**Annex 18. Item 5.1. – Chapter 3.10.8. Toxoplasmosis**

###### 2	MEETING OF THE BIOLOGICAL STANDARDS COMMISSION

3	**Paris, 4–8 September 2023**

4

5	**CHAP T E R  3 . 1 0 . 8 .**

# 6	T OXOPL A SMOSI S

7	**SUMMARY**

8 *Toxoplasmosis is a zoonotic infection of animals caused by the protozoan parasite*Toxoplasma gondii*. This*

9 *parasite has the potential to infect all warm-blooded animals. Although infection does not result in clinical*

10 *illness in the majority of animal species, in some it causes acute life-threatening disease. In some animals,*

11 *particularly small ruminants,*Toxoplasma *infection may manifest itself as a disease of pregnancy by*

12 *multiplying in the placenta and fetus. In these animals it can result in abortion or the birth of weak offspring.*

13 *Human infections are generally asymptomatic, but they can cause abortion in pregnant women, ocular*

14 *disease, hydrocephalus or intracranial calcifications in congenitally infected children, ocular toxoplasmosis*

15 *in immunocompetent individuals, and serious symptoms and even death in severely immunocompromised*

16 *patients.*

17 Toxoplasma gondii *is an obligate intracellular parasite that has a sexual cycle in Felidae and a two-stage*

18 *asexual cycle in all warm-blooded animals. Globally the*T. gondii *population structure is diverse and the*

19 *various genotypes are associated with the extent of virulence in particular hosts. In the acute phase of*

20 *infection, tachyzoites multiply in host cells to cause varying degrees of tissue destruction. With the onset of*

21 *an immune response, tachyzoites transform into bradyzoites that multiply slowly in cells to produce tissue*

22 *cysts.*

23 ***Detection of the agent:****In aborted fetuses and placenta,*T. gondii *is often difficult to find histologically, but*

24 *is more likely to be seen in tissue sections of brain and placenta. Parasitic stages can be identified by*

25 *immunohistochemistry, while nucleic acid-based assays might be used to confirm presence of parasite DNA*

26 *in tissues and may allow genotyping of the parasite in biological specimens.*In-vitro *isolation of*T. gondii

27 *from host samples is expensive, time consuming and rarely used.*

28 *The sexual part of the life-cycle of*T. gondii *takes place exclusively in epithelial cells of the feline intestine*

29 *and can result in the excretion of large numbers of oocysts in the faeces. Oocysts may remain viable in the*

30 *environment for many months. Oocysts of*T. gondii *~~morphologically~~resemble those from*Hammondia

31 hammondi*, a related but non-virulent parasite that also uses cats as definitive hosts. Nucleic acid-based*

32 *molecular tests are available to distinguish between these related parasites.*

33 ***Serological tests:****Among the easy-to-perform serological tests, the indirect fluorescent antibody test*(*IFAT*)

34 *and the direct agglutination test*(*DAT*) *allow the titration of sera and the establishment of appropriate cut-*

35 *offs to ensure diagnostic sensitivity and specificity. The IFAT can be used to differentiate IgM and IgG*

36 *antibodies. The DAT is fast and requires no complex laboratory facilities. Enzyme-linked immunosorbent*

37 *assays*(*ELISA*) *require more sophisticated laboratory equipment but can process large numbers of samples*

38 *and are easier to standardise.*

39 ***Requirements for vaccines:****A vaccine composed of live*T. gondii *tachyzoites is available commercially for*

40 *use in sheep in certain countries. The vaccine is supplied as a concentrated suspension of tachyzoites with*

41 *an approved diluent and delivery system. The vaccine must be handled strictly according to the*

42 *manufacturer’s instructions as it can be hazardous to the user and has a very short shelf life.*

## 43	A. INTRODUCTION

44 *Toxoplasma gondii*is a zoonotic, obligate intracellular protozoan parasite with the capacity to infect all warm-blooded

45 animals, including birds. Although clinical toxoplasmosis seldom occurs in the majority of animal species, acute life-

46 threatening disease has been reported in some animals. In small ruminants, in particular, it manifests itself as a disease

47 of pregnancy by multiplying in the placenta and fetus. Acute, potentially fatal, infections have been recorded from a range

48 of wild or zoo animals (Dubey, 2022)*.*Infected humans often show no symptoms, but congenital toxoplasmosis, postnatally

49 acquired ocular toxoplasmosis in immunocompetent individuals, or toxoplasmosis in severely immunocompromised

50 patients represent serious threats (EFSA, 2018).

51 *Toxoplasma gondii*has a two-stage asexual cycle in warm-blooded animals and a sexual cycle in Felidae. A systematic

52 review and meta-analysis have reported a global seroprevalence of 38% in domestic cats and 64% wild felids (Hatam-

53 Nahavandi *et al.*, 2021). The genetic diversity of *T. gondii*is complicated; three archetypal clonal lineages (I, II, and III)

54 prevail in Europe and North America; in South America, Asia, and Africa, much greater genetic diversity is apparent and,

55 furthermore, fewer clonal and non-clonal lineages have been genotyped (Lorenzi *et al.*, 2016). Transport between

56 continents via animal migration, including birds, and human activity such as trade may have contributed to the genetic

57 population structures of *T. gondii*in different geographical regions (Shwab *et al.*, 2018).

