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Introduction
The larvae of the genus Baylisascaris can cause tissue damage in the paratenic hosts, and are considered to be causative agents of visceral, ocular or neural larva migrans in mammals and birds (Sapp et al. 2017; Bauer 2013; Gavin et al. 2005). Baylisascaris procyonis of raccoons are known to cause avian cerebrospinal nematodiasis leading to mortality, and the larvae had been found in central nervous system of chickens (Richardson et al. 1979), emus (Kazacos et al. 1981), partridges (Sass 1978), bobwhites quails (Reed et al. 1981) or Australian brush turkeys (Kazacos et al. 1982). The roundworm of kinkajous was previously thought to be B. procyonis (Kazacos et al. 2011; Overstreet 1970). Genetic and morphological analysis revealed that the roundworm of kinkajous differed from B. procyonis (Taira et al. 2013), and has been newly described as B. potosis (Tokiwa et al. 2014). Kinkajous (Potos flavus) belong to the family Procyonidae and are closely related to the raccoon (Procyon lotor), which is the natural final host of B. procyonis.
Kinkajous is kept as pets in household in some countries. In Japan in 2016, 8 out of 29 (27.6 %) imported captive kinkajous, the majority from the Republic of Guyana, were positive for Baylisascaris eggs in the feces (Tokiwa et al. 2016). The close genetic relationship between B. potosis and B. procyonis as well as between their hosts underline a potential risk of larva migrans both in humans and birds.
Experimental study of B. potosis larvae in mice, rats and rabbits was revealed that the migration activity of larvae in host tissues is not as high as the larvae of B. transfuga, the roundworm of bears (Taira et al, 2018). The authors suggested that the pathogenicity of B. potosis larvae would be lower than that of B. procyonis, the roundworm of raccoons. There are no reports on the migration and the clinical sings of hosts by B. potosis larvae in chickens. Chickens is known to be a paratenic host of ascarid larvae such as Toxocara canis and T. cati, the round worm of dogs and cats (Taira et al. 2003; Taira et al. 2011), is able to transmit the larvae to humans through the ingestion of raw or undercooked meat, and consequently cause larva migrans syndrome in humans.
The aims of this study are to investigate B. potosis larval migration in chickens and the associated clinical manifestations of host.
Material and Methods
Parasites
Eggs of B. potosis were collected from feces of naturally infected kinkajous, which were imported from Guyana to Japan, by a sugar-salt flotation technique (Taira et al., 2018) and cultured in 0.5 % formalin solution at 25°C for about 1 month for embryonation. The embryonated eggs were preserved in 0.5 % formalin solution at 10°C for up to 3 months. The eggs were washed twice with tap water prior to inoculation to remove formalin.
Experimental animals
Thirty six 3-week-old chickens (Boris-Brown breed) of both sexes and seven 5-week-old male mice (ICR strain, outbred) were used in the study. Chickens were hatched in our laboratory from fertilized eggs purchased from a commercial farm, and kept in our laboratory. Mice were purchased from a commercial supplier of experimental animals (Japan SLC, Inc., Shizuoka, Japan). All animals were kept at 25°C and provided with commercial feed (Bird food, Pet’s One Japan, Inc. or CLEA Rodent Diet CE-2, CLEA Japan, Inc.) and water ad libitum. Animals were acclimatized for 1 week prior to the experimental infection.
Design of experiment
Chickens were orally inoculated with 3,000 (2918.3 ± 193.7 (95 % confidence interval) B. potosis embryonated eggs/chick by a 1 ml pipet, and 6 birds were euthanized by intracranial injection of 70 % ethanol and necropsied at days 1, 2, 3, 7, 30 and 90 post inoculation (PI), respectively, for larval counts.
Mice were inoculated with 2,000 (1941.0 ± 135.3 (95 %CI)) embryonated B. potosis eggs/mouse to serve as a control for the infectivity of eggs. The eggs were of the same batch as those used to inoculate the chickens. Inoculation in mice was conducted by a stomach tube attached to a 1 ml syringe. The infected mice were euthanized by giving isoflurane anesthesia followed by cervical dislocation and necropsied at days 28 or 60 PI for larval counts.
Animals were monitored daily for clinical symptoms during routine animal care. In particular, they were carefully observed for the onset of neurological signs, such as torticollis, ataxia, circling, extensor rigidity and paralysis.
Recovery of larvae
For chickens, the liver, lungs, breast muscles, hind-limb muscles, eyes, heart, kidneys, and spleen were removed individually, and digested for larval counts. For mice, the whole body without stomach, intestines, skin, tail, tips of limbs and tips of nose were digested for larval counts.
The digestion was done according to Taira et al., (2018). Briefly, each organ was minced and digested in an 1 % of HCl (37 %) - pepsin (1:10,000) solution at 37°C for 2 h under constant stirring. The ratio of tissue (g) to digestive fluid (ml) was approximately 1:10. Following digestion, the fluid were settled for 1 h at 37°C for sedimentation of larvae. Then, the sediment was filtered through a 42-mesh metal sieve into a centrifugal tube with 37°C saline, and allowed to settle for 1 h. The number of larvae in the sediment was counted under a light microscope within 24 hours after digestion.
