Spirurid infection Rictularia affinis jaegerskiold, 1909 ... · Abu Zanada., 1993) to identify the...

Danish Journal of Medical and Biology Sciences , June, 2015, Pages: 15-28

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Spirurid infection Rictularia affinis jaegerskiold, 1909 (Spirurida:Rictulariidae) from Egyptian fox Vulpes niloticus E.Geoffroy Saint Hilaire,1803 (Mammalia:Canidae)

Khalaf Nour Abd el-Wahed Ammar Zoology Dept., Faculty of Science; South Valley University, Qena, Egypt

A r t i c l e I n f o r m a t i o n A b s t r a c t

Article history: Received: 1 May Received in revised form: 28 May Accepted: 6 June Available online: June Keywords: Rictularia fox Vulpes niloticus Mammalia Upper Egypt SEM Carnivorous.

Corresponding Author: Khalaf Nour Abd el-Wahed [email protected] © 2015 Danish Journals All rights reserved

A spirurid parasite Rictularia affinis was detected in the small intestine of four Egyptian fox from nine dissected (36%). The males measured 7 to 9 mm but the females were 26 to 30 mm. The eggs were thick shelled. Species was redescribed by light and scanning electron microscopy, compared with the other related species of the same genus. Observed using SEM the body ornamented with paired combs, sclerotized spine, subventrally commencing immediately behind the helmet capsule. Buccal capsule inclined subterminal, hemicircular; dorsal rim with teeth, ventral rim with large cutting plate; two rings of cephalic papillae were observed: the internal ring, consisting of 6 papillae and external ring consisting of 4 papillae, amphidial pores and a spherical swellings deirids are present. There are numerous sclerotized teeth like denticles arranged in two rows (11-13 ventral denticles), situated in the buccal cavity around the mouth opening. The denticles are better developed on the ventral side of the mouth. Male spicular sheath (genital cone) well developed, ring-formed with numerous papillae on the surface, there are 5 to 6 well developed semicircular fan-shaped., 4 preanal; 2 adanal and 8 postanal pedunculated caudal pappilae, gubernaculum not discernible. In female2 phasmide present in posterior end and having a spine in the distal extremity Protruded, vulva without lip, situated at the posterior of esophageal-intestinal junction.

To Cite This Article: Khalaf Nour Abd el-Wahed Ammar,Zoology Dept., Faculty of Science; South Valley University, Qena, Egypt,, Danish journal of psychology, 15-28, 2015

Introduction Foxes live in all around the world in different climates in even in the North Pole, fox is an important reservoir host of parasites, which can be spread to another animals and humans Thompson, et al 2009.it is definitive host of the cestode Echinococcus multilocularis, this helminth is mainly responsible for highly pathogenic alveolar echinococcosis in humans Khan mohammadi, et al., 2011. Interestingly Echinococcus spp., Toxocara spp., Trichinella spp., and Dipylidium caninum etc. were very common in high percentages in foxes in different regions and climates of the world, the fox parasites have vital zoonotic importance for both human and domestic animals.Although foxes are considered as hosts for parasites of potential zoonotic and veterinary significance, few data are available relating to parasites of this wild canid in Egypt and about their role in the natural nidality and propagation of these pathogens, knowledge that is necessary to develop prevention strategies., appreciative efforts have been done by (Meyers et al., 1962, Mickhail 1967, Fahmy et al., 1971, Ashour 1980, and Omar and Abu Zanada., 1993) to identify the rictulariid from small mammals in Egypt. Zoonoses with wildlife reservoirs represent a major public health problem affecting all continents, therefore the importance and recognition of wildlife as a reservoir is increasing. Study on the helminth infections of foxes, have been the subject of several surveys around the world (Taira et al., 2002; Smith et al., 2003; Kruse et al., 2004; Vervaeke et al., 2005; Gorski et al., 2006., Thompson et al.,2009). Dalimi et al., 2006 mentioned that several canine parasites are zoonotic and are considered to public health, particularly helminth, protozoon and ectoparasites, adding Rictularia affinis are the most abundant spiruride parasites in fox because it has been recorded from this canide in its natural habitat, thought desirable not to assign any specific status to it. Willingham, et al., 1996 and liccioli.,et al.,2012 are recorded that animal carnivore may play a role in the circulation of parasites that can have implications on the health of humans and domestic animals, but can also be affected by pathogens transmitted from domestic reservoirs. Lately, the increase in the incidence of zoonoses, diseases transmissible from animals to humans, has been registered. The causative agents can be transferred by direct contact with infected animals, their excreta, by parasites or indirectly through contaminated environment (soil, food and water).It is believed that there are more than 200 registered zoonoses Klimpek et al., 2007. Rictulariid nematodes are mainly parasites of rodents and carnivorous mammals Quentin 1969, among the 29 species of wild carnivores reported from Iran by Ziaie, 1996 jackals and foxes are the two most abundant species with the ability to adopt a variety of habitats and human proximity. Therefore, they are a potential source for producing parasitic infections in other animals and man. It has been known that rictulariids cause a