58 In the asexual part of the lifecycle, the two developmental stages are the rapidly multiplying tachyzoite and the slowly

59 multiplying bradyzoite. In acute infection, tachyzoites actively penetrate host cells where they multiply, causing the cell to

60 rupture and release organisms locally and into the circulation. As the host develops immunity, the parasite retains its overall

61 size and shape, but transforms into the bradyzoite stage and multiplies more slowly within tissue cysts to establish a

62 persistent infection. These microscopic tissue cysts occur most frequently in brain and skeletal muscle and represent the

63 quiescent stage of the parasite within the host. Viable tissue cysts within muscle (meat) are a significant source of human

64 infection, and ingestion of bradyzoites in prey is probably the main route of infection to predators, including the felid

65 definitive host. In animals that succumb to acute infection, tachyzoites may be demonstrated in ascitic fluid or in lung

66 impression smears, as well as in tissue sections of the liver and other affected organs.

67 Abortions in sheep and goats due to *T. gondii*are of particular veterinary importance. Toxoplasmosis in small ruminants

68 must be differentiated from diseases caused by other infectious agents, including infections with *Chlamydophila abortus*

69 (see Chapter 3.8.5 *Enzootic abortion of ewes*), *Coxiella burnetii*(see Chapter 3.1.17 *Q fever*), *Brucella melitensis*(see

70 Chapter 3.1.4 *Brucellosis*[*Brucella abortus*, *B. melitensis*and *B. suis*]), *Campylobacter foetus*(see Chapter 3.4.4 *Bovine*

71 *genital campylobacteriosis*), *Salmonella*spp. (see Chapter 3.10.7 *Salmonellosis*), and the viruses that cause border

72 disease (see Chapter 3.8.1 *Border disease*), bluetongue (see Chapter 3.1.3), Wesselsbron’s disease, and Akabane

73 disease (see Chapter 3.10.1). In pigs, *Brucella suis*(see Chapter 3.1.4) may also cause fetal death, mummification, and

74 abortion.

75 The sexual part of the lifecycle occurs in enteroepithelial cells of the feline definitive host, and results in the production of

76 *T. gondii*oocysts. Following primary infection of a cat, oocysts may be shed in the faeces for several days, with large

77 numbers contaminating the environment; up to one billion oocysts from domestic cats, and probably similar numbers from

78 wild felids (Shapiro *et al.*, 2019). The oocysts sporulate in the environment over the next 1–5 days (depending on aeration,

79 humidity, and temperature), at which time they become infective. The structure of *T. gondii*oocysts results in extreme

80 resistance to environmental conditions, with the polymeric structure of the walls ~~giving~~providing strength against

81 mechanical forces and protection against chemical agents (Shapiro *et al.*, 2019). This results in prolonged survival, up to

82 18 months in water at 4°C and, once sporulated, can persist in damp soil for as long, at temperatures ranging from −20°C

83 to 35°C. Sporulated oocysts are 11 × 13 μm in diameter and each contains eight sporozoites, four in each of two sporocysts

84 (Dubey, 2022). When a susceptible animal ingests sporulated oocysts, the sporozoites are released to penetrate the

85 intestinal lining, become tachyzoites, and establish an infection.

#### 86 1.	Human health risks

87 *Toxoplasma gondii*is a zoonotic parasite and readily infects people. While human infection, as determined by seropositivity,

88 is moderately common globally (local prevalence ranges from under 10% to over 90% [Pappas *et al.*, 2009]), clinical illness

89 is relatively uncommon. The immunosuppressed are particularly at risk of developing clinical illness. In patients being

90 treated with immunosuppressive drugs, toxoplasmosis may occur due to new infection or activation of chronic infection. In

91 addition, the parasite can pose a serious threat to an unborn child if the mother becomes infected for the first time while

92 pregnant. The *T. gondii*genotype is also relevant, and outbreaks of clinical infection with some non-archetypal exotic

93 strains have occurred in people with no apparent immune deficiency. Toxoplasmosis usually manifests as general malaise,

94 fever, and lymphadenopathy. However, more severe symptoms may occur, including ocular problems, such as

95 retinochoroiditis, potentially resulting in loss of vision, pneumonitis, and also toxoplasmic encephalitis. The main burden of

96 human disease, based on disability-adjusted life years (DALYs), ranks as having a high contribution to disease burden

97 globally (Torgerson *et al.,*2015).

98 As with animal infections, people may be infected by ingestion of bradyzoites in raw or lightly cooked meat containing live

99 *T. gondii*tissue cysts or by ingestion of sporulated oocysts. These may be as contaminants of water or of raw or lightly

100 cooked fresh produce; less commonly, people can be infected by ingestion of tachyzoites in non-heat-treated milk. In

101 addition, transmission via blood transfusion or organ transplantation is also possible. Outbreaks of both waterborne and

102 foodborne toxoplasmosis have been described (EFSA, 2018; Shapiro *et al.,*2019). The largest outbreak to date occurred

103 in Santa Maria, Brazil in 2018, and is considered to be due to contamination of the water supply with oocysts of a virulent

104 strain of *T. gondii*; at least 930 confirmed cases occurred, among which 8% required hospitalisation, with three fetal deaths,

105 10 abortions, and 29 cases of congenital transmission, with 19 infants with ocular lesions (Dubey, 2021).

106 Clearly, *T. gondii*represents a human health risk and all laboratory manipulations with live organisms should be handled

107 with appropriate measures determined by risk analysis as described in Chapter 1.1.4 *Biosafety and biosecurity: Standard*

108 *for managing biological risk in the veterinary laboratory and animal facilities.*

## 109	B. DIAGNOSTIC TECHNIQUES

110	***Table 1.****Test methods available for the diagnosis of toxoplasmosis and their purpose*

111	Key: +++ = recommended for this purpose; ++ recommended but has limitations;

112	+ = suitable in very limited circumstances; – = not appropriate for this purpose.