The brain and the eyes were removed individually and pressed between two slide-glasses to count the larvae under a light microscope.
Ethical Approval and/or Informed Consent
This study was approved by the Institutional Animal Care and Use Committee of Azabu University with the reference number 130207-3, and the experimental animals were kept according to the rules and regulations.
Results
Larvae in chickens
No neurological symptoms nor abnormal behaviors were observed in chickens throughout the study. Larvae were detected in the liver, lungs and breast muscles of 13/36 (36.1 %) chickens (Table 1). The mean total number of larvae in the liver, lungs and breast-muscles at days 1, 2, 3, 7, 30 and 90 PI were 0.34, 0.17, 1.66, 1.01, 0.17 and 0, respectively. No larvae were found from the hind-limb muscles, brain, eyes, heart, kidneys and spleen. The mean recovery of larvae were less than 0.055 % for all the groups of chicken sacrificed at each day.
Number of larvae recovered from 3 week-old chickens (n=6/group) inoculated with 3,000 Baylisascaris potosis eggs/chick.
Days PI1)
Mean number of larvae recovered
Recovery (%)3)
Liver
Lungs
Breast muscles
Others2)
Total
1
0.17
0.17
0
0
0.34
0.011
2
0.17
0
0
0
0.17
0.006
3
0.33
1.33
0
0
1.66
0.055
7
0.17
0.67
0.17
0
1.01
0.034
30
0.17
0
0
0
0.17
0.006
90
0
0
0
0
0
0
1) Post inoculation
2) Hind-limb muscles, heart, brain, eyes, kidneys and spleen
3) Total recovered larvae / 3,000 (inoculum) x 100
Larvae in mice
No neurological symptoms nor abnormal behaviors were observed in the mice throughout the study. There are no statistical differences in the number of larvae between mice necropsied at days 28 and 60 PI (t=-.0205, df=4.47, n.s.: Welch’s t-test) (Table 2). The mean number of larvae recovered was 328.9 (n=7), and the mean recovery of larvae was 16.4 %.
Number of larvae recovered from mice inoculated with 2,000 Baylisascaris potosis eggs/mouse.
Mouse ID
Days PI1)
Recovered larvae
Recovery (%)2)
1
28
181
9.1
2
363
18.2
3
437
21.9
4
60
135
6.8
5
104
5.2
6
378
18.9
7
704
35.2
Mean (n=7)
328.9
16.4
1) Post inoculation
2) Recovered larvae / 2,000 (inoculum) x 100
Discussion
The results of the present study suggested that the chicken can be a paratenic host for B. potosis, because larvae could still be found in tissues of experimentally infected chickens up to days 30 PI. The result implies a risk in public health since the chicken sashimi (raw chicken meat) is a delicacy in some regions in Japan and Korea. Kazacos and Wirtz (1982) reported that 3-day-old chickens orally inoculated with 400 to 3,200 infective eggs/chick of B. procyonis, the round worm of raccoons, showed clinical signs of central nervous system (CNS) such as torticollis, ataxia, circling, extensor rigidity and paralysis. The onset of the CNS signs was at an average of day 20.4 PI, and the duration of CNS signs varied from less than 1 to 23 days. They also reported that chickens receiving higher dosages exhibited much more severe clinical signs, had higher mortality rates, and survived for a shorter duration after the onset of the signs than those receiving lower dosages. Richardson (1979) reported an outbreak of clinical case of fatal encephalitis by B. procyonis larvae in 1 to 7-week-old White-Leghorn chickens in Indiana, USA.
In our present study, 3-week-old chickens inoculated with 3,000 embryonated eggs of B. potosis neither died nor had shown any symptoms, and no larvae were recovered from the brain of chickens. Comparing the results of our study and those of Kazacos and Wirtz (1982) and Richardson (1979), it is suggested that B. potosis larvae is less aggressive in tissue migration in chickens than that of B. procyonis larvae.
We also observed that the recovery of larvae from mice was 16.4 %, while it was less than 0.06 % from chickens. This disparity might be due to the physiological difference between mammals and birds, such as difference in body temperature and immunological responses. Difference between rodent and birds in coevolutionary status with B. potosis may also be a factor of the disparity (Gernick 1992). It is also possible to speculate that most of embryonated egg failed to hatch in the gut of the chickens or those that hatched were not able to penetrate through the intestinal wall. Further studies are needed to elucidate the factors that led to the difference in the larval migration activity of B. potosis in mice and chickens.
In conclusion, the present study demonstrated that B. potosis larvae can infect chickens, and the result suggested that the chicken can be a paratenic host for B. potosis. The result may underline a public health importance of B. potosis infection as a potential foodborne disease in humans.
