Refinement of techniques...

Acta Tropica 97 (2006) 364–369

Refinement of techniques for the propagation of Leishmania donovani in hamsters Susan Wyllie, Alan H. Fairlamb ∗ Division of Biological Chemistry and Molecular Microbiology, Wellcome Trust Biocentre, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK Received 1 October 2005; received in revised form 5 January 2006; accepted 9 January 2006 Available online 7 February 2006

Abstract Improved animal models are urgently required for drug and vaccine development against visceral leishmaniasis. Here we report refinements to the hamster model of infection that reduce the severity of the disease as well as the number of animals required to maintain infection while improving parasite yields. A comparison between infection via the intracardiac and intraperitoneal routes showed that the less commonly used intraperitoneal route is the simpler and preferred method. The KAtex latex agglutination test for visceral leishmaniasis accurately detected Leishmania donovani antigen in hamster urine as early as 6 weeks post-inoculation. With modification, this assay could be an important tool in the evaluation of experimental drugs and vaccines. © 2006 Elsevier B.V. All rights reserved. Keywords: Leishmania donovani; Animal model; Hamster; Intraperitoneal; KAtex

1. Introduction The intracellular protozoan parasite Leishmania donovani is the causative agent of the visceral form of leishmaniasis, commonly known as kala azar. This progressive infection, invariably fatal if untreated, is characterized by long-term fever, hepatosplenomegaly, anaemia, leukopenia and severe weight loss. The World Health Organisation reports over 12 million cases of all forms of leishmaniasis worldwide with 500,000 new infections each year resulting in an annual death toll of 60,000 (WHO, 1999). Despite the recent advances in understanding the biochemistry and molecular biology of Leishmania species, chemotherapeutic treatment of

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Corresponding author. Tel.: +44 1382 38 5155; fax: +44 1382 34 5542. E-mail address: [email protected] (A.H. Fairlamb). 0001-706X/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2006.01.004

visceral leishmaniasis remains problematic. Pentavalent antimonial preparations, such as sodium stibogluconate (Pentostam), are the mainstay of treatment for the visceral disease despite several harmful side effects and the emergence of significant levels of drug resistance (Sundar et al., 2000; Croft et al., 2006). Identifying novel drug targets and more effective chemotherapies has now become a matter of urgency and has resulted in a proliferation of anti-Leishmania drug development and screening studies. Undoubtedly, drug development programs are facilitated by the ability to reproduce infection in an appropriate animal model. To date, the models of choice for visceral leishmaniasis have been the mouse and hamster, with L. donovani infection of BALB/c mice being the most widely studied. Mice are known to be either genetically susceptible or resistant to L. donovani. However, even susceptible strains can control and clear the infection through their immnune responses (Murray et al.,

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1987). Consequently, mouse models are far from ideal in studying the progressive and disseminating visceral infection most commonly seen in the human disease. Perhaps the most appropriate model to study progressive visceral leishmaniasis is in the hamster where L. donovani infection leads to heptosplenomegaly, hypoalbuminemia and pancytopenia closely mimicking the human disease (Melby, 2002). Another advantage of the hamster model is the sheer number of splenic parasites produced which can be used in subsequent biochemical or pathological studies. However, the hamster is by no means the perfect animal model. Firstly, due to their comparatively high body mass, they require larger quantities of drugs than mice in drug efficacy tests. Secondly, there is no obvious way to monitor progressive visceral leishmaniasis in hamsters until the very late stages of infection when the most notable feature is a significant weight loss. Thirdly, since there is no intravenous route with which to infect hamsters, inoculation is most commonly achieved via the intracardiac route which is both technically difficult and highly hazardous for the animal. With this in mind, in this study we investigate ways to optimize and improve the current L. donovani hamster model (Croft and Yardley, 1999). In addition, we look for ways to monitor infection prior to termination of the animal which might be of future use in expediting drug discovery. 2. Materials and methods 2.1. Animals Commercially bred, male Golden hamsters (Mesocricetus auratus, Harlan Laboratories, Germany) weighing 40–60 g were used in this study. They were housed in temperature-controlled accommodation, fed with standard rodent dried food and provided with water ad libitum. Nesting materials and activity toys were also provided in each cage. 2.2. L. donovani infection in hamsters Hamsters were inoculated, under halothane anaesthesia, with hamster spleen-derived L. donovani (LV9 strain; WHO designation: MHOM/ET/67/HU3) amastigotes. Infection was achieved by either intracardial inoculation with 2 × 108 amastigotes (0.2 ml inoculum per animal) or by intraperitoneal injection with 2 × 108 amastigotes (1.0 ml inoculum per animal). The weight of each animal was closely monitored following infection and the end-point of the infection was judged to be a weight loss of either 25% or 10% of the animal’s maximum weight, as described. In cases where no

