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 Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 8  |  Issue : 2  |  Page : 107-114

Effect of nitroxynil (fasciolid) on adult Fasciola gigantica and Fasciola hepatica in infected cows


Department of Medical Parasitology, Faculty of Medicine, Sohag University, Sohag, Egypt

Date of Submission12-Dec-2014
Date of Acceptance25-May-2015
Date of Web Publication27-Jan-2016

Correspondence Address:
Eman K Omran
PhD, Department of Medical Parasitology, Faculty of Medicine, Sohag University, Sohag 2630
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1687-7942.175008

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  Abstract 

Background
Drug resistance to treatment of fascioliasis with triclabendazole (TCBZ) has emerged in Sohag Governorate, Egypt. Nitroxynil belongs to the halogenated phenol group of fasciolicides. It is highly active against adult liver flukes. A nitroxynil metabolite is produced in the liver parenchyma adding to its flukicidal activity and augmenting its efficacy against late immature flukes that migrate through the liver tissues. Treatment with nitroxynil may be an effective replacement for therapy with TCBZ in cases of resistance.
Objective
The aim of this study was to evaluate the efficacy of nitroxynil in the treatment of fascioliasis by assessing its effect on teguments and durability of adult Fasciola gigantica and Fasciola hepatica worms.
Materials and methods
Infected cows were selected on the basis of clinical signs, and infection was confirmed by detection of Fasciola eggs in their stools. Nitroxynil was administered as recommended in two doses 15 days apart, and the animals were slaughtered 15 days after treatment. Fasciola worms collected from the bile ducts were identified and prepared for electron microscopy. Tegument changes were examined with scanning electron microscopy.
Results
The removed adult flukes of both species were moving sluggishly and appeared pale with no evidence of gut content. Scanning electron microscopy examination of these flukes revealed evidence of swelling of the tegument that showed regional variation in its severity. Loss of spines was also observed.
Conclusion
The present study demonstrated the flukicidal properties of nitroxynil, proving that the tegument is an important target for its action. Disruption of the fluke's main line of defense allowed the drug access to other internal tissues, leading to more widespread damage. Nitroxynil may be successfully used for treatment in case of resistance to TCBZ.

Keywords: adult Fasciola, in vivo treatment, flukicides, nitroxynil (flukicide), scanning electron microscope


How to cite this article:
Omran EK, Ahmad NS. Effect of nitroxynil (fasciolid) on adult Fasciola gigantica and Fasciola hepatica in infected cows. Parasitol United J 2015;8:107-14

How to cite this URL:
Omran EK, Ahmad NS. Effect of nitroxynil (fasciolid) on adult Fasciola gigantica and Fasciola hepatica in infected cows. Parasitol United J [serial online] 2015 [cited 2017 Oct 22];8:107-14. Available from: http://www.new.puj.eg.net/text.asp?2015/8/2/107/175008


  Introduction Top


Fascioliasis is one of the most important parasitic diseases in domestic ruminants throughout the world caused by the trematodes Fasciola gigantica and Fasciola hepatica. Both flukes are economically significant parasites in livestock constituting one of the important causes of zoonotic infections. In Egypt, fascioliasis has been recorded as an important clinical problem, particularly among school-aged children living in rural areas of the Nile Delta [1],[2] . Animal as well as human fascioliasis is a growing problem, as it has been recorded in almost all governorates, especially those of the Nile Delta in Lower Egypt [3] . A large variety of animals, such as sheep, goats, cattle, buffalo, horses, donkeys, camels, and rabbits, showed Fasciola infection rates that may reach 90% in some areas [4] . Among these animal species, fascioliasis is highly endemic in sheep as indicated by macroscopic detection of liver flukes in slaughtered sheep during abattoir surveys (20.6%) [5] . In another report from North Sinai, 12.7% of Fasciola spp. were recorded in sheep and goats by microscopic detection of eggs [6] . In the same year, infection was found to occur mostly in sheep (17.84%), followed by cows (12.31%), buffaloes (9.73%), and lastly goats (5.40%) [7] . Reported prevalence rates for Fasciola infections have reduced in recent years due to control measures enforced in the Egyptian governorates, including triclabendazole (TCBZ) administration [2] .

