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Detection of Trypanosoma evansi in brains of the naturally infected hog deer by streptavidine–biotin immunohistochemistry
Veterinary Parasitology 87 (2000) 223–230
Short communication
Detection of Trypanosoma evansi in brains of the naturally infected hog deer by streptavidine–biotin immunohistochemistry D. Tuntasuvan a,∗ , S. Mimapan a , N. Sarataphan a , L. Trongwongsa a , R. Intraraksa a , A.G. Luckins b b
a National Institute of Animal Health, Kasetklang, Jatujak, Bangkok 10900, Thailand Centre for Tropical of Veterinary Medicine, University of Edinburgh, Roslin, Edinburgh EH25 9RG, UK
Received 17 February 1999; accepted 6 July 1999
Abstract Twenty-four percent of hog deer (Cervus porcinus) that ranged free on a farm in Samut Prakarn province, Thailand, died showing nervous signs between September 1997 and February 1998. The nervous signs shown by most of them included ataxis, paresis of hind limbs, lateral recumbency, excitation and convulsion. Six animals and one carcass were submitted for diagnosis at the National Institute of Animal Health, Bangkok. Trypanosoma evansi was detected in blood and cerebrospinal fluid of four and five animals, respectively. Antibodies to T. evansi were found in all the hog deer by indirect enzyme-linked immunosorbent assay. Histopathological observation revealed a generalised non-suppurative meningoencephalitis affecting the white and grey matter at all levels of the brain. Typically, there were broad perivascular cuffs of mononuclear inflammatory cells, including lymphocytes, and some Mott cells. No trypanosomes were found in any tissue examined by conventional histopathology. However, numerous T. evansi were demonstrated by streptavidine–biotin immunohistochemistry in neuropil and Virchow-Robin spaces of brain in three animals. ©2000 Elsevier Science B.V. All rights reserved. Keywords: Trypanosoma evansi; Brain; Hog deer; Streptavidine–biotin immunohistochemistry
∗ Corresponding author. Tel.: +662-579-8908; fax: +662-579-8918. E-mail address: [email protected] (D. Tuntasuvan)
0304-4017/00/$ – see front matter ©2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 9 9 ) 0 0 1 6 4 - 8
224
D. Tuntasuvan et al. / Veterinary Parasitology 87 (2000) 223–230
1. Introduction Trypanosoma evansi has been reported in different parts of Thailand as a cause of the disease in horses (Boonyawong et al., 1975), buffaloes (Matthias and Muangyai, 1980; Lohr et al., 1985), cattle (Chaichanapunpol et al., 1985; Trisanarom et al., 1987), pigs (Teeraprasert et al., 1984), dogs (Kashemsant et al., 1989) and deer (Indrakamhang et al., 1996). The clinical manifestations vary considerably: infected horses show an increase in body temperature, anaemia, ventral oedema, lethargy and ataxia; abortion occurs in pregnant sows (Teeraprasert et al., 1984) and cows (Lohr et al., 1986); and infected cattle sometimes show nervous signs. Tuntasuvan et al. (1997) observed that Thai cattle (Bos indicus) showed nervous signs, including circling, jumping, lateral recumbency, excitation, and convulsion, and finally died. Trypanosoma evansi was detected, by mouse inoculation, in their blood, cerebrospinal fluid (CSF) and brains. This paper reports the presence of T. evansi, detected by immunohistochemistry, in the brains of hog deer that died with nervous signs on a farm in Thailand.
