Histopathologic changes and larval recovery of Toxocara cati in experimentally infected chickens
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- Azizi, S., Oryan, A., Sadjjadi, S.M. et al. Parasitol Res (2007) 102: 47. doi:10.1007/s00436-007-0722-5
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This study was made to determine the distribution pattern of Toxocara cati larvae in chickens as a paratenic host and its potential zoonotic risk by consuming infected chickens. Two groups of chickens were fed with 1,000 and 3,000 embryonated eggs of T. cati. The chickens were necropsied 3, 7, 14, and 21 days postinfection. The liver, lungs, kidneys, spleen, small intestine, and half of all the striated muscles were digested for larval recovery. Squash method was used for brain. Larvae were recovered from the liver and brain of infected chickens with 1,000 embryonated eggs. Samples of these tissues were prepared for histopathologic studies. Experimental chickens exhibited hemorrhages in the liver, lungs, and kidneys on all days postinfections (dpi). White spots on the liver surfaces that showed necrotic foci, infiltration of eosinophils, and a few lymphocytes around necrotic areas were seen on 14 and 21 dpi. Remains of larvae were present in the liver on 14 dpi. Pathologic findings showed that larvae migrated in different organs of chickens. We suggest that chickens could be paratenic hosts, and human infection with T. cati might occur after consumption of raw or undercooked meat of infected chicken with T. cati.
Toxocara cati (T. cati) and Toxocara canis (T. canis) are cosmopolitan zoonotic nematodes of the family Ascaridae, whose adult forms inhabit the proximal small intestine of their mammalian definitive hosts of canides and felids. They have a wide range of paratenic hosts, including birds, rodents, human beings, and other mammals, where larvae migrate to various tissues and survive for a long period.
These parasites are the causative agents of human toxocariasis (Magnaval et al. 2001; Akao and Nobuo 2007). Systemic infection with this nematode is relatively common in humans with seroprevalance rates varying from 3.6 to 86% in different countries (Thompson et al. 1986; Magnaval et al. 1994; Taylor 2001). Sadjjadi et al. (2000) investigated the seroprevalence of toxocara infection in school children in Shiraz, southern Iran and gained 30.15 and 20.2% seropositivity in urban and in rural residents, respectively. Considering the high prevalence of T. cati (52.8%) in stray cats in Shiraz (Sadjjadi et al. 1998), it is possible that domestic poultries reared outside of the poultry house, or from wild ones, as nutritional sources for humans, can be contaminated with the parasite. Direct contact of children with cats could be another reason for the high incidence of human toxocariasis in this region. The incidence of human toxocariasis due to larval migration of T. cati and T. canis is increasing nowadays all over the world (Glickman and Schantz 1981; Dunne and Gill 1987; Kondo 1988; Shigeno et al. 1988).
Human beings become infected by ingesting infective eggs or eating the meat of paratenic hosts containing encapsulated larvae. Beaver (1956) first hypothesized that organs or tissues of infected animals can serve as sources of Toxocara infections for humans. There are reports indicating infection of human with toxocariasis after eating raw or undercooked infected chicken meat (Ito et al. 1986; Nagakura et al. 1989), raw beef liver (Aragane et al. 1999), raw pig liver (Stürchler et al. 1990), and raw lamb liver (Salem and Schantz 1992). Inoue (1987) experimentally inoculated T. canis eggs into the gizzard of chickens and observed migrating larvae in their liver; the infected liver then were fed to mice and were capable of infecting them.
There are few experimental studies on migrating pattern of T. canis larvae in chicken as paratenic host and its potential zoonotic risk, but, there is no report showing migration of T. cati larvae in the visceral organs of this animal and showing potential zoonotic risk of this species in human beings. No experiment is conducted as yet to show the pattern of T. cati larvae migration in food animals, including chicken. Most attention has focused on the zoonotic potential of T. canis (Fisher 2003), and compared to T. canis, there are only little information about migration of T. cati in the paratenic host (Hrčkova et al. 2001). It is possible that the distribution patterns for T. cati and T. canis in tissues and organs of paratenic hosts are not similar, and this might result in variations in the host inflammatory responses. Therefore, the present study was undertaken to investigate the pathogenesis of migrating larvae of T. cati and its distribution pattern in chicken by histopathological changes and larval recovery from tissues.
Materials and methods
Recovery of embryonated eggs from the infected cats
Stray cats were captured with cage at different areas of Shiraz city in Fars Province, southern Iran, and were autopsied after they were euthanized by standard methods of Iranian Veterinary Organization Rule at autopsy room of Pathology Department of Shiraz Veterinary School for collecting T. cati from their small intestines. Eggs of T. cati were collected by dissecting adult female worms. The eggs were then transferred into 2% neutral formalin saline. The specimens were then kept at 27°C for 2–4 months until they were embryonated (Zibaei et al. 2007).
Inoculation of T. cati eggs to chickens
The eggs were washed three times with normal saline before inoculation. Twelve 15-day-old broiler chickens were divided into three equal groups. The animals of groups A and B were inoculated using gastric tube with 1,000 and 3,000 eggs of embryonated T. cati eggs, respectively. The chickens of group C were used as uninfected controls. After inoculation, one chicken of each group was euthanized by standard methods of Iranian Veterinary Organization Rule at 3, 7, 14, and 21 dpi and necropsied.
Digestive method (larval recovery)
The liver, lungs, kidneys, spleen, small intestine, and half of all the striated muscles of different groups of animals were finely minced, then were added to a solution containing 1% pepsin (1:10,000), 1% HCl (37%) in distilled water and were incubated at 46°C for 4 h with constant stirring. After incubation, the digests were filtered through a system of sieves with numbers 70, 200, 400, and 500. The sedimental liquids then were centrifuged for 2 min at 1,500 rpm. The sediments were then collected, transferred to a petri dish, and viewed under a stereoscopic microscope (Wang et al. 1983; Taira et al. 2004). Squash method between two slides was applied for brain.