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apparent weight loss accompanied infection, hamsters were culled 165 days post-infection by halothane anaesthesia followed by cervical dislocation. The spleens of infected animals were aseptically removed, weighed and homogenized in Dulbecco’s modified essential medium (D-MEM, Sigma) containing 10% (v/v) foetal calf serum, 20 mM l-glutamine and 10 mM sodium pyruvate. The homogenate was then centrifuged at 100 × g for 5 min at 4 ◦ C to remove large cell debris, supernatant collected and centrifuged at 2000 × g for a further 10 min at 4 ◦ C. The resulting pellet was resuspended in culture medium containing 0.05% (w/v) saponin and incubated at room temperature for 5 min. Following centrifugation (2000 × g, 10 min, 4 ◦ C), the amastigote-enriched pellet was washed twice in fresh culture medium before being resuspended in a final volume of 2 ml. The amastigote suspension was passed through a 23-G needle several times to disperse clumps prior to counting. Amastigotes were subsequently used in studies on the mode of action of antimonial drugs (Wyllie et al., 2004). 2.3. Latex agglutination test for the diagnosis of visceral leishmaniasis Urine was collected from both infected and uninfected hamsters over a 12 h period using a model MTB0210 metabolic cage (Nalgene). The individual urine samples were then analyzed using the KAtex latex agglutination test for the diagnosis of visceral leishmaniasis (Kalon Biological Ltd., Aldershot, UK) as per manufacturer’s instructions with minor modifications. Briefly, 0.25 ml of each hamster urine sample was transferred into a sterile sample tube and boiled for 5 min. After allowing samples to cool to an ambient temperature, the treated urine was diluted 1:10 in phosphate buffered saline. Fifty microlitres of the diluted urine was then added to one drop of the test latex on a glass slide and mixed thoroughly by continuous rotation of the slide. After 2 min, the degree of agglutination was assessed as compared to positive and negative reference samples provided in the kit. 2.4. Cryostorage of L. donovani amastigotes Freshly harvested spleen-derived L. donovani amastigotes were resuspended at a final concentration of 2 × 108 parasites/ml in D-MEM containing 10% (v/v) glycerol prior to long-term storage in liquid nitrogen. Each inoculum was then removed from storage, rapidly thawed at 37 ◦ C and used directly to inoculate a juvenile hamster via the IP route.

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Table 1 Comparison of intracardiac and intraperitoneal routes of L. donovani inoculation in hamsters Route (n)

Time (days)

Weight loss (g)

Spleen weight (g)

Amastigotes (×1010 /spleen)

Amastigotes (×1010 /g wet weight spleen)

IP (11)a IC (9)b

139 ± 30 117 ± 32

18.3 ± 11.1 20.5 ± 3.8

1.63 ± 0.1c 1.56 ± 0.1c

3.1 ± 1.0 2.3 ± 0.85

1.90 ± 0.86 1.50 ± 0.76

All values are means and standard deviations. a One hamster lost due to ascites. b Two hamsters lost during infection and one hamster lost due to ascites. c The average spleen weight of five uninfected control hamsters was 0.15 ± 0.02 g.

3. Results 3.1. Comparison of intracardiac and intraperitoneal routes of L. donovani inoculation in hamsters The intracardiac (IC) route of infecting hamsters with L. donovani parasites, first demonstated by Stauber et al. (1958), has become the preferred method for many researchers since it is generally believed to result in a more rapid and productive infection (Croft and Yardley, 1999). However, this procedure requires a high degree of technical skill and can be extremely hazardous for the infected animal due to cardiac arrest, cardiac tamponade or haemorrhage. In order to directly assess the advantage of the IC route of L. donovani infection, two groups of hamsters were infected either by IC or by the less commonly used intraperitoneal (IP) route and several parameters of infection were monitored (summarized in Table 1). Underlining the technical difficulties associated with IC, two hamsters were lost immediately following the infection procedure. Post-mortem analysis revealed the deaths were due to haemorrhaging into the thoracic cavity. In addition, one hamster from each infected group developed ascites mid-way through the infection. These animals were culled and not included in the correlated data detailed in Table 1. The severity limit for the termination of the experiment was initially set as the loss of not more than 25% of the animal’s maximum body weight. Hamsters inoculated via the IC route developed a more rapid infection, reaching the end-point, on average, 22 days quicker than animals infected via the IP route. Infected spleens were then recovered from each hamster, weighed and the number of Leishmania amastigotes per spleen determined. Notably, the spleens of IP-infected hamsters had an average wet weight higher than those of the IC-infected animals. This increased wet weight correlated directly with a marked increase in the yield of amastigotes per gram of spleen, 1.9 × 1010 amastigotes in IP-hamsters as compared to 1.5 × 1010 amastigotes in IC-hamsters.