2. Materials and methods 2.1. Hog deer Between September 1997 and February 1998, 24% (47/200) of free-ranging hog deer on a farm in Samut Prakarn province, Thailand, died. Most of them showed nervous signs before death, and several pregnant hog deer aborted. One carcass and six sick hog deer from the farm were submitted for diagnosis at the National Institute of Animal Health (NIAH), Bangkok, and the sick were observed for clinical signs until they died. They were five females and two males, aged between eight months and two years. Blood and CSF were collected and examined for trypanosome by wet preparation, thin smear, microhematocrit centrifuge test, and mouse inoculation (Paris et al., 1982). At post-mortem, pathological lesions were observed. Sections of brain, spinal cord, liver, heart, lung, kidney and lymph nodes were examined for trypanosomes by impression smear and mouse inoculation. The organs were examined for pathological bacteria by culture on MacConkey agar. The possible presence of pathological viruses in tissues was examined by passage in BHK-21 cell culture. Organs were preserved in 10% buffered neutral formalin for study by conventional histopathology (hematoxylin and eosin staining) and immunohistochemistry. 2.2. Immunohistochemical examination Streptavidine–biotin (SAB) immunohistochemistry technique for the detection of T. evansi was modified from Sueyoshi et al. (1995) and Kaku et al. (1999). The formalin-fixed, preserved tissues of the hog deer were processed, embedded in paraffin, and cut into 5 m sections. The tissue slide was coated with 3-aminopropyl triethoxy silane (APS) and incubated with normal goat serum before incubation with rabbit anti- T. evansi hyperim-
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mune serum. After washing with phosphate buffer saline (PBS), pH 7.4, it was incubated with biotin-labelled rabbit anti-goat conjugate and SAB complex (Histofine SAB-PO kit, Nichirei, Japan). Finally, it was stained with 3, 3 diaminobenzidine tetrahydrochloride (DAB) and examined by light microscopy. The anti-T. evansi hyperimmune serum was prepared by subcutaneous injection of 2 × 106 T. evansi isolated from the hog deer into a New Zealand White rabbit. On day 10 after inoculation, the rabbit was treated with diminazine (Berenil® , Hoechst) 7 mg/kg, and serum was collected from the rabbit on day 17 after inoculation. 2.3. Antibody detection Sera from the six hog deer were examined for the antibodies against T. evansi by an indirect enzyme-linked immunosorbent assay (ELISA) using sonicated antigen prepared from T. evansi. According to the technique of Tuntasuvan et al. (1996), protein-G horseradish peroxidase conjugate (Zymed) was used. For control purposes, a negative control serum was obtained from a hog deer in Chonburi province which had been shown free from trypanosome infection.
3. Results The clinical signs shown by the six infected hog deer were emaciation, depression, weakness, ataxia and anaemia. The mean packed cell volume (PCV) was 18.2% (15–23%). Five animals had fever, with a mean body temperature of 38.8◦ C (36.9–39.4◦ C). Four showed nervous signs, including paresis of the hind limbs, incoordination, lateral recumbency, and convulsion, and then died. Three deer had their heads twisted back. Whilst under observation, one animal aborted in a late stage of pregnancy. All the observed animals died within 2–14 days after admission at NIAH. T. evansi was detected in five of the six animals by parasitological techniques. It was noticed that the mice inoculated with the positive blood or CSF showed nervous signs before death. Antibodies to T. evansi were detected in all deer by indirect ELISA. The result of trypanosomosis examination of blood and CSF by various techniques are shown in Table 1. At post-mortem, all carcasses were anaemic. No specific pathological lesions were observed in any organs, except in that of two animals; blood congestion and petechial haemorrhage were seen in the brains. Histopathological studies showed that there was a generalised Table 1 Trypanosomosis examination by various methods Sample
Thin smeara
HCTb
MITc
Ab-ELISAd
Blood CSF
1/6 0/6
3/6 5/6
4/6 5/6
6/6 0/6
a Number
of positive sample/total samples. centrifuge test. c Mouse inoculation test. d Antibody detection by ELISA. b Hematocrit
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non-suppurative meningoencephalitis and myelitis affecting all levels of the cerebrum, cerebellum and pons and diffuse gliosis. Typically, there were broad perivascular cuffs of inflammatory mononuclear cells and Mott cells. Severe vacuolar degeneration of white matter and myelopathy were found in the spinal cords of six animals. The kidneys of three animals showed renal nephrosis and proteinaceous material in renal tubules. Some animals showed severe haemorrhage in liver and spleen, lymphoid depletion, interstitial pneumonia and epicarditis. However, no trypanosomes were found in any tissue examined by histopathological technique. The presence of T. evansi in organs of hog deer detected by various techniques are shown in Table 2. Moreover, T. evansi was detected by impression smears in the brains of two animals, and in the aqueous humour of one animal; but by using mouse inoculation techniques, more positive animals could be detected. The SAB-immunohistochemistry technique demonstrated numerous T. evansi in the intravascular and extravascular spaces of brains in three animals. The parasites were demonstrated in dark brown colour in neuropil and Virchow-Robin spaces of cerebrum, cerebellum and pons (Figs. 1 and 2). No pathogenic bacteria or viruses were isolated from any of the culture preparation organs of the hog deer. Table 2 Detection of T. evansi in organs of hog deer by various techniques Animal No. T. evansi detection by
1 2 3 4 5 6 7
Impression smear Mouse inoculation test
SAB-immunohistochemistry
+ (in cerebrum) not done – – + (in cerebrum) – –
+ (in cerebrum, cerebellum and pons) – + (in cerebrum, cerebellum and pons) – – – + (in cerebrum, cerebellum and pons)
+ (in cerebrum, cerebellum and pons) not done + (in cerebrum) – + (in cerebrum) + (in cerebrum) –
Fig. 1. T. evansi shown in neuropil of cerebrum by SAB-immunohistochemistry technique.
