Immunization of lambs with the S48 strain of Toxoplasma gondii reduces tissue cyst burden following oral challenge with a complete strain of the parasite

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Immunization of lambs with the S48 strain of Toxoplasma gondii reduces tissue cyst burden following oral challenge with a complete strain of the parasite Frank Katzer a,∗ , German Canton a,b , Alison Burrells a , Javier Palarea-Albaladejo c , Ben Horton a , Paul M. Bartley a , Yvonne Pang a , Francesca Chianini a , Elisabeth A. Innes a , Julio Benavides d a b c d

Moredun Research Institute, Pentlands Science Park, Bush Loan, Edinburgh EH26 0PZ, Scotland, UK Instituto Nacional de Tecnología Agropecuaria (INTA), EEA Balcarce CC276, Argentina Biomathematics & Statistics Scotland, The King’s Buildings, Edinburgh EH9 3JZ, Scotland, UK Instituto de Ganadería de Monta˜ na (CSIC-ULE), 24346 Grulleros (León), Spain

a r t i c l e

i n f o

Article history: Received 5 May 2014 Received in revised form 27 June 2014 Accepted 1 July 2014 Keywords: Toxoplasmosis Zoonosis Transmission Vaccine Ovine Tissue cysts

a b s t r a c t This study evaluates the influence of immunizing lambs with the incomplete S48 strain of Toxoplasma gondii, on parasite dissemination following a live oral challenge with a complete strain of T. gondii (M4). Lambs were culled at 14, 28 and 42 days post challenge. Parasite DNA was detected at significantly (p < 0.0001) lower levels in samples from the vaccinated/challenged group (0% in heart and 5.9% in skeletal muscles), when compared to the non-vaccinated/challenged animals (75% heart, 87.9% skeletal muscle). S48 T. gondii DNA was found in muscle or lymph nodes until 42 days post infection, suggesting that parasite DNA or tachyzoites could persist longer after immunization than previously thought. Non-vaccinated/challenged animals showed more frequent lesions in muscles and central nervous system than the vaccinated animals. These results demonstrate that vaccination of lambs with the incomplete S48 T. gondii strain, can protect against establishment of tissue cysts following challenge with a complete strain of T. gondii. Consumption of undercooked meat containing T. gondii cysts is a major route of transmission to people, therefore vaccination of food animals may improve the safety of meat for human consumption. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Toxoplasma gondii infection is widely distributed and poses a major health threat to many warm blooded animals and humans, causing abortions in pregnant females or severe disease in immunocompromised individuals (Dubey, 1996, 2009; Dubey and Jones, 2008). Toxoplasma

∗ Corresponding author. E-mail address: [email protected] (F. Katzer).

infection is very common, and causes significant economic losses, in the ovine livestock industry, as it is one of the major abortifacient agents affecting sheep (Buxton et al., 2007; Katzer et al., 2011). Although congenital infection, ingestion of infected tissues and ingestion of oocysts are the three main modes of transmission of T. gondii to humans (Remington et al., 1995; Dubey and Jones, 2008), the exogenous infection through contaminated food is considered as the main source of infection. Specifically, inadequately cooked meat (lamb, pork or game) infected with T. gondii was identified a major

http://dx.doi.org/10.1016/j.vetpar.2014.07.003 0304-4017/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Katzer, F., et al., Immunization of lambs with the S48 strain of Toxoplasma gondii reduces tissue cyst burden following oral challenge with a complete strain of the parasite. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.07.003

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Table 1 Experimental design of the study. Group