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severe, sometimes fatal, disease in primates. Lubimov (1933) recovered about 1,700 individuals of Pterygodermatites (Mesopectines) alphi Lubimov, 1933 from 1 capuchin monkey, Cebus apella, which died in the Moscow Zoo. Lindquist et al. (1980) considered that the cause of death of a white-handed gibbon, Hylobates lar lar, was intestinal in tussusception and chronic parasitism with Pterygodermatites sp. Rictularia, is a cosmopolitan genus of spirurid nematodes that includes species with a prominent buccal capsule and conspicuous two row of spines along the surface of the cuticle. These nematodes occur in the digestive tract of mammals and show a heteroxenous pattern of transmission (Quentin, 1967). Froelich, 1802 created the genus Rictularia with R. cristata from Mus sylvaticus as the type species. Hall, 1913 accepted it as the type genus of a new sub-family Rictulariinae, which was raised three years later to the status of a family by Railliet, 1916.Though presenting some unmistakable points of resemblance with the Thelaziinae, the systematic relationships of this genus are still very uncertain. Representatives of the genus are parasitic in the small intestine of mammals-carnivores, bats and rodents. The worm has its cuticle armed along the sides with two sub-ventral series of large, flattened, comb-like spines. Towards the hinder end, the spines become sparse and diminish in size. The subterminal, transversely elongated and dorsally directed mouth is bordered by small denticles. The small, strongly chitinous, buccal capsule is armed at its base with teeth and spines. The caudal end of the male is conical. Several pairs of pre-and postanal papillae are present. The spicules are short and an accessory piece is present. The female genital opening is situated at about the level of the posterior end of the esophagus, which is simple and slightly club-shaped. The uterine branches are parallel and are posteriorly directed. The eggs contain embryos when laid. Rictularia cahirensis has been previously recorded from the intestine of stray cat and dog in Egypt, Russian Turkestan and Palestine, from civet cat in India and from the South American Azaras fox in captivity in England.The parasite requires two intermediate hosts for the completion of its lifecycle. The first intermediate host is some invertebrate while the second is a lizard or a snake. Material and method Nine foxes were collected by hunters from different localities, in Qena, Upper Egypt from May to October 2013. Foxes were taken to the laboratory alive where they were serially pithead and autopsied. All foxes were examined for helminth infection within 24 hours of capture. Parasites were collected from the duodenum. They were fixed in 70% ethanol. The isolated nematodes were washed in saline solution (0.9%) and fixed in 4% buffered formalin. Nematodes were cleared in lactophenol and examined as wet mounts. Drawings were made with the aid of a drawing tube attached to a microscope (Camera Lucida), connected to a wild bright field microscope. All measurements were given in millimeters except otherwise mentioned. To obtain histological preparations, small pieces of parasites were fixed in10% phosphate buffered formalin for 24 hours, dehydrated in a graded series of alcohols, cleared in three changes of methyl benzoate for 3 days, embedded in paraffin wax and sectioned at 6-8 um . Alternative sections were stained with Haematoxylin and Eosin combination. The stained sections were examined under research microscope and then photographed. For SEM studies, recovered species were washed in phosphate buffered saline and fixed overnight in 2.5% gluteraldehyde (pH 7.4) at 4 ºC. Specimens were washed three times in phosphate buffer and post fixed in 1% osmium tetraoxide in 0.1 M phosphate buffer and dehydrated through a graded ethanol series. Complete dehydration was performed in two changes of absolute ethyl alcohol and dried using CO2 in an Emitech K850 critical point dryer. Specimens were then mounted on stubs with double adhesive tape, coated with gold. Coated samples were examined with a high-resolution scanning electron microscope (JEOL SEM T330) operating at 20 Kev. Result Total of 9 individuals were dissected and examined; 4 of these showed the presence of specimen. The degree of infestation was therefore (36%). The spirurid parasite are classified and described as follows: Order: Spirurida Family; Rictulariidae, Railliat, 1916 Subfamily: Rictulariinae Hall, 1913 Genus: Rictularia Froelich, 1802 Rictularia affinis jaegerskiold, 1909 The recovered worms were usually inserted into the intestinal wall. Their anterior ends were bent dorsally and narrower than the posterior ends. Small nematodes with annulated, ornamented cuticle. Along particularly the entire ventral surface there are two rows of cuticular combs or spines. Buccal capsule, always surrounded by a circlet of (11- 13) denticles or teeth. Esophagus slightly club shaped, divided into muscular and glandular portions. Stoma semicircular, subterminal, the oral opening was transverse and dorsally located. The buccal capsule was well sclerotized and provided with a single dorsal esophageal tooth. Lips not well defined,


