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Biosystems
Diversity
ISSN 2519-8513 (Print) ISSN 2520-2529 (Online) Biosyst. Divers., 2019, 27(1), 85-98 doi: 10.15421/011913
Morphological variation of four species of Strongyloides (Nematoda, Rhabditida) parasitising various mammal species
Y. A. Gugosyan*, O. O. Boyko*, V. V. Brygadyrenko*, **
*Dnipro State Agrarian and Economic University, Dnipro, Ukraine **Oles Honchar Dnipro National University, Dnipro, Ukraine
Article info
Received 19.01.2019 Received in revised form
07.02.2019 Accepted 10.02.2019
Dnipro State Agrarian and Economic University, SergiyEfremov st., 25, Dnipro, 49000, Ukraine. Tel.: +38-066-112-95-05. E-mail:
Oles Honchar Dnipro National University, Gagarin av., 72, Dnipro, 49010, Ukraine. Tel.: +38-050-93-90-788. E-mail: [email protected]
Introduction
Gugosyan, Y. A., Boyko, O. O., & Brygadyrenko, V. V. (2019). Morphological variation of four species of Strongyloides (Nematoda, Rhabditida) parasitising various mammal species. Biosystems Diversity, 27(1), 85-98. doi:10.15421/011913
Study of the morphologic peculiarities of free-living stages of nematodes of the Strongyloides genus is important in differential diagnostics of pathogens. We studied the parasite of goats (Capra aegagrus hircus Linnaeus, 1758) - Strongyloides papillosus (Wedl, 1856); the parasite of horses (Equus Jerus caballus Linnaeus, 1758) - S. westeri Ihle, 1917; the parasite of swine (Sus scroJa Linnaeus, 1758 ) - S. ransomi Schwartz & Alicata, 1930; the parasite of dogs (Canis lupus Jamiliaris Linnaeus, 1758) - S. stercoralis (Bavay, 1876). Rhabditiform larvae (L1, L2), filarial larvae (L3), mature males (M) and free-living females (F) were analyzed for each of the four species by five morphological parameters: body length (Lb), total maximum body width (Sb), length of the esophagus (Lo), length of the intestine (Le), length of the tail end (Lc); for females, we additionally examined length from the head end to the vulva (Lv) and the number of formed eggs in the uterus cavity (E), and for males - length of spicules (Ls). First stage larvae of S. papillosus and S. stercoralis significantly differed in all parameters, and first stage larvae of S. westeri and S. stercoralis significantly differed in four out of five parameters. By indices of ratios of total maximum body width (Sb) to body length (Lb), length of the esophagus (Lo) to length of the intestine (Le), length of the tail end (Lc) to body length (Lb), length of the intestine (Le) to body length (Lb), a reliable difference was recorded between Li of S. papillosus and S. ransomi, L3 of S. papillosus and S. westeri, S. westeri and S. ransomi, between males of S. papillosus and S. westeri, S. westeri and S. stercoralis, between females of S. papillosus and S. westeri, S. papillosus and S. stercoralis. All four examined species were observed to have reliable differences of ratio of length of esophagus to length of intestine in third stage larvae.
Keywords: morphometric parameters; Strongyloides papillosus; S. westeri; S. ransomi; S. stercoralis.
Nematodes of the Strongyloides genus parasitize the small intestine of many species of wild and domestic mammals. Strongyloides species inhabiting Ukraine include Strongyloides papillosus Wedl, 1856, parasite of ruminants, rabbits and guinea pigs; S. ransomi Schwartz & Alicata, 1930, parasite of swine; S. westeri Ihle, 1917, parasite of horses; S. stercoralis (Bavay, 1876), parasite of carnivores and humans. In conditions of low intensity of the invasion, parasitization by nematodes of the Strongyloides genus does not lead to emergence of manifested clinical signs and the course of the disease is accompanied with no symptoms. However, high parameters of intensity of Strongyloides infestation can in most cases cause disorders of metabolism in the organism, functions of the gastrointestinal tract, organs of the respiratory system, and also reduction of animals' productivity. As a result, farms contaminated with stron-gyloidiases suffer significant economic losses.
