- Email: [email protected]
Sequential testicular and epididymal damage in Zebu bulls experimentally infected with Trypanosoma vivax
Veterinary Parasitology 143 (2007) 29–34 www.elsevier.com/locate/vetpar
Sequential testicular and epididymal damage in Zebu bulls experimentally infected with Trypanosoma vivax S. Adamu a, M.Y. Fatihu a, N.M. Useh a,*, M. Mamman b, V.O. Sekoni c, K.A.N. Esievo a a
Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Nigeria b Department of Veterinary Physiology and Pharmacology, Ahmadu Bello University, Zaria, Nigeria c Department of Animal Reproduction, National Animal Production Research Institute, Shika, Nigeria Received 6 February 2006; received in revised form 18 July 2006; accepted 20 July 2006
Abstract Six Zebu bulls aged between 31 and 34 months exhibiting good libido were used to study sequential testicular and epididymal damage in Trypanosoma vivax infection. Three bulls were infected with T. vivax, while the other three served as controls. All infected bulls became parasitaemic by day 5 post-infection and developed clinical trypanosomosis with rapidly developing anaemia. Representative bulls, one from each of the infected and control groups, were sacrificed on days 14, 28 and 56 postinfection. Testes and epididymides from these animals were studied histopathologically after processing and staining with haematoxylin and eosin (H and E). Testicular degeneration developed in all the infected bulls characterized by depletion of spermatogenic cells and destruction of interstitial tissue. The most severe testicular degeneration occurred in the bull that was sacrificed 56 days post-infection. Epididymal sperm reserves were 36%, 4% and 0%, respectively, in infected bulls that were sacrificed on days 14, 28 and 56 post-infection. The 0% epididymal sperm reserve may suggest complete cessation of spermatogenesis. It was concluded from this study that T. vivax infection of Zebu bulls could cause severe testicular and epididymal damage that may result in infertility or even sterility of the affected animals at early infection stages not previously thought. # 2006 Elsevier B.V. All rights reserved. Keywords: Trypanosomosis; Bulls; Testes; Epididymides; Trypanosoma vivax; Histopathology
1. Introduction In spite of decades of investigations on animal trypanosomosis, the disease has continued to remain an important, if not most important constraint to livestock and mixed crop-livestock farming in tropical Africa (Kristjanson et al., 1999; Irungu et al., 2002). Over 45 million cattle and 60 million people live at risk of * Corresponding author. Tel.: +234 8037032523; fax: +234 69332412. E-mail address: [email protected] (N.M. Useh). 0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2006.07.022
getting infected with trypanosomes; while each year 3 million cattle and other domestic livestock are lost through deaths caused by the parasites (FAO, 2002). Three important pathogenic species of trypanosomes are responsible for the disease in ruminants; these are Trypanosoma vivax, T. congolense and T. brucei. In Nigeria, the incidence of animal trypanosomosis is on the increase. This is because for decades there has not been large-scale surveillance and control programme to curtail the menace of the disease (Esuruoso, 1973; Ikede and Taiwo, 1985; Daniel et al., 1993). Livestock owners are, thus, left with curative and preventive treatments as
30
S. Adamu et al. / Veterinary Parasitology 143 (2007) 29–34
the only means of controlling this highly devastating disease. African livestock producers administer an estimated 35 million curative and preventive treatments annually (Geerts and Holmes, 1998). This is therefore costing producers and government colossal amount of money (Kristjanson et al., 1999). It therefore becomes imperative to make re-assessment of the pathologies produced by trypanosomosis in animals with the view to establishing when, at the different stages of infection such pathologies become very severe as this could serve as a guide in knowing when intervention with treatment could be of little or no value. In bulls, for example, trypanosomosis has been shown to cause testicular and epididymal damage that could result in infertility and, probably, sterility (Isoun et al., 1975; Sekoni et al., 1988, 1990). Although some data are already available about the effects of chronic T. vivax infection (Sekoni et al., 1990), there is however dearth of information on the degree of testicular and epididymal damage at different stages of the acute infection. The objective of this study therefore was to sequentially follow the development testicular and epididymal damage in T. vivax infected Zebu bulls as the information that could be obtained from this study may be of value in the continued use of trypanosomeinfected bulls for breeding purposes. 2. Materials and methods 2.1. Experimental animals Six normal healthy Zebu bulls of White Fulani breed aged between 31 and 34 months were used. These bulls were purchased from a local farm in an apparently tsetse-free area of Kaduna State of Nigeria. On arrival they were individually accommodated in a fly proof experimental house and fed concentrates, groundnut hay, fresh pastures, salt licks and water ad-libitum. They were dewormed using albendazole1 (Pentax, Holland B.V.). The bulls were allowed to acclimatize for about 6 months during which they were examined regularly and exposed to routine handlings such as collection of blood samples twice a week for parasite screening and routine haematological analyses. Prior to commencement of the experiment, they were certified free of trypanosomes based on weekly haematocrit centrifuge tests (Woo, 1969). 2.2. Trypanosome stock The parasite, T. vivax used in the study was an isolate from a natural infection of cow in Kudaru village in
Northern Nigeria. The parasite was identified and confirmed to be T. vivax using morphological characteristics described by Soulsby (1968). Infective blood from the cow was first inoculated into a donor calf in which parasitaemia was detected 4 days after infection. 2.3. Animal allocation and infection On the day of infection (day 0), bulls were allocated to two groups of three infected and three control animals. These groups were closely matched on the basis of live mass (infected 198.0 9.4 kg, control 191.5 10.1 kg) and haematocrit index (infected 29.1 0.3%, control 28.4 0.8%). Bulls in the infected group were each injected intravenously with 2 ml of blood, from donor calf, containing approximately 2 106 trypanosomes. The number of trypanosomes was estimated using haemocytometer (Coulter Electronics, Hearts, England). Control bulls were uninfected. 2.4. Routine blood examination Starting from day 5 before infection, blood (3 ml) was obtained from each bull by jugular venepuncture in test tubes containing disodium salt of ethylene diaminetetraacetic acid as anticoagulant every other day until the day of infection for estimation of packed cell volume (PCV) using standard microcapillary method. After infection, jugular blood samples were taken daily from infected bulls and examined for the presence of trypanosomes using buffy coat-dark ground method described by Murray et al. (1977). Once parasitaemia was detected, blood samples were taken every other day for estimation of PCV and parasitaemia levels. Parasitaemia was estimated by the scoring method described by Parris et al. (1982) at 400 magnification. The experiment lasted for 56 days. 2.5. Histopathological studies Two bulls, each time, one from infected group and the other from control group were sacrificed at 14,28 and 56 days post-infection. At necropsy, samples were taken from the testes and epididymides of all the six bulls. The tissues taken were fixed in Bouin’s solution for 72 h, processed according to standard histopathological techniques and stained with haematoxylin and eosin (H and E). The presence and conditions of the germinal epithelia and sertoli cells were studied microscopically. The interstitial tissues were equally examined. Occurrence of lesions and their frequency were recorded.