Splenomegaly is a characteristic of visceral leishmaniasis and the spleens of infected hamsters were on average 10 times heavier than those of uninfected control hamsters (0.15 g). In a small minority of animals, L. donovani infection was asymptomatic and not accompanied by severe weight loss. In these cases, found in both IC and IP groups, the end-point of the infection was set at 165 days post-inoculation. A notable feature of these asymptomatic infections was the increased numbers of parasites recovered from the spleens of these hamsters when compared to those showing profound weight loss (Fig. 1). Indeed, total parasite yield was found to be inversely proportional to hamster weight loss with a negative correlation coefficient of 0.81.

Fig. 1. Analysis of L. donovani parasite yield against total hamster weight loss. Two groups of juvenile Golden hamsters were inoculated with 2 × 108 spleen-derived L. donovani amastigotes either via an intraperitoneal or intracardiac route of infection, as detailed in Section 2. Infections were terminated following a maximum 25% total weight loss, or in cases where severe weight loss was not apparent, 165 days post-inoculation. The total final weight loss of each animal was plotted against the number of parasites recovered from their spleens. Intraperitoneal inoculated hamsters; closed circles, intracardiac inoculated hamster; closed circles. The line shows a linear regression fitted to these data.

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Table 2 Analysis of L. donovani infection in hamsters following a 10% symptomatic weight loss Hamster (n)

Time (days)

Weight loss (g)

Spleen weight (g)

Amastigotes (×1010 /spleen)

Amastigotes (×1010 /g wet weight spleen)

5

98 ± 8

12.4 ± 0.4

1.69 ± 0.35

3.41 ± 0.50

2.02 ± 0.68

Animals were infected on day 0 by intraperitoneal inoculation with

2 × 108

3.2. Analysis of maximal 10% hamster weight loss Having noted that severe weight loss is not conducive to the maximum yield of amastigotes from hamsters, infection was monitored in an IP-infected group where the end-point was moderated to a 10% loss in body weight (Table 2). In comparison to the 25% weight loss groups, considerably higher yields of amastigotes were isolated from these hamster spleens. Specifically, the amastigote yield/spleen improved marginally from an average of 3.1 × 1010 amastigotes in the 25% group to 3.4 × 1010 in the 10% group. The stress and impact of infection on individual hamsters was greatly reduced in the 10% group with the average total weight loss reduced from 20.5 to 12.4 g. In addition, the total time of infection was reduced from an average of 139 days (IP) in the 25% group to 98 days in the 10% group. The absence of clinical signs of visceral leishmaniasis in hamsters is problematic for researchers. Infections often last in excess of 120 days often with no outward or measurable signs of a successful infection evident until 80–100 days post-inoculation. In this study, we directly compared the weight profiles of infected and

amastigotes.

uninfected hamsters for 110 days, or until a 10% weight loss, to monitor for any early definitive signs of infection (Fig. 2). The average starting weights of the uninfected and infected hamster groups were nearly identical at 50 and 52 g, respectively. The infected animals gained weight at a markedly slower rate than their uninfected counterparts, reaching 100 g in an average of 41 days compared with 31.2 days for uninfected hamsters. While uninfected animals gained weight steadily throughout the 110 days of the study, the weight of the infected hamsters began to plateau at approximately 50 days postinoculation and decrease thereafter. 3.3. Analysis of the KAtex agglutination test in measuring levels of L. donovani infection in hamster urine In an attempt to accurately measure levels of parasitaemia throughout a visceral leishmaniasis infection in hamsters, freshly voided urine was collected from three IP-inoculated animals at 3-week intervals and tested for L. donovani antigen using the KAtex agglutination test. This assay has been successfully used to diag-

Fig. 2. Comparative weight profiles of L. donovani-infected and uninfected hamsters. Five juvenile hamsters were inoculated with 2 × 108 spleenderived L. donovani amastigotes via the intraperitoneal route. The weights of each hamster were monitored until the end-point of infection, determined to be a maximum of 10% weight loss. Weight profiles for each animal were plotted over time (A). In addition, the weight profiles of an uninfected control group of hamsters were also plotted against time (B).