According to Roesel et al. (2016), in the territory of East Africa, swine-breeding complexes infested with helminthiases, suffer economic losses as a result of reduction of the output volumes, and also increase in production costs. In most farms of East Africa, swine are recorded to be infested with nematodes of the Strongylida order, parasites of the gastrointestinal tract, Metastrongylus spp., Ascaris suum Goeze, 1782, S. ransomi, and also Trichuris suis Schrank, 1788. In Indonesia, swine-breeding in rural areas is an important part of livestock rearing. However, the high level of infestation and mortality of swine obstructs the development of the production in the country. Studies by Nugroho et al. (2015) indicate the spread of the following parasites among swine: nematodes of the Strongylida order, T. suis, A. suum, S. ransomi, and also coccidi-ans. In Tanzania, swine are also ubiquitously recorded to host helminths
of the gastointestinal tract (infection rate of helminthiases equaled 63.7%): Oesophagostomum spp., Trichostrongylus spp., A. suum, T. suis and S. ransomi with extensity of infestation equaling 57.4%, 17.5%, 5.3% and 1.1% respectively (Kabululu et al., 2015). Nganga et al. (2007) studied abundance of different species of helminths among swine in Kenya. Nematodes were identified in 67.8% of swine, out of which 31.3% of animals had mixed infestations: 10 species of helminths were found: Oesophagostomum dentatum (39.1%), T. suis (32.2%), A. suum (28.7%), Oesophagostomum quadrispinulatum (14.8%), Trichostrongylus colubri-Jormis (10.4%), T. axei (4.3%), S. ransomi (4.3%), Hyostrongylus rubi-dus (1.7%), Ascarops strongylina (1.7%) and Physocephalus sexalutus (0.9%). In farms in Kenya, where swine are kept free-range, nematode infestations are also often a serious problem for swine-breeding. On such farms, the extensity of infestation reaches 84.2%, and mean intensity of nematode infestation equals 2,355 eggs/g of feces. Kagira et al. (2011) found Oesophagostomum spp. in 75% of swine, and S. ransomi in 37.0%. Other species of swine nematodes recorded on free-range farms in Kanya are rarer; A. suum (18%), Metastrongylus spp. (11%), T. suis (7%) and P. sexalatus (3%). According to Tamboura et al. (2006), intensity of infestation of swine with gastrointestinal nematodes depends on the type of system of production of animals. The examination of swine in Burkina Faso revealed that out of 383 examined swine, 91% were infested with A. suum helminths (extensivity of the infestation was 40%; intensity of infestation - 100-1400 EPG (eggs per gram of feces)) -commonest species, other species were S. ransomi (21%; 100-4200 EPG), Oesophagostomum spp. (18%; 100-1000 EPG), H. rubidus (11%; 1001800 EPG), Globocephalus spp. (10%; 100-400 EPG) and T. suis (1%; 100-200 EPG). The literature contains information on intensity of infestation and severity of the course of strongyloidiasis for parasitization by
S. papillosus in different species of hosts: depending on their age, geographic lange, season and many other factors (Singh et al., 1997; Bekele, 2002; Agyei, 2003; Jäger et al., 2005; Eysker et al., 2005; Boyko et al., 2009). During the larva stage, S. papillosus infests the gastrointestinal tract, and the respiratory organs of mammals. In an experimental infestation of rabbits, Nakamura et al. (1994) observed decrease in body weight to 44% compared to the initial weight and consequent exhaustion of the animals. As a result of parasitization of S. papillosus in calves, Kvac et al. (2007) recorded mortality in 25% of animals. At autopsy, all the dead calves were observed to have pathological changes in the lungs, which were related to migration of larvae. Studies by Wymann et al. (2008) also indicate age peculiarities of vulnerability to this species of nema-todes. Currently, new methods against S. papillosus and control of its abundance in animals and environment are being developed (Boyko & Brygadyrenko, 2016; Boyko et al., 2016).
Studies by Dewes et al (1990) on percutaneous penetration ofS. westeri indicate that animals become anxious, hyperactive, develop swelling of the lower part of their limbs and dermatitis on the skin of legs, stomach and snouts during 2-3 weeks. Through this path of infestation, broodmares can develop mastitis as a result of concentration of larvae in mammary gland during migration (Greiner et al., 1991). In the case of an extreme peroral infestation of animals with hundreds of thousands of invasive larvae of S. westeri, Greer et al. (1974) observed animals to have diarrhea, periodic cramps, anemia of mucus membranes. Changes in hematological parameters were characterized by increase in the number of leukocytes with increase in the percent of eosinophiles, neutro-phils and lymphocytes, reduction of content of albumins and increase in ß-globulins; the peak of changes occurred on the sixth week after the experiment started. In the duodenum, there were found moderate inflammation and hyperemia. Histological studies revealed atrophy of the villi of the intestine, and accumulation of lymphatic cells, neutrophils and eosinophils were found around the parasites. In Brazil, lethal cases of strongyloidiasis were described among colts. Pathological autopsy revealed subcutaneous edemas, ascites, hydrothorax, inflammation and hyperemia of the duodenum, and histological examination revealed depressions in the upper part of the villi of the intestine, filled with hermaphrodite females of Strongyloides. Numerous small concentrations of atelectases and blunted edges of diaphragm lobes were observed in all lung lobes and under the pleura (Lucena et al., 2012).
No less common is S. stercoralis. Parasitization by this nematode species can also lead to various disorders in the host organism. Carnivores suffering strongyloidiases were observed to have symptoms of acute watery diarrhea, pain during defecation, intermittent cough, vomiting and exhaustion (Umur et al., 2017). According to Jaleta et al. (2017) and Nagayasu et al. (2017), S. stercoralis of dogs has two populations. One is specific only to dogs, and the other population of S. stercoralis can also parasitize humans. Such studies indicate the high risk for humans who are in contact with dogs. According to Catalano et al. (2017), S. stercoralis colitis is a severe disease with a high level of mortality, but is easily curable. Strongyloidiasis in humans can remain for up to several decades and can lead to chronic colitis. Chronic colitis is related to heightened risk of development of colorectal cancer; quite possibly chronic colitis induced by strongyloidiasis poses a similar risk as well. Poveda et al. (2017) have also recorded colitis in patients diagnosed with strongyloi-diasis. Studies by Politis (2017) indicate presence of diarrhea in people with S. stercoralis. People parasitized by this species of helminth have nocturnal dyspnea, sweatiness, productive cough, and also constrictive pericarditis (Lee et al., 2017). The study by Nabeya et al. (2017) described cases of manifestations in the lungs, including acute respiratory distress syndrome, bacterial pneumonia and lung bleeding.