A B C D

Immunization and/or challenge

No animals culled per timepoint

−28 dpi immunization

0 dpi challenge

14 dpi

28 dpi

42 dpi

– Immunized Immunized –

5 × 105 oocysts 5 × 105 oocysts – –

4 4 2 1

4 4 2 1

4 4 2 1

risk for humans (Kapperud et al., 1996; Cook et al., 2000; Kijlstra and Jongert, 2008, 2009). Toxoplasma infection is widely prevalent in adult sheep and although the prevalence in lambs is not known, viable T. gondii tissue cysts are found in many edible tissues of congenitally infected lambs (Dubey and Kirkbride, 1989). Exposure to T. gondii may be increased in countries with a significant sheep or pig industry as outdoor reared pork and lamb carry a higher risk of infection than beef or poultry (Dubey, 1996; Cook et al., 2000). Animals that are kept outdoors, like sheep reared for meat production, may be at greater risk of environmental exposure than animals reared indoors (Kijlstra and Jongert, 2009). Vaccination has been proposed as the best method to control the occurrence of T. gondii abortions in sheep (Innes et al., 2009a,b), but also to reduce the rate of congenital infection in lambs (Wilkins et al., 1988), which would in turn result in a reduced risk of food-borne transmission of T. gondii to humans (Dubey, 1994; Innes et al., 2011). There is currently no non-viable, effective vaccine to prevent T. gondii infection and/or disease in animals or humans. Recently, it has been shown that immunization of lambs with ME-49, a complete T. gondii strain, with low virulent in mice, can protect against the formation of parasite cysts following a challenge with the M3 strain of Toxoplasma (Falcon and Freyre, 2009). Similar studies have been done to reduce the formation of tissue cysts in rats or pigs (Garcia et al., 2005; Freyre et al., 2008), showing that immunization with low-virulent strains of T. gondii confers more protection than immunization with killed subunit vaccines. A recent study has also shown that vaccination with double knock-out (mic1 and mic3 genes) tachyzoites of T. gondii confers a degree of protection against abortion in sheep and also reduces the burden of tissue cysts in congenitally infected lambs (Mevélléc et al., 2010). A vaccine to prevent Toxoplasma abortion in sheep is commercially available (Wilkins et al., 1988; Buxton, 1993; Buxton and Innes, 1995). This vaccine is based on the “incomplete” S48 strain, which is not able to form tissue cysts or oocysts, the main infective parasite stages. After inoculation into the host, the tachyzoites of the S48 strain undergo limited intracellular multiplication that primes a protective immune response (Innes et al., 2009b). Tissues from S48 immunized lambs did not transmit the infection to susceptible mice or cats if ingested (Wilkins et al., 1988). In pigs, a vaccine which was comprised of another stage-deficient Toxoplasma strain (RH strain), prevented the formation of tissue cysts in 58% of the pigs following challenge (Dubey et al., 1991; Kringel et al., 2004). The aim of this study was to investigate the effect of immunization of lambs with the S48 T. gondii strain on

the development of lesions and parasite distribution over different skeletal muscles of commercial importance and other tissues following oral challenge with the complete M4 isolate. The study also addresses whether this vaccination approach will reduce the T. gondii tissue cyst burden in vaccinated animals making their meat safer for human consumption. 2. Materials and methods 2.1. Animals, inoculum and experimental design Thirty-three Suffolk lambs aged 4 months, seronegative for T. gondii by ELISA (Buxton et al., 1988) were selected for the experiment. Until the beginning of the experimental challenge, the lambs remained with their mothers on pasture. One week before the start of the experiment, lambs were moved to containment facilities and were observed daily throughout the experiment, recording rectal temperature and clinical signs until 14 days post-infection (dpi), and then every 2 days thereafter until 29 dpi. Blood samples were collected twice weekly from 1 day before challenge until the end of the experiment by jugular venepuncture into 5 ml Venosafe® tubes (Terumo Europe) to monitor humoral immune responses. Lambs were randomly allocated into each experimental group and then each group was housed separately in pens until the time of culling. 2.2. Experimental design Lambs were randomly divided into 4 groups (Table 1). Twelve (Group B) and 6 (Group C) lambs were immunized intramuscularly with 1 × 105 tachyzoites of the S48 T. gondii strain, 4 weeks before the oral challenge with M4 oocysts. The 12 Group A and 3 Group D lambs remained unvaccinated. At day 0, lambs from Groups A and B were each dosed through a gastric tube with a suspension containing 500,000 sporulated T. gondii oocysts of the M4 isolate (as previously described by Benavides et al., 2011) in 5 ml of PBS. Groups C and D lambs remained un-challenged, receiving only PBS by the same route. The M4 isolate was obtained from a natural ovine abortion in 2004 and had been maintained since then by serial passage within mice (Benavides et al., 2011). Four animals from Groups A and B, 2 animals from Group C and 1 animal from Group D were culled at 14, 28 and 42 dpi using an intravenous barbiturate overdose and examined at post mortem (Table 1). All procedures were approved by the Moredun Research Institute experiments and ethics committee and complied fully with the regulations laid down by the Home Office of