In the absence of any effective vaccines, the principal method of control remains to be the use of drugs. Treatment with TCBZ, which is a member of the benzimidazole family of anthelminthics, is the main therapy for control of fascioliasis morbidity [8] . It works by preventing the polymerization of the tubulin molecule into the cytoskeletal microtubule structures [9],[10] and is particularly active against both juvenile and adult flukes [11] . Likewise, closantel, nitroxynil, rafoxanide, and TCBZ are among the few flukicides that are active against both mature and immature liver flukes. Other flukicides such as albendazole, clorsulon, and oxyclozanide are active against adult liver fluke only [12] .

However, resistance of F. hepatica to TCBZ has been registered since 1990 [13] and has since been reported in Australia [14] , Ireland [15] , the Netherlands [16] , and southern Wales [17] . It is now known to exist in a number of European countries as well [9],[18] , and in Egypt [19] . A number of fasciolicides (including nitroxynil) have been evaluated against TCBZ-resistant flukes [20] .

Nitroxynil (4-hydroxy-3-iodo-5-nitrobenzonitrile), which is a halogenated phenol [21],[22] , acts by stopping oxidative phosphorylation in the cell mitochondria. This disturbs the production of ATP, thus impairing motility of the parasites [23],[24] . Nitroxynil binds very strongly (97-98%) to plasma proteins [25] , indicating that oral ingestion is the most likely route. Its nematocidal activity against adult and larval stages of Haemonchus contortus in sheep and Haemonchus placei, Oesophagostomum radiatum, and Bunostomum phlebotomum in cattle was previously recorded [26] . It was reportedly active against TCBZ-resistant F. hepatica [27] and was found synergistically active when therapy was tested in combination with clorsulon or closantel in the treatment of salicylanilide-resistant flukes, even when administered in lower than recommended doses [28] . Therapy with nitroxynil gave 100% efficacy against Fasciola spp. in all post-treatment observations [29],[30] . Efficacies around 100% have also been described under field [31] and experimental [27] conditions.

The integrity of the surface plasma membrane and the tegumental syncytium is essential as a first line of defense for flukes. The tegument is the primary drug target immediately exposed to anthelminthics. In 2010, Toner et al. [32] observed that, due to the severity of changes produced by TCBZ treatment, the flukes become eliminated from the host through the gall bladder and that their migration corresponds with the degenerative changes. In their detailed report, they described progressive disruption of the tegumental system and musculature, which became severe with time of exposure, up to complete sloughing of the tegument and degeneration of the underlying tissues. Disruption of the gastrodermis also increased in severity over time, although it was not as severely affected as the tegument. They added that, notably, the recovered flukes displayed disruption of the subtegumental muscle bundles that appeared widely separated, as well as progressive loss of fibers that rendered the muscle blocks almost empty and barely recognizable [32] . It was noted that, on lengthening the period of treatment with 40 mg/kg nitroxynil, the activity of the adult fluke was affected up to immobility [33] . The earliest tegumental changes were recorded after 24 h from onset of treatment.

Most recently, in 2015, Junquera [34] stated that, in contrast to other anthelminthics (e.g. imidazothiazoles, benzimidazoles, and tetrahydropyrimidines), nitroxynil has a residual effect i.e., it not only kills the parasites present in the host at the time of treatment, but also protects against reinfestation for a period of up to several weeks, depending on the dose and the specific parasite. This further collaborates the former declaration that nitroxynil can be used as an alternative to TCBZ in therapy of resistant cases [35] .

Scanning electron microscopy (SEM) has been successfully used for evaluating anthelminthic effect on changes of tegumental surface of flukes. SEM allows scanning of the whole-body surface before examination with transmission electron microscope of internal structure changes [33] , revealing the location and possible variation of any disruption caused by a drug [36] . Therefore, the aim of the present study was to evaluate the efficacy of nitroxynil for the treatment F. gigantica and F. hepatica in naturally infected cows, by noting its action on the tegument and durability after treatment.


  Materials and methods Top


Type of study

This is a clinical veterinary trial.

Animal source of flukes

Infected cows were selected from Tahta Veterinary Clinic on the basis of clinical signs such as foul-smelling watery diarrhea, anorexia, and hypothermia with abrupt decrease in milk yield. Natural infection was determined by detection of Fasciola eggs in the cows' stools using direct smear and sedimentation methods.