Identification of larvae and mature specimens of Stmngyloides genus by morphological parameters poses difficulties for parasitologists. Therefore, the objective of this paper was to determine variability of metric parameters and morphometric indices for four of the commonest species of this genus, which are in most frequent contact with pets and humans.
Materials and methods
The research was conducted in 2018 at the Department of Parasitology and Veterinary-Sanitary Examination of the Dnipro State Agrarian
and Economic University. For the experiment, we selected animals infested with Strvngyloides spp. pathogen. Diagnosis was based on finding eggs of Strvngyloides using the McMaster method. Samples of the animals' feces were taken separately, preventing their contamination with soil nematodes. Cultivation of eggs and larvae was performed in a thermostat at temperature of 23-25 °C over 1-7 days. Obtaining cultures of larvae and free-living generations of Strvngyloides spp. was made using the Baermann method, and identification - in accordance with notable morphological signs (Van Wyk et al., 2004; Van Wyk & Mayhew, 2013). During the experiment, we identified and examined the following species: S. papillosus (Wedl, 1856), S. westeri (Ihle, 1917), S. ransomi (Schwartz & Alicata, 1930), S. stercoralis (Bavay, 1876).
Larvae at different stages of the development and free-living generations of Stmngyloides were differentiated by taking into account morphological peculiarities of structure of the head and tail ends, presence of widening of the esophagus. Metric parameters ofpostembryonic stages of the development of Strongyloides spp. were determined using an object-micrometer and a Sigeta CAM MD-300 3 Mpix digital camera (China). In all studied objects, we determined total length of body (Lb), maximum body width (Sb), length of the esophagus (Lo), length of the intestine (Le), length of the tail end (Lc), separately for free-living females we measured length from the head end to the vulva (Lv), number of developed eggs in the cavity of the womb (E), and separately for the males we measured length of the spicules (Ls). We calculated the indices of total maximum body width (Sb) to body length (Lb); length of esophagus (Lo) to length of intestine (Le); length of tail end (Lc) to body length (Lb); length of intestine (Le) to body length (Lb).
In total, 20 individuals of each species were analyzed for each parameter for each post-embryonic stage of the development for each of the four host animal species. Differences between the selections were considered statistically significant at P < 0.05. The data were compared using Tukey's test (with consideration of the Bonferroni correction). The data was analyzed in Statistica 8 (StatSoft Inc., USA). In the diagrams the small squares show the median, the large rectangles show the 25% and 75% quartiles, the vertical lines show 95% of the variation, the stars and circles show the outliers.
Results
The appearance and morphological features of larvae and mature nematodes are demonstrated in Figures 1, 3, 5, 7 and 9. The examination of body length of larvae of the first stage of the development of nematodes of Strongyloides genus from ruminants, horses, swine, and dogs (on average body length equals 210-310 pm) revealed reliable difference by the parameters between S. papillosus and S. stercoralis, S. wes-teri and S. ransomi, S. westeri and S. stercoralis, and also S. ransomi and S. stercoralis (Fig. 2a). Width of the body of first stage larva ranged within 12-19 pm. Parameter of total maximum body width reliably differed for S. papillosus and S. ransomi, S. papillosus and S. stercoralis. We observed no reliable difference in this parameter for S. papillosus and S. westeri, S. westeri and S. ransomi, S. westeri and S. stercoralis, and also S. ransomi and S. stercoralis (Fig. 2b).
Length of the esophagus significantly differed in first stage larvae of S. papillosus and S. westeri, S. papillosus and S. ransomi, S. papillosus and S. stercoralis. We also recorded reliable difference in this parameter for S. westeri and S. ransomi, S. westeri and S. stercoralis. Average value of this morphometric parameter equaled 60-119 pm (Fig. 2c). Parameters of length of the intestine in four species of nematodes of the Strongyloides genus ranged within 100-160 pm. No significant differences were determined in length of the intestine for three examined species of nematodes: S. papillosus, S. westeri, S. ransomi. Compared to other three species, length of the intestine significantly differed only in first stage larvae ofS. stercoralis nematodes (Fig. 2d).
Length of the tail end of L! S. westeri differed from that of S. ransomi and S. stercoralis. For nematode larvae of the Strongyloides genus, this parameter ranged within 31-49 pm (Fig. 2e). Average body length of larvae of the second stage ranged within 325-475 pm. Significant difference in parameters of body length was recorded only for L2 S. stercora-lis. The rest of the studied species did not reliably differ one from ano-
ther by this parameter (Fig. 4a). Measurements of body width showed reliable difference also for L2 S. stercoralis. Insignificant difference in body width was observed only between L2 S. stercoralis and S. westeri. L2 S. papillosus, S. westeri, S. ransomi did not have significant difference in body width - 18-25 цт (Fig. 4b).