Please cite this article in press as: Katzer, F., et al., Immunization of lambs with the S48 strain of Toxoplasma gondii reduces tissue cyst burden following oral challenge with a complete strain of the parasite. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.07.003

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Great Britain and Northern Ireland for compliance with the Animals (Scientific Procedures) Act 1986. 2.3. Collection of tissue samples Post mortem examinations of the lambs were carried out immediately after euthanasia and carcasses were examined for gross pathology. Central nervous system (CNS) samples from 10 different areas from frontal cerebral cortex to lumbar spinal cord were collected as previously described (Buxton et al., 1997). Samples from skeletal muscle with a high commercial value including chop (Longissimus dorsi), loin (Psoas major), right and left forelimb (Triceps femoralis), right and left hind limb (Semitendinosus), neck (Trapezius), as well as diaphragm and tongue were taken. Heart, lung, liver, kidney and lymph nodes (right prescapular and distal jejunal) were also sampled. Tissue samples, 2 cm × 2 cm approx., were collected and fixed in 10% buffered formaline solution during 5 days and later processed by standard histopathological methodologies for haematoxylin and eosin staining and immunohistochemistry (IHC). Samples of the same fresh tissues were frozen during post mortem examination, then stored at −20 ◦ C prior DNA extraction.

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During histopathological examination, numbers of glial foci and perivascular cuffs were recorded in each area of CNS studied for each animal. The same protocol was followed to evaluate the IHC labelled slides, where positively labelled T. gondii-like structures (tachyzoites and tissue cysts) were quantified in each section. An animal was considered as positive by IHC when positive labelling of tissue cysts or tachyzoites were found in at least one of its tissue sections.

2.7. DNA extraction DNA was extracted from approximately 1 g of homogenized tissue as described previously (Burrells et al., 2013; Bartley et al., 2013). Briefly, thawed tissue was transferred into a CK28 Precellys tissue homogenizer tube (Cepheid, Stretton Derbyshire, UK), containing 1 ml Nuclei Lysis Solution (Promega, Madison WI, USA). Samples were homogenized for 2× 50 s at 380 × g using a Precelys 24 tissue homogenizer (Cepheid). The homogenate was processed using the Wizard® genomic DNA purification protocol (Promega), and volumes were scaled up to allow for 1 g of starting material. The final DNA pellet was resuspended in 200 ␮l of DNase/RNase free water and stored at −20 ◦ C prior to PCR analysis.

2.4. Serology Collected blood samples were allowed to clot, serum was removed following centrifugation (800 × g) and stored at −20 ◦ C until they were tested using an IgG ELISA test (Buxton et al., 1988). Seroconversion was considered to have occurred when the optical density (OD) of a sample was greater than 25% of the OD of the standard high titre positive control serum (Buxton et al., 1988). Similarly, OD values for each group were averaged and a significant increase was considered when the mean value was above 25% of the initial (0 dpi) OD mean value. All animals included in the experiment were checked to be seronegative to T. gondii before any experimental procedure was applied. 2.5. Immunohistochemistry Sections were cut from the formalin-fixed tissues and were labelled for the detection of Toxoplasma antigen using a specific polyclonal antibody raised against T. gondii tachyzoites and bradyzoites (Buxton et al., 1988). The IHC procedure was carried out using Dako EnVision® + System-HRP (Dako North America Inc., Carpinteria, USA) as previously described by Benavides et al. (2011). 2.6. Quantification of histopathological lesions and IHC labelling Haematoxylin–eosin stained and IHC treated slides were randomized, blind-coded and examined under optical microscopy conditions using various magnifications (10×, 20× and 40×). To eliminate inter-operator error all slides were read by a single investigator.

