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J. Med. M icrobiol.- ol. 46 (1997), 263-2661997 The Pathological Society of Great Britain and IrelandTECHNICAL NOTE

A mouse model of recrudescence of Toxoplasmagondi i infectionS. NICOLL, S. WRIGHT*, S. W. MALEY* , S. BURNST and D . BUXTON*Centre for Tropical Veterinary Medicine, Easter Bush, Rosl in, Midlothian EH25 9RG, * Moredun ResearchInst i tute, 408 Gilmerton Road, Edinburgh EH 17 7JH and 7Regional Virus Laboratory, City Hospital , GreenbankDrive, Edinburgh EH9 5SB

Dexamethasone was given to mice infected with Tuxuplasma gon dii to provide a model ofrecrudescence of infection in immunocompromised patients and to permit investigationof the interaction between parasite and host.

IntroductionToxoplusmu go ndii affects c. 50 0 million people world-wide [ I ] . Although it causes a subclinical infection inmost individuals, reactivation (or recrudescence) oftissue cysts is an increasing problem in immunosup-pressed patients. Clinical toxoplasmosis occurs in 33%of AlDS patients with evidence of past toxoplasmainfection [2 ] with the development of encephalitis,chorioretinitis and lymphadenopathy. The polymerasechain reaction (PCR) has been used to detect 7: gondiiDNA in peripheral blood samples from shcep [3]. Thetechnique has also been used to detect the parasite intissue from AIDS patients before clinical manifesta-tions, suggesting that parasitaemia occurs duringreactivation, although it may be transient [4].The aim of this study was to develop a mouse modelof i': gondii recrudescence. Previous studies have usedanimals, including athymic (nude) and SCID mice, toinvestigate interactions between parasite and hostduring acute infection [5-91. Howev er, both nudeand SCID mice are difficult to work with because oftheir severely impaired immune systems and therequirement for them to be kept in sterile conditionsto prevent opportunist infections. Dexamethasone wasadministered to clinically normal mice in a study ofPneurnocystis carinii [101 and this technique wasapplied to mice persistently infected with 7: gondiiwith a view to inducing recrudescence of infection.Materials and methodsAnimalsA pool of 60 chronically infected Porton mice (30females and 30 males) was established by intraper-Received 9 Apr. 1996; revised version accepted 13 Aug.1996.Corresponding author: Dr S. Nicoll.

itoneal inoculation of each with 30 7: gondii tissuecysts of the M3 isolate [ 113. A further 40 Porton mice(20 females and 20 males) were kept as uninfectedcontrols. Groups of mice were caged separately and fedwith an approved standard ration. Six weeks afterinfection, two groups of infected mice (groups 1 and 2)were given dexamethasone 8 an d 4 mg/L, respectively,in drinking water, and 20 infected mice (group 3)received untreated water. Two uninfected controlgroups were given dexamethasone 8 mg/L (group 4)or untreated water (group 5 ) . Treated and untreatedwater was changed every second day. All groupsreceiving dexamethasone also received oxytetracyline1 mg/L in drinking water for the duration of the studyto reduce the risk of contracting other opportunistinfections. Blood samples were collected from allgroups by tail bleeding before treatment commenced,and weekly thereafter. Mice were monitored daily over8 weeks for clinical signs of toxoplasmosis; any thatshowed signs of illness were killed immediately withCOZ gas and samples of brain, blood and heart weretaken for analysis. At the end of the 8-week studyperiod all remaining mice were killed and examined inthe same way.PathologyThe brains were removed from all mice post mortemwith stringent aseptic precautions to avoid cross-contamination. Half the brain was placed in a sterileMicrofuge tube and stored at -20C until required forPC R analysis. The other half was placed in form01saline 10% for 7 days. Each was then sectionedcoronally and four blocks were dehydrated throughgraded alcohols and toluene before embedding inparaffin wax. Sections 5 p m thick were cut and stainedwith haematoxylin and eosin (HE) and then examinedfor lesions. Sections with minimal to mild histopatho-logical lesions were scored +, and those with moderateto severe change were scored ++.

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26 4 S . N I C O L L E T . 4 L .PCR analysis and seipologySamples of blood taken weekly, and blood, brain andheart obtained pos t inorten?were examined by PCR forthe presence of the B 1 gene of 7: gondii [121. Plasmasamples collected on day 0 and post niortem weretested for IgG against 7: gondii by an ELISA modifiedfor use in mice [131.

Pa tho og yExamination of brain showed three patterns ofneuropathological lesions; ( i ) focal necrosis thatoccurred in the thalamus or cerebral cortex or both;(ii) foci of gliosis that ranged from small to largeclusters of cells; (iii) lymphoid inflammation withperivascular cuffing by lymphoid cells as well asmeningeal accumulations of similar cells (Fig. 1 ) . Th eresults are summarised in Table 1 .

Statistical unalysisThe effect of dexamethasone on the morbidity rate andon the presence or absence of the parasite in sampleswas analysed by Fisher's exact test.