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Fig. 2. T. evansi in the Virchow-Robin spaces of cerebrum by SAB-immunohistochemistry technique.
Lead poisoning was considered as a possible cause of death in two animals that had brain oedema, liver and kidney degeneration. Some chemical levels in blood and lead level in liver, kidney and stomach content of the animals showed that the amounts of lead were at normal levels (Tables 3 and 4). Lead content in grass and drinking water from the farm, where the animals fed, were determined. Lead in grass and in water was 4.647 and 0.044 ppm, respectively, which is lower than the maximum values permitted by the US Environmental Protection Agency, 1973, and National Academy of Science, 1974. The maximum values of lead in grass and in water are 3–7 and 0.1 ppm, respectively. Table 3 Blood chemistry values of two T. evansi infected hog deer Animal SGOT No. (IU/l) 1 3 Na
SGPT BUN TP (% g) P (% mg) Ca (mg/dl) Mg (mg/dl) Cu (mg/dl) Zn (mg/dl) Se (mg/dl) (IU/l) (% mg) (% g)
88.8 9.12 241.7 >105.6 47.1 69.1 49−123 14−43 8−20 a Blood
5.4 6.7 6−7.9
9.05 13.89 5−7.3
4.3 7.1 11−12
8.3 2.5 2−2.8
72.14 93.28 58−160
66.4 48.8 –
3.37 2.11 –
chemistry values of normal sheep (Kaneko, 1989).
Table 4 Lead values in liver, kidney and stomach contents of three T. evansi infected hog deer Sample examined
Livera Kidneya Stomach content a The
Lead values (ppm) Animal No. 1
Animal No. 3
0.09 0.153 0.28
0.74 1.13 0.56
maximum values of lead in organs is <10 ppm (Fraser, 1991).
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4. Discussion The present study has shown that T. evansi can cause nervous symptoms and death in the naturally infected hog deer, and this was supported by the demonstration of the parasites in the brains by impression smear, mouse inoculation test and SAB-immunohistochemistry. No pathogenic bacteria or viruses were isolated from the organs of the animals. This study supports the earlier work in Thailand by Tuntasuvan et al. (1997), who detected T. evansi in the brains using impression smear and mouse inoculation of the Thai cattle that died with nervous signs. In China, T. evansi has been detected in a number of species of deer. Lun et al. (1993) reviewed that about 23% of reindeer died during 1978–1979 of surra, characterised clinically by emaciation, oedema and nervous signs. Various other species of wild animals have been shown to be susceptible to infection with T. evansi, including capybara (Hydrochoerus hydrochoerus), wild dogs (Canis azarae) and deer (Axis axis, Rusa timorensis). Such infections are fatal frequently (Clark and Dunn, 1933). Our findings differ from those published by Sudarto et al. (1990) and Ngeranwa et al. (1993). Sudarto et al. (1990) found scattered T. evansi in the Virchow-Robin spaces and neuropil of the brain of a buffalo (Bubalus bubalis) using avidine–biotin complex immunohistochemistry. However, the buffalo did not show nervous signs. Similarly, in studies of Ngeranwa et al. (1993), on the pathogenesis of T. evansi in small East African goats, T. evansi was detected by direct microscopy in extravascular locations, including CSF, but without any nervous signs. Moreover, all of the hog deer examined showed signs of anaemia with a low average PCV of 18.2% compared with the study of Indrakamhang et al. (1996), which found the average PCV of the infected deer to be 26.6%. All the infected hog deer showed a generalised non-suppurative meningoencephalitis with broad perivascular cuffings. Similar histopathological lesions have been seen in infected horses, although no hemosiderin deposits were detected in phagocytic cells of the spleen, lymph nodes, liver or lung (Seiler et al., 1981); but similar lesions have been found in T. evansi infected pigs, which showed nervous signs (Teeraprasert et al., 1984). The parasite strain appeared to be virulent. After passaging heparinized blood of mouse