S. Adamu et al. / Veterinary Parasitology 143 (2007) 29–34
31
Degrees of testicular degeneration were graded according to the method described by Sekoni et al. (1990). Sections of epididymides were similarly studied and lesions were graded according to the method described by Sekoni et al. (1990). 2.6. Estimation of epididymnal sperm reserve At least 50 cross sections of epididymal tubules were observed at 100 for determining spermatozoa content. The epididymal sperm reserve was then expressed as a percentage of number of tubules with normal sperm reserve out of the total examined. 2.7. Statistical analysis Values of PCV in both infected and control bulls were pooled together as pre- and post-infection means. Comparison between the post-infection means in the two groups of animals was made using Student’s t-test. 3. Results 3.1. Clinical observations Trypanosomes were identified in the blood of infected bulls within 4–5 days after infection. The first peak parasitaemia (mean score 6.0 0.1) was recorded 6 days after infection and the infected bulls remained parasitaemic, though intermittently, throughout the course of the experiment which lasted for 56 days. PCV values declined rapidly in infected bulls from the mean pre-infection value of 29.0 0.5% to 16.0 2.9% on day 20 post-infection. By day 56 post-infection, the pooled mean post-infection PCV (19.3 0.5%) in infected bulls was significantly lower (P < 0.05) than that in control bulls (27.3 1.5%). 3.2. Histopathological findings 3.2.1. Testes Control bulls had normal testes with seminiferous tubules containing full complement of spermatogenic cells, representing different phases in the development of spermatozoon (Fig. 1a). The interstitial tissues of the testes had full complement of Leydig cells and were normal. Varying degrees of testicular degeneration were however observed in the T. vivax-infected Zebu bulls. The testicular degenerations were generalized in nature; hence, a uniform distribution of affected tubules and Leydig cells.
Fig. 1. (a) Testis of a control Zebu bull showing normal seminiferous tubule containing full complement of spermatogenic cells (H and E, 200). (b) Epididymis of a control Zebu bull showing normal tubules with normal epididymal sperm reserve (H and E, 153).
The infected bull that was sacrificed 14 days after infection had moderate to severe testicular degeneration. In some tubules germinal epithelial layers were reduced to one or two due to depletion of spermatogenic cells (Fig. 2a, Table 1). There were focal areas of necrosis of Leydig cells in the interstitial tissue. The infected bull that was sacrificed 28 days after infection had severe testicular degeneration (Fig. 3a, Table 1). Germinal epithelial layers were reduced to one or two, with many sertoli types of cells. Some of Table 1 Grades of teticular damage in Trypanosoma vivax-infected Zebu bulls Bull no.
No. of STCS examined
No. of STCS affected
%STCS affected
Grades
1A 8A 6A
50 50 50
35 47 50
70 94 100
Severe Severe Severe
STCS: seminiferous tubule cross sections. 1A: T. vivax-infected Zebu bull that was sacrificed on day 14 post-infection, 8A: T. vivax-infected Zebu bull that was sacrificed on day 28 post-infection, 6A: T. vivaxinfected Zebu bull that was sacrificed on day 56 post-infection.
32
S. Adamu et al. / Veterinary Parasitology 143 (2007) 29–34
Fig. 2. (a) Testis of the Trypanosoma vivax-infected Zebu bull that was sacrificed 14 days post-infection showing depletion of spermatogenic cells and reduction in germinal epithelial layers of the seminiferous tubules(s) (H and E, 153). (b) Epididymis of the same bull showing reduction in epididymal sperm reserve (H and E, 200).
Fig. 3. (a) Testis of the T. vivax-infected Zebu bull that was sacrificed 28 days post-infection showing severe degeneration with germinal epithelial layers reduced to one in the seminiferous tubules(s) (H and E, 153). (b) Epididymis of the same bull showing absence of epididymal sperm reserve (H and E, 153).
the seminiferous tubules contained proteinaceous material representing remnant of necrotic cells. Most cellular structures within the interstitial tissue were depleted. The most severe testicular lesions were observed in the infected bull that was sacrificed 56 days after infection (Fig. 4a, Table 1). Seminiferous tubules here showed germinal hypoplasia with complete depletion of spermatogenic cells. Sertoli cells were also depleted. Most of the Leydig cells had undergone karyolysis and the few discernible ones were at various phases of necrosis.
