used

for medication at appropriate dosage.

Dosage of Extracts

Methanolic extracts of H. rhamnoides, H. salicifolia and P.

longum were administered to the mice of the respective

immunomodulated group, orally @ 500 mg/kg body

weight [11]. Levamisole was orally administered to the

mice @ 2.5 mg/kg body weight [12], in aqueous solution.

All the immunomodulators were administered at their

respective dose rates for three consecutive days.

Method of Infection

The experimental animals were infected orally with 500

infective ova of T. canis. Following each infection, a few

drops of distilled water were sucked into the syringe

(without needle), shaken and any remaining eggs left, were

flushed into the mouth of the experimental animal (Table 1).

Assessment of Immunomodulatory Efficacy

Tissue samples of experimental rats were fixed in 10 %

neutral buffered formalin. The 5 l thick sections were cut

and stained with haematoxylin and eosin for the histopa-

thological studies as per the method of Luna and Lee [13].

Results and Discussion

Histopathological studies revealed inflammatory changes

in various organs, mainly liver, kidneys, lungs and muscles.

The lesions were scored and categorized into four levels:

normal (0), mild (?), moderate (??) and severe (???).

Various parameters targeted to adjudge the lesion score in a

particular organ were: level of inflammatory reaction,

degenerative changes, necrotic area, hemorrhages and

presence of migrated larval stages in the organ.

On day 14 post infection, in infected control group I,

liver showed granulomatous infiltrations, representing

severe inflammatory reaction (???) (Fig. 1), moderate

degenerative changes (??) and severe necrosis (???).

Kidneys, lungs and muscles also showed severe inflam-

matory reaction (???). These observations were in

agreement with the observations of Fan et al. [14], who

observed that histopathological changes were moderate to

severe in the liver and lungs, mild to moderate in the

muscles, and only normal to mild in the brain throughout

the trial. However, Agnihotri [15] reported maximum

damage in the liver due to T. canis larvae migration at day

2 post infection. In group II, (levamisole immunomodu-

lated and T. canis infected) liver, kidneys and muscles

showed mild (?) inflammatory reaction, whereas, lungs

showed severe inflammatory reaction (???). There were

mild (?) degenerative changes and necrosis in liver. The

mild inflammatory reaction observed in vital organs as

compared to group I could be attributed to the established

immunomodulatory efficacy of levamisole [12]. In various

organs of groups III and IV, (H. salicifolia and H.

rhamnoides immunomodulated and T. canis infected) only

mild (?) to moderate changes (??) were revealed. These

observations were indicative of better immune conference

in H. salicifolia and H. rhamnoides which establishes them

as better immunomodulators. It has been reported that H.

rhamnoides extract and oil [16] stimulate cellular and

A. D. Moudgil et al.

123

humoral immunity in rabbits and mice, supporting the

observations of the present study. In liver, kidneys and

lungs of the group V (P. longum immunomodulated and T.

canis infected), showed severe (???) inflammatory

reaction and also moderate (??) degenerative changes and

necrosis. In this group larvae were recovered at certain foci

in muscles ascertaining its poor immunomodulatory effect.

On day 28 post infection, severe (???) inflammatory

reaction was recorded in liver, lungs and muscles of

infected control group I. There were moderate (??)

degenerative changes and necrosis in liver and muscles

only. In the levamisole immunomodulated and T. canis

infected group II, moderate (??) to severe (???)

inflammatory reaction was recorded in every organ,

accompanied by mild (?) to moderate (??) degenerative

changes and necrosis in liver (Fig. 2) and muscles. Mild

(?) to moderate (???) inflammatory changes were

recorded in almost every organ in the groups III and IV

Table 1 Experimental design depicting grouping of mice

S.

no

Group Number of

mice

Details

1 I 8 Toxocara canis infected control

2 II 8 Levamisole immunomodulated and T. canis infected

3 III 8 Hippophae rhamnoides immunomodulated and T. canis infected

4 IV 8 Hippophae salicifolia immunomodulated and T. canis infected

5 V 8 Piper longum immunomodulated and T. canis infected

6 VI 16 Sixteen mice of this group were divided into four subgroups (each containing four mice). Each subgroup was

immunomodulated with H. rhamnoides, H. salicifolia, P. longum and levamisole without infection

7 VII 4 Healthy control (non-infected and non-immunomodulated)

Fig. 1 Microphotograph of section of liver, showing granulamatous

inflammatory reaction, induced by larvae of T. canis H and E 9132

Fig. 2 Microphotograph of section of liver showing transversely cut

larva (L) of T. canis H and E 9132

Fig. 3 Microphotograph of section of lungs, showing longitudinally

cut larva of T. canis (L) H and E 9132

Assessment of Comparative Efficacy of Immunomodulators

123

(H. salicifolia and H. rhamnoides immunomodulated and

T. canis infected). This could be attributed to immuno-

modulatory and anti-oxidant properties of plants of genus

Hippophae [17–19]. Larvae were also recorded at many

foci in the group III (H. rhamnoides immunomodulated and

T. canis infected) (Fig. 3). However, mild (?) to severe

(???) inflammatory changes along with presence of lar-

vae were recorded in the organs like kidneys, muscles and

brain of the mice of group V (P. longum immunomodulated

and T. canis infected group) (Figs. 4–6). These observa-

tions were in partial corroboration with the observations of

Agnihotri [14], who reported lesser degenerative reaction

in later phase of the infection in the lungs but more neu-

ronal degeneration, gliosis and satellitosis in brain tissue in

Golden hamster.

The results of the present study projecting H. rhamno-

ides and H. salicifolia to be better immunomodulators than

established allopathic immunomodulator levamisole are in

complete corroboration with the findings of Moudgil et al.

[20].

Future Perspective

The immunomodulatory efficacy of the herbal extracts

against the visceral larva migrans due to T. canis larvae in

the paratenic host was evaluated in the present study.

Further studies on anthelmintic efficacy of these herbal

extracts by means of in vitro and in vivo studies in the

definitive hosts are warranted to adjudge the efficacy of

Hippophae species against adult and larval stages of T.

canis.

Conclusion

On the basis of histopathological observations i.e., micro-

scopic changes in various organs of mice belonging to

different groups, at days 14 and 28 post infection, it can be

proposed that H. salicifolia and H. rhamnoides are superior

immunomodulators followed by levamisole and P. longum

for controlling larval toxocarosis in Swiss albino mice

model. Thus, these herbal immunomodulators can be opted

as an alternative for controlling visceral larva migrans in

adult dogs and accidental hosts especially human beings.

Acknowledgments Authors are thankful to the Dean, Dr. G.C. Negi

College of Veterinary and Animal Sciences, Palampur for providing

the necessary facilities for undertaking this investigation.

Fig. 4 Microphotograph of section of kidneys, showing longitudi-

nally cut larva (L) H and E 9132

Fig. 5 Microphotograph of section of muscles showing transversally

cut larva (L) of T. canis, without any inflammatory zone. H and E

9132

Fig. 6 Microphotograph of section of brain, showing transversely cut

larva (L) of T. canis H and E 9132

A. D. Moudgil et al.

123

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