2.8. Detection of T. gondii DNA by PCR Nested PCR targeting the multicopy internal transcribed spacer (ITS1) region of the parasite was used to detect T. gondii DNA. Primers and methodology were described by Burrells et al. (2013). Briefly, each 20 ␮l reaction contained 2 ␮l 10× PCR mix (45 mM Tris–HCl, 11 mM (NH4 )2 SO4 , 4.5 mM MgCl2 , 0.113 mg/ml BSA, 4.4 ␮M EDTA and 1.0 mM dATP, dATC, dGTC, dTTP), 0.75 units BioTaq (Bioline, London, UK), and 2 ␮l DNA. First round reactions contained 0.05 ␮M of forward primer and reverse primers NN1 5 -TCAACCTTTGAATCCAAA-3 , NN2 5 -CGAGCCAAGACATCCATT-3 (Buxton et al., 1997), reactions were made to a final volume of 20 ␮l with DNase/RNase free water. To improve the sensitivity of the technique, each reaction was carried out in triplicate. Cycling conditions were 5 min at 95 ◦ C, followed by 35 cycles of 1 min at 95 ◦ C, 1 min at 55 ◦ C, 1 min at 72 ◦ C, and a final extension period of 5 min at 72 ◦ C. First round PCR products were diluted 1:5 with dH2 O, 2 ␮l of diluted product was used in the second round reaction. The conditions for each second round were identical to the first however contained 0.05 ␮M of internal forward and reverse primers Tg-NP1 5 -GTGATAGTATCGAAAGGTAT-3 , Tg-NP2 5 -ACTCTCTCTCAAATGTTCCT-3 (Hurtado et al., 2001). The nested PCR products (10 ␮l) were electrophoresed on a 2% (w/v) agarose gel incorporating Gel RedTM (Biotum, Hayward, CA, USA), and visualized under UV light. A sample was regarded positive if an amplicon of 227 bp was present in any triplicate reaction. A positive and multiple negative controls (dH2 O) were included in each PCR run and results were only accepted if no product was amplified in these reactions.

Please cite this article in press as: Katzer, F., et al., Immunization of lambs with the S48 strain of Toxoplasma gondii reduces tissue cyst burden following oral challenge with a complete strain of the parasite. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.07.003

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2.9. Genetic characterization of T. gondii using SAG3 and GRA6 nested PCR-RFLP Genetic characterization was performed using two markers SAG3 (Prestrud et al., 2008) and GRA6 (Su et al., 2006; Prestrud et al., 2008) which are spread over two different loci of the T. gondii genome. The first round of each 20 ␮l multiplex PCR reaction contained 2 ␮l 10× custom PCR mix (45 mM Tris–HCl. 11 mM (NH4 )2 SO4 , 4.5 mM MgCl2 , 0.113 mg/ml BSA, 4.4 ␮M EDTA and 1.0 mM dATP, dATC, dGTP, dTTP), 0.10 ␮M of forward and reverse external primers for both SAG3 and GRA6, 0.75 units BioTaq (Bioline), 5.85 ␮l dH2 O and 2 ␮l DNA. To improve the sensitivity of the technique each reaction was carried out in quadruplicate. First round cycling conditions were 4 min at 95 ◦ C, followed by 25 cycles of 30 s at 94 ◦ C, 1 min at 53.7 ◦ C, and 2 min at 72 ◦ C. First round PCR products were diluted 1:1 with dH2 O. The nested second round reaction used separate internal primers for each marker and used the diluted first round PCR products instead of DNA. Each individual 20 ␮l second round reaction contained 2 ␮l 10× custom PCR mix (see above), 0.30 ␮M primers (individual forward and reverse internal markers for SAG3 and GRA6), 0.75 units BioTaq (Bioline), 13.85 ␮l DNase/RNase free water, and 2 ␮l of diluted first round PCR product. Cycling conditions were 4 min at 95 ◦ C, followed by 40 cycles of 30 s at 94 ◦ C, 1 min at 60 ◦ C, and 1.5 min at 72 ◦ C. A positive control and multiple negative controls were incorporated amongst samples to be genotyped. RFLP typing of positive samples was achieved by preparing an enzyme digest which contained 2 ␮l of the second round product, 2 ␮l of 10× NEB Buffer 4 (New England Biolabs, Herts, UK), 4 units of the appropriate restriction enzyme as described by Prestrud et al. (2008), made up to a final volume of 20 ␮l in dH2 O. Each digest was incubated for 1 h at the appropriate temperature for the restriction enzyme. Digested products were visualized by electrophoresis using a 3% Metasieve Agarose (Flowgen Bioscience) gel incorporating Gel RedTM (Biotum). PCR-RFLP patterns were compared to those of control reference strains B1 (Type I), M4 (Type II) and NED (Type III) (B1 and NED obtained from BRC Toxoplasma, Limoges, France).

