Pathogenic Potential of Fresh, Frozen, and Thermally Treated Anisakis spp. Type II (L3) (Nematoda: Anisakidae) after Oral Inoculation into Wistar Rats: A Histopathological Study

Fish examination and parasitological study: Seventy specimens of the European seabass Dicentrarchus labrax (family: Moronidae) were collected during the period from July to December, 2015 from boat landing sites and fishermen at the coasts of Alexandria city along the Mediterranean Sea in Egypt. Live fish were transported to the laboratory of Parasitology, Zoology Department, Faculty of Science, Cairo University; they were identified according to Randall (1992). Nematodes (Fig. 1A,B) were collected as encapsulated larvae from the surface of visceral organs such as the stomach, intestine, and muscles; subsequently, they were rinsed in phosphate-buffered saline for further experiments. For morphological characterization by light microscopy, some of the collected worms were fixed in hot 70 % ethanol (60°C).

FIG. 1.

Scanning electron microscopy: Parasite specimens were prepared according to Madden and Tromba (1976); worms were fixed in 3% phosphate-buffered glutaraldehyde (pH 7.3) for 18–24 hr, washed in the same buffer, and dehydrated in ascending alcohol series. After passing through an ascending series of Genosolv-D, they were processed in a critical point drier “Bomer-900” with freon 13 and sputter coated with gold–palladium in a Technics Hummer V and examined with an Etec Autoscan at 20 kV Jeol scanning EM.

Experimental study: Thirty-two male Wistar rats, 6–8 wk old and 130–150-g weight provided from (National Research Centre animal house, Cairo, Egypt) were kept in suitable cages with perforated covers at a temperature of 22 ± 2 °C, 55% ± 5% humidity, and 12-hr dark/light cycle. Rats were fed on standard food and water ad libitum. Animals were determined to be free of parasites as shown by repeated stool examination and applying direct wet saline smear and flotation techniques. All experimental procedures were performed following the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, USA.

Rats were divided into four groups (eight rats each):

Gastric intubation method: After washing the isolated worms from the host fish with 0.85 NaCl, they were transported into a petri dish containing 5 ml of 50 mM HCl solution. Larvae were orally inoculated into rat stomach by the gastric intubation method according to Figueiredo et al. (2012). This step was applied under anesthesia of rats with sodium pentobarbital (50 mg/kg).

The experimental rat groups II, III, and IV were inoculated with L3 larvae according to the time line as shown in Figure 1.

After the end of the experiment, rats were euthanized under an overdose of sodium pentobarbital (100 mg/kg) after 14 d of the second administration.

Histological examination: The thymus and spleen from the experimental rat groups were isolated, washed, and fixed in 10% buffered formalin until sectioned. Tissues were dehydrated in series of alcohols, cleared in xylol, embedded in paraffin wax, and sectioned by a microtome at 5 μm thick. The tissue sections were processed and stained with eosin and hematoxylin according to Drury and Wallington (1967). The stained sections were examined and photographed using a Zeiss research photomicroscope (Carleton, 1967) supplied with a Canon digital camera.

Image morphometry: The morphometric analysis was performed at the Pathology Department, National Research Center, using routine hematoxylin and eosin stained slides and a Leica Qwin 500 Image Analyzer (LEICA Imaging Systems Ltd., Cambridge, England) which consists of a Leica DM-LB microscope with a JVC color video camera attached to a computer system Leica Q 500IW.

Statistical analysis: Kolmongrov–Smirnov analysis confirmed that the data obtained for the thymus and spleen from the experimental rat groups were non-normally distributed and consequently analyzed by Kruskal–Wallis analysis and Mann–Whitney U test. Data were expressed as mean ± SEM.

Morphological characterization of Anisakis Type II (L3): Thirty fish specimens of seventy (42.9%) were found to be naturally infected by nematode worms as encapsulated larvae recovered from the peritoneal cavity of the infected fish with an intensity ranged from 13 to 20 worms per fish (Fig. 2A,B). Morphological and morphometric characterization generally showed that all the recovered and examined parasite specimens closely resembled Anisakis spp. Identification was performed according to the key published by Rocka (2004). The recorded larvae (Fig. 1C–H) had a slender body 0.6–3.5 cm long and 0.3–1 mm wide. Prominent boring teeth were observed at the anterior extremity of the mouth area with four small papillae (two dorsolateral and two ventrolateral) surrounding the triangular mouth. The cuticle had transverse striations that started from the cephalic region and extended to the anus. The postanal tail was rounded without a terminal mucron.

FIG. 2.

Thymus: Light microscopic examination of the histological sections from the control rat group treated with saline revealed the normal size of thymus (Fig. 3A,B) with a normal distribution of thymus lobules. The ratio between cortex and medulla was normal (2: 1). Rare apoptotic bodies were seen in some control sections.

FIG. 3.