113 PCR = polymerase chain reaction; LAMP = loop mediated isothermal amplification; IFAT = indirect fluorescent antibody test;

114 ELISA = enzyme-linked immunosorbent assay; DAT = direct agglutination test; MAT = modified agglutination test.

115 (a)In IFAT and ELISA, detection of *Toxoplasma*-specific IgG and IgM antibodies may permit some discrimination between acute and

116 chronic cases of infection. In ELISA, assays assessing the avidity of an IgG response to *T. gondii*may provide information regarding

117 how recently the tested animals have experienced a primary *T. gondii*infection.

#### 118 1.	Detection of the agent

##### 119 1.1.	Histopathology

120 In animals that die with acute toxoplasmosis, focal mononuclear inflammation, with or without focal necrosis, may be

121 seen in a number of tissues, including the liver, heart, and lungs. The latter may be oedematous. Lymph nodes may

122 have undergone expansion and there may or may not be focal necrosis with or without haemorrhage. Typically, *T.*

123 *gondii*tachyzoites may be demonstrable in association with necrosis and inflammation. In fatal cases, tachyzoites

124 may be demonstrated in ascitic fluid or in lung impression smears.

125 In cases of abortion and stillbirth in small ruminants, affected placental cotyledons typically contain large foci of

126 coagulative necrosis that may have become mineralised with time. Any associated inflammation is characteristically

127 slight and nonsuppurative. Well-preserved samples of placental cotyledons may show moderate oedema of the

128 mesenchyme of the foetal villi, with a diffuse hypercellularity due to the presence of large mononuclear cells. Small

129 numbers of intracellular and extracellular stages are sometimes visible, usually on the periphery of a necrotic area or

130 in a villus that is in the early stages of infection. The *T. gondii*tachyzoites appear ovoid, 2–6 μm long, with nuclei that

131 are moderately basophilic and located centrally or towards the posterior end.

132 In the fetal brain, primary and secondary lesions may develop. Microglial foci, typically with a necrotic and sometimes

133 mineralised centre, and often associated with a mild focal lymphoid meningitis, represent a fetal immune response

134 following direct damage by local parasite multiplication. *Toxoplasma gondii*tissue cysts are only rarely found, usually

135 at the periphery of these lesions. Focal leukomalacia is also common and is considered to be due to fetal anoxia in

136 late gestation caused by advanced lesions in the placentome preventing sufficient oxygen transfer from mother to

137 fetus. Such foci most commonly occur in the cerebral white matter cores, but sometimes also in the cerebellar white

138 matter. Focal leukomalacia on its own suggests placental disease or acute insufficiency, but the two types of

139 neuropathological change seen together are characteristic of *T. gondii*infection.

##### 140 1.2.	Immunohistochemistry

141 Confirmation of the identity of *T. gondii*-like structures in tissue sections from such cases, as well as from instances

142 of acute toxoplasmosis, may be achieved by immunohistochemistry that labels intact *T. gondii*or antigenic debris

143 using polyclonal or monoclonal *T. gondii*specific antibodies (Dubey, 2022). The antigen-antibody reaction can be

144 visualized by avidin-biotin-complex (ABC) or indirect immune-peroxidase and the peroxidase–antiperoxidase (PAP)

145 technique. The method is both convenient and sensitive and is used with fixed tissues (including archived tissues)

146 that may also exhibit a degree of decomposition, where isolation would not be appropriate or possible. However,

147 cross-reactions with related parasites like *Neospora caninum*are possible.

##### 148 1.3.	Detection of oocysts

149 *Toxoplasma gondii*oocysts can be detected in stools of felids, as well as contaminating different environmental

150 matrices, such as soil and water, or food, such as molluscs and fresh produce. The low quantity or sparse distribution

151 of oocysts in the contaminated matrix, as for water and fresh produce, means that an initial procedure to concentrate

152 the oocysts from a large volume of sample is needed. Chemical flocculation (e.g. using ferric or aluminium sulphate

153 or calcium carbonate), filtration by cellulose acetate or polycarbonate membranes or cartridge filters and flotation with

154 sucrose or caesium chloride gradient have been widely used for water samples. Washing with appropriate buffer(s)

155 and pelleting by centrifugation is often used for fresh produce (Shapiro *et al.*, 2019; Slana *et al.*, 2021).

156 Although the autofluorescence of *T. gondii*oocysts, pale blue under UV light, facilitates detection by microscopy, this

157 property is shared with oocysts and sporocysts of other related coccidian parasites (e.g. *Hammondia hammondi*)

158 (Lindquist *et al.*, 2003). As a commercially available antibody specific for *T. gondii*oocysts for microscopy detection

159 is currently lacking, molecular assays are usually used to confirm *T. gondii*identification in field samples. Molecular

160 methods are needed to assess or confirm the identity of oocysts~~observed~~.

##### 161 1.4.	Molecular methods – detection of nucleic acids

162 The presence of *T. gondii*(tachyzoites, tissue cysts, oocysts) can be assessed by detecting the parasite genomic

163 DNA using several molecular techniques, including conventional polymerase chain reaction (PCR), nested PCR and

164 loop-mediated isothermal amplification (LAMP) (Table 1). No standard methods are available and many of the

165 published protocols are not yet sufficiently validated (for details refer to Chapter 1.1.6. *Principles and methods of*

166 *validation of diagnostic assays for infectious disease*and Chapter 2.2.3. *Development and optimisation of nucleic*

167 *acid detection assays*).