inoculated with T. evansi from hog deer to ten mice, 70% of the experimentally infected mice developed a high parasitaemia by day 3 after inoculation, and died with nervous signs. Glaucoma, or keratitis, was found in three hog deer, but only one hog deer showed numerous trypanosomes in aqueous humour by impression smear. It is important to note that three of the females that showed severe disease were pregnant. Lohr et al. (1985) reported that stress conditions, such as fasciolosis and malnutrition, could cause serious trypanosomosis in buffalo and cattle, and it is possible that pregnancy also induces stress, leading to serious trypanosomosis. One infected hog deer aborted during the observation, similar to reports of the infected buffalo, cattle and pigs (Teeraprasert et al., 1984; Chaichanapunpol et al., 1985; Lohr et al., 1986). In conclusion, surra can cause fever, severe anaemia, abortion, nervous signs and death in hog deer. The nervous signs are similar to those shown by horses infected with T. evansi, that is muscular weakness and progressive paralysis of the hind limbs (Luckins, 1994). The SAB-immunohistochemistry markedly increased the sensitivity of the detection of T. evansi in brain compared with conventional histopathological examination. In addition, the indirect ELISA is found to be very valuable in cerebral trypanosomosis cases, when
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the parasites accumulate in nervous tissues and cannot be detected in blood. The ability of T. evansi to survive in an extravascular location is of considerable importance in the context of chemotherapy and studies on sub-species of cerebral T. evansi.
Acknowledgements We thank the staff of the parasitology, bacteriology, virology and pathology sections of the National Institute of Animal Health, Thailand, for excellent laboratory support, and also thank the staff of the epidemiology section for their kind co-operation.
References Boonyawong, T., Chantaraprateep, P., Muangyai, M., 1975. Surra in horses. Thai J. Vet. Med. 5, 665–672. Chaichanapunpol, I., Sirirangkamanont, T., Trongwongsa, L., Suvanwasi, P., 1985. Observations on trypanosomiasis in a native bull. Kasetsart Vet. 6, 1–9. Clark, H.C., Dunn, L.H., 1933. Animal susceptibility to Trypanosoma hippicum, the equine trypanosome of Panama. Am. J. Trop. Med. 13, 273. Fraser, C.M., 1991. The Merck Veterinary Manual, 7th ed. Merck & Co., Rahway, p. 969. Indrakamhang, P., Mohkaew, K., Roonguthai, P., 1996. Preliminary report on Trypanosoma sp. in native Sambar deer and imported Rusa deer raised in a deer farm in Thailand. In: Proc. 16th Ann. Livestock Conf., Dept. Livestock Development, Thailand, 3–4 May 1996, pp. 55–67. Kaku, T., Kamura, T., Hirakawa, T., Sakai, K., Amada, S., Kobayashi, H., Nakano, H., 1999. Endometrial carcinoma associated with hyperplasia-immunohistochemical study of angiogenesis and p53 expression. Gynecol. Oncol. 72 (1), 51–55. Kaneko, J.J., 1989. Clinical Biochemistry of Domestic Animals, 4th ed. Academic Press, New York, pp. 886–891. Kashemsant, A., Pholpark, M., Pholpark, S., Srihakim, S., Leidl, K., 1989. Epidemiological pattern of Trypanosoma evansi in northeast Thailand and control measures. J. Thai Vet. Med. Assoc. 40 (3–4), 84–92. Lohr, K.F., Pholpark, S., Srikitjakarn, L., Betterman, G., Staak, C., 1985. Trypanosoma evansi infection in buffaloes in north-east Thailand. 