Histological examination of thymus sections of the experimental rat group orally inoculated with freshly isolated larval worms (Fig. 3D,F) revealed their atrophy (Fig. 3D) and a striking tinctorial change which was obvious especially in the cortical region because of marked apoptosis of the lymphocytes in this region. However, there was a robust recovery of the medullary thymocytes evidenced by the darker medullary region. The cortical–medullary ratio in these sections was difficult to be determined. Focal hemorrhages and more apoptotic lymphocytes were seen in the examined sections (Fig. 3E,F).

Thymus sections of the experimental rats orally inoculated with frozen larvae (Fig. 4A–D) showed a dramatic decrease in the overall size of the organ (Fig. 4A) and were severely affected by necrosis and inflammation of the cortical tissue in addition to focal hemorrhage (Fig. 4B,C). Also, it was observed that several apoptotic lymphocytes with intracytoplasmic apoptotic bodies were spread in the medulla region (Fig. 4D).

FIG. 4.

In contrast to the control, histological sections of thymus isolated from the experimental rat group orally inoculated with thermally treated larvae (Fig. 4E–G) showed no marked change in the size of the organ (Fig. 4E). However, a decreased cellularity of the medulla region led to difficulty in determining the ratio between cortex and medulla (Fig. 4F). Oncotic necrosis and lymphocyte depletion were observed in the cortical region in addition to the presence of a large number of apoptotic bodies, blood vessel congestions, and hemorrhages (Fig. 4G).

Spleen: Histological sections from the spleen of control rats (Fig. 5A–C) inoculated orally with saline showed its normal architecture with a normal ratio between white and red pulps (Fig. 5A,B). Also, the periarteriolar lymphatic sheath surrounding artery was observed with a normal distribution. The white pulp was dark because of the presence of predominant small lymphocytes and lymphoid follicles. Rare apoptotic cells were observed (Fig. 5C) and no congestion or hemorrhages were seen in the examined sections.

FIG. 5.

Examination of the spleen sections from the rat group orally inoculated with freshly isolated larval worms of Anisakis spp. Type II (Fig. 5D–F) revealed a severe red pulp congestion (Fig. 5D,E) with a severe recovery of lymphocytic cells in the periarteriolar lymphoid sheath (PALS) in addition to the distribution of large number of apoptotic cells with aggregates of pigmented materials of apoptotic bodies (Fig. 5F).

The spleen sections from the experimental rat group orally inoculated with frozen larvae (Fig. 6A–D) revealed different grades of lymphoid follicular atrophy; follicles were with indistinct germinal center (Fig. 6A). Also, severe congestion of the red pulp and lymphocyte infiltrations in the PALS were observed (Fig. 6A,B). Macrophages with aggregates of pigmented materials in addition to lymphocyte necrosis (Fig. 6C) were also observed in the examined sections.

FIG. 6.

The histological examination of the spleen sections from the experimental rat group orally inoculated with thermally treated larvae revealed an abnormal distribution of white and red pulps accompanied with severe red pulp congestion (Fig. 6E-G). The number of lymphocytes in the PALS was decreased. The distribution of scattered, occasional macrophages with aggregates of pigmented materials in addition to oncotic necrosis was also observed.

Image analysis: Histological sections from thymus (Table 1) revealed that there was a marked reduction in the cortical and medulla regions of the three parasite inoculated rat groups in addition to a significant change in the lymphocyte count compared with the control. The rat group orally inoculated with fresh larvae showed a significant decrease (P < 0.01) in the lymphocyte count compared with the control. At the same time, a significant increase (P < 0.05 and P < 0.01) was observed in the lymphocyte count of the rat groups orally inoculated with frozen and thermally treated larvae, respectively. Also, the follicular area of the spleen white pulp which is the key part of immune response was affected by a nonsignificant increase in its area fraction in case of fresh and frozen L3 inoculated rats in comparison with the control group. The lymphocyte count was significantly increased (P < 0.01) in the follicular area of all of the experimental rat groups (thermally treated L3 inoculated rats > L3 fresh inoculated rats > frozen L3 inoculated rats).

In the present study, the recovered worms from the examined fish were identified belonging to the genus Anisakis Dujardin (1845). They possessed all the general morphological and morphometric characteristic features of that genus according to the key proposed by Pippy and Van Banning (1975) including inconspicuous three lips with a prominent boring tooth on the anterior end; a straight anterior gut consisting of esophagus, ventriculus, intestine; the posterior end terminated with or without mucron; and the cuticle is obviously transversely striated. As there are two morphotypes of that genus Anisakis: Type I and II, the isolated worms in the present study are morphologically identified as Anisakis Type II according to the key described by Berland (1961), where it is characterized by a triangular mouth in the head region surrounded by four papillae with no lips; the postanal tail is rounded without terminal mucron. By contrast, the first type is characterized by an anterior head with a rounded mouth opening with dorsal and ventrolateral lips equipped by papillae; the postanal region was terminated with a small mucron.