Resu ItsSigns of illness included a tottering gait coupled with ahunched appearance and evidence of emaciation anddehydration. Eight of 20 (40 %) mice given dexam etha-sone at 8 mg/L developed clinical signs and werekilled, compared with two (10%) of 20 given 4 mg/L(p = 0.049), and none of the control groups ( p =0.002 7). Uninfected control mice did not show signs oftoxoplasmosis, but one uninfected mouse given dex-amethasone died suddenly. Parasite DNA was detectedby PCR in nine samples of brain, six samples of heartand three samples of blood (no sample was obtainedfrom one mouse) collected from the 10 mice thatdeveloped clinical signs of toxoplasmosis. All samplesfrom infected mice that did not receive dexamethasone(group 3) and uninfected controls (group 4 and 5) gavenegative results.Blood samples collected at weekly intervals werepositive by the PCR in 49 ( 38.9 %) of 126 samplesfrom group 1 (dexamethasone 8 mg/L), 32 (23.4% ) of137 samples from group 2 (dexamethasone 4 mg/L),and 12 (8.6%) of 140 samples in group 3 (n odexamethasone). The results of group 1 v e i w s group2 (p = 0.0064), group 1 versus group 3 (p < 0.0001)and group 2 vemts group 3 (p = 0.008) were highlysignificant.Blood samples taken from survivors at the end of theexperiment were positive by the PCR in four (33.3%)of 12 mice from group 1 , five (27.7%) of 18 fromgroup 2, and three (15 %) of 20 from group 3(differences were not significant). All 12 samples ofbrain from mice given dexamethasone at 8 mg/L(grou p 1) were positive, compared w ith 16 (88.8%)of 18 that received dexa meth asone at 4 mg/L (group2), and 15 (75% ) of 20 that received no dexametha-sone (group 3). These differences were not significant.Similarly, all 12 samples of heart were positive ingroup 1 , 15 (83.3%) of 18 in group 2, and nine (45%)of 20 in group 3 (significance between groups 1 an d

Necrosis was observed in two mice with no evidenceof recrudescence (group 1 ) and in five infected micewith clinical signs of infection; all of them had beengiven dexamethasone. Gliosis was observed in 14(70%) of 20 samp les from group 1, 13 (65% ) of 20samples from group 2, and 15 (75%) of 20 samplesfrom group 3, and was more frequently observed inclinically affected than in clinically normal animals.The incidence of lymphoid inflammation was similarin groups 1 , 2 and 3 (85%, 90% and 1009'0,respectively) but tended to be more severe in group 3.7: gondii tissue cysts were observed in brains ofinfected mice (40%, 30% and 35% of mice,

Fig. 1. ( A ) Lymphoid meningi t i s and an associa ted gl ia lresponse in the cerebral cortex of a mouse infected wi th7: gondi i and t reated wi th dexamethasone ( 4 m g i L indr inking water ) ( H E X 56) . ( B ) Acute necros i s in thecerebral cortex of a mouse infected with 7: gondi i andt reated wi th dexamethasone (4 mg/L in dr inking water )-3; p = 0.0006). ( H E X 5 6 ) .

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M O D E L O F 7: G O N D / / R E C R U D E S C E N C E 26 5Table 1. Results of pathological examination of brain sections tested

Number of mice withNumber Necrosis Gliosis Lymphoid inflammationof mice

Experimental group examined + ++ + ++ + ++20 (8 ) 5 ( 3 ) 1 ( 1 ) 12 ( 5 ) 2 ( 1 ) 16 (7) 1 ( 1 )20 (2 ) 0 (0 ) 1 (1 ) 1 1 ( I ) 2 ( I ) 15 ( I ) 3 ( 1 )20 (0 ) 0 0 13 (0) 2 (0) 14 (0) 6 (0 )

Infected, dexamethasone 8 mg/L (group 1 )Infected, dexamethasone 4 mg/L (group 2 )Infected, no dexamethasone (group 3 )Uninfected, dexamethasone 8 mg/L (group 4) 20 0 0 0 0 0 0Uninfected no dexamethasonc (group 5 ) 20 0 0 0 0 0 0Numbers in parentheses indicate those mice in groups I , 2 and 3 showing clinical signs of illness.

respectively, in groups 1-3), but not in uninfectedcontrol mice. Tissue cysts in groups 1 and 2 seemedto be adjacent to areas of inflammation whereas ingroup 3 they were generally unassociated with otherchanges.

S e m og yBlood samples obtained on day 0 from infected micewere positive by IgG ELISA (O D values: 119.7-128.6), whereas those from uninfected mice werenegative (O D values

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26 6 S. NICOLL E T A L .Toxoplasma gondii and their immunity to challenge when 27 : 1787- 1792.pregnant. Vet Re c 1991; 129: 89-93. 13. Buxton D, Blewett DA, Trees AJ, McColgan C, Finlayson J.12 . Burg JL , Grover CM, Pouletty P, Boothroyd JC. Direct and Further studies in the use of Monen sin in the control ofsensitive detection of a pathogenic protozoan, Toxoplasma experimental ovine toxoplasmosis. J Comp Puthol 1988; 98:gondii, by polymerase chain reaction. J Clin Microbiol 1989; 225-236.