Coenurosis is a major zoonotic parasitic infection, which is caused by
The cerebral and cerebellar tissues of 15 sheep and lambs of different breeds and ages were submitted to the Pathology Department of Harran University, Faculty of Veterinary Medicine for necropsy. These tissues, necropsied between 2012 and 2016 in the Sanliurfa region of Turkey, constituted the material of the study.
During macroscopic examination parasite cysts were found in the cerebral and cerebellar tissues. The size of the cerebral and cerebellar cysts was measured.
All the cysts contained protoscolices. The protoscolices were first fixed in 70% ethanol, then stored at –20°C. A PureLinkTM Genomic DNA Kit (Invitrogen, USA) was used for DNA extraction. The 446 bp-cytochrome c oxidase subunit I (CO1) gene region of
The brain and cerebellum tissues were fixed in 10% formalin. The samples were processed routinely and stained with haematoxylin and eosin.
Inflammatory lesions caused by the parasite were stained immunohistochemically using the streptavidin-biotin-peroxidase method. Stainings performed for the measurement of the inflammatory response demonstrated the presence of anti-CD3 antibody T cells, CD79+ B cells, and CD163+ macrophages around the cyst. An ovine mediastinal lymph node was used as the positive control in the immunohistochemical staining and an antibody diluent not containing primary antibodies was used as the negative control. Glial fibrillary acidic protein (GFAP) antibodies produced by astrocytes, which are activated in neurodegenerative disorders, were also used.
For immunohistochemistry, monoclonal rabbit anti-human CD3 diluted 1:150, (Invitrogen, USA), monoclonal mouse anti-human CD79α diluted 1:50 (DAKO, Germany), monoclonal mouse anti-human CD163 diluted 1:50 (Invitrogen, USA), and polyclonal mouse anti-human GFAP diluted 1:300 (DAKO, Germany) comprised the primary antibodies, and polyclonal biotinylated goat anti-polyvalent was the secondary antibody (TP-125-BN, Lab Vision, USA). The antibodies differed for the immunohistochemical staining procedure. These differences are presented in Table 1. All immunohistochemically stained sections were examined under a light microscope. In view of the distribution and intensity of the immunohistochemical staining, five regions were randomly selected in each section using a 40× objective, and the positively stained cells were counted in these regions.
Differences in immunohistochemical staining protocol
CD3
CD79α
CD163
GFAP
Antigen retrieval
20 min, 120°C, 1 atm, autoclave, citrate buffer
20 min, 120°C, 1 atm, autoclave, citrate buffer
10 min, 800 watt, microwave, citrate buffer
15 min, room temperature, microwave, proteinase K
H2O2
30 min
25 min
30 min
15 min
Primer antibody application, time and temperature
1 h, room temperature
1 h, room temperature
30 min, room temperature
1 h, 37°C
3,3’-diaminobenzidine tetrahydrochloride (DAB)
10 min
10 min
7 min
3 min
The arithmetic means of the numbers of positively stained cells were used for statistical analyses, counted in five different regions in the sections prepared from each sample. The normality of the data was assessed using the Shapiro-Wilk test. Differences between the cells with respect to the numbers of cells stained positively for different antibodies were tested for significance using one-way analysis of variance for repeated measures.
Pairwise comparison for antibodies was made using the Bonferroni test. Pearson’s correlation test was applied to determine whether a correlation existed between the numbers of cells stained positively for different antibodies. For statistical analyses, the Statistical Software Package for Social Sciences, version 11.0 (SPSS 11.0, IBM-SPSS, USA) was used.
Parasite cysts were localised only in the cerebral tissue in 13 animals and in the cerebral and cerebellar tissues in 2 animals. The localisations of the cysts in the cerebrum and cerebellum were superficial and the cysts were of varying size, had a fluctuant consistency, and contained many white coloured scolices (Fig. 1A). the number and location of the parasite cysts and the number of scolices are given in Table 2. Overall, 33 parasite cysts were detected in the cerebra and cerebella. Seven (21.21%) cysts were located in the right and left frontal cerebral lobes, six (18.18%) in the right and left parietal lobes, two (6.06%) in the right and left temporal lobes, and one (3.03%) in an occipital lobe. Two (6.06%) cysts were detected in the cerebellar tissue. The largest cyst measured 6 × 5 cm and the smallest one was 2 × 2 cm in size.