Drug

Nitroxynil 1 ml/25 kg body weight was given orally to two naturally infected cows according to the instructions of Chemical Industries Development under license of Pharmachim Bulgaria. Two doses were given 15 days apart in an attempt to avoid any side effects.

Parasite collection

Flukes identified as adult F. gigantica and F. hepatica were recovered from the bile ducts of slaughtered cows 15 days after treatment and washed in several changes of warm (37°C) sterile physiological saline to remove any attached particles.

Scanning electron microscopy studies

Preparation of samples for SEM was carried out as described [37] . Washed flukes were fixed in 5% glutaraldehyde for 24-72 h and washed four times in sodium cacodylate buffer (pH 7.3) for about 15 min. Each postfixation was carried out by adding 1% osmium tetroxide for 2 h and then the samples were washed three times in the same buffer. Dehydration was carried out in ascending concentrations of ethanol (30%, 50%, 70%, and 90%), each for 30 min, and in double changes of absolute ethanol for 24-48 h. The samples were incubated at 20-25°C on double-sided carbon scotch tape and coated with gold. The samples were prepared, examined, and photographed at the SEM Unit, Assuit University.


  Results Top


Flukes recovered from the bile ducts of cows 15 days after treatment were moving sluggishly and appeared pale with no evidence of gut contents. The SEM examination revealed evidence of swelling of the tegumental surfaces, with regional variation in the severity of the swelling. On the ventral surfaces of the flukes, moderate swelling of the teguments was observed around the oral and ventral suckers ([Figure 1] and [Figure 2]). In the central regions of the oral cones, the teguments were severely swollen. The swelling was less severe along the lateral margins of the oral cones. At the same time, the spines were barely visible ([Figure 3] and [Figure 4]). In the anterior mid-body regions, directly behind the ventral suckers, no spines were visible as they had become completely submerged by the externally swollen teguments ([Figure 5] and [Figure 6]). The mid-body regions displayed widespread swelling and extensive furrowing of the teguments. The spines were almost submerged by the surrounding swollen teguments ([Figure 7] and [Figure 8]). The dorsal surfaces were similarly acutely affected as the ventral surfaces. The spines appeared to be flaking off, and there were multiple pores and furrowing of the teguments ([Figure 9] and [Figure 10]).
Figure 1: SEM of F. gigantica after treatment in vivo with Nitroxynil 1 cc/25 kg BW. Ventral Surface: The apical cone and the anterior mid-body regions revealed tegumental swelling with regional variations in severity surrounding the oral and ventral suckers (arrow).

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Figure 2: SEM of F. gigantica after treatment in vivo with Nitroxynil 1 cc/25 kg BW. Ventral Surface: In the central region of the oral cone the tegument was severely swollen and the spines were barely visible (arrows).

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Figure 3: SEM of F. gigantica after treatment in vivo with Nitroxynil 1 cc/25 kg BW. Ventral Surface: In the anterior mid-body region, directly behind the ventral sucker, no spines were visible as they had become completely submerged by the extremely swollen tegument.

Click here to view
Figure 4: SEM of F. gigantica after treatment in vivo with Nitroxynil 1 cc/25 kg BW. Ventral Surface: In the mid-body region, the spines appeared almost submerged by the swollen tegument surrounding them.

Click here to view
Figure 5: SEM of F. gigantica after treatment in vivo with Nitroxynil 1 cc/25 kg BW. Dorsal Surface: The spines appeared to be fl aking off, with multiple pores and furrowing of the tegument (arrows).

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Figure 6: Identified SEM of F. hepatica after treatment in vivo with Nitroxynil 1 cc/25 kg BW. Ventral surface. Tegumental swelling of the apical cone, showed regional variations in severity surrounding the oral and ventral suckers (arrows).

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Figure 7: SEM of F. hepatica after treatment in vivo with Nitroxynil 1 cc/25 kg BW. Ventral surface. Identified area between the oral and ventral sucker showed displaced spines (arrow).

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Figure 8: SEM of F. hepatica after treatment in vivo with Nitroxynil 1 cc/25 kg BW. Ventral surface. The mid-body region laterally showed displaced spines directly behind the ventral sucker, no spines were visible as they had become completely submerged by the extremely swollen tegument.