effects and the smooth terms. Besides, pairwise comparisons between experimental groups were performed by defining adequate tests of linear contrasts, with the associated p-values being adjusted for false discovery rate (FDR). Statistical significance was assessed at the 5% level in all cases. In the serological data analysis, pre-inoculation OD values per group were compared by heterocedastic ANOVA fitted using generalized least squares. Then, tests of linear contrasts (FDR-adjusted p-values) were performed to compare subsets of groups of interest. The same approach was applied to compare OD responses after 21 days post-inoculation. In this case, a first order autoregressive residual correlation structure was fitted to account for the temporal component. Finally, the association between experimental groups and the output of the PCR analysis (counts of positive/negative cases) was tested using a Fisher’s exact test. Correspondence analysis was used to investigate the nature of the association. 3. Results 3.1. Clinical observations Mean rectal temperature of the M4 T. gondii orally challenged lambs (Groups A and B) showed an increase by 3 dpi (Fig. 1). Group B mean temperature returned to control levels (below 40.5 ◦ C) by 4 dpi while Group A mean temperature increased and peaked by 5–6 dpi. This was followed by a fall of the mean temperature reaching control temperature on 10 dpi. A second small temperature peak was detected in Group A lambs at 15 dpi (Fig. 1). The mean temperature of the A group was statistically higher than any of the others (p < 0.0001). Although less evident, statistically significant differences in mean temperatures were also found, between group B and groups C and D (p < 0.0257). During the febrile period (4–10 dpi), the lambs in group A appeared subdued and had an increased breathing rate. Among the non-challenged lambs (Groups C and D), mean rectal temperature showed a statistically constant trend at physiological levels throughout the monitoring period, with no significant differences (p > 0.2100).

2.10. Statistical analysis 3.2. Serology Generalized additive models (GAM) were used to model rectal temperature and serological data trends over time. Restricted maximum likelihood (REML) was used as estimation method, and model selection was based on the Akaike’s information criteria (AIC). For rectal temperatures, the model included experimental groups (A–D) as main effect and separate spline-based smooth terms for each group in order to fit non-linear relationships of the response with time. A first order autoregressive structure accounted for serial residual correlation. Finally, heterogeneous within-group variances were allowed. For serological data, in addition to the above, the model included baseline (pre-inoculation) optical density (OD) values as main effect. From the model estimates, analysis of variance (ANOVA) based on the F-statistic was carried out to determine the statistical significance of the main

Pre-challenged (0 dpi) combined mean OD for Groups A and D animals (non-vaccinated) was statistically significantly lower (p < 0.0001) compared with Groups B and C (vaccinated), demonstrating that the vaccine administration (4 weeks before challenge) effectively induced specific T. gondii antibody production (Fig. 2). Statistically significant overall differences in mean OD values during the period were observed between all four groups (p < 0.0035). Between 10 and 14 dpi mean antibody titres increased steadily until 21 dpi (peak of 9.45-fold increase from baseline at 21 dpi) in the Group A lambs, and remained increased until the last animal of this group was culled. Group B lambs showed an increase of more than 25% of the initial mean OD value after 7 dpi and remained elevated until the end of the experiment (peak of 2.35-fold