168 Appropriate protocols may allow detection of *T. gondii*DNA from circulating tachyzoites (acute infection) or

169 bradyzoites in tissue cysts (latent infection) and in different biological samples, including animal and human

170 tissues (e.g. heart and skeletal muscle, placenta, brain) and body fluids (e.g. blood, urine, aqueous humour,

171 cerebrospinal fluid, amniotic fluid, milk). In addition, DNA from oocysts in stool (only felids), food and

172 environmental samples (fresh produce, water, soil) can be detected (Slana *et al.*, 2021). No standard method

173 for extracting *T. gondii*DNA exists, but suitable DNA extraction protocols, based on both in-house protocols and

174 commercial kits have been developed (Dzib Paredes *et al.*, 2016). Sample preparation and DNA extraction

175 procedures are likely to have a considerable impact on the sensitivity of the test. Sensitivity is generally higher

176 in DNA-poor matrices than DNA-rich ones (e.g. tap water vs meat samples). Moreover, inhibitors of DNA

177 amplification differ and are related to sample type. Inhibition of DNA amplification can be avoided by DNA

178 extraction optimised for sample type or by using appropriate additives (e.g. bovine serum albumin) during DNA

179 amplification. A specific concentration of the parasite stage (e.g. oocysts) or its DNA (e.g. by magnetic capture)

180 from the matrix might be required prior to DNA extraction as reported for oocysts from water, fresh produce, or

181 faeces (Slana *et al.*, 2021). A validated protocol for DNA extraction from meat and meat products using a

182 commercial kit is available at the website of the European Union Reference Laboratory for Parasites (EURLP) 48.

183 An example of in-house method for DNA extraction from pig tissue (Jauregui *et al.*, 2001) is reported below.

###### 184 1.4.1.	DNA extraction from animal tissue

185 **Procedure**

186 i)	Homogenate sample (e.g. 50 g of brain or tongue, or 1 g of muscle) in a blender with 5 volumes

187 of sterile saline solution (phosphate-buffered saline [PBS]: 300 mM NaCl, 2.7 mM KCl, 10 mM

188 Na2HPO4, 1.7 mM NaH2PO4).

189 ii)	Digest sample with an equal volume of warm (37°C) pepsin-HCl (1.4 mg of pepsin and 10 mg

190 of NaCl per ml in 0.1 N HCl) for 1 hour at 37°C in a shaking water bath.

191 iii)	Neutralise mixture by two washes with 0.1 M Tris buffer (pH 8.0).

192 iv)	Centrifuge mixture aliquots for 10 minutes at 1180 ***g***.

193 v)	For each aliquot, digest the post centrifugation pellet overnight at 55°C with DNA digestion

194 buffer (0.5% sodium dodecyl sulphate [SDS], 25 mM ethylene diamine tetra-acetic acid [EDTA],

195 100 mM NaCl, 20 mM Tris-HCl [pH 8.0], and proteinase K [0.1 mg/ml final concentration]).

196 vi)	Extract with one volume of phenol-chloroform-isoamyl alcohol (25:24:1).

197 vii)  Precipitate DNA in 0.3 M sodium acetate (final concentration) with 2.5 volumes of 100% ethanol.

198 viii) Resuspend DNA pellets in TE buffer (10 mM Tris-HCl, 1 mM EDTA). Store DNA at –20°C until

199 use.

200 PCR-based assays are commonly applied for the molecular detection of *T. gondii*genomic DNA (Dzib

201 Paredes *et al.*, 2016; Robert *et al.*, 2021; Slana *et al.*, 2021).

###### 202 1.4.2.	DNA extraction from oocysts

203 Although DNA detection is considered highly specific, cross reactivity has been observed between

204 *T. gondii*and *H. hammondi*, a non-zoonotic coccidian that also uses felids as definitive hosts and

205 cannot be differentiated based on oocyst morphology. A real-time PCR targeting a repetitive element

206 of *H. hammondi*(HhamREP-529) has been demonstrated to be highly sensitive and efficient in

207 distinguishing between the two parasites (Schares *et al.*, 2021).

208 Detection of DNA from *T. gondii*oocysts may present additional challenges because of inhibitors in

209 faecal matter, vegetable or water sediment, and difficulty of extracting DNA from the oocysts. Options

210 for ~~an~~efficient breaking of oocyst~~s~~walls include bead-beating, freeze–thaw cycles, heating or

211 chemical/enzymatic treatments (Slana *et al.*, 2021). An in-house method is detailed below for

212 preparation of oocysts and extraction of DNA. An example of a validated method using a commercial

213 kit and a bead-beating-based DNA extraction is available (Lalle *et al.*, 2018).

214 **Procedure**

215 i)	Wash oocysts four times in 15 ml PBS (300 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.7 mM

216 NaH2PO4) in a 15 ml centrifugation tube, with centrifugation between washes (1100 g for

217 7 minutes without braking).

42 https://www.iss.it/documents/5430402/5722370/MI_12_rev._1.pdf/a82a4078-f511-affe-8f90-cabc397bc8ce?t=1620381672663

218 ii)	Incubate pellet (up to 0.5 ml) in 2 ml 5.75 % sodium hypochlorite (sodium hypochlorite, aqueous

219 solution, ≥ 4% as active chlorine) for 30 minutes at 37°C.

220 iii)	Add double-distilled H2O up to 15 ml.

221 iv)	Centrifuge supernatant (1100 g for 7 minutes without use of brake) and mix the pellet with PBS.

222 Wash the pellet three times with PBS (1100 ***g***for 7 minutes without brake).

223 v)	After final centrifugation, re-suspend pellet in 1 ml PBS, transfer into a 1.5 ml reaction tube and

224 spin down (1100 ***g***for 7 minutes without brake).

225 vi)	Remove as much supernatant as possible and apply three freeze–thaw cycles (10 minutes at

226 –20°C followed by 2 minutes at room temperature) to the pellet.

227 vii)  Re-suspend pellet in 100 μl OOC lysis buffer (600 mM EDTA, 1.3% [v/v] N-lauroylsarcosine, 2

228 mg/ml proteinase K, pH 9.5) for 45 minutes, at 65°C.