(Fig. 2b, Table 2). Reduction in epididymal sperm reserve was however evident. The infected bull that was sacrificed 28 days after infection had moderate degeneration and 4% epididymal sperm reserve (Fig. 3b, Table 2). The most severe epididymal degeneration was observed in the infected bull that was sacrificed 56 days after infection. More than 60% of the epididymides were abnormal (Fig. 4b, Table 2). Some of the epididymal
3.2.2. Epididymides The epididymides and epididymal sperm reserve, which was 100%, were normal in the control Zebu bulls (Fig. 1b). However, various degrees of epididymal degeneration were observed in the infected bulls. The infected bull that was sacrificed 14 days after infection had mild epididymal damage, as most of the tubules were normal
Table 2 Epididymal sperm reserve in T. vivax-infected Zebu bulls Bull no.
No. of ETCS
No. of ETCS with normal ESR
%ESR
1A 8A 6A
50 50 50
18 2 0
36 4 0
ETCS: epididymal tubule cross sections. ESR: epididymal sperm reserve. 1A: T. vivax-infected Zebu bull that was sacrificed on day 14 post-infection, 8A: T. vivax-infected Zebu bull sacrificed on day 28 post-infection, 6A: T. vivax-infected Zebu bull sacrificed on day 56 post-infection.
S. Adamu et al. / Veterinary Parasitology 143 (2007) 29–34
Fig. 4. (a) Testis of the T. vivax-infected Zebu bull that was sacrificed 56 days post-infection showing very severe degeneration in the seminiferous tubules(s) with the only remaining basal germinal epithelial layer showing vacuolations (H and E, 153). (b) Epididymis of the same bull showing absence of epididymal sperm reserve in the tubules (H and E, 153).
lesions include focal areas of necrosis, squamous metaplasia of the epididymal epithelia with loss of stereocillia and presence of submucosal fibroplasias. Epididymal sperm reserve was 0%. 4. Discussion The findings in the present study have demonstrated that the devastating effects of T. vivax infection on male reproductive organs reported earlier by Isoun et al. (1975) and Sekoni et al. (1990) could occur early in the infection. Infected bulls in the present study had severe testicular degeneration by 28 days post-infection and by 56 days, epididymal sperm reserve was 0%, thus, suggesting complete cessation of spermatogenesis. The degenerative changes observed in the testes and epididymides of infected bulls are similar to those reported by Sekoni et al. (1990) who examined lesions that were present between 12 and 30 weeks post-infection, but did occur much earllier in the infection in the present study. The complete
33
depletion of spermatogenic cells by 56 days after infection and the consequent 0% epididymal sperm reserve may also seem to suggest that T. vivax infection of bulls could cause sterility not necessarily in long standing cases as reported by Isoun et al. (1975) and Sekoni et al. (1990). Decrease or cessation of gonadal sperm production due to impairment of spermatogenesis was most probably the cause of the decrease or absence of epididymal sperm reserves observed in the present study, as previously observed (Isoun and Anosa, 1974; Anosa and Isoun, 1980). Although semen characteristics have not been studied in the present study, it is highly probable that the degree of testicular damage observed by 14 days after infection could result in deterioration of semen characteristics comparable with that reported by Sekoni (1992) in T. vivax - infected rams 3 weeks after infection. It may be reasonable to say that the degree of testicular and epididymal damage observed 56 days after infection in the present study may be difficult to reverse. This is because chemotherapy has been reported to be of little or no value when genital lesions are very severe (Akpavie et al., 1987; Grundler et al., 1988; Sekoni, 1994). The pathophysiological mechanisms involved in the trypanosome-induced testicular dysfunction are not quite understood. The possibility that pituitary damage during trypanosomosis may result in testicular dysfunction as a consequence of disruption of LH and FSH secretion (Apted, 1970; Ikede and Losos, 1975) has been disproved by reports of Boly et al. (1994) and Mutayoba et al. (1994) who reported that pituitary function, as observed by the measurement of LH response to GnRH injection, was not affected. However, destruction of Leydig cells, as observed in the present study, could result in decline in testicular steroidogenesis. This would consequently aggravate the degenerative changes in the seminiferous tubules with subsequent depletion of spermatogenic cells, since spermatogenesis depends on normal androgen secretion (Setchell, 1978; Cameron et al., 1993). Many pathophysiological mechanisms might have been involved in the development of testicular and epididymal damage in the T. vivax-infected Zebu bulls which include anoxia due to anaemia, pyrexia, capacity of trypanosomes to cause extensive and generalized tissue and organ damage, immunological factors, ability of trypanosomes to produce biologically active and toxic substances (Morrison et al., 1981; Esievo and Saror, 1991; Logan-Henfrey et al., 1992; Sileghem et al., 1993; Sekoni, 1987, 1994). In conclusion, this study has demonstrated that T. vivax infection of Zebu bulls could result in very severe testicular and epididymal damage that could result in infertility or even sterility of the affected animals at
34
S. Adamu et al. / Veterinary Parasitology 143 (2007) 29–34