Click here to view
Figure 9: SEM of F. hepatica after treatment in vivo with Nitroxynil 1 cc/25 kg BW. Ventral surface. The central mid-body region showed severe swelling and furrowing of the tegument (arrow).

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Figure 10: SEM of F. hepatica after treatment in vivo with Nitroxynil 1 cc/25 kg BW. Dorsal surface. The spines appeared to be fl aking off with multiple pores and furrowing of the tegument (arrow).

Click here to view



  Discussion Top


Treatment of animal fascioliasis is imperative for the management of economic losses and for avoiding zoonotic infections. Cows and sheep pose as the main hosts for both Fasciola spp. and are responsible for passing the infection to man [38] . The effective control of these infections with anthelminthic drugs is therefore vital for both animal welfare and productivity [21],[39],[40] . The drug of choice in the treatment of fascioliasis has been TCBZ, until the flukes started to exhibit their resistance to it [13] . Resistance of liver flukes to treatment with nitroxynil has not reached the scale experienced with nematodes, but it has been reported for salicylanilides, rafoxanide, and closantel, with cross resistance to nitroxynil. Another report on treatment with closantel and nitroxynil was published in SCOPS [41] .

It was postulated that the administration of these flukicides to animals used for human consumption can lead to transfer of drug residues to human food [12],[21],[42] . In 2013, all drug products containing the active ingredients of clorsulon, closantel, nitroxynil, rafoxanide, and TCBZ were prohibited for use in lactating animals producing milk for human consumption. The reason presented was that residues in undetectable concentrations in milk could be concentrated during manufacturing, and become detectable in milk products [12] . The researchers recommended treatment of dairy cows during the dry period, when no milk is being produced for human consumption, and advised that an adequate withdrawal period is allowed for the elimination of drug residues before resumption of milking.

In vitro studies have shown great drug disruption of worms following treatment with clorsulon and nitroxynil [33],[43] . Besides ingestion through the gut, the tegument presents an absorptive surface for the uptake of drugs by the fluke. The adult fluke is bathed in bile when in the bile ducts of the host, which are its preferential dwelling site, and becomes exposed to drugs excreted in the flow of bile. Thus, because the tegument is immediately exposed to anthelminthics, it is a crucial drug target. The drug-induced damage of the tegument facilitates drug penetration to deeper-lying tissues, thus leading to serious effects on the flukes. It was deduced by McKinstry et al. [33] that, in the in vivo situation, the surfactant action of bile and the immune response aggravates the damage, leading to the discoloration of the flukes observed after 72 h treatment. Nitroxynil is excreted mostly unchanged through the liver giving rise to high concentrations in the bile ducts. Recent research showed that a metabolite of nitroxynil produced in the liver parenchyma added to its flukicidal activity and explained its efficacy against late immature flukes that migrate through the liver tissues [34] .

In the present study, the flukes recovered from the bile ducts of nitroxynil-treated cows 15 days after treatment were not dead and moved sluggishly. They appeared pale with no evidence of gut contents. As described, healthy flukes are grayish-brown in color, with a flattened spiny tegument, through which the dark outline of blood-filled ceca is evident [44] . The pallor and empty guts of flukes, observed in our study confirms that penetration and autophagia of substances such as bile and drugs takes place [32] . As regards other flukicides, it was reported that praziquantel produces flaccid paralysis of the fluke, starting with initial increase followed by decrease in muscle tone [45] . The researchers added that oxyclozanide, rafoxanide, niclofolan, bithionol, and hexachlorophene also induce rapid spastic paralysis of the fluke. Nitroxynil produces a similar effect, which explains the sluggish movement recorded in our study. By discontinuing oxidative phosphorylation in the cell mitochondria, it disrupts the production of ATP, thus causing a long-term suppression of movement [23],[24] . In another report, the flukes after treatment in vivo with nitroxynil showed little or no sign of movement and appeared yellow in color, with no evidence of gut contents. Swelling of the mid-body was visible to the naked eye [36] .