229 viii) Add 400 μl OOC-CTAB buffer (2% [v/v] cetyl-trimethyl ammonium bromide, 1.4 M NaCl, 0.2 %

230 [v/v] mercaptoethanol, 20 mM EDTA, 100 mM tris[hydroxymethyl]aminomethane) for 60

231 minutes at 60°C.

232 ix)	Mix with 500 μl phenol/chloroform/isoamyl alcohol (25/24/1) by inverting 50 times. Centrifuge

233 for 7 minutes at 13,000 ***g***.

234 x)	Transfer supernatant to fresh tube and mix with 500 μl phenol/chloroform/isoamyl alcohol

235 (25/24/1) by inverting 50 times. Centrifuge for 7 minutes at 13,000 ***g***.

236 xi)	Transfer supernatant to a fresh tube and add 0.04 volumes of 4 M NaCl and 2–3 volumes of –

237 20°C cold 96% (v/v) ethanol to precipitate DNA (keep at least 20–30 minutes at –20°C).

238 xii)  Centrifuge for 15 minutes at 13,000 ***g***. Decant the supernatant.

239 xiii) Wash the pellet using 70% (v/v) ethanol and centrifuge for 15 minutes at 13,000 ***g***.

240 xiv) Discard the ethanol solution and air dry the pellet.

241 xv)  Solve DNA in double-distilled water for at least 12 hours at 4°C.

242 xvi) Use 2.5–10 μl aliquots for PCR (see Section B.1.2 above).

243 The same PCR-based and LAMP assays detailed in Section B.1.2 have been also used for oocyst

244 detection, with B1 gene and the 529RE being targets of choice (Slana *et al.*, 2021).

245 Another important issue is the possibility of combining detection with information on oocyst viability.

246 Bioassays, currently the only definitive way to detect viable oocysts, are expensive and relatively few

247 laboratories have the necessary facilities. Reverse transcription (RT) real-time PCR (real-time RT-

248 PCR) or propidium monoazide-based real-time PCR have shown some promise for assessing oocyst

249 viability in complex sample matrices (Kim *et al.*, 2021; Rousseau *et al.*, 2018).

###### 250 1.4.3.	Nucleic acid detection methods

251 Although single copy genes (e.g. SAG1, SAG2, SAG3, GRA6, and GRA7) have been used, multi-

252 copy genes or genetic elements (e.g. 18S rDNA, B1, ITS1, 529RE) are preferred as they provide a

253 higher sensitivity (Dzib Paredes *et al.*, 2016; Slana *et al.*, 2021). For instance, 35 copies of the B1

254 gene and 200–300 copies of the 529 bp repetitive element (529RE) are present in the *T. gondii*

255 genome, and 10 to 100-fold higher sensitivity is generally observed in amplification targeting the

256 529RE compared with B1, although this also reflects the type of assay and sample being analysed

257 (Belaz *et al.*, 2015). In addition, some strains may have partially lost, or have a mutated, 529RE, and

258 this could compromise diagnostic sensitivity (Wahab *et al.*, 2010).

259 Conventional PCR targeting B1 was the first to be used in clinical diagnostics in people (Burg *et al.*,

260 1989). To obtain details on *T. gondii*genotype (e.g. for outbreak investigation, infection source

261 tracing) the methods of choice are multi-locus PCR combined with restriction fragment length

262 polymorphism (PCR-RFLP) or sequencing and multi-locus microsatellite typing (Ajzenberg *et al.*,

263 2010; Su *et al.*, 2006).

264 To increase sensitivity, several nested PCR assays have been implemented (Dzib Paredes *et al.*,

265 2016). The reaction consists of two successive rounds of amplification. The product of the first

266 amplification serves as template for the second amplification, using one or two internal primers. The

267 risk of cross- and carry-over contamination and false positives is increased with nested PCR, and

268 precautions should be taken to mitigate the risk (Dzib Paredes *et al.*, 2016).

269 There are several real-time PCR protocols and real-time PCR in combination with a hydrolysis probe

270 is the most frequently applied (Slana *et al.*, 2021). This has largely improved both sensitivity and

271 specificity of detection of *T. gondii*DNA, with the advantage of avoiding post-amplification

272 manipulations and thus limiting the risk of carry-over contamination. Although sensitivity can be

273 satisfactory with both conventional and real-time PCR using pure genomic *T. gondii*DNA, assay

274 specificity might be affected when testing field samples. Conventional PCR can result in non-specific

275 amplification, whereas this is not detected by real-time PCR due to the probe detection, despite the

276 amplified target being the same. Furthermore, real-time PCR can be multiplexed and simultaneous

277 amplification of an internal amplification control (a heterologous DNA fragment) can be used to

278 monitor for the presence of inhibitors. In addition, amplifying two *T. gondii*-specific targets at once

279 may increase sensitivity. Another advantage of real-time PCR is the possibility of quantification of *T.*

280 *gondii*DNA.

281 A selective enrichment of target DNA combined with real-time PCR (i.e., magnetic capture PCR) has

282 been reported to increase *T. gondii*detectability in animal samples (Gisbert Algaba *et al.*, 2017). The

283 principle relies on separating and concentrating *T. gondii*DNA from sample DNA by specific DNA

284 probes, complementary to the targeted parasite 529RE genomic region, which are conjugated to

285 magnetic beads and followed by real-time PCR (Gisbert Algaba *et al.*, 2017). However, this method

286 is expensive, time consuming, and requires further expertise, so might be not suitable for routine

287 analysis or large surveys.