early infection stages not previously thought. The information gotten from this study may therefore be of considerable economic importance in exploiting the use of bulls for breeding purposes in tsetse endemic areas. Acknowledgement The authors owe a lot of gratitude to the Board of Research of Ahmadu Bello University, Zaria, Nigeria for partly funding the research. References Akpavie, S.O., Ikede, B.O., Egbunike, G.N., 1987. Ejaculate characteristics of sheep infected with T. brucei and T. vivax: changes caused by treatment with diminazene aceturate. Res. Vet. Sci. 42, 1–6. Anosa, V.O., Isoun, T.T., 1980. Further observations on testicular pathology in T. vivax infection of sheep and goats. Res. Vet. Sci. 28, 151–160. Apted, P.I.C., 1970. Clinical manifestations and diagnosis of sleeping sickness. In: Mulligan, E.W., Potts, W.H. (Eds.), The African Trypanosomiasis. Allen and Unwin, London, pp. 661–683. Boly, H., Humblot, P., Tillet, Y., Thibier, M., 1994. Effects of Trypanosoma congolense infection on the pituitary gland of Baoule bulls: Immunohistochemistry of LH and FSH - secreting cells and response of plasma LH and testosterone to combined dexamethasone and GnRH treatment. J. Rep. Fer. 100, 157–162. Cameron, D.F., Muffy, K.E., Naziam, J., 1993. Testosterone stimulated spermatid binding to competent sertoli cells In vitro. End. J. 1, 61–65. Daniel, A.D., Dadah, A.J., Kalejaiye, J.O., Dalhatu, A.D., 1993. Prevalence of bovine trypanosomosis in Gongola State of Northern Nigeria. Rev. Med. Vet. Trop. 46 (4), 71–574. Esievo, K.A.N., Saror, D.I., 1991. Immunochemistry and immunopathology of animal trypanosomiasis. Vet. Bull. 61 (8), 765–777. Esuruoso, G.O., 1973. The epiziotiology,prevalence and economic aspects of bovine trypanosomiasis in Nigeria. In: Proceedings of a Meeting on US Animal Health Assistants, vol. 77. pp. 160–175. FAO, 2002. Food, Agriculture and Food Security: The Global Dimention, WFSO2/Tech/Advanced unedited version, FAO, Rome, pp 19-28. Geerts, S., Holmes, P.H., 1998. Drug management and parasite resistance in bovine trypanosomiasis in Africa. PAAT Techn. Scientific Ser. 1, 31. Grundler, G., Diabakou, K., Hanichen, T., Adomefa, K., 1988. Leisions testiculaires des bovines infestes avec T. congolense. Tryp. Prod. Anim. 5, 17–21. Ikede, B.O., Losos, G.J., 1975. Pathogenesis of T. brucei infection in sheep; hypophyseal and other endocrine lesions. J. Comp. Pathol. 85, 37–44. Ikede, B.O., Taiwo, V.O., 1985. Prevalence of bovine trypanosomiasis in sedentary Zebu and trypanotolerant breeds in South Western and NorthWestern Nigeria. In: International Scientific Council for
Trypanosomiasis Research and Control, 18th Meeting, Harare, Zimbabwe, Nairobi, OAU/STRC (Publication No. 113), p. 146. Irungu, P., Nyamwaro, S.O., Masiga, D.K., 2002. Financial implications of rearing sheep and goats under natural trypanosomosis challenge at Galana Ranch. Kenya Trop. Anim. Hlth. Prod. 34 (22), 503–513. Isoun, T.T., Anosa, V.O., 1974. Lesions in the reproductive organs of sheep and goats experimentally infected with T. vivax. Trop. Med. Parasitol. 25, 469–476. Isoun, T.T., Akpokodje, J.U., Anosa, V.O., 1975. Testicular changes in White Fulani (Bunaji) cattle experimentally infected with T. vivax: a preliminary report. Nig. Vet. J. 4, 107–108. Kristjanson, P.M., Swallow, B.M., Roulands, G.J., Kruska, R.L., de Leeuw, P.N., 1999. Measuring the cost of African animal trypanosomosis, the potential benefits of control and returns to research. Elsevier Agric. Sys. 59, 79–98. Logan-Henfrey, L.L., Gardiner, P.R., Mahmoud, M.M., 1992. In: Kreier, J.P., Baker, J.R. (Eds.), Parasitic Protozoa, 2. Sandiego Academic Press, pp. 157–275. Morrison, W.I., Murray, M., Sayer, P.D., Prestone, J.M., 1981. The pathogenesis of experimentally induced T. brucei infection in dog: Tissue and organ damage. Am. J. Pathol. 102, 168–181. Murray, M., Murray, P.K., McIntyre, W.I.M., 1977. An improved parasitological technique for the diagnosis of African trypanosomiasis. Trans. R. Soc. Trop. Med. Hyg. 71, 325–326. Mutayoba, B.M., Eckersall, P.D., Jeffcoate, I.A., Cestnik, V., Holmes, P.H., 1994. Effects of Trypanosoma congolense infection in rams on the pulsatile secretion of testosterone and response to injection of GnRH. J. Rep. Fer. 102, 425–431. Parris, J., Murray, M., McOdimba, F., 1982. A comparative evaluation of parasitological techniques currently available for the diagnosis of African trypanosomiasis in cattle. Acta Trop. 39, 307–316. Sekoni, V.O., 1987. Effects of Trypnosoma vivax and Trypanosoma congolense infections and season on reproduction in the bull, Ph.D Thesis, Ahmadu Bello Universitty, Zaria, Nigeria. Sekoni, V.O., 1992. Effect of Trypanosoma vivax on semen characteristics of Yankasa rams. Br. Vet. J. 148, 501–506. Sekoni, V.O., 1994. Reproductive disorders caused by animal trypanosomiasis: a review. Theriogenology 42, 557–570. Sekoni, V.O., Kumi-Diaka, J., Njoku, C.O., 1988. The effect of T. vivax and T. congolense infections on the reaction time and semen characteristics in Zebu bulls. Br. Vet. J. 144, 388–394. Sekoni, V.O., Njoku, C.O., Kumi-Diaka, J., Saror, D.I., 1990. Pathological changes in male genitalia of cattle infected with T. vivax and T. congolense. Br. Vet. J. 146, 175–180. Setchell, B.P., 1978. Endocrinology of testis. In: Setchell, B.P. (Ed.), The Mammalian Testis. Paul Elec, London, pp. 234–570. Sileghem, M.R., Flynn, S.R., William, D.J.Z., 1993. Secretion of costimulatory cytokines by monocytes and macrop[ages during infection with T. congolense in susceptible and trypanotolerant cattle. Vet. Immunol. Immunopathol. 37, 123–134. Soulsby, E.J.L. (Ed.), 1968. Helminths, Arthropods and Protozoa of Domesticated Animals. The Williams and Wilkins Company, Baltimore, USA, p. 809. Woo, P.T.K., 1969. The haematocrit centrifuge techniques for the detection of trypanosomes in blood. Can. J. Zool. 43, 921–923.