A similar picture was also observed during comparison of morphometric parameters in relation to length of the esophagus. L2 S. papillosus, S. westeri, S. ransomi had also no reliable differences between one another.
Length of the esophagus in L2 S. stercoralis was significantly different compared to the other examined species of Strongyloides nematodes. On average, this parameter ranged within 85-120 цт (Fig. 4c). In the second stage Strongyloides larvae, length of the esophagus on average equalled 190-310 ^m. The highest parameters were recorded in S. westeri, the lowest - in S. stercoralis (Fig. 4d). Reliable differences by this parameter were determined between S. papillosus and S. stercoralis, S. papillosus and S. westeri, S. ransomi and S. stercoralis, S. westeri and S. stercoralis.
Fig. 1. Rhabditiform larvae (L1): a - S. papillosus, b - S. westeri, c - S. ransomi, d- S. stercoralis; 1 - bulbus-like widening in the esophagus, 2 - the tail end; bar=50 ^m
380 г 360 340 320 300 280 260 240 220 200 180 160
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S. ransomi S. stercoralis
S. papillosus S. westeri S. ransomi S. stercoralis
S. papillosus S. westeri
S. ransomi S. stercoralis
Fig. 2. Morphological variability of rhabditiform larvae (Lj) of S. papillosus, S. westeri, S. ransomi and S. stercoralis: a - body length (Lb), b - maximum body width (Sb), c - length of esophagus (Lo), d - length of intestine (Le), e - length of tail end (Lc)
Lb ab
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Length of the tail end of second stage larvae ranged within 40-80 ^m. The lowest parameters were recorded for L S. westeri, the highest - for L2 S. papillosus. Also a significant difference by this parameter was observed for L2 S. papillosus and S. westeri, S. papillosus and S. stercora lis, S. westeri and S. ransomi, S. ransomi and S. stercoralis (Fig. 4e). Examination of morphometric parameters in third stage larvae revealed
reliable differences in body length between S. westeri and S. stercoralis, S. westeri and S. ransomi. Body size of the larvae on average was 510690 ^m (Fig. 6a). The lowest parameters of total maximum body width were observed in L3 S. westeri, the highest - in L3 S. stercoralis. Reliable differences by this parameter were recorded for L3 S. westeri. Other species did not reliably differ between one another in body width.
Fig. 3. Rhabditiform larvae (L2): a - S. papillosus, b - S. westeri, c - S. ransomi, d- S. stercoral pointers indicate presence of two bulbuses in the esophagus; bar = 50 ^m
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Lc a
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S. papillosus S. westeri S. ransomi S. stercorals
Fig. 4. Morphological variability of rhabditiform larvae (L2) of S. papillosus, S. westeri, S. ransomi and S. stercoralis: a - body length (Lb), b - maximum body width (Sb), c - length of esophagus (Lo), d- length of intestine (Le), e - length of tail end (Lc)
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S. papillosus
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The lowest length of the esophagus was determined for L3 S. papillosus, which reliably distinguished it from the larvae of other studied species. By this parameter, L3 S. westeri, S. ransomi and S. stercoralis had no reliable differences between one another. On average, length of the esophagus of the third stage larvae ranged within 180-270 ЦШ (Fig. 6c). The lowest length of the esophagus was observed in L3 S. westeri, which reliably distinguishes this species from the other nematode larvae we studied. L S. papillosus, S. ransomi and S. stercoralis did not reliably differ from one another by this parameter (Fig. 6d). The longest tail end of third stage larvae was recorded in S. papillosus. Length of tail ends of
L3 S. ransomi and S. stercoralis and S. westeri did not reliably differ between one another and on average equaled about 100 ^m. Difference by this parameter between L3 S. papillosus and larvae of the remaining species equaled around 30 ^m (Fig. 6e).
Measurements of morphometric parameters of males indicate absence of reliable differences by length of the body and the esophagus in the four studied species of nematodes. Average values of body length were within 700-790 ^m, length of the esophagus - 140-170 ^m (Fig. 8a, с). The lowest total maximum body width was recorded in S. westeri. This value reliably differed from S. ransomi and S. stercoralis (Fig. 8b).