Samples of the lungs, liver, brain, kidneys, spleen, heart, and skeletal muscles from the infected and control groups were fixed in 10% neutral buffered formalin, dehydrated with graded ethanols, and embedded in paraffin. Tissue sections of 5 μm thickness were stained with haematoxylin and eosin and studied with ordinary light microscopy.
Larval recovery results
The remarkable gross pathologic changes in the experimental chickens infected with 1,000 and 3,000 eggs were focal to linear hemorrhages in the liver parenchyma and to a lesser extent in lungs and kidneys of all the experimentally infected chickens in both groups on day 3 and persisted until day 14 dpi. Remains of larvae were seen in the liver parenchyma at 14 days dpi in two chickens infected with 1,000 embryonated eggs.
There are evidences in the literature that shows T. cati as a zoonotic nematode (Fisher 2003). Human infection can occur as a result of ingesting embryonated eggs from the environment such as contaminated vegetables and fruits (Dubinský et al. 1995; Magnaval et al. 2001; Talvik et al. 2006), although the possibility of ingestion of larvae within small paratenic hosts, such as beetles, or in uncooked meat, cannot be excluded (Fisher 2003).
The present study is the first report of the experimental infection of chickens with T. cati embryonated eggs that shows extraintestinal migration of larvae in the organs and tissues.
Similar studies have taken place in chickens using T. canis embryonated eggs and demonstrated larval migration in different tissues of this animal as paratenic host (Galvin 1964; Agnihotri et al. 1987; Maruyama et al. 1994; Taira et al. 2003).
In the studies conducted in chickens infected with different doses of embryonated T. canis eggs, it is showed that the larvae always accumulated in very great numbers in liver. In our study, the larvae were recovered from liver and brain. Furthermore, the most prominent pathologic lesions due to migration of larvae were observed in liver (Agnihotri et al. 1987; Maruyama et al. 1994; Okoshi and Usui 1968; Sharma and Bhatia 1983; Galvin 1964; Gargili et al. 1999). Hrčkova et al. (2001) investigated histopathologic changes of infected mice with T. cati eggs and showed that remarkable histopathologic changes were seen in liver and lungs. Morimatsu et al. (2006) recently reported visceral toxocariasis in a family after consumption of raw chicken livers.
In the present study, no larva was detected in lungs, kidneys, and muscles in both larval recovery and histopathological methods. It is stated that because of their mobility, larvae are not always found in histopathologic lesions (Fenoy et al. 2001). However, larvae were recovered from liver and brain of the infected chickens. Previous studies with T. canis reported presence of a few larvae in the brain and other organs such as spleen, kidneys, heart, and muscles at 6 dpi and afterwards (Galvin 1964; Okoshi and Usui 1968; Maruyama et al. 1994; Gargili et al. 1999).
Similar to our findings, it was previously shown that the percentage of total larval recoveries was different widely among chickens, irrespective of the date of necropsy postinfection or egg dose levels (Taira et al. 2003).
Focal hemorrhages in the liver and lung parenchyma might indicate that T. cati migrated in the body by other routes, in addition to blood-borne route (Smith 1993). Histopathologic findings including hemorrhagic tracts, tissue necrosis, and infiltration of eosinophils indicate a pattern of parasitic migration similar to that observed in infected chickens with T. canis in other studies (Gargili et al. 1999; Taira et al. 2003), but it is unknown why larvae were not seen in lungs, kidneys, and muscles. It is also unclear why liver and brain infection was mild and very few larvae were retrieved from their parenchyma. It may be due to the brief persistence of these larvae in these tissues (Buijs et al. 1994), or larvae migrated in tissues undergoing fixation, which influenced histological finding (Prociv 1989). The number of inoculated eggs could also be another reason of absence of the migrating larvae in muscles, heart, lungs, and kidneys and presence of a few larvae in brain and liver parenchyma. Possibly, the chicken hosts receive more embryonated eggs and larva in nature specially by feeding on earthworms. Eating earthworms, which ingest soil and are covered in soil, is certainly a risk for infection with Toxocara. (Pahari and Sasmal 1991). Due to ingesting large numbers of eggs in the naturally infected chickens, different tissues become seriously infected. In this case, by inoculation of higher numbers of eggs in experimental models, it is possible to detect the migrating larvae in other tissues and higher rate of infection of brain and liver parenchyma. The effects of gizzard’s muscular pressure on the embryonated eggs and the enzymatic and hormonal secretions of the proventriculus, crop, gizzard, and small intestine on the viability of the embryonated eggs need further attention. Further studies need to clarify different aspects of the T. cati biology. The severity of this disease may be dose-dependent. To date, there have not been studies on infected chickens with embryonated eggs of T. cati as paratenic host to answer these questions. Detailed studies with a larger dose of T. cati embryonated eggs are necessary to investigate the larval migration behavior.
We suggest a potential risk in eating chicken particularly the liver and brain, as causative agent of human toxocariasis due to T. cati, because this study has shown larval migration in chicken as a paratenic host. Therefore, people must be aware of the danger of ingesting raw chickens.
The authors would like to appreciate the financial support of Shiraz University, Shiraz, Iran. We also would like to thank Mr. L. Shirvani, Mr. G. Yussefi, Mr S. A. Roueentan, Dr N. Tanideh, Mr. B. Farhangmehr, and Mrs S. Kazemian for their technical assistance.