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amphidial (a) pores present on the lateral lip-like structures; deirids (d) spike-like, a spherical swelling at junction between muscular and glandular portions of oesophagus.Vulva anterior, near the posterior end of the esophagus. In the oviparous females, the gravid uterus was occupying the posterior two thirds of the body. Tail in both sexes conical. Female (Figs.1, 3, 4): Length of body, 26 - 30 mm, maximum width0.0 3–0.04mm. Buccal capsule, 57–77µm long, measured from ventral rim of capsule to basis, by 56–69 µm wide. Two rows of subventral cuticular extend practically throughout the length of the body; cuticular spines are more sparse and shorter towards posterior end. Fifty three paired prevulvar processes. Combs 85-126 µm long by 32–41µm wide at basis. Muscular portion of esophagus0.01 –0.02 mm. long, glandular portion 0.01-0.03mm. long. Distance of nerve ring, excretory pore and deirids, 0.024–0.025µm, 0.0 30-0.032µm and 0.034–0.044µm from anterior end, respectively. Vulva pre equatorial protruded without lip, 2.41–2.58 µm from anterior end. Eggs oval or almost rounded, 31-42 µm by 24–31 µm, containing first-stage larva. Anal opening with cuticular flanges. Tail length 87–116 µm. It was observed that the thorns in the female varies in shape ,type and size at the front of the body to be weak and short while in an area of the vagina increased in number and size, sclerotized and then weaken after and less in number (figs 2c&e.,figs. 4d&e&g). These comb-like spines became carver and smaller behind the vulva. The prevulvular spines were chitinous cuticular appendages that carried from 44-53 pairs. The excretory pore was located behind the buccal capsule. Spines behind the vulva were not adjacent and less in number and each spine 0.093-0.099mm and 0.13-mm at the prevulvular part. The female tail has a spine-like terminal spike. Male (Figs.2, 3, 5, 6): Length of body, 7–9mm, and maximum width 0.8–0.11 mm. Buccal capsule 13–22 µm by 18–25 µm wide. Two subventral rows of combs including 46-53 pairs, with sharp points projecting posterior; Length of combs 58–95 µm, width at base 29–39µm. Esophagus divided into muscular portion and glandular portion. Muscular portion of esophagus 14–21 µm long; glandular portion 45–63 µm long. Distance of nerve ring (nr), excretory pore (e) and deirids (d), 15–19 µm, 32–33 µm and 28–31 µm from anterior end, respectively. Spicules curved and equal, 30–50 (36 µm) long, with tapering distal end. Gubernaculum not discernible, 5-6 fan-like cuticular processes, semicircular, not are overlapping, anterior to cloacal opening, 42–72 µm long, by 10–29 µm wide. 7 pairs of caudal papillae (Two preanal, pair's adanal, 4 pairs postanal ).Tail conical with blunt tip. Genital cone well developed (gc), ring-formed with numerous papillae on the surface. The histological study: (Figure3 d&e) comprised consecutive cross sections of the middle parts of the female worm (in vulvular region), Examination demonstrated a multilayered cuticle measuring about 16-48 µm in thickness with three