Fig. 5. Filariform larvae (L3): a - S. papillosus, b - S. westeri, c - S. ransomi, d - S. stercoralis; pointers indicate the place where the esophagus turns into the intestine; bar = 50 ^
b
s
^__
S. papillosus S. westeri S. ransomi S. stercoralis
S. papillosus S. westeri S. ransomi S. stercoralis
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S. papillosus S. westeri S. ransomi S. stercoralis
S. papillosus S. westeri S. ransomi S. stercoralis
Fig. 6. Morphological variability of filariform larvae (L3) of S. papillosus, S. westeri, S. ransomi and S. stercoralis: a - body length (Lb), b - maximum body width (Sb), c - length of esophagus (Lo), d- length of intestine (Le), e - length of tail end (Lc)
Lb
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Fig. 7. Free-living male: a, e - S. papillosus; b, f- S. westeri; c, g - S. ransomi
S. stercoralis; pointers indicate the spicules; bar = 50 цш
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S. papillosus S. westeri
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S. papillosus S. westeri S. ransomi S. stercoralis
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S. papillosus S. westeri S. ransomi S. stercoralis
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T
S. papillosus S. westeri S. ransomi S. stercoralis
S. papillosus S. westeri S. ransomi S. stercoralis
Fig. 8. Morphological variability of free-living male of S. papillosus, S. westeri, S. ransomi and S. stercoralis: a - body length (Lb), b - maximum body width (Sb), c - length of esophagus (Lo), d - length of intestine (Le), e - length of tail end (Lc), f- length of the spicules (Ls)
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Length of the intestine much lower compared to the rest of the species was observed in S. papillosus (Fig. 8d). Length of the tail end of males reliably differed between S. papillosus and S. westeri, S. westeri and S. ransomi. Average value of this parameter ranged within 59-120 pm (Fig. 8e). Length of the spicules of four species of nematodes ranged within 31-37 pm. This parameter reliably differed only between S. ransomi and S. stercoralis (Fig. 8f).
Body length of females on average ranged within 850-950 pm. By this parameter, we found no reliable differences among the four species of nematodes (Fig. 10a). Similar results in morphometry were obtained also for length of the esophagus (Fig. 10c). Average values of this parameter were within 130-180 pm. Total maximum body width reliably differed in S. papillosus and S. stercoralis, S. westeri and S. stercoralis. This parameter was within 37-56 pm (Fig. 10b).
Fig. 9. Free-living female: a - S. papillosus, b - S. westeri, c - S. ransomi, d- S. stercoralis, e - area of the vulva,/- uterus with formed eggs, pointers indicate the vulva slit; bar = 50 pm
Table 1
The results of morphometric analysis of three larva stages, free-living males and females of different species of the genus Strongyloides
Stage of larva de- Total number The number Characteristics by which species reliably differed
velopment, sex Species of the parameters of statistically reliably from three other examined species
of adult nematode compared differing parameters S. papillosus S. westeri S. ransomi S. stercoralis
S. papillosus 5 - Lo, Lc Sb, Lo Lb, Sb, Lo, Le, Lc
L1 S. westeri 5 4 - - Lo, Lc Lb, Lo, Le, Lc
S. ransomi 5 - - - Lb, Le
S. stercoralis 5 - - - -
S. papillosus 5 - Le, Lc * Lb, Sb, Lo, Le, Lc
L2 S. westeri 5 4 - - Lc Lb, Lo, Le
S. ransomi 5 - - - Lb, Sb, Lo, Le, Lc
S. stercoralis 5 - - - -
S. papillosus 4 - Sb, Lo, Le, Lc Lo, Lc Lo, Lc
L3 S. westeri 5 5 - - Lb, Sb, Le Lb, Sb, Le
S. ransomi 5 - - - *
S. stercoralis 5 - - - -
S. papillosus 2 - Le, Lc Le Le
Male S. westeri 6 3 - - Sb, Lc Sb
S. ransomi 3 - - - Ls
S. .stercoralis 3 - - - -
S. papillosus 4 - Le, Lv, E E Sb, Le, E
Female S. westeri 7 5 - - Lc Sb, E
S. ransomi 2 - - - *
S. stercoralis 3 - - - -
Note: * - no reliably differing characteristics.
1200 -1100 1000 900 800 700 600 500 400 300 -
a 260 240 220 200 180 160 140 120 100 80
c
280 260 240 220 200 180 160 140 120 100 80 60 40 20
T
S. papillosus S. westeri S. ransomi S. stercoralis
65 -
60
55
50
45
40
35
30
25 -b
S. papillosus S. westeri S. ransomi S. stercoralis
' b ' b
ab
S. papillosus S. westeri S. ransomi S. stercoralis
S. papillosus S. westeri S. ransomi S. stercoralis
S. papillosus S. westeri S. ransomi S. stercoralis 7 r 6 5 4 3 2 1
S. papillosus S. westeri S. ransomi S. stercoralis
S. papillosus S. westeri S. ransomi S. stercoralis
Fig. 10. Morphological variability of free-living female of S. papillosus, S. westeri, S. ransomi and S. stercoralis: - body length (Lb), b - maximum body width (Sb), c - length of esophagus (Lo), d - length of intestine (Le), e - length of tail end (Lc), f - length from the head end to the vulva (Lv), g - number of developed eggs in the uterus cavity (E)
Measurement of the intestine revealed reliable difference between S. papillosus and S. westeri, S. papillosus and S. stercoralis (Fig. 10d). This parameter in the four species of nematodes ranged within 480-700 цш Length of the tail end of females had reliable difference only between S. westeri and S. ransomi (Fig. 10e). On average, this parameter equaled 105-140 цш. Length from the head end to vulva reliably differed between females of S. papillosus and S. westeri. This parameter on average ranged within 400-480 цш (Fig. 10f). The number of eggs in the uterus ranged from one to four. Their lowest number was recorded in S. papillosus, the highest - in S. stercoralis (Fig. 10g). A reliable difference by this parameter was recorded between S. papillosus and S. westeri, S. papillosus and S. stercoralis, S. papillosus and S. ransomi, S. westeri and S. stercoralis.