Sequential testicular and epididymal damage in Zebu bulls experimentally infected with Trypanosoma vivax S. Adamu a, M.Y. Fatihu a, N.M. Useh a,*, M. Mamman b, V.O. Sekoni c, K.A.N. Esievo a a
Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Nigeria b Department of Veterinary Physiology and Pharmacology, Ahmadu Bello University, Zaria, Nigeria c Department of Animal Reproduction, National Animal Production Research Institute, Shika, Nigeria Received 6 February 2006; received in revised form 18 July 2006; accepted 20 July 2006
Abstract Six Zebu bulls aged between 31 and 34 months exhibiting good libido were used to study sequential testicular and epididymal damage in Trypanosoma vivax infection. Three bulls were infected with T. vivax, while the other three served as controls. All infected bulls became parasitaemic by day 5 post-infection and developed clinical trypanosomosis with rapidly developing anaemia. Representative bulls, one from each of the infected and control groups, were sacrificed on days 14, 28 and 56 postinfection. Testes and epididymides from these animals were studied histopathologically after processing and staining with haematoxylin and eosin (H and E). Testicular degeneration developed in all the infected bulls characterized by depletion of spermatogenic cells and destruction of interstitial tissue. The most severe testicular degeneration occurred in the bull that was sacrificed 56 days post-infection. Epididymal sperm reserves were 36%, 4% and 0%, respectively, in infected bulls that were sacrificed on days 14, 28 and 56 post-infection. The 0% epididymal sperm reserve may suggest complete cessation of spermatogenesis. It was concluded from this study that T. vivax infection of Zebu bulls could cause severe testicular and epididymal damage that may result in infertility or even sterility of the affected animals at early infection stages not previously thought. # 2006 Elsevier B.V. All rights reserved. Keywords: Trypanosomosis; Bulls; Testes; Epididymides; Trypanosoma vivax; Histopathology
1. Introduction In spite of decades of investigations on animal trypanosomosis, the disease has continued to remain an important, if not most important constraint to livestock and mixed crop-livestock farming in tropical Africa (Kristjanson et al., 1999; Irungu et al., 2002). Over 45 million cattle and 60 million people live at risk of * Corresponding author. Tel.: +234 8037032523; fax: +234 69332412. E-mail address: [email protected] (N.M. Useh). 0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2006.07.022
getting infected with trypanosomes; while each year 3 million cattle and other domestic livestock are lost through deaths caused by the parasites (FAO, 2002). Three important pathogenic species of trypanosomes are responsible for the disease in ruminants; these are Trypanosoma vivax, T. congolense and T. brucei. In Nigeria, the incidence of animal trypanosomosis is on the increase. This is because for decades there has not been large-scale surveillance and control programme to curtail the menace of the disease (Esuruoso, 1973; Ikede and Taiwo, 1985; Daniel et al., 1993). Livestock owners are, thus, left with curative and preventive treatments as
30
S. Adamu et al. / Veterinary Parasitology 143 (2007) 29–34
the only means of controlling this highly devastating disease. African livestock producers administer an estimated 35 million curative and preventive treatments annually (Geerts and Holmes, 1998). This is therefore costing producers and government colossal amount of money (Kristjanson et al., 1999). It therefore becomes imperative to make re-assessment of the pathologies produced by trypanosomosis in animals with the view to establishing when, at the different stages of infection such pathologies become very severe as this could serve as a guide in knowing when intervention with treatment could be of little or no value. In bulls, for example, trypanosomosis has been shown to cause testicular and epididymal damage that could result in infertility and, probably, sterility (Isoun et al., 1975; Sekoni et al., 1988, 1990). Although some data are already available about the effects of chronic T. vivax infection (Sekoni et al., 1990), there is however dearth of information on the degree of testicular and epididymal damage at different stages of the acute infection. The objective of this study therefore was to sequentially follow the development testicular and epididymal damage in T. vivax infected Zebu bulls as the information that could be obtained from this study may be of value in the continued use of trypanosomeinfected bulls for breeding purposes. 2. Materials and methods 2.1. Experimental animals Six normal healthy Zebu bulls of White Fulani breed aged between 31 and 34 months were used. These bulls were purchased from a local farm in an apparently tsetse-free area of Kaduna State of Nigeria. On arrival they were individually accommodated in a fly proof experimental house and fed concentrates, groundnut hay, fresh pastures, salt licks and water ad-libitum. They were dewormed using albendazole1 (Pentax, Holland B.V.). The bulls were allowed to acclimatize for about 6 months during which they were examined regularly and exposed to routine handlings such as collection of blood samples twice a week for parasite screening and routine haematological analyses. Prior to commencement of the experiment, they were certified free of trypanosomes based on weekly haematocrit centrifuge tests (Woo, 1969). 2.2. Trypanosome stock The parasite, T. vivax used in the study was an isolate from a natural infection of cow in Kudaru village in
Northern Nigeria. The parasite was identified and confirmed to be T. vivax using morphological characteristics described by Soulsby (1968). Infective blood from the cow was first inoculated into a donor calf in which parasitaemia was detected 4 days after infection. 2.3. Animal allocation and infection On the day of infection (day 0), bulls were allocated to two groups of three infected and three control animals. These groups were closely matched on the basis of live mass (infected 198.0 9.4 kg, control 191.5 10.1 kg) and haematocrit index (infected 29.1 0.3%, control 28.4 0.8%). Bulls in the infected group were each injected intravenously with 2 ml of blood, from donor calf, containing approximately 2 106 trypanosomes. The number of trypanosomes was estimated using haemocytometer (Coulter Electronics, Hearts, England). Control bulls were uninfected. 2.4. Routine blood examination Starting from day 5 before infection, blood (3 ml) was obtained from each bull by jugular venepuncture in test tubes containing disodium salt of ethylene diaminetetraacetic acid as anticoagulant every other day until the day of infection for estimation of packed cell volume (PCV) using standard microcapillary method. After infection, jugular blood samples were taken daily from infected bulls and examined for the presence of trypanosomes using buffy coat-dark ground method described by Murray et al. (1977). Once parasitaemia was detected, blood samples were taken every other day for estimation of PCV and parasitaemia levels. Parasitaemia was estimated by the scoring method described by Parris et al. (1982) at 400 magnification. The experiment lasted for 56 days. 2.5. Histopathological studies Two bulls, each time, one from infected group and the other from control group were sacrificed at 14,28 and 56 days post-infection. At necropsy, samples were taken from the testes and epididymides of all the six bulls. The tissues taken were fixed in Bouin’s solution for 72 h, processed according to standard histopathological techniques and stained with haematoxylin and eosin (H and E). The presence and conditions of the germinal epithelia and sertoli cells were studied microscopically. The interstitial tissues were equally examined. Occurrence of lesions and their frequency were recorded.