1. Field investigation. Trop. Anim. Hlth. Prod. 17, 121–125. Lohr, K.F., Pholpark, S., Upatum, N., Leidl, K., Staak, C., 1986. Trypanosoma evansi infection, a frequent cause of abortion in buffaloes. Trop. Anim. Hlth. Prod. 18, 103–108. Luckins, A.G., 1994. Equine trypanosomiasis. Equine Vet. Ed. 6, 259–262. Lun, Z.R., Fang, Y., Wang, C.J., Brun, R., 1993. Trypanosomiasis of domestic animals in China. Parasitol. Today 9, 41–45. Matthias, E., Muangyai, M., 1980. Trypanosoma evansi infection in a swamp buffalo calf. Thai. J. Vet. Med. 10, 47–54. Ngeranwa, J.J., Gathumbi, P.K., Mutiga, E.R., Agumbah, G.J., 1993. Pathogenesis of Trypanosoma (brucei) evansi in small East African goats. Res. Vet. Sci. 54, 283–289. Paris, J., Murray, M., McOdimba, F., 1982. Comparative evaluation of the parasitological techniques currently available for the diagnosis of African trypanosomiais in cattle. Acta Tropica 39, 307–316. Seiler, R.J., Omar, S., Jackson, A.R.B., 1981. Meningoencephalitis in naturally occurring Trypanosoma evansi infection (surra) of horses. Vet. Pathol. 18, 120. Sudarto, W.M., Tabel, H., Haines, D.M., 1990. Immunohistochemical demonstration of Trypanosoma evansi in tissues of experimentally infected rats and a naturally infected water buffalo (Bubalus bubalis). J. Parasitol. 76, 162–167. Sueyoshi, M., Tsuda, T., Yamazaki, K., Yoshida, K., Nakazawa, M., Sato, K., Minami, T., Iwashita, K., Watanabe, M., Suzuki, Y., 1995. An immunohistochemical investigation of porcine epidemic diarrhoea. J. Comp. Pathol. 113 (1), 59–67.
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Teeraprasert, E., Chaichanapunpool, I., Trongwongsa, L., 1984. A case report of Trypanosoma evansi infection in pigs. In: Proc. 11th Ann. Livestock Conf., Dept. Livestock Development, Thailand, 12–14 December 1984, pp. 43–64. Trisanarom, A., Markmee, S., Pedugsorn, C., 1987. Trypanosoma evansi infection in Chiengmai dairy cattle. In: Proc. 6th Ann. Livestock Conf., Dept. Livestock Development, Thailand, 18–19 May 1987, pp. 1–12. Tuntasuvan, D., Chompoochan, T., Wongpakorn, M., Mohkaew, K., 1996. Development on the detection of Trypanosoma evansi in pigs using enzyme-linked immunosorbent assay. J. Thai Vet. Med. Assoc. 47, 45– 53. Tuntasuvan, D., Sarataphan, N., Nishikawa, H., 1997. CNS trypanosomiasis in native cattle. Vet. Parasitol. 73, 357–363.
Short communication
Detection of Trypanosoma evansi in brains of the naturally infected hog deer by streptavidine–biotin immunohistochemistry D. Tuntasuvan a,∗ , S. Mimapan a , N. Sarataphan a , L. Trongwongsa a , R. Intraraksa a , A.G. Luckins b b
a National Institute of Animal Health, Kasetklang, Jatujak, Bangkok 10900, Thailand Centre for Tropical of Veterinary Medicine, University of Edinburgh, Roslin, Edinburgh EH25 9RG, UK
Received 17 February 1999; accepted 6 July 1999
Abstract Twenty-four percent of hog deer (Cervus porcinus) that ranged free on a farm in Samut Prakarn province, Thailand, died showing nervous signs between September 1997 and February 1998. The nervous signs shown by most of them included ataxis, paresis of hind limbs, lateral recumbency, excitation and convulsion. Six animals and one carcass were submitted for diagnosis at the National Institute of Animal Health, Bangkok. Trypanosoma evansi was detected in blood and cerebrospinal fluid of four and five animals, respectively. Antibodies to T. evansi were found in all the hog deer by indirect enzyme-linked immunosorbent assay. Histopathological observation revealed a generalised non-suppurative meningoencephalitis affecting the white and grey matter at all levels of the brain. Typically, there were broad perivascular cuffs of mononuclear inflammatory cells, including lymphocytes, and some Mott cells. No trypanosomes were found in any tissue examined by conventional histopathology. However, numerous T. evansi were demonstrated by streptavidine–biotin immunohistochemistry in neuropil and Virchow-Robin spaces of brain in three animals. ©2000 Elsevier Science B.V. All rights reserved. Keywords: Trypanosoma evansi; Brain; Hog deer; Streptavidine–biotin immunohistochemistry
∗ Corresponding author. Tel.: +662-579-8908; fax: +662-579-8918. E-mail address: [email protected] (D. Tuntasuvan)
0304-4017/00/$ – see front matter ©2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 9 9 ) 0 0 1 6 4 - 8