distinct visible layers. The sections showed a heteroxenous pattern of transmission, the presence of pseudocoelum having paired uterine tubes containing large numbers of unsegmented eggs and a large digestive tube. The pronounced feature of the cuticle was the presence of fine regularly spaced low longitudinal ridges on the external layer and distinct radial striations of the layer immediately underneath. The external longitudinal ridges were 4 µm in height, more or less regularly spaced. By scanning electron microscope: releaved that the body ornamented with paired combs subventrally commencing immediately behind the buccal cavity. The buccal capsule is rounded, distinctly separated from the body. Its anterior surface is oblique.The head terminates in a thick lip, rounded in the form of a helmet, and bears 10 small papillae. Mouth opening is subterminal and opens dorsally. It is large, oval, elongated transversally. There are numerous sclerotized teeth like denticles arranged in two rows (11-13 ventral denticles), situated in the buccal cavity around the mouth opening. The denticles are better developed on the ventral side of the mouth. Opening seems to be somewhat sclerotized (or pseudochitinized, according to Bursa and Tenora 1967). Buccal cavity contains one large buccal teeth extending anteriorly from its floor about one-half its depth. Two rings of cephalic papillae were observed: the internal ring, consisting of 6 papillae and external ring consisting of 4 papillae. Papillae of internal ring are situated around the mouth opening. They are sessile, bulky, rounded in shape. There are two dorsal, two ventral and two lateral papillae in the internal ring, all are similar in shape and size. Papillae of the external ring (two dorso lateral and two ventrolateral) are subapical, sessile, transversally elongated, surrounded by a shallow groove. Apmhids are small, possessing a well defined opening. They are situated on opposite sides of the buccal capsule, close to the internal cephalic papillae of the inner ring. Deirids are situated laterally on both sides of the body. Observed using scanning electron microscope the spine comb sclerotized, change its size and shape in the first trimester to be thin and upright as we go head to the posterior end. In the anterior part of the body are similar in both sexes. They are leaf- shaped, slightly overlapping one another. Thereafter, however, the shape of the cuticlar elements changes in different way in males and females. In males, the combs in the middle and posterior part of the body still overlap one another. They are more or less rectangular in shape, with pointed posterior ends of their upper edges. In females; the combs gradually acquire the spine like shape so that it is difficult to indicate the strict border between combs. Just anterior to the vulvar opening they are already completely transformed into thick, but sharply pointed spines, not overlapping one another. More posteriorly, they are diminishing and disappear approximately at level of border between the third and fourth quarters of the body