288 As an alternative to PCR, LAMP has been considered for *T. gondii*DNA detection, and diagnostic

289 purposes, in environmental, veterinary, and human samples. LAMP takes advantage of a DNA

290 polymerase (originally Bst) having both high strand displacement and replication activities. Nucleic

291 acid amplification is performed under isothermal conditions (60–65°C), without the need of a DNA

292 denaturation step. Both, B1 and 529RE have been widely used as targets in different LAMP assays,

293 and LAMP is reported as comparable to real-time PCR for the detection of *T. gondii*in blood and

294 animal tissues (Robert *et al.*, 2021). Although LAMP provides an opportunity for development of point-

295 of-care testing or implementation of molecular tests in settings with limited facilities, there are several

296 drawbacks including design of appropriate primers and the high risk of carry-over contamination.

297 Commercial assays for both real-time PCR and LAMP are available for clinical diagnosis of

298 toxoplasmosis.

299 Overall, the reported sensitivity of published molecular methods can be as low as one (or even less)

300 genome equivalent per reaction. However, this largely depends on sample type, DNA extraction,

301 copies of the targeted gene or sequence, amplification and detection reagents, procedures and

302 platforms. The lack of accepted standard methods prevents robust comparison of sensitivity and

303 specificity of the currently applied molecular tests.

#### 304 2.	Serological tests

305 There are several serological tests available for the detection of *T. gondii*antibodies (Table 1). All serological tests have

306 limitations in diagnostic sensitivity and specificity and need proper validation to ensure confidence in results (refer to

307 Chapter 1.1.6. *Principles and methods of validation of diagnostic assays for infectious disease*and Chapter 2.2.1.

308 *Development and optimisation of antibody detection assays*).

309 The Sabin-Feldman dye test (DT) is a reference serological test for *T. gondii*antibody in humans (Dubey, 2022). Although

310 the DT appears both specific and sensitive in humans, it is not extensively validated in other species. In addition, it is

311 potentially hazardous as live parasite is used, is expensive, and requires a high degree of technical expertise.

##### 312 2.1.	Preparation of antisera and antigens

313 Antisera to *T. gondii*and conjugated antisera for use in IFAT and ELISA, to allow screening of a variety of animal

314 species, may be obtained commercially. International standards for animal sera are not available.

315 Below are protocols for the preparation of tachyzoite antigen for use in the IFAT and ELISA. Tachyzoites may be

316 grown in tissue culture and retained as whole parasites for the IFAT, or prepared as soluble antigen for the ELISA.

##### 317 2.2.	Preparation of frozen stabilates of *T. gondii*tachyzoites

###### 318 2.2.1.	Test procedure

319 i)	Produce tachyzoites in tissue cell culture as described. Suitable *T. gondii*strains, able to multiply

320 in cell culture, like the RH strain are available in a number of repositories; e.g. at the American

321 Tissue Cell Culture Collection (ATCC 49).

322 ii)	Centrifuge three times at 500 ***g***for 5 minutes and resuspend tachyzoites in Iscove’s modified

323 Dulbecco’s medium (IMDM) or any other cell culture medium suitable to cultivate *T. gondii.*Final

324 concentration of the tachyzoite suspension should be approximately 1.5 × 108 tachyzoites/ml.

325 iii)	Combine dimethyl sulphoxide (DMSO), normal horse serum (free from antibody to *T. gondii*)

326 and the tachyzoite suspension to give these final concentrations: 10% DMSO, 20% normal

327 horse serum, 70% tachyzoite suspension; this gives a final concentration of approximately 1 ×

328 108 tachyzoites/ml.

329 iv)	Allow the preparation to stand on the bench for 1 hour (4-10°C; optimally on ice).

330 v)	Dispense into 1-ml aliquots using screw-topped tubes appropriate for liquid nitrogen storage.

331 vi)	Put the tubes into a small container, wrap in thick insulating material (e.g. paper towels) and

332 place in –70°C freezer to allow the tachyzoites to freeze gradually.

333 vii)  The next day transfer to liquid nitrogen, keeping well insulated while transferring.

334 viii) These stabilates may then be used for tissue culture growth of the parasite. When removing

335 from storage, thaw the sample rapidly in a water bath (37°C).

336 ix)	Centrifuge three times at 500 ***g***for 5 minutes and resuspend tachyzoites in Iscove’s modified

337 Dulbecco’s medium (IMDM) or any other cell culture medium suitable to cultivate *T. gondii*and

338 add suspension to cell culture.

##### 339 2.3.	Production of *Toxoplasma*tachyzoites in cell culture

###### 340 2.3.1.	Test procedure

341 i)	*Toxoplasma gondii*can be grown and maintained in tissue culture by twice-weekly passage in

342 African green monkey kidney (Vero) cells. Other cell lines (e.g. MARC145 cells) are also

343 suitable. Cell lines are available from repositories (e.g. ATCC).

344 ii)	Cells and parasite are grown in IMDM supplemented with 50 IU/ml penicillin, 50 μg/ml

345 streptomycin, and 2% fetal bovine serum; there are other cell culture media suitable as well.

346 iii)	Tachyzoites are harvested from tissue culture flasks by scraping the cell monolayer using a

347 sterile cell scraper.

348 iv)	Using 25 cm2 vented tissue culture flasks that have each been seeded with 1 × 105 Vero cells,

349 add tachyzoites at the rate of two tachyzoites per monolayer cell and incubate at 37°C in a 5%

350 CO2 humidified chamber. Harvest, when 2/3 of the cell layer was destroyed by tachyzoite

351 multiplication, usually after 3–4 days.

##### 352 2.4.	Preparation of whole tachyzoites for IFAT and agglutination

###### 353 2.4.1.	Test procedure

354 i)	Produce 4 × 107/ml suspension of *T. gondii*tachyzoites in PBS.