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1. Introduction Trypanosoma evansi has been reported in different parts of Thailand as a cause of the disease in horses (Boonyawong et al., 1975), buffaloes (Matthias and Muangyai, 1980; Lohr et al., 1985), cattle (Chaichanapunpol et al., 1985; Trisanarom et al., 1987), pigs (Teeraprasert et al., 1984), dogs (Kashemsant et al., 1989) and deer (Indrakamhang et al., 1996). The clinical manifestations vary considerably: infected horses show an increase in body temperature, anaemia, ventral oedema, lethargy and ataxia; abortion occurs in pregnant sows (Teeraprasert et al., 1984) and cows (Lohr et al., 1986); and infected cattle sometimes show nervous signs. Tuntasuvan et al. (1997) observed that Thai cattle (Bos indicus) showed nervous signs, including circling, jumping, lateral recumbency, excitation, and convulsion, and finally died. Trypanosoma evansi was detected, by mouse inoculation, in their blood, cerebrospinal fluid (CSF) and brains. This paper reports the presence of T. evansi, detected by immunohistochemistry, in the brains of hog deer that died with nervous signs on a farm in Thailand.
2. Materials and methods 2.1. Hog deer Between September 1997 and February 1998, 24% (47/200) of free-ranging hog deer on a farm in Samut Prakarn province, Thailand, died. Most of them showed nervous signs before death, and several pregnant hog deer aborted. One carcass and six sick hog deer from the farm were submitted for diagnosis at the National Institute of Animal Health (NIAH), Bangkok, and the sick were observed for clinical signs until they died. They were five females and two males, aged between eight months and two years. Blood and CSF were collected and examined for trypanosome by wet preparation, thin smear, microhematocrit centrifuge test, and mouse inoculation (Paris et al., 1982). At post-mortem, pathological lesions were observed. Sections of brain, spinal cord, liver, heart, lung, kidney and lymph nodes were examined for trypanosomes by impression smear and mouse inoculation. The organs were examined for pathological bacteria by culture on MacConkey agar. The possible presence of pathological viruses in tissues was examined by passage in BHK-21 cell culture. Organs were preserved in 10% buffered neutral formalin for study by conventional histopathology (hematoxylin and eosin staining) and immunohistochemistry. 2.2. Immunohistochemical examination Streptavidine–biotin (SAB) immunohistochemistry technique for the detection of T. evansi was modified from Sueyoshi et al. (1995) and Kaku et al. (1999). The formalin-fixed, preserved tissues of the hog deer were processed, embedded in paraffin, and cut into 5 m sections. The tissue slide was coated with 3-aminopropyl triethoxy silane (APS) and incubated with normal goat serum before incubation with rabbit anti- T. evansi hyperim-
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225
mune serum. After washing with phosphate buffer saline (PBS), pH 7.4, it was incubated with biotin-labelled rabbit anti-goat conjugate and SAB complex (Histofine SAB-PO kit, Nichirei, Japan). Finally, it was stained with 3, 3 diaminobenzidine tetrahydrochloride (DAB) and examined by light microscopy. The anti-T. evansi hyperimmune serum was prepared by subcutaneous injection of 2 × 106 T. evansi isolated from the hog deer into a New Zealand White rabbit. On day 10 after inoculation, the rabbit was treated with diminazine (Berenil® , Hoechst) 7 mg/kg, and serum was collected from the rabbit on day 17 after inoculation. 2.3. Antibody detection Sera from the six hog deer were examined for the antibodies against T. evansi by an indirect enzyme-linked immunosorbent assay (ELISA) using sonicated antigen prepared from T. evansi. According to the technique of Tuntasuvan et al. (1996), protein-G horseradish peroxidase conjugate (Zymed) was used. For control purposes, a negative control serum was obtained from a hog deer in Chonburi province which had been shown free from trypanosome infection.