Thus, first stage larvae reliably differed by all five parameters between S. papillosus and S. stercoralis, and by four out of five parameters between S. westeri and S. stercoralis. Second stage larvae also had reliable differences by all five parameters in S. papillosus and S. stercoralis. At this stage, only S. ransomi and S. stercoralis significantly differed in all parameters. Beginning from the third stage of the development, the number of reliable differences by morphometric parameters decreased (Table 1).
Using the results of our research, we calculated the indices of total maximum body width (Sb) to body length (Lb), length of esophagus (Lo) to length of intestine (Le), length of tail end (Lc) to body length (Lb), length of intestine (Le) to body length (Lb) for the four species of the Strongyloides genus (Fig. 11-15).
b
Lb
Sb
800
Lo
700
600
500
400
300
200
d
Lc
Lv
200
e
be
g
a
Sb/Lb a
i.....
S. papillosus S. westeri S. ransomi S. stercoralis
26 24 22 20 18 16 14 12 10 8 6 -
T
b
66 r
64 62 60 58 56 54 52 50 48 46 44 42
S. papillosus S. westeri S. ransomi S. stercoralis
T
S. papillosus S. westeri S. ransomi S. stercoralis
S. papillosus S. westeri S. ransomi S. stercoralis
Fig. 11. Morphometric indices (%) of rhabditiform larvae (L1) of four species of Strongyloides genus: a - ratio of total maximum body width (Sb) to body length (Lb), b - ratio of length of the esophagus (Lo) to length of intestine (Le), c - ratio of length of the tail end (Lc) to body length (Lb), d - ratio of length of the intestine (Le) to body length (Lb)
7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5
T T
S. papillosus S. westeri S. ransomi S. stercoralis
S. papillosus S. westeri S. ransomi S. stercoralis
26 24 22 20 18 16 14 12 10 8 6 -
S. papillosus S. westeri S. ransomi S. stercoralis
S. papillosus S. westeri S. ransomi S. stercoralis
Fig. 12. Morphometric indices of rhabditiform larvae (L2) of four species of Strongyloides genus: a-d - for explanations see Fig. 11
8.5
100
a
90
b
80
70
b
60
b
50
40
3.5
30
a
28
a
a
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7.5
65
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60
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3.0
25
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44
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2.0 a
S. papillosus S. westeri S. ransomi S. stercoralis
34 32 30 28 26 24 22 20 18 16 14 12
S. papillosus S. westeri S. ransomi S. stercoralis
S. papillosus S. westeri S. ransom S. stercoralis
S. papillosus S. westeri S. ransomi S. stercoralis
Fig. 13. Morphometric indices of filariform larvae (L3) of four species ofStrongyloides: a-d - for explanations see Fig. 11
в
d
c
a
36
c
bc
10
28
d
c
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5
a
24 22 20 18 16 14 12 10 8 6 4 2
c
Fig. 14. Morphometric indices of free-living male of four species of Strongyloides genus: a-d - for explanations see Fig. 11
55 г
50
45
40
35
30
25
20
15
10 -
T
S. papillosus S. westeri S. ransomi S. stercoralis ь S. papillosus S. westeri S. ransom S. stercoralis
S. papillosus S. westeri S. ransom S. stercoralis d S. papillosus S. westeri S. ransom S. stercoralis
a
a
a
c
bc
b
85
b
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75
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S. papillosus S. westeri S. ransom S. stercoralis
1
S. papillosus S. westeri S. ransom S. stercoralis
X
I
S. papillosus S. westeri S. ransomi S. stercoralis
S. papillosus S. westeri S. ransomi S. stercoralis
Fig. 15. Morphometric indices of free-living females of four species of Strongyloides genus: a-d - for explanations see Fig. 11
Among larvae of the first stage, reliable differences by the first index were observed between S. papillosus and S. westeri, and also between S. papillosus and S. ransomi (Fig. 11a). Index ofratio oflength of esophagus to length of intestine statistically differed only in S. ransomi (Fig. 11b). No reliable differences by this index were observed in other species. The same was observed for ratio of length of tail end to body length (Fig. 11c). Significant differences were observed in S. ransomi and S. stercoralis by ratio oflength of the intestine (Le) to body length (Lb) (Fig. 11d).
Second stage larvae of all four studied species of nematodes had no reliable differences by the first two indices (ratio of total maximum body width to body length, length of the esophagus to length of the intestine) (Fig. 12a, b). By the other two indices (ratio of length of the tail end to body length and length of the intestine to body length) statistically reliable difference was observed only for S. westeri (Fig. 12c, d).