S. Adamu et al. / Veterinary Parasitology 143 (2007) 29–34
31
Degrees of testicular degeneration were graded according to the method described by Sekoni et al. (1990). Sections of epididymides were similarly studied and lesions were graded according to the method described by Sekoni et al. (1990). 2.6. Estimation of epididymnal sperm reserve At least 50 cross sections of epididymal tubules were observed at 100 for determining spermatozoa content. The epididymal sperm reserve was then expressed as a percentage of number of tubules with normal sperm reserve out of the total examined. 2.7. Statistical analysis Values of PCV in both infected and control bulls were pooled together as pre- and post-infection means. Comparison between the post-infection means in the two groups of animals was made using Student’s t-test. 3. Results 3.1. Clinical observations Trypanosomes were identified in the blood of infected bulls within 4–5 days after infection. The first peak parasitaemia (mean score 6.0 0.1) was recorded 6 days after infection and the infected bulls remained parasitaemic, though intermittently, throughout the course of the experiment which lasted for 56 days. PCV values declined rapidly in infected bulls from the mean pre-infection value of 29.0 0.5% to 16.0 2.9% on day 20 post-infection. By day 56 post-infection, the pooled mean post-infection PCV (19.3 0.5%) in infected bulls was significantly lower (P < 0.05) than that in control bulls (27.3 1.5%). 3.2. Histopathological findings 3.2.1. Testes Control bulls had normal testes with seminiferous tubules containing full complement of spermatogenic cells, representing different phases in the development of spermatozoon (Fig. 1a). The interstitial tissues of the testes had full complement of Leydig cells and were normal. Varying degrees of testicular degeneration were however observed in the T. vivax-infected Zebu bulls. The testicular degenerations were generalized in nature; hence, a uniform distribution of affected tubules and Leydig cells.
Fig. 1. (a) Testis of a control Zebu bull showing normal seminiferous tubule containing full complement of spermatogenic cells (H and E, 200). (b) Epididymis of a control Zebu bull showing normal tubules with normal epididymal sperm reserve (H and E, 153).
The infected bull that was sacrificed 14 days after infection had moderate to severe testicular degeneration. In some tubules germinal epithelial layers were reduced to one or two due to depletion of spermatogenic cells (Fig. 2a, Table 1). There were focal areas of necrosis of Leydig cells in the interstitial tissue. The infected bull that was sacrificed 28 days after infection had severe testicular degeneration (Fig. 3a, Table 1). Germinal epithelial layers were reduced to one or two, with many sertoli types of cells. Some of Table 1 Grades of teticular damage in Trypanosoma vivax-infected Zebu bulls Bull no.
No. of STCS examined
No. of STCS affected
%STCS affected
Grades
1A 8A 6A
50 50 50
35 47 50
70 94 100
Severe Severe Severe
STCS: seminiferous tubule cross sections. 1A: T. vivax-infected Zebu bull that was sacrificed on day 14 post-infection, 8A: T. vivax-infected Zebu bull that was sacrificed on day 28 post-infection, 6A: T. vivaxinfected Zebu bull that was sacrificed on day 56 post-infection.
32
S. Adamu et al. / Veterinary Parasitology 143 (2007) 29–34
Fig. 2. (a) Testis of the Trypanosoma vivax-infected Zebu bull that was sacrificed 14 days post-infection showing depletion of spermatogenic cells and reduction in germinal epithelial layers of the seminiferous tubules(s) (H and E, 153). (b) Epididymis of the same bull showing reduction in epididymal sperm reserve (H and E, 200).
Fig. 3. (a) Testis of the T. vivax-infected Zebu bull that was sacrificed 28 days post-infection showing severe degeneration with germinal epithelial layers reduced to one in the seminiferous tubules(s) (H and E, 153). (b) Epididymis of the same bull showing absence of epididymal sperm reserve (H and E, 153).
the seminiferous tubules contained proteinaceous material representing remnant of necrotic cells. Most cellular structures within the interstitial tissue were depleted. The most severe testicular lesions were observed in the infected bull that was sacrificed 56 days after infection (Fig. 4a, Table 1). Seminiferous tubules here showed germinal hypoplasia with complete depletion of spermatogenic cells. Sertoli cells were also depleted. Most of the Leydig cells had undergone karyolysis and the few discernible ones were at various phases of necrosis.
(Fig. 2b, Table 2). Reduction in epididymal sperm reserve was however evident. The infected bull that was sacrificed 28 days after infection had moderate degeneration and 4% epididymal sperm reserve (Fig. 3b, Table 2). The most severe epididymal degeneration was observed in the infected bull that was sacrificed 56 days after infection. More than 60% of the epididymides were abnormal (Fig. 4b, Table 2). Some of the epididymal
3.2.2. Epididymides The epididymides and epididymal sperm reserve, which was 100%, were normal in the control Zebu bulls (Fig. 1b). However, various degrees of epididymal degeneration were observed in the infected bulls. The infected bull that was sacrificed 14 days after infection had mild epididymal damage, as most of the tubules were normal
Table 2 Epididymal sperm reserve in T. vivax-infected Zebu bulls Bull no.