355 ii)	Add formaldehyde (40%) to give a final concentration of 0.2% (v/v).

356 iii)	Incubate at 4°C overnight and divide into aliquots in suitable tubes and store frozen until

357 required (–20°C).

358

43 American Type Culture Collection, P.O. Box 1549, Manassas, Virginia 20108, United States of America.

##### 359 2.5.	Production of soluble antigen for ELISA

###### 360 2.5.1.	Test procedure

361 i)	Produce a suspension of *T. gondii*tachyzoites in PBS.

362 ii)	Centrifuge at 2000 ***g***for 15 minutes, retain the pellet and resuspend it in nine times its volume

363 of distilled water.

364 iii)	Lyse the tachyzoites by freezing and thawing three times.

365 iv)	Sonicate the antigen preparation for 20 seconds at 4°C at an amplitude of 20 microns.

366 v)	Remove any cellular debris by centrifugation at 10,000 ***g***for 30 minutes at 4°C.

367 vi)	Retain the supernatant and store at –20°C until required; protein estimation should be between

368 2 and 4 μg/ml.

##### 369 2.6.	Indirect fluorescent antibody test

370 The indirect fluorescent antibody test (IFAT) (Dubey, 2022) is a simple and widely used method. Whole, killed

371 *Toxoplasma*tachyzoites are incubated with diluted test serum, the appropriate fluorescent labelled secondary

372 antibody is added, and the result is then viewed with a fluorescence microscope. Fluorescent-labelled species-

373 specific secondary antibodies are available commercially, the method is relatively inexpensive, and kits are

374 commercially available. However, the results are read by eye, so subjective variation may occur. It may be difficult to

375 find some species-specific conjugates and there is a risk of possible cross-reactivity with rheumatoid factor and anti-

376 nuclear antibodies. The following is a protocol for carrying out an IFAT for anti-*Toxoplasma*IgG antibodies in sheep

377 serum. It only requires minor modifications for testing different species or for measuring IgM antibody.

###### 378 2.6.1.	Test procedure

379 i)	Clean the required number of multi-well immunofluorescence assay slides (e.g. 10–21 well-

380 slides with wells of 4–6 mm in diameter are suitable) and allow to dry.

381 ii)	Apply 5 μl of a whole tachyzoite preparation (Section B.2.4.1 above) on to each well and allow

382 to air dry.

383 iii)	Fix in methanol for 10 minutes.

384 iv)	Wash twice (10 minutes for each wash) in 0.3 M PBS, pH 7.4.

385 v)	Prepare serial dilutions of the test sera in PBS (e.g. 1/16, 1/32, etc. up to 1/1024).

386 vi)	Add 5 μl of the given test sheep serum (diluted in PBS) to each well. Ensure that positive and

387 negative control sera are included in each test as well as a ‘PBS-only’ sample. Incubate for

388 30 minutes at room temperature.

389 vii)  Wash twice (10 minutes each time) in PBS.

390 viii) Add 5 μl of an appropriate dilution of rabbit-anti-sheep IgG conjugated to fluorescein

391 isothiocyanate, diluted in 0.2% filtered Evans blue dye in PBS (filtered through a 0.45 μm sterile

392 filter), to each well and incubate for 30 minutes at room temperature.

393 ix)	Wash three times for 10 minutes each time in PBS.

394 x)	Mount the slides under cover-slips with buffered glycerol (nine parts PBS, one part glycerol).

395 xi)	Examine using a fluorescence microscope, fitted with ×20 and ×40 objective lenses.

396 With a negative test serum result, the whole parasites will appear red due to the autofluorescence of

397 the Evans blue dye. They may also present with a green fluorescent cap at the parasite pole

398 (nonspecific polar fluorescence). With a positive test serum, the parasites will fluoresce red and at

399 least 80% of them within a given well will be surrounded by an unbroken band of green fluorescence.

400 In an adult sheep/goat a positive titre could be defined as ≥1/64 and a negative titre as ≤1/32. For

401 lamb/kid and fetal sera, respective titres could be defined as ≥1/32 and ≤1/16. These cut off values

402 should be validated locally as results may vary between laboratories, depending on, e.g., the

403 fluorescence microscope and the operator. Optimally, each slide should include positive control and

404 negative controls.

##### 405 2.7.	Modified agglutination test

406 The modified agglutination test (MAT) (Dubey, 2022) is both sensitive and specific. Formalinised *Toxoplasma*

407 tachyzoites are added to U-shaped well microtiter plates and dilutions of test sera are then applied. Positive samples

408 will produce agglutination that can be graded, whereas negative samples will produce a ‘button’ of precipitated

409 tachyzoites at the bottom of the well. The test is simple and easy to perform although relatively large amounts of

410 antigen are required. Kits are commercially available. It is important to treat sera with 0.2 2-mercaptoethanol to avoid

411 false positives due to non-specific IgM. The MAT has been used extensively for detection of *T. gondii*antibodies in

412 sera of many animal species and the procedure is detailed below. A commercially available latex agglutination test

413 is also available, but this test is regarded relatively insensitive compared with MAT or IFAT.

###### 414 2.7.1.	Serum-diluting buffer

415 i)	Dissolve 42.5 g NaCl, 1.54 g NaH2PO4, and 5.4 g Na2HPO4 in 900 ml deionised water.

416 ii)	Adjust the pH to 7.2. Bring the volume to 1 litre with deionised water.

417 iii)	Store in a refrigerator. This is the 5× stock solution.

418 iv)	Dilute this stock solution 1/5 to give 0.01 M PBS (working serum-diluting buffer: 1 part stock and

419 4 parts deionised water). PBS should be filtered through a 0.22 μM filter immediately prior to

420 use.