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length. In addition, the males possess a short row of four unpaired, ventral, precloacal plates, oval in shape (Figs.2b, 6c&d). Also, noted the presence of two rows of sensual papillae in anterior third region of dorsal male, and spicular sheath or genital cone in posterior end, boasts an array of arrangement, numerous, rows' of small sexual papillae researcher believes that her role in the secretion of chemotaxis substances during sexual intercourse mating (copulation).The rugose area is a modification of the cuticular striation pattern, composed of long stripes of cuticular ridges obliquely placed in a longitudinal band at the ventral posterior coiled region. Cuticular transverse striae are occurring along the entire body seen posterior to the excretory pore.The width of these striae was 18μm. They appear highly corrugated towards the posterior end giving the body a general rough appearance. Discussion Foxes have importance for carrying some parasites whose larvae can infect both human and domestic animals. Therefore, presence of foxes and their roles in zoonotic infections in Egypt have been well documented. Nematodes of the family Rictulariidae are divided into two genera, Rictularia Froelich, 1802 and Pterygodermatites Wedl, 1861. The buccal opening of the genus Rictularia is dorsally positioned and transverse with a single pharyngeal tooth, and the number of prevulvar armaments is lower than or equal to 34 pairs. In Pterygodermatites, the buccal opening is axial or slightly dorsal but never completely dorsal or transverse, with three pharyngeal teeth, and the number of prevulvar armaments ranges from 29 to 56 pairs. Based on different characters including the extent of the dorsal displacement of the buccal opening, the number of cephalic papillae and peribuccal denticles, arrangement of caudal papillae, and an increase in the number of prevulvar armaments, the species of Pterygodermatites are divided into five subgenera: Paucipectines Quentin, 1969, Neopaucipectines Quentin, 1969, Pterygodermatites Quentin, 1969, Mesopectines Quentin, 1969 and Multipectines Quentin, 1969. Diouf, et al., 2013. The specific diagnosis of the various species of the genus Rictularia rests, as pointed out by Dollfus and Desporte,1945. , Trener. 1948 and Dollfus 1960, 'primarily on the position of the oral opening and the number of combs or spines, particularly the prevulvar ones. In both R. ratti as well as the present form the oral opening is inclined dorsally and the number of the prevulvar combs is (40 – 53). A slight variation is. However, noticeable in the lengths of the entire worm and the tail, positions of the vulva and the nerve ring and size of the eggs. It is also interesting to note that Khera, 1954 described R. ratti from Rattus novegicus while the specimen under discussion was collected from vulpis niloticus. Ashour, 1980 redescribed the Rictulariid nematode from Nemiechinus auritus from Abu- Rawash ,Egypt. He considered the parasite identified by Meyers et al., 1962, Mickhail 1967 and Fahmy et al., 1971 from hedgahoge, as Pterygodermatities plagiostoma and not Rictularia falax. Identification of Ashour 1980 was based on the structure and position of the oral opening, the number of prevulvular spines and the esophageal teeth. For Rictularia the oral opening is dorsal in position and transverses, a single dorsal esophagus tooth present and the prevulvular spines are less than 34 pairs in number, these are the same morphological characters given for the present specimens except that the spines are 44-53 pairs. However, in Pterygodermatities onychomis there are 26- 27 pairs, although in all the other Pterygodermatities spp. There are more than 34 pairs of prevulvular spines. On the other hand, the number varies in a single species. Pterygodermatities affinis where 42-57 pairs of prevuvular spines are found Sandground, 1935; Rao, 1965. The specimen described above resembles very much Rictularia ratti Khera, 1954 in both the oral opening is inclined dorsally and the number of combs is not much different. Khera, 1954 reported 56 pairs of combs in R. ratti as compared to 53 on the present specimen. The latter is, however, much smaller and differs from the former in many details. It has only sixth fans while R. rattí possesses five. Allowing even for some variability in the number of fans, as pointed out by Trener, 1948 in Ríctularia citelli MCLeod, 1933, the number and arrangement of papillae in the two is quite different. In R. ratti there are six pairs of papillae, all post cloacal, while in the present specimen there are seven pairs arranged in three groups. Rictullaria balli Sandground, 1935, described from gray squirrels, Sciurus cardinensis leucotis, and regarded as con-specific with R. citelli McLeod, 1933 by Trener 1948, possesses a dorsally inclined oral opening and at least two males of R. citelli have been reported by Trener, 1948 to have only four fans, but six fans in the present specimen, though the number varies from six to four. however, differs from it in having a larger number of combs.(34-39 vs. 53).Rictularia onycbomis Cuckler, 1939 , recorded, amongst others, from Sciurus niger, is reported to have a comb range of 56-64 pairs which would cover the 53 pairs of the specimen under discussion. R.onycbomis, however. Has the oral opening more anterior than dorsal as compared to the distinctly dorsally inclined oral opening in the present specimen. Rictularia ratti Khera, 1954 is reported, with certain differences from the original description, from Rattus rattus a new host. Rictularia cahirensis jaegerskiold, 1904, a parasite of Felis domesticus, is recorded for the first time from Rajasthan Desert in India. The differences from the original description have been noted.