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Table 3 Analysis of L. donovani infection in hamsters using the KAtex latex agglutination for visceral leishmaniasis Hamster

1 2 3

Amastigote yield (×1010 /spleen)

Latex agglutination 0 days post-infection

50 days post-infection

90 days post-infection

− − −

++ + +

+++ ++ +++

3.6 1.4 3.2

(−) no agglutination compared to the negative control; (+) agglutination can just be discerned when compared to the negative control; (++) agglutination can clearly be discerned; (+++) greater agglutination evident than in the positive control.

nose infection in dogs, humans and cotton rats (Attar et al., 2001; Sarkari et al., 2002), but has not previously been tested against hamsters. In each case, neat urine from the infected hamsters tested negative for agglutination in the KAtex test. However, following dilution in phosphate buffered saline (PBS) (1:10 dilution), urine samples were re-tested and found to be strongly positive for visceral leishmaniasis infection (Table 3). PBS alone had no effect on agglutination. The degree of agglutination in each sample appeared to be directly related to the levels of L. donovani infection in each hamster. Urine collected from hamster 1, found to be heavily infected with L. donovani post-mortem, tested strongly positive for agglutination on days 50 and 90 post-infection. In contrast, hamster 3 which yielded significantly fewer splenic amastigotes, tested only mildly positive. Infections could be detected in hamsters as early as 6 weeks post-inoculation. Urine from an uninfected control hamster was collected in an identical manner to the infected animals and found to be negative by KAtex agglutination over a period of 100 days (data not shown). 3.4. Analysis of the virulence of L. donovani amastigotes in hamsters following prolonged cryostorage In order to reduce the number of animals required to continuously maintain L. donovani infections, we investigated whether amastigotes could retain virulence following a prolonged period of cryostorage. Spleenderived amastigotes, cryopreserved in DMEM containing 10% glycerol, were stored in liquid nitrogen for 6 weeks. Following thawing, these amastigotes were used to inoculate three hamsters and several parameters of infection were monitored. Infection in these hamster progressed in an identical manner to that previously seen in animals inoculated with fresh amastigotes. The average length of infection (98 ± 6 days) and the final amastigote yield (3.3 (±0.2) × 1010 /spleen) for these hamsters compared extremely well with the “freshly” inoculated group.

4. Discussion It is the responsibility of every scientist involved in animal studies to constantly investigate ways of improving and refining current animal methodology. In cases where the use of an animal model is unavoidable, every effort must be made to minimize the number of animals needed and to use the most appropriate techniques. With this in mind, in this study we have directly compared the intracardiac and intraperitoneal routes of infecting hamsters with L. donovani as a model for visceral leishmaniasis. Surprisingly, we found that in almost every parameter of infection measured, the less commonly used IP route of inoculating hamsters was at least as effective as the IC route. Although IC infection resulted in a marginally quicker infection, IP-infected hamsters yielded on average higher numbers of Leishmania amastigotes per spleen. Hamsters infected by both methods were equally susceptible to developing ascites (∼10% incidence). Most significantly, the loss of two animals during intracardiac inoculation underlined the technical difficulties associated with procedure. In light of the data presented in this study, it is apparent that the severe risks associated with IC infection outweigh any perceived benefits of this method. Indeed, in studies where parasite yield is the primary concern the IP route of inoculating hamsters should now be considered as the method of choice. Weight loss is virtually the only measurable indicator of infection in live hamsters and is often used to determine the end-point of infection. In this study, we have noted that severe weight loss, in the region of 25% of the hamster’s body weight, is not conducive to the maximum yield of Leishmania parasites per spleen. In experiments where the end-point was determined to be 10% body weight loss, amastigote yields were on average 10% higher than in animals suffering a more severe weight loss. This refined protocol has several inherent advantages. Not only are parasite yields per animal greatly improved but the time required to reach the endpoint of infection is greatly reduced. Importantly, the