The present study has confirmed that nitroxynil causes swelling and severe disruption of the tegumental surface of Fasciola, as well as swelling of the basal in-folds in the tegumental syncytium, edematous flooding of the parenchyma, and mitochondrial deformation in the syncytium and tegumental cells [46],[47] . In our study, no spines were visible in the anterior mid-body region, directly behind the ventral sucker, as they had become completely submerged by the externally swollen tegument. The mid-body region also displayed widespread swelling and extensive furrowing of the tegument, and the spines were almost submerged by the swollen tegument surrounding them. From previous studies, disruption to the spines seems to be a particular feature of nitroxynil action [33],[44],[45] . When due to the action of the drug, the thin tegument covering the spines splits and sloughs off and the spines are lost, the holes thus formed in the syncytium allow more access of the drug to internal tissues. This becomes especially promoted by the surfactant action of bile in vivo [33] . The cause for disruption of spine structure observed in our study and reported with other fasciolicides is unknown [48],[49],[50] .

In the present study, the dorsal surface disruption was as severe as that seen on the ventral surface. The spines appeared to be flaking off, and multiple pores and furrowing of the tegument were detected. This outcome of in vivo treatment with nitroxynil was previously defined as regional differences in tegumental disruption that is more severe on the dorsal than on the ventral surface, and on the anterior than on the posterior region of the fluke [33] . In their investigation, the researchers also described extensive swelling and blebbing of the tegument on both surfaces, and at high magnification microvillus-like projections were evident with some tegumental loss in the oral region of the fluke. In our point of view, greater affection of the dorsal surface resulted from higher exposure to the drug as the flukes are folded ventrally when in the bile ducts.

McKinstry et al. [36] stated that, after 24 h of in vivo treatment with 40 mg/kg of nitroxynil, the recovered flukes were moving normally but appeared pale, with no evidence of gut contents. After 48 h, the flukes were less active, and surface swelling could be observed. After 72 h, the flukes showed little or no sign of movement; all appeared yellow with empty gut contents. Swelling of the mid-body was visible to the naked eye. In comparison, another report found that after the full course of in vivo treatment with TCBZ the recovered flukes remained active for 48 h displaying limited morphological disruption, but they were all dead after 72 h. The researchers described some blebbing and sloughing of the tegument around the oral sucker, when an extra layer had apparently been deposited on the fluke surface, giving it a flattened appearance [51] . A relatively recent investigation indicated that each of TCBZ-susceptible and TCBZ-resistant isolates were affected by nitroxynil treatment in vitro and in vivo, showing ultrastructural changes similar to those previously seen in the Cullompton TCBZ-susceptible isolate [36] . In the present study, 15 days after treatment the flukes were still alive, but suffered severe swelling of the tegument, accompanied by isolated areas of flattening along the lateral margins of the flukes and in the tail region. Our SEM examination of nitroxynil-treated flukes showed limited areas of tegument sloughing in the tail region. In more seriously affected specimens, the syncytium appeared stripped away to reveal the basal lamina. Some deeper lesions were also observed as the loss of tegument facilitated penetration of the drug to the exposed deeper tissues of the flukes. There was disruption of the subtegumental tissues with spacing or flooding between the cells and breakdown of the cell bodies, to the extent that often they were difficult to identify. Similar findings were described [32] in TCBZ-treated flukes. The flooding feature was also recorded in other drug studies on F. hepatica using sulfoxide metabolite of the compound alpha [50] and metabolites of TCBZ as sulfoxide (TCBZSO) and sulfone (TCBZSO 2 ) [52] . A very recent publication in 2015 [53] revealed that, in sheep with a high fluke burden, TCBZ was ineffective in treating chronic fasciolosis indicating TCBZ resistance. At the same time nitroxynil and closantel proved to be wholly active against TCBZ-resistant flukes in chronically infected sheep with high chronic fluke burdens.

In conclusion, our study has confirmed previous SEM studies with nitroxynil in that the drug effectively disrupts the tegument of Fasciola. In addition, it causes disruption to the gut following in vivo treatment, an observation that is consistent with the oral uptake of the drug as well. Giving similar therapeutic results with little resistance, nitroxynil therapy of fascioliasis in rural animals may be considered a safe replacement for TCBZ treatment.

Acknowledgements

Author contribution:
EK Omran and NS Ahmad proposed the research idea, conducted the work, and shared in writing and revision of the manuscript.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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