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The surface topography of parasites seems to be especially important in the intricate relationship between these organisms and their hosts. The chitinous buccal cavity are probably used during penetration into and migration thought the intestine wall of the fox host; while the rows of sensitive papillae in male to orientation. The cuticle have rugae or folds which also been described as transverse ridges and external raised annulations; they are incomplete branches and interrupted on the cuticle surface. The female tail has a pair of sensory papillae situated in a ventro lateral position, which represent the phasmids and they are considered to be comparable to the amphids seen on the head and may have both a glandular and sensory function Melarn, 1976. The ventral genital cone with ventral papillae helps in attachment during copulation. The posterior end of male supported with 5 fans that may assist in creeping during copulation. It is worth mentioning that in this work, much concern was directed towards the anterior end and its teeth pattern which was lacking in previous works described mainly the posterior end. Nematode parasite has at least three sensory responses: chemical, mechanical and thermal. The structures of the sensilla on the head of the present specimen suggest which of them could detect these attractants; Chemoreceptive neurons must either directly contact the environment surrounding the nematode or contact another specialized cell which then contacts the surround. All of the neurons in the amphid meet these criteria Amphids have been assumed to be chemoreceptors because they open to the outside; the female tail has a pair of sensory organs or papillae situated in a lateral or venterolateral position. Their form and appearance varies among the different species of nematode. McLaren 1976 considers that they represent the phasmids. At the present time our knowledge of the phasmids in animal parasitic nematodes is limited and only a small number of species have been examined at the electron microscopically level, Phasmids are considered to be comparable to the amphids seen on the head and may have both a glandular and sensory function, the phasmids posses a short cuticle- lined canal which opens to the exterior via a small pore., the deirids are located at the level of the fifth pair of combs in P. mexicana, whereas these structures are located at the level of the eight pair in R. nana.where is in the present specimens in six pair. The presences of large pedunculated cephalic papillae in the specimens under investigation coincide with that given before by Chitwood, 1952 for Rictullaria halli. The number of cloacal papillae 14 and the presence of gubernaculums in the male worms confirm the difference with Pterygodermatities species where Ashour 1980 mentioned that there are 15 cloacal papillae and the gubernaculums is very rudimentary. From the above argument, clear differentiation between Pterygodermatities and Rictullaria species can be made on the basis of the position of the oral opening and structures of the buccal capsule, the number of spines is not a definite criterion for differentiating the two genera. Concerning the measurenments reported of the present specimens may are closely similar to that of P.plagiostoma by Ashour 1980 but with light variations. The sex ratio of the present species is approximately the same of Pterygodermatities coloradensis and P. affinis recorded by Trener, 1948 and Rao 1965 respectively. However, Quentin 1969 recorded greater ratios varying from 12-18 By SEM observation, Gardiner and Imes (1987) found prominent dorsal cuticular ridges just posterior to the oral aperture. , such ridges were also observed in the present material.The spicular sheath or spinous tube seen covering part or all of the spicule. It is sometimes referred to in the literature as cirrus. The spicular sheath is formed from the cloacal cuticle which is everted through the cloacal opening Wright 1978 observed in trichuris muris that the loose, probably fluid, median zone of the cuticle enabled the layers of the cuticle to split so that only the outer layers are everted as the sheath. He considered that the spicular sheath and spicule are probably everted directly into the female vagina which has a rugose cuticular lining, thus providing a grip for the spines on the sheath. The spicule passes through the centre of the spicular sheath and is partially converted even when fully everted. The inclusion of the species in the subgenus Rictularia Jagerskiöld, 1904 is proposed based on four sets of characters: the subterminal oriented stoma; the presence of irregularly-spaced small denticles in the buccal cavity; total number of combs and spines prevulvar are 53 pairs, prevulvar cuticular processes, the position of the vulva relative to the cuticular processes and the sublateral orientation of pedunculated caudal papillae pairs (2 preanal; 1 adanal and 4 postanal ). The main distinguishing characters of R. affinis are markedly short spicules; deirids situated posterior to the oesophago-intestinal junction; phasmide in posterior end; marked sclerotizations in the oesophago-intestinal junction; and esophagus divided in two distinct portions muscular and glandular. A unique feature of the present specimen seems to be the number of fans and two rows of sensitive papillae in males. In addition, the transver striation. (Figs.5a&6e). Triner, 1948 notes that rictularia spp. In American rodents are of two types. One in which the oral opening is circular and more anteriorly directed (R.coloradoensis) and another in which it is narrow, transverse and dorsally directed (R.citelli). In australian R. mackerrasae differs from other species in the number, size and arrangement of teeth on the anterior border of the buccal capsule.