Reliable difference for all four species was recorded for third stage larvae by index of length of the esophagus to length of the intestine (Fig. 13b). By ratios of total maximum body width to body length and length of tail end to body length, statistical difference was observed for S. papillosus, S. westeri and S. ransomi (Fig. 13a, c). By index of ratio of total maximum body width to body length, reliable differences were observed for S. stercoralis and S. westeri, and also S. stercoralis and S. ransomi (Fig. 13a). By index of length of the tail end to body length, we recorded reliable difference for S. stercoralis and S. papillosus (Fig. 13c). Difference in values of index of the intestine to body length in third stage larva were reliable between S. papillosus and S. westeri, S. papillosus and S. stercoralis, S. westeri and S. stercoralis, S. ransomi and S. westeri, S. ransomi and S. stercoralis. No differences by this index were observed in S. papillosus and S. ransomi (Fig. 13d).
In males, ratio of total maximum body width to body length unreliably differed for S. papillosus, S. ransomi and S. stercoralis. The only statistically reliable difference was observed for S. westeri compared to other species of the Strongyloides genus (Fig. 14a). Reliable difference in ratio of length of the esophagus to length of intestine was observed for S. papillosus, S. westeri and S. stercoralis, and also for S. papillosus and S. ransomi (Fig. 14b). For males of S. papillosus and S. westeri, we
also found statistically reliable differences in ratio of length of tail end to body length. In males of S. stercoralis, S. westeri and S. ransomi, this index differed as well (Fig. 14c). Other results were obtained for comparing ratio of length of the intestine to body length. This index was reliably different was between S. papillosus, S. westeri and S. ransomi, and also between S. ¡papillosus and S. stercoralis, S. westeri and S. stercoralis (Fig. 14d).
In females of the four species of nematodes of Strongyloides genus, reliable differences by total maximum body width to body length were observed in S. papillosus, S. westeri and S. stercoralis, and also S. westeri and S. ransomi (Fig. 15a). Index of length of esophagus to length of intestine reliably differed in S. papillosus (Fig. 15b). Also, females of S. papillosus differed from S. westeri and S. stercoralis by ratio of length of the tail end to body length. For females of S. ransomi, we also recorded reliable differences compared to S. westeri and S. stercoralis by this index (Fig. 15c). Females of S. papillosus, S. westeri and S. ransomi, and also S. papillosus and S. stercoralis had statistically significant differences in ratio of length of the intestine to body length (Fig. 15d).
Thus, by all four indices, we observed reliable difference in Lj between S. papillosus and S. ransomi, in L3 - S. papillosus and S. westeri, S. westeri and S. ransomi, in males - S. papillosus and S. westeri, S. westeri and S. stercoralis, in females of S. papillosus and S. westeri, S. papillosus and S. stercoralis. For four species of Strongyloides genus, the ratio of length of the esophagus to length of the intestine statistically differed in third stage larvae (Table 2).
Discussion
Studies accompanied by examination of metric parameters of the body of representatives of the Strongyloididae family are undertaken mostly for the purpose of description of new species (Dos Santos et al., 2010; Huang et al., 2016; Spratt, 2018). The data we presented are focused on morphology of common species of Strongyloides which parasitize domestic animals in Ukraine. In territory of our country, such studies have not been performed since the early XX century. In turn, we should mention presence of variability characteristic of all living orga-
8
55
50
7
bc
c
45
6
40
a
5
35
30
4
b
b
25
3
20
2
15
b
a
a
b
76
bc
74
a
b
b
54
52
50
d
c
nisms, including by their morphological and metric parameters. Peculiarities of adaptive and evolutionary changes of the development of ne-matodes of the Strongyloides genus help to improve the understanding of the processes of parasitism (Lok et al., 2017; Hunt et al., 2018).
In this article, we determined differential metric features of the structure of postembryonic stages of the development of S. papillosus, S. westeri, S. ransomi, S. stercoralis. The obtained data prove the changes of size of the body of larvae over the development and transition into
invasive form or free-living generation. The results we obtained coincide with studies by different authors. As for comparison of metric parameters of S. papillosus, obtained over our studies, with the studies by Basir (1950), we should mention absence of differences in sizes of L1, L2 and free-living females. At the same time, average length of body of L3, according to our data equaled 515 pm, which is insignificantly lower than what is shown by the data of Basir (575-640 pm); average length of body of free-living males was also lower by 25 pm.