###### 421 2.7.2.	Antigen-diluting buffer

422 i)	Prepare a stock of borate buffer: dissolve 7.01 g sodium chloride, 3.09 g boric acid, 2.0 g sodium

423 azide in 900 ml deionised water.

424 ii)	Add 24 ml 1 N NaOH and adjust the pH to 8.9.

425 iii)	Bring the volume to 1 litre. This is the stock solution and can be stored at room temperature.

426 iv)	For the working antigen-diluting buffer, dissolve 0.4 g bovine serum albumin in 100 ml borate

427 buffer. Store at 4°C.

###### 428 2.7.3.	Serum dilutions

429 i)	Dilute serum samples with working serum-diluting buffer (Section B.2.7.1 above) in small test

430 tubes (1.2 ml in strips of 8 or 12) with a multichannel pipette, starting at 1/25.

431 Note: Microtiter plates may also be used for making serum dilutions.

###### 432 2.7.4.	Preparation of antigen mixture

433 i)	For each plate, mix 2.5 ml working antigen-diluting buffer (see Sections B.2.4.1 and B.2.7.2

434 above), 35 μl 2-mercaptoethanol, 50 μl Evans blue dye solution (2 mg/ml water) and 0.15 ml

435 antigen (formalin-fixed whole parasites).

###### 436 2.7.5.	Agglutination procedure

437 Agglutination is done in U-bottom 96-well microtiter plates.

438 i)	Pipette 25 μl antigen mixture to each well immediately after mixing.

439 ii)	Pipette 25 μl serum dilutions into the wells and mix gently with the antigen by repeated pipetting

440 action.

441 iii)	A positive control should be included in each plate. The control should have a titre of 1/200, and

442 two-fold dilutions from 1/25 to 1/3200 should be used.

443 iv)	Cover the plates with sealing tape and incubate overnight at 37°C.

444 v)	Read results using a magnifying mirror. A blue button at the bottom of the well means negative.

445 A clear bottom means positive.

##### 446 2.8.	Enzyme-linked immunosorbent assay

447 One of the first *T. gondii*enzyme-linked immunosorbent assay (ELISA) (Voller *et al*., 1976) used a soluble antigen

448 preparation made from *T. gondii*RH strain tachyzoites (as described below) and layered into wells in an ELISA

449 microtiter plate. Test sera are added, followed by a species-specific secondary antibody conjugated with a reactive

450 enzyme, such as horseradish peroxidase. Protein A/G conjugates were used to replace species-specific antibody

451 conjugates, making ELISAs applicable to more than one animal species. Any conjugated enzyme causes a colour

452 change in the substrate that is directly related to the amount of bound antibody, and which can be read with a

453 spectrophotometer at the absorbance wavelength specific to the substrate used. The assay is simple, can readily test

454 a large number of samples, and is easy to perform with the chosen anti-species conjugate. Defined anti-species

455 conjugates, substrates, and whole kits are commercially available. The ELISA is well suited for analysing large

456 numbers of samples. A large number of species-specific or multi-species ELISAs are commercially available to detect

457 *T. gondii*antibodies.

458 To improve the specificity of the conventional ELISA, native purified *T. gondii*-specific antigens have been used

459 (Basso *et al.*, 2013). In addition, severam recombinant antigens have been established, and these seem suitable for

460 replacing native antigens for serological diagnostic tests. For many of these recombinant antigen ELISAs, thorough

461 validation is lacking.

462 Clinically, there is a need to distinguish between recent (acute) and long-standing (chronic) infections. With the

463 conventional ELISA, detection of *Toxoplasma*-specific IgG and IgM antibodies, along with IgA, may permit some

464 discrimination between acute and chronic *T. gondii*infection. Assays assessing the avidity of an IgG response to

465 *T. gondii*have been applied in sheep and pigs. However, such avidity tests were used for research purpose only.

## 466	C. REQUIREMENTS FOR VACCINES

467 Currently there is only one commercially available live vaccine, which is licensed for use in breeding sheep in some regions

468 (Europe and New Zealand) to reduce the effects of *T. gondii*infection (e.g. early embryonic death, abortion). It consists of

469 ≥ 105 tachyzoites of the S48 strain of *T. gondii*that has been attenuated by multiple passages in mice. The vaccine

470 stimulates effective protective immunity for at least 18 months following a single intramuscular injection given at least 4

471 weeks prior to mating and only for use in healthy, non-pregnant female sheep. Despite the acknowledged importance of

472 human toxoplasmosis, human vaccines are currently unavailable, and the vaccine for sheep has disadvantages, such as

473 a short shelf-life (10 days), strict storage conditions, and, as a live vaccine, potential risk to operators. Information on the

474 production details of this vaccine and QC requirements are not available.

475 Although the importance of a killed or non-live vaccine is acknowledged – for vulnerable humans (e.g. women before they

476 are pregnant), for reducing abortions in sheep, for reducing tissue cysts in meat animals (pigs, cattle, chickens, etc.), and

477 for limiting oocyst shedding from kittens – to date this remains elusive (Innes *et al.,*2019). However, with recent scientific

478 advances, including availability of genetic, transcriptomic, and metabolomic data, the potential for developing knockout

479 variants, and other new technologies suggest new possibilities for development of such a vaccine (Mevelec *et al.,*2020;

480 Zhang *et al.*, 2022).

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555	*

556	* *

557	**NB:**At the time of publication (2024) there were no WOAH Reference Laboratories

558	for toxoplasmosis (please consult the WOAH Web site:

559	https://www.woah.org/en/what-we-offer/expertise-network/reference-laboratories/#ui-id-3).

560	**NB**: FIRST ADOPTED IN 2004. MOST RECENT UPDATES ADOPTED IN 2017.