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Inglis and Ogden 1965 suggest that the extent to which the mouth is directed dorsally may depend on the degree to which it is opened or closed. This temporary movement however would not account for the greatly thickened cuticle anteriorly. Or for the greater length of the median dorsal teeth on the anterior border of the buccal capsule, which appear to be associated with the more dorsal slite like mouth in the species listed above.

Figure 1. Camera lucida drawing of female Rictularia affinis. (a): Anterior end the buccal capsule was well chitinized esophagus divided into muscular (m) and glandular (g) portions, nerve ring (nr) located at posterior end of muscular; (b): magnified anterior portion note that lips not well defined, four pairs of papillae arranged in an outer circle, two dorsal and two ventral, amphide lateral lip-like structures, deirids present below the fifth pair of cuticular comb-like processes; (C):Posterior end of female provided with pair of phasmide (ph), tail conical and has a spine-like terminal spike ( d): Vulvular region; (e): thick shell ebmryonated egg.


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Figure.2: Camera lucida drawing of Rictularia affinis. (a) :whole mount of male, the body ornamented with paired combs, sclerotized spine, subventrally commencing immediately behind the helmet capsule the entire ventral surface there are two rows of cuticular combs or spines. (b): magnified posterior end showing spicular sheath or genital cone (gc) well developed, ring-formed with numerous papillae on the surface, well developed semicircular fan-shaped (f)., equal spicules (s), 4 preanal; 2 adanal and 8 postanal pedunculated caudal pappilae, gubernaculum not present. (C&e): different types of spines prevulvular spines, vulvular spines respectively. (d): pedunculated caudal papillae.


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Figure 3 : Photomicrographs of unstained anterior ends of female (a) and male (b) showing annulated, ornamented cuticle stoma semicircular, subterminal, opens dorsally, well developed sclerotized buccal capsule; one tooth-like projections attached at base. buccal capsule, surrounded by a circlet of 13 denticles, and with its base armed with teeth and spines two rows of cuticular combs or spines. (4x). (c) :Photomicrographs of posterior part of female showing intestine (i), uterus filled by eggs (u), and tail have a spine-like terminal spike and phasmide (ph).(4x). (d): Photomicrographs of the cross section of female worm observed a heteroxenous pattern of transmission,thick cuticle(tegument) with longitudinal ridges, hypodermis and muscle cells. There is one intestinal tube and paired uterine tubes containing immature eggs. (e): High magnification showing illustrating fine regularly spaced low longitudinal ridges and three distinct layered cuticle, two placoid shape spine. (f) : Photomicrographs of Thick shell embryonated egg (100 x).


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Figure 4. Scanning electron microscopy of female Rictularia affinis (a): anterior portion, (v) Valvular region . (b): Cephalic extremity, (arrows indicate amphidial pores) two dorsal and two ventral; amphidial pores present on the lateral lip-like structures, deirids present below the fifth pair of cuticular comb-like processes. (c): high magnification of helmet chitinized capsule, provided with a single dorsal esophageal tooth. Lips not well defined, four pairs of papillae arranged in an outer circle. (d): spine in valvular region. (e): spine in prevalvular region. (f): Caudal extremity, phasmide (ph). (g): spine postvalvular region with transverstriation.


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Figure 5: Scanning electron micrographs of male R. affinis showing. (a): Male, general view. (b): cephalic extremity, subdorsal view (arrows indicate amphidial pores) (c): high magnification of anterior end with helmet buccal capsule and papillae (p). (d): high magnification of genital cone, ring-formed with numerous papillae on the surface. (e): magnified anterior portion note that lips not well defined, four pairs of papillae arranged in an outer circle, two dorsal and two ventral, amphide (a) lateral lip-like structures, deirids (d) present below the fifth pair of cuticular comb-like processes, excretory pore (e).


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Figure 6: Scanning electron micrographs of male R. affinis showing. (a&b): lateral view of posterior extremity with six fan-shaped preanal protrusions (f), spicules curved and equal (s),transverstriation, genital cone (gc), seven pairs of caudal papillae (p) plus a terminal pair of phasmids (ph). (Two pairs of papillae preanal, pair's adanal, four pairs postanal in position). Tail conical with blunt tip. (c): fan-like cuticular processes, semicircular, not overlapping, anterior to cloacal opening. (e): two rows of sensual papillae in anterior third region of dorsal male surface.


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