No. of ETCS
No. of ETCS with normal ESR
%ESR
1A 8A 6A
50 50 50
18 2 0
36 4 0
ETCS: epididymal tubule cross sections. ESR: epididymal sperm reserve. 1A: T. vivax-infected Zebu bull that was sacrificed on day 14 post-infection, 8A: T. vivax-infected Zebu bull sacrificed on day 28 post-infection, 6A: T. vivax-infected Zebu bull sacrificed on day 56 post-infection.
S. Adamu et al. / Veterinary Parasitology 143 (2007) 29–34
Fig. 4. (a) Testis of the T. vivax-infected Zebu bull that was sacrificed 56 days post-infection showing very severe degeneration in the seminiferous tubules(s) with the only remaining basal germinal epithelial layer showing vacuolations (H and E, 153). (b) Epididymis of the same bull showing absence of epididymal sperm reserve in the tubules (H and E, 153).
lesions include focal areas of necrosis, squamous metaplasia of the epididymal epithelia with loss of stereocillia and presence of submucosal fibroplasias. Epididymal sperm reserve was 0%. 4. Discussion The findings in the present study have demonstrated that the devastating effects of T. vivax infection on male reproductive organs reported earlier by Isoun et al. (1975) and Sekoni et al. (1990) could occur early in the infection. Infected bulls in the present study had severe testicular degeneration by 28 days post-infection and by 56 days, epididymal sperm reserve was 0%, thus, suggesting complete cessation of spermatogenesis. The degenerative changes observed in the testes and epididymides of infected bulls are similar to those reported by Sekoni et al. (1990) who examined lesions that were present between 12 and 30 weeks post-infection, but did occur much earllier in the infection in the present study. The complete
33
depletion of spermatogenic cells by 56 days after infection and the consequent 0% epididymal sperm reserve may also seem to suggest that T. vivax infection of bulls could cause sterility not necessarily in long standing cases as reported by Isoun et al. (1975) and Sekoni et al. (1990). Decrease or cessation of gonadal sperm production due to impairment of spermatogenesis was most probably the cause of the decrease or absence of epididymal sperm reserves observed in the present study, as previously observed (Isoun and Anosa, 1974; Anosa and Isoun, 1980). Although semen characteristics have not been studied in the present study, it is highly probable that the degree of testicular damage observed by 14 days after infection could result in deterioration of semen characteristics comparable with that reported by Sekoni (1992) in T. vivax - infected rams 3 weeks after infection. It may be reasonable to say that the degree of testicular and epididymal damage observed 56 days after infection in the present study may be difficult to reverse. This is because chemotherapy has been reported to be of little or no value when genital lesions are very severe (Akpavie et al., 1987; Grundler et al., 1988; Sekoni, 1994). The pathophysiological mechanisms involved in the trypanosome-induced testicular dysfunction are not quite understood. The possibility that pituitary damage during trypanosomosis may result in testicular dysfunction as a consequence of disruption of LH and FSH secretion (Apted, 1970; Ikede and Losos, 1975) has been disproved by reports of Boly et al. (1994) and Mutayoba et al. (1994) who reported that pituitary function, as observed by the measurement of LH response to GnRH injection, was not affected. However, destruction of Leydig cells, as observed in the present study, could result in decline in testicular steroidogenesis. This would consequently aggravate the degenerative changes in the seminiferous tubules with subsequent depletion of spermatogenic cells, since spermatogenesis depends on normal androgen secretion (Setchell, 1978; Cameron et al., 1993). Many pathophysiological mechanisms might have been involved in the development of testicular and epididymal damage in the T. vivax-infected Zebu bulls which include anoxia due to anaemia, pyrexia, capacity of trypanosomes to cause extensive and generalized tissue and organ damage, immunological factors, ability of trypanosomes to produce biologically active and toxic substances (Morrison et al., 1981; Esievo and Saror, 1991; Logan-Henfrey et al., 1992; Sileghem et al., 1993; Sekoni, 1987, 1994). In conclusion, this study has demonstrated that T. vivax infection of Zebu bulls could result in very severe testicular and epididymal damage that could result in infertility or even sterility of the affected animals at
34
S. Adamu et al. / Veterinary Parasitology 143 (2007) 29–34