Table 2
Morphometric indices of three larva stages, free-living males and females of different species of the genus Strongyloides
Stage of larva Total number The number of Index by which a species reliably differed from three other examined species
development, sex of adult nematode of the parameters compared Species statistically reliably differing parameters S. papillosus S. westeri S. ransomi S. stercoralis
S. papillosus 3 - Sb/Lb, Lc/Lb Sb/Lb, Lo/Le *
L1 4 S. westeri 3 - - Lo/Le, Lc/Lb Lc/Lb
S. ransomi 4 - - - Lo/Le, Le/Lb
S. stercoralis 3 - - - -
S. papillosus 2 - Lc/Lb, Le/Lb * *
L2 4 S. westeri 2 - - Lc/Lb, Le/Lb Lc/Lb, Le/Lb
S. ransomi 2 - - - *
S. stercoralis 2 - - - -
S. papillosus 4 Sb/Lb, Lo/Le, Lc/Lb, Le/Lb Sb/Lb, Lo/Le, Lc/Lb Lo/Le, Lc/Lb, Le/Lb
L3 4 S. westeri 4 - - Sb/Lb, Lo/Le, Lc/Lb, Le/Lb Sb/Lb, Lo/Le, Le/Lb
S. ransomi 4 - - - Lo/Le, Le/Lb
S. stercoralis 4 - - - -
S. papillosus 4 Sb/Lb, Lo/Le, - Lc/Lb, Le/Lb Lo/Le, Le/Lb Lo/Le, Le/Lb
Male 4 S. westeri 4 - - Sb/Lb, Lc/Lb, Le/Lb Sb/Lb, Lo/Le, Lc/Lb, Le/Lb
S. ransomi 4 - - - Lc/Lb
S. stercoralis 4 - - - -
S. papillosus 4 Sb/Lb, Lo/Le, Lc/Lb, Le/Lb Lo/Le, Le/Lb Sb/Lb, Lo/Le, Lc/Lb, Le/Lb
Female 4 S. westeri 4 - - Sb/Lb, Lc/Lb, Le/Lb Sb/Lb
S. ransomi 4 - - - Lc/Lb
S. stercoralis 4 - - - -
Note: * - no reliably differing characteristics.
Comparing sizes of S. westeri with the studies by Schuurmans-Ste-khoven (1930), we found support for our data. Differences were found in sizes of rhabditiform larvae and we consider this related to the fact that the authors had not divided the larvae into L1 and L2. Our previous studies proved that metric parameters of embryonic stages of the development of S. westeri have significant differences and depend on the stage of the development of helminth. At the same time, regulation of the formation of filarial larvae and free-living generations of males and females can be controlled using changes in temperatures during their cultivation (Gugosyan et al., 2018).
Schwartz & Alicata (1930) indicate a possibility of parasitism of different species of Strongyloides genus in swine. The main differential features are metric parameters of the body of helminths. After we had analyzed results of these studies, we determined that the body length of free-living males of S. ransomi according to our studies is lower. At the same time, sizes of rhabditiform and filarial larvae significantly did not differ. Huong Giang et al. (2017) continued the study of parasitism of Strongyloides in swine, comparing morphological and molecular characteristics of S. ransomi, obtained from the territory of different countries of Asia. They determined that compared to other species of Strongyloides genus, S. ransomi is morphologically similar to S. papillosus, but at molecular analysis of sequence ofDNA, it was close to S. venezuelensis.
Eberhardt et al. (2008) indicate presence of two species of the Stron-gyloides genus which parasitize agricultural ruminants. The helminths they isolated belonged to certain genetically isolated populations. Based on the results of molecular studies, these authors suggest using name S. pa-pillosus for Strongyloides of sheep, and S. vituli for Strongyloides of cattle. Currently, in Ukraine, there are no data on parasitism of different species of Strongyloides in ruminants, and the name S. papillosus is generally accepted. Differential diagnosis of different species of Strongy-
loides is also possible on the basis of morphological differences of the head ends of parasitic females (Sato et al., 2007).
The data we obtained for length of body of free-living males and females of S. stercoralis were lower than the same parameters presented by Little (1966). Variability of sizes is especially characteristic of free-living males of Strongyloides, though length of the spicules of these helminths remains relatively constant. Diagnosis and differentiation of Strongyloides is often complicated, at the same time parasitization can be lethal (Eydal & Skirnisson, 2016). For identification and isolation of S. stercoralis, researchers (Jongwutiwes et al., 1999; Khanna et al., 2015) recommend using agar-plate culture. Lopez et al. (2000) suggested a method of differentiation of rhabditiform L1 and L2 larvae using count of cells in this primordial organ over their development. Our studies can help conducting efficient differentiation of larvae and free-living generations of Strongyloides using metric parameters and by comparing mor-phometric indices.
Conclusions
Metric parameters of postembryonic stages of the development of Strongyloides have characteristic differences and depend on the species and stage of the development of the helminth. In our study, we demonstrated possibility of differential diagnosing of species on the basis of determining morphometric indices. We obtained new metric data on the structure of S. papillosus, S. westeri, S. ransomi and S. stercoralis, which parasitize domestic animals.
We measured body length (Lb), total maximum body width (Sb), length of the esophagus (Lo), length of the intestine (Le), length of the tail end (Lc); additionally for females we measured length from the head end to vulva (Lv) and the number of formed eggs in the uterus
cavity (E), and additionally for males we measured length of spicules (Ls). According to morphometric parameters, there are significant differences for L1, L2 S. papillosus and S. stercoralis, and also L2 S. ransomi and S. stercoralis. We analyzed ratio of total maximum body width (Sb) to body length (Lb), length of the esophagus (Lo) to length of intestine (Le), length of tail end (Lc) to body length (Lb), length of intestine (Le) to body length (Lb) and determined a reliable difference by these indices in L1 of S. papillosus and S. ransomi, L3 - S. papillosus and S. wes-teri, S. westeri and S. ransomi, in males - S. papillosus and S. westeri, S. westeri and S. stercoralis, in females of S. papillosus and S. westeri, S. papillosus and S. stercoralis. Larvae of all four examined species had reliable differences by index of ratio of length of the esophagus to length of the intestine.
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