3. Results The clinical signs shown by the six infected hog deer were emaciation, depression, weakness, ataxia and anaemia. The mean packed cell volume (PCV) was 18.2% (15–23%). Five animals had fever, with a mean body temperature of 38.8◦ C (36.9–39.4◦ C). Four showed nervous signs, including paresis of the hind limbs, incoordination, lateral recumbency, and convulsion, and then died. Three deer had their heads twisted back. Whilst under observation, one animal aborted in a late stage of pregnancy. All the observed animals died within 2–14 days after admission at NIAH. T. evansi was detected in five of the six animals by parasitological techniques. It was noticed that the mice inoculated with the positive blood or CSF showed nervous signs before death. Antibodies to T. evansi were detected in all deer by indirect ELISA. The result of trypanosomosis examination of blood and CSF by various techniques are shown in Table 1. At post-mortem, all carcasses were anaemic. No specific pathological lesions were observed in any organs, except in that of two animals; blood congestion and petechial haemorrhage were seen in the brains. Histopathological studies showed that there was a generalised Table 1 Trypanosomosis examination by various methods Sample
Thin smeara
HCTb
MITc
Ab-ELISAd
Blood CSF
1/6 0/6
3/6 5/6
4/6 5/6
6/6 0/6
a Number
of positive sample/total samples. centrifuge test. c Mouse inoculation test. d Antibody detection by ELISA. b Hematocrit
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non-suppurative meningoencephalitis and myelitis affecting all levels of the cerebrum, cerebellum and pons and diffuse gliosis. Typically, there were broad perivascular cuffs of inflammatory mononuclear cells and Mott cells. Severe vacuolar degeneration of white matter and myelopathy were found in the spinal cords of six animals. The kidneys of three animals showed renal nephrosis and proteinaceous material in renal tubules. Some animals showed severe haemorrhage in liver and spleen, lymphoid depletion, interstitial pneumonia and epicarditis. However, no trypanosomes were found in any tissue examined by histopathological technique. The presence of T. evansi in organs of hog deer detected by various techniques are shown in Table 2. Moreover, T. evansi was detected by impression smears in the brains of two animals, and in the aqueous humour of one animal; but by using mouse inoculation techniques, more positive animals could be detected. The SAB-immunohistochemistry technique demonstrated numerous T. evansi in the intravascular and extravascular spaces of brains in three animals. The parasites were demonstrated in dark brown colour in neuropil and Virchow-Robin spaces of cerebrum, cerebellum and pons (Figs. 1 and 2). No pathogenic bacteria or viruses were isolated from any of the culture preparation organs of the hog deer. Table 2 Detection of T. evansi in organs of hog deer by various techniques Animal No. T. evansi detection by
1 2 3 4 5 6 7
Impression smear Mouse inoculation test
SAB-immunohistochemistry
+ (in cerebrum) not done – – + (in cerebrum) – –
+ (in cerebrum, cerebellum and pons) – + (in cerebrum, cerebellum and pons) – – – + (in cerebrum, cerebellum and pons)
+ (in cerebrum, cerebellum and pons) not done + (in cerebrum) – + (in cerebrum) + (in cerebrum) –
Fig. 1. T. evansi shown in neuropil of cerebrum by SAB-immunohistochemistry technique.
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Fig. 2. T. evansi in the Virchow-Robin spaces of cerebrum by SAB-immunohistochemistry technique.
Lead poisoning was considered as a possible cause of death in two animals that had brain oedema, liver and kidney degeneration. Some chemical levels in blood and lead level in liver, kidney and stomach content of the animals showed that the amounts of lead were at normal levels (Tables 3 and 4). Lead content in grass and drinking water from the farm, where the animals fed, were determined. Lead in grass and in water was 4.647 and 0.044 ppm, respectively, which is lower than the maximum values permitted by the US Environmental Protection Agency, 1973, and National Academy of Science, 1974. The maximum values of lead in grass and in water are 3–7 and 0.1 ppm, respectively. Table 3 Blood chemistry values of two T. evansi infected hog deer Animal SGOT No. (IU/l) 1 3 Na
SGPT BUN TP (% g) P (% mg) Ca (mg/dl) Mg (mg/dl) Cu (mg/dl) Zn (mg/dl) Se (mg/dl) (IU/l) (% mg) (% g)
88.8 9.12 241.7 >105.6 47.1 69.1 49−123 14−43 8−20 a Blood
5.4 6.7 6−7.9
9.05 13.89 5−7.3
4.3 7.1 11−12
8.3 2.5 2−2.8
72.14 93.28 58−160
66.4 48.8 –
3.37 2.11 –
chemistry values of normal sheep (Kaneko, 1989).