early infection stages not previously thought. The information gotten from this study may therefore be of considerable economic importance in exploiting the use of bulls for breeding purposes in tsetse endemic areas. Acknowledgement The authors owe a lot of gratitude to the Board of Research of Ahmadu Bello University, Zaria, Nigeria for partly funding the research. References Akpavie, S.O., Ikede, B.O., Egbunike, G.N., 1987. Ejaculate characteristics of sheep infected with T. brucei and T. vivax: changes caused by treatment with diminazene aceturate. Res. Vet. Sci. 42, 1–6. Anosa, V.O., Isoun, T.T., 1980. Further observations on testicular pathology in T. vivax infection of sheep and goats. Res. Vet. Sci. 28, 151–160. Apted, P.I.C., 1970. Clinical manifestations and diagnosis of sleeping sickness. In: Mulligan, E.W., Potts, W.H. (Eds.), The African Trypanosomiasis. Allen and Unwin, London, pp. 661–683. Boly, H., Humblot, P., Tillet, Y., Thibier, M., 1994. Effects of Trypanosoma congolense infection on the pituitary gland of Baoule bulls: Immunohistochemistry of LH and FSH - secreting cells and response of plasma LH and testosterone to combined dexamethasone and GnRH treatment. J. Rep. Fer. 100, 157–162. Cameron, D.F., Muffy, K.E., Naziam, J., 1993. Testosterone stimulated spermatid binding to competent sertoli cells In vitro. End. J. 1, 61–65. Daniel, A.D., Dadah, A.J., Kalejaiye, J.O., Dalhatu, A.D., 1993. Prevalence of bovine trypanosomosis in Gongola State of Northern Nigeria. Rev. Med. Vet. Trop. 46 (4), 71–574. Esievo, K.A.N., Saror, D.I., 1991. Immunochemistry and immunopathology of animal trypanosomiasis. Vet. Bull. 61 (8), 765–777. Esuruoso, G.O., 1973. The epiziotiology,prevalence and economic aspects of bovine trypanosomiasis in Nigeria. In: Proceedings of a Meeting on US Animal Health Assistants, vol. 77. pp. 160–175. FAO, 2002. Food, Agriculture and Food Security: The Global Dimention, WFSO2/Tech/Advanced unedited version, FAO, Rome, pp 19-28. Geerts, S., Holmes, P.H., 1998. Drug management and parasite resistance in bovine trypanosomiasis in Africa. PAAT Techn. Scientific Ser. 1, 31. Grundler, G., Diabakou, K., Hanichen, T., Adomefa, K., 1988. Leisions testiculaires des bovines infestes avec T. congolense. Tryp. Prod. Anim. 5, 17–21. Ikede, B.O., Losos, G.J., 1975. Pathogenesis of T. brucei infection in sheep; hypophyseal and other endocrine lesions. J. Comp. Pathol. 85, 37–44. Ikede, B.O., Taiwo, V.O., 1985. Prevalence of bovine trypanosomiasis in sedentary Zebu and trypanotolerant breeds in South Western and NorthWestern Nigeria. In: International Scientific Council for
Trypanosomiasis Research and Control, 18th Meeting, Harare, Zimbabwe, Nairobi, OAU/STRC (Publication No. 113), p. 146. Irungu, P., Nyamwaro, S.O., Masiga, D.K., 2002. Financial implications of rearing sheep and goats under natural trypanosomosis challenge at Galana Ranch. Kenya Trop. Anim. Hlth. Prod. 34 (22), 503–513. Isoun, T.T., Anosa, V.O., 1974. Lesions in the reproductive organs of sheep and goats experimentally infected with T. vivax. Trop. Med. Parasitol. 25, 469–476. Isoun, T.T., Akpokodje, J.U., Anosa, V.O., 1975. Testicular changes in White Fulani (Bunaji) cattle experimentally infected with T. vivax: a preliminary report. Nig. Vet. J. 4, 107–108. Kristjanson, P.M., Swallow, B.M., Roulands, G.J., Kruska, R.L., de Leeuw, P.N., 1999. Measuring the cost of African animal trypanosomosis, the potential benefits of control and returns to research. Elsevier Agric. Sys. 59, 79–98. Logan-Henfrey, L.L., Gardiner, P.R., Mahmoud, M.M., 1992. In: Kreier, J.P., Baker, J.R. (Eds.), Parasitic Protozoa, 2. Sandiego Academic Press, pp. 157–275. Morrison, W.I., Murray, M., Sayer, P.D., Prestone, J.M., 1981. The pathogenesis of experimentally induced T. brucei infection in dog: Tissue and organ damage. Am. J. Pathol. 102, 168–181. Murray, M., Murray, P.K., McIntyre, W.I.M., 1977. An improved parasitological technique for the diagnosis of African trypanosomiasis. Trans. R. Soc. Trop. Med. Hyg. 71, 325–326. Mutayoba, B.M., Eckersall, P.D., Jeffcoate, I.A., Cestnik, V., Holmes, P.H., 1994. Effects of Trypanosoma congolense infection in rams on the pulsatile secretion of testosterone and response to injection of GnRH. J. Rep. Fer. 102, 425–431. Parris, J., Murray, M., McOdimba, F., 1982. A comparative evaluation of parasitological techniques currently available for the diagnosis of African trypanosomiasis in cattle. Acta Trop. 39, 307–316. Sekoni, V.O., 1987. Effects of Trypnosoma vivax and Trypanosoma congolense infections and season on reproduction in the bull, Ph.D Thesis, Ahmadu Bello Universitty, Zaria, Nigeria. Sekoni, V.O., 1992. Effect of Trypanosoma vivax on semen characteristics of Yankasa rams. Br. Vet. J. 148, 501–506. Sekoni, V.O., 1994. Reproductive disorders caused by animal trypanosomiasis: a review. Theriogenology 42, 557–570. Sekoni, V.O., Kumi-Diaka, J., Njoku, C.O., 1988. The effect of T. vivax and T. congolense infections on the reaction time and semen characteristics in Zebu bulls. Br. Vet. J. 144, 388–394. Sekoni, V.O., Njoku, C.O., Kumi-Diaka, J., Saror, D.I., 1990. Pathological changes in male genitalia of cattle infected with T. vivax and T. congolense. Br. Vet. J. 146, 175–180. Setchell, B.P., 1978. Endocrinology of testis. In: Setchell, B.P. (Ed.), The Mammalian Testis. Paul Elec, London, pp. 234–570. Sileghem, M.R., Flynn, S.R., William, D.J.Z., 1993. Secretion of costimulatory cytokines by monocytes and macrop[ages during infection with T. congolense in susceptible and trypanotolerant cattle. Vet. Immunol. Immunopathol. 37, 123–134. Soulsby, E.J.L. (Ed.), 1968. Helminths, Arthropods and Protozoa of Domesticated Animals. The Williams and Wilkins Company, Baltimore, USA, p. 809. Woo, P.T.K., 1969. The haematocrit centrifuge techniques for the detection of trypanosomes in blood. Can. J. Zool. 43, 921–923.