The thymus is a primary or central lymphoid organ in which T lymphocytes undergo differentiation and maturation autonomously within the cortex. It has been shown to be a sensitive target organ following exposure to pathogens (Verinaud et al., 2004; Pearse, 2006). Therefore, changes in thymus histology and architecture are considered to be of particular relevance for immunotoxicity screening. The spleen is the largest secondary lymphoid organ; it is dark red to blue–black color located adjacent to the greater curvature of the stomach and containing about one-fourth of the body’s lymphocytes and initiates immune responses to bloodborne antigens (Nolte et al., 2002; Balogh et al., 2004; Haley et al., 2005). It comprises two functionally and morphologically distinct compartments, the red pulp and the white pulp. The red pulp is a blood filter that removes foreign material and damaged erythrocytes as well as particulate materials and circulating bacteria from the blood supply (Suttie, 2006; Mebius and Kraal, 2005). The white pulp is composed of three subcompartments: the PALS, the follicles, and the marginal zone (Cesta, 2006).

In the present study, most of the examined thymus sections obtained from experimental rat groups inoculated with fresh, frozen, and thermally treated L3 Anisakis Type II larvae showed congestion of blood vessels with an excess amount of blood in addition to the dilation of some capillaries and veins. These observations agreed with Stefanski et al. (1990) and AL-kindi et al. (2009). It is also well-known that decreased cellularity of the thymus is the most frequently encountered histological finding associated with worm-induced effects. Elmore (2006) mentioned that the decreased cellularity is often associated with the presence of dead lymphocytes. Determination of the cell death type is important because it may provide insight into the pathogenesis of the lesion. The STP Committee on the Nomenclature of Cell Death recommends the use of the term “necrosis” to describe findings comprising dead cells in histological sections, regardless of the pathway by which the cells died (Levin et al., 1999). They also recommend the use of the modifiers “apoptotic” and “oncotic” to specify the predominant morphological cell death pathway. Oncotic necrosis is the cellular process that can be seen in areas of thymus infarction (death of tissue due to an absence of blood flow which is often due to obstruction of a blood vessel). However, as in the present study, apoptotic necrosis, on the other hand, is characterized by cell shrinkage, nuclear fragmentation, extrusion of membrane-bound cytoplasm, and nuclear debris in the form of small dense apoptotic bodies. The lymphocytic depletion and necrosis of the thymus may be related to its functional role or to the degree of lymphocytic maturation, which was greatly observed in both the cortex and medulla regions of thymus section from all of the experimentally inoculated rat groups. Therefore, cooking or freezing treatments did not protect the experimental rats against infection by anisakid larvae. These findings agreed with the previous study by Audicana and Kennedy (2008). Rat groups that were inoculated with fresh and frozen larvae revealed the atrophy of the thymus with striking tinctorial change and severe lymphocytic depletion and necrosis in both cortical and medulla areas with increased number of apoptotic lymphocytes that appeared at high magnification. These observations also agreed with Dutz et al. (1973) and Smythe et al. (1971) who stated that temporary thymus atrophy or necrosis may be due to nutritional or infectious stress in animals and may cause prolonged and profound changes in the immune status and host defense. This atrophy is characterized by intense structural and morphological alterations associated with an increased level of apoptosis (Suttie, 2006; Verinaud et al., 2004).

As the largest secondary lymphoid organ in the body, the spleen is involved in the capture and destruction of pathogens as well as the induction of immune responses (Cesta, 2006). Most of the spleen sections obtained from experimental rat groups inoculated with fresh, frozen, cooked, or thermally treated L3 showed a severe congestion of the red pulp with excessive distension of its sinuses by erythrocytes in accordance with (Stefanski et al., 1990; Ward et al., 1999). Also, pigmented materials in the red pulp were observed in some spleen sections, in accordance with Stefanski et al. (1990); Ward et al. (1999) and Suttie (2006) who stated that ceroid/lipofuscin or hemosiderin pigments is usually harbored in macrophages and may be present in the marginal zone of the red pulp following immune response eliciting. Most of the spleen sections from rat group inoculated thermally treated L3 revealed severe congestion of red pulp, and no demarcation between red pulp and white pulp was detected. Also, inflammatory cell infiltrations in splenic capsule and inflammatory lesions were observed. These observations coincide with those reported by AL-kindi et al. (2009) who concluded that the inflammatory lesions and degenerations of epithelial cells produced as a result of parasitic antigen administration is related to mononuclear cell infiltrates and migration of white blood cells mainly macrophages and lymphocytes to the immune organs for attacking and clearing parasite antigen debris.

The data obtained from the oral inoculation of Anisakis Type II (L3) into Wistar rats revealed that cooking and freezing of worms might not protect against stimulation of immune responses inducing allergic and hypersensitivity reactions as well as histological alterations with various degrees. So, it is important to note that although cooking or freezing kills the parasite, these procedures are not necessarily destroying the allergenic capacity of Anisakis Type II (L3). Anisakis Type II (L3) can not only cause symptoms directly associated with the gastrointestinal infection but also cause histopathological changes in the thymus and spleen which reflects to the body immune response against L3 larvae. Thus, it is important to create a wider awareness of this potential risk to human health.