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impact of infection on the animal itself, as determined by length of infection and severity of symptoms, is greatly reduced. In all studies this should be a primary concern of researchers. Careful monitoring of hamster weights proved to be a good indicator of infection in the early stages of visceral leishmaniasis. Uninfected juvenile hamsters were found to gain weight significantly faster than their infected counterparts. In addition, the weights of infected hamsters were found to plateau mid-way through the 100+ day infection. In future experiments, it may be possible to use the comparative weight of uninfected control hamsters to confirm infection in the early stages. However, a more convenient method of monitoring infection may involve the use of the KAtex agglutination test (Attar et al., 2001), developed to diagnose visceral leishmaniasis in dogs, but also shown to identify infections in cotton rats and humans. This assay, based upon the detection of a structurally uncharacterized low molecular weight glycoconjugate in urine (Sarkari et al., 2002), proved effective in the diagnosis of L. donovani infection hamsters. Interestingly, hamster urine only tested positive in the KAtex test following dilution in PBS. This observation may be explained by the concentrated nature and high osmolality (3000 mOsmol) of hamster urine (Minsky and Chlapowski, 1978). Golden hamsters are desert dwelling animals known to concentrate their urine as a mechanism of avoiding dehydration. In doing so they are likely to significantly concentrate any antigen present in the urine. Vast excesses of antigen can result in apparent negative agglutination tests (Lim and Choy, 1988), explaining the requirement for urine dilution in this study. Further work is required in this area. However, our preliminary studies suggest that this assay could be an important tool in monitoring of infection in live animals, for parasite production, for in vitro studies and in the evaluation of experimental drugs and vaccines. In conclusion, our findings demonstrate that IP inoculation of hamster with L. donovani amastigotes has several clear advantages over the more frequently used IC route and should perhaps be considered as the method of choice in future studies. We have demonstrated that an ideal end-point of infection is a moderate 10% symptomatic weight loss in animals rather than the more commonly used 25% weight loss. In addition, we have modified the KAtex agglutination test to allow detection of L. donovani infection in hamsters. This data has lead to a clear refinement in the methods used to establish visceral leishmaniasis in hamsters and will lead to

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a reduction in the number of animals required for future studies. Acknowledgements We would like to thank the staff of the Wellcome Trust Resource Unit for their assistance. In addition, we would also like to thank Dr. Luke Newman for helpful discussion and the UK Home Office inspector for suggesting publication of this study. This work was funded by a grant from the Burroughs Wellcome Fund and Wellcome Trust Infectious Disease Initiative. References Attar, Z.J., Chance, M.L., el-Safi, S., Carney, J., Azazy, A., El-Hadi, M., Dourado, C., Hommel, M., 2001. Latex agglutination test for the detection of urinary antigens in visceral leishmaniasis. Acta Trop. 78, 11–16. Croft, S.L., Yardley, V., 1999. Animal models of visceral leishmaniasis. In: Zak, O., Sande, M.A. (Eds.), Handbook of Animal Models of Infection. Academic Press, London, pp. 783–787. Croft, S.L., Sundar, S., Fairlamb, A.H., 2006. Drug resistance in Leishmaniasis. Clin. Microbiol. Rev. 19, 111–126. Lim, P.L., Choy, W.F., 1988. A spectrophotometric method for evaluating a latex agglutination assay of Salmonella typhi lipopolysaccharide. J. Immunol. Methods 115, 269–274. Melby, P.C., 2002. Recent developments in leishmaniasis. Curr. Opin. Infect. Dis. 15, 485–490. Minsky, B.D., Chlapowski, F.J., 1978. Morphometric analysis of the translocation of lumenal membrane between cytoplasm and cell surface of transitional epithelial cells during the expansion–contraction cycles of mammalian urinary bladder. J. Cell Biol. 77, 685–697. Murray, H.W., Stern, J.J., Welte, K., Rubin, B.Y., Carriero, S.M., Nathan, C.F., 1987. Experimental visceral leishmaniasis: production of interleukin 2 and interferon-gamma, tissue immune reaction, and response to treatment with interleukin 2 and interferongamma. J. Immunol. 138, 2290–2297. Sarkari, B., Chance, M., Hommel, M., 2002. Antigenuria in visceral leishmaniasis: detection and partial characterisation of a carbohydrate antigen. Acta Trop. 82, 339–348. Stauber, L.A., Franchino, E.M., Grun, J., 1958. An eight-day method for screening compounds against Leishmaina donovani in the Golden hamster. J. Protozool. 5, 269–273. Sundar, S., More, D.K., Singh, M.K., Singh, V.P., Sharma, S., Makharia, A., Kumar, P.C.K., Murray, H.W., 2000. Failure of pentavalent antimony in visceral leishmaniasis in India: report from the center of the Indian epidemic. Clin. Infect. Dis. 31, 1104– 1107. WHO, 1999. Tropical Disease Research: Progress 1997–98, Fourteenth Programme Report edition. UNDP/World Bank/WHO Special Programme for Reasearch & Training in Tropical Diseases (TDR). Geneva, Switzerland. Wyllie, S., Cunningham, M.L., Fairlamb, A.H., 2004. Dual action of antimonial drugs on thiol redox metabolism in the human pathogen Leishmania donovani. J. Biol. Chem. 279, 39925–39932.