Cyst location and scolex numbers
No
Sex
Age
Cyst location
Size of cyst
Scolex number
1
Male
1 year
Left parietal lobe
6×5 cm
53
Right parietal lobe
5×4 cm
47
2
Female
6 months
Left parietal lobe
4×3 cm
38
Right parietal lobe
4×3 cm
36
3
Male
9 months
Left frontal lobe
5×4.5 cm
49
Right frontal lobe
3×2 cm
26
Right parietal lobe
3×2 cm
29
4
Female
2 years
Left parietal lobe
6×5 cm
55
Right parietal lobe
5×4 cm
45
5
Male
6 months
Right parietal lobe
3×2.5 cm
32
Right temporal lobe
3×2 cm
24
Left frontal lobe
3×2 cm
25
Left parietal lobe
3×2 cm
28
6
Male
1 year
Left frontal lobe
5×4.5 cm
38
Right frontal lobe
5×5 cm
48
7
Female
1 year
Left frontal lobe
6×5 cm
53
Right frontal lobe
4×5 cm
50
8
Female
4 years
Left frontal lobe
4×3
32
Right frontal lobe
4×3
30
9
Male
6 months
Left frontal lobe
3×3
28
Right frontal lobe
3×3
26
Left parietal lobe
2×2.5
21
Right parietal lobe
2×2.5
24
10
Male
8 months
Left temporal lobe
4×3
36
11
Male
6 months
Left temporal lobe
3×3
33
Right temporal lobe
3×2
21
12
Female
2.5 years
Left occipital lobe
3×3
33
Right occipital lobe
3×2
25
13
Male
7 months
Right frontal lobe
3×3.5 cm
38
Cerebellum
2×2 cm
30
14
Male
7 months
Cerebellum
3×2 cm
30
15
Female
1 year
Left frontal lobe
5×6 cm
47
Right frontal lobe
4×3 cm
35
The highest number of scolices was 55 and the lowest was 21. The number of rostellar hooks ranged between 22 and 30. Fresh unstained samples of the internal fluid of the cyst revealed the rose thorn protoscolices typical for
The histopathological examination of the sections prepared from the cerebral and cerebellar tissues demonstrated hyperaemia and mononuclear cell infiltration in the regions in the proximity of the cysts. Furthermore, foreign body giant cells, moderate mononuclear cell infiltration, and calcification in some of the sections were observed in the periphery of the cyst wall (Fig. 1B). Perivascular cuffing in the white matter, mild microgliosis and astrocytosis, demyelination, degeneration of a few neurons, necrosis, non-purulent meningitis (Fig. 1C), and hyperaemia of the meningeal blood vessels were observed in the area surrounding the cysts.
The CD3 (Figs 3 A and B) and CD79α (Figs 3 C and D) positively stained cells displayed dark brown stained cell membranes and the CD163 positively stained cells displayed dark brown stained cytoplasm. Positive stained cells were macrophages and giant cells located in the perivascular cuffing areas and around the cyst (Figs 3 E, F).
Positively stained cells displayed a diffuse distribution in the cerebral and cerebellar parenchyma and were present in neurons and astrocytes (Fig. 3G). Staining was observed as a dark brown colour of the cytoplasm and cytoplasmic extensions (Fig. 3H).
Microscopic examination of the immunohisto-chemically stained sections showed that, quantitatively, the rank order of staining intensity was as follows: GFAP>CD163>CD3>CD79α.
The differences between the number of positive cells in the sections stained with different antibodies were found to be statistically significant (P < 0.001). The difference detected between the antibodies for the number of positive cells on the basis of multiple comparison was also found to be statistically significant (Table 3, P < 0.001).
Difference between positive stained cell numbers using different antibodies Different letters indicate that the difference between the different lines is statistically significant
Antibody
X ±SE
Min
Max
CD3
31.68 ± 2.362a
22.6
51.2
CD79
18.627 ± 0.705b
13.4
24.2
CD163
57.147 ± 3.68c
30.4
80.0
GFAP
85.347 ± 0.609d
80.0
89.0
P < 0.001
The correlation between the numbers of positive cells obtained with different antibodies is shown in Table 4. Accordingly, the correlation between the groups was ascertained to be statistically insignificant (P > 0.05).
Correlation between positive stained cell numbers using different antibodies
Antibody
CD3
CD79
CD163
CD3
CD79
0.351
CD163
–0.218
–0.059
GFAP
0.0214
–0.024
0.14
Coenurosis, which is generally observed in small ruminants, remains a major and common parasitic infection (19). Coenurosis is a zoonotic parasitic disease of sheep and goats, which is caused by
Some researchers have reported that the parasite cysts are mostly localised to the parieto-occipital region (10). The present study demonstrated that the cysts were located in the frontal and parietal lobes of the cerebral tissue. This finding suggests that the parasite cysts may develop in any region of the central nervous system. The size of
Several literature reports are available on the histopathological investigation of parasite cysts localised to the cerebral and cerebellar tissues and the inflammatory lesions in the periphery of these cysts. In agreement with previous research (11, 14, 23), in the present study histopathological examination demonstrated a marked hyperaemia around the cyst, foreign body giant cells, moderate mononuclear cell infiltration, and in some sections calcification in the periphery of the cysts, as well as perivascular cuffing, mild microgliosis and astrocytosis in the white matter near the cysts, and also demyelination, degeneration of a few neurons, necrosis, non-purulent meningitis, and hyperaemia of the meningeal blood vessels.
To the authors’ knowledge, there is no previous microscopic study on the inflammatory cells surrounding coenurosis lesions in the central nervous system. In order to reach the goal of demonstrating the distribution of inflammatory cells in the parasitic granulomatous inflammation due to
It is hoped that the results of the present study will light the way for future pathological and parasitological research to be conducted on this subject.