Table 4 Lead values in liver, kidney and stomach contents of three T. evansi infected hog deer Sample examined
Livera Kidneya Stomach content a The
Lead values (ppm) Animal No. 1
Animal No. 3
0.09 0.153 0.28
0.74 1.13 0.56
maximum values of lead in organs is <10 ppm (Fraser, 1991).
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4. Discussion The present study has shown that T. evansi can cause nervous symptoms and death in the naturally infected hog deer, and this was supported by the demonstration of the parasites in the brains by impression smear, mouse inoculation test and SAB-immunohistochemistry. No pathogenic bacteria or viruses were isolated from the organs of the animals. This study supports the earlier work in Thailand by Tuntasuvan et al. (1997), who detected T. evansi in the brains using impression smear and mouse inoculation of the Thai cattle that died with nervous signs. In China, T. evansi has been detected in a number of species of deer. Lun et al. (1993) reviewed that about 23% of reindeer died during 1978–1979 of surra, characterised clinically by emaciation, oedema and nervous signs. Various other species of wild animals have been shown to be susceptible to infection with T. evansi, including capybara (Hydrochoerus hydrochoerus), wild dogs (Canis azarae) and deer (Axis axis, Rusa timorensis). Such infections are fatal frequently (Clark and Dunn, 1933). Our findings differ from those published by Sudarto et al. (1990) and Ngeranwa et al. (1993). Sudarto et al. (1990) found scattered T. evansi in the Virchow-Robin spaces and neuropil of the brain of a buffalo (Bubalus bubalis) using avidine–biotin complex immunohistochemistry. However, the buffalo did not show nervous signs. Similarly, in studies of Ngeranwa et al. (1993), on the pathogenesis of T. evansi in small East African goats, T. evansi was detected by direct microscopy in extravascular locations, including CSF, but without any nervous signs. Moreover, all of the hog deer examined showed signs of anaemia with a low average PCV of 18.2% compared with the study of Indrakamhang et al. (1996), which found the average PCV of the infected deer to be 26.6%. All the infected hog deer showed a generalised non-suppurative meningoencephalitis with broad perivascular cuffings. Similar histopathological lesions have been seen in infected horses, although no hemosiderin deposits were detected in phagocytic cells of the spleen, lymph nodes, liver or lung (Seiler et al., 1981); but similar lesions have been found in T. evansi infected pigs, which showed nervous signs (Teeraprasert et al., 1984). The parasite strain appeared to be virulent. After passaging heparinized blood of mouse inoculated with T. evansi from hog deer to ten mice, 70% of the experimentally infected mice developed a high parasitaemia by day 3 after inoculation, and died with nervous signs. Glaucoma, or keratitis, was found in three hog deer, but only one hog deer showed numerous trypanosomes in aqueous humour by impression smear. It is important to note that three of the females that showed severe disease were pregnant. Lohr et al. (1985) reported that stress conditions, such as fasciolosis and malnutrition, could cause serious trypanosomosis in buffalo and cattle, and it is possible that pregnancy also induces stress, leading to serious trypanosomosis. One infected hog deer aborted during the observation, similar to reports of the infected buffalo, cattle and pigs (Teeraprasert et al., 1984; Chaichanapunpol et al., 1985; Lohr et al., 1986). In conclusion, surra can cause fever, severe anaemia, abortion, nervous signs and death in hog deer. The nervous signs are similar to those shown by horses infected with T. evansi, that is muscular weakness and progressive paralysis of the hind limbs (Luckins, 1994). The SAB-immunohistochemistry markedly increased the sensitivity of the detection of T. evansi in brain compared with conventional histopathological examination. In addition, the indirect ELISA is found to be very valuable in cerebral trypanosomosis cases, when
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the parasites accumulate in nervous tissues and cannot be detected in blood. The ability of T. evansi to survive in an extravascular location is of considerable importance in the context of chemotherapy and studies on sub-species of cerebral T. evansi.
Acknowledgements We thank the staff of the parasitology, bacteriology, virology and pathology sections of the National Institute of Animal Health, Thailand, for excellent laboratory support, and also thank the staff of the epidemiology section for their kind co-operation.
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