Please cite this article in press as: Katzer, F., et al., Immunization of lambs with the S48 strain of Toxoplasma gondii reduces tissue cyst burden following oral challenge with a complete strain of the parasite. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.07.003

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Fig. 1. Mean rectal temperatures of lambs. Mean maternal rectal temperatures (± standard error bars) over time for challenged (groups A and B) and un-challenged lambs (groups C and D). Group A (non-vaccinated and challenged) showed elevation of temperature from 4 to 10 dpi and a second peak at 15 dpi, while Group B only showed one peak of high temperature at 3 dpi. Mean temperatures of Group C (vaccinated, non challenged) and Group D (non-vaccinated, non-challenged) control animals remained low for the whole experiment.

increase at 27 dpi). From 21 dpi, no statistically significant differences between groups A and B (p = 0.4867) were observed. Mean OD Toxoplasma ELISA values of Group C lambs achieved an increase of 25% of the 0 dpi value at 14 dpi, with two peaks of mean values at 17 and 38 dpi (peak of 1.59 and 1.62-fold increase, respectively). Lastly, mean OD values for the Group D lambs remained at the same low levels (below positive cut off value) during the duration of the experiment. 3.3. Detection of T. gondii DNA by PCR The results of PCR detection of T. gondii are summarized in Table 2. 3.3.1. CNS Two different tissue samples from internal capsule and hypothalamus collected from each lamb of Groups A–D were analyzed by PCR in triplicate. At least one T. gondii positive sample was detected in 11 out of 12 lambs from Group A (91.7% of animals). The highest percentage of positive samples was found at 14 dpi, and from there on, the percentage declined towards the end of the experiment. No PCR positive results were obtained in any of the CNS samples collected from the Groups B–D lambs.

3.3.2. Heart and skeletal muscle All the Group A lambs, with the exception of one culled at 42 dpi tested PCR positive for T. gondii in the heart samples. From all the analyzed myocardial samples (tested in triplicate for each sample), 75% of PCRs were positive, showing a similar tendency as the CNS samples, where the highest percentage of positive reactions were found at 14 dpi, and from then on, the percentage decreased. All the hearts of Groups B–D tested negative by PCR. Skeletal muscles samples analyzed by PCR were positive for T. gondii DNA in all Group A animals (similarly to CNS and heart samples, each of the 9 muscle samples collected from each lamb was analyzed in triplicate). A high frequency of positive reactions was observed among the samples collected from Group A lambs culled at 14 and 28 dpi, and a reduction was observed in the lambs culled in this group at 42 dpi (92.59%, 97.22% and 74.07%, respectively). No clear differences were observed among the studied muscles of the lambs from this group, showing a similar percentage of T. gondii positivity. When the muscles from Group B lambs were analyzed, at least one positive sample was found in 9 out of the 12 lambs; however, the percentage of positive PCR reactions was significantly reduced when compared to Group A, with only 6.17% of the analyzed muscles testing positive

Please cite this article in press as: Katzer, F., et al., Immunization of lambs with the S48 strain of Toxoplasma gondii reduces tissue cyst burden following oral challenge with a complete strain of the parasite. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.07.003

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Fig. 2. Mean OD values for ELISA. Mean OD values for IgG ELISA. Pre-challenged (0 dpi) combined means OD for Groups A and D animals (un-vaccinated) were lower than Groups B and C (vaccinated). Between 7 and 21 dpi mean antibodies titres increased in the challenged groups (A and B), and then remains steady. Group C (vaccinated, non-challenged) mean OD increases from 0 to 14 dpi and OD from Group D (non-vaccinated, non-challenged) control animals remained low for the whole experiment.

for T. gondii. In this group, in contrast to group A, the highest percentage of positive reactions (7.40%) was found at 42 dpi. Most of these positive results from Group B lambs were obtained in Trapezius samples. Four out of the 6 lambs from Group C tested positive in at least one of the muscular samples, although a very low detection rate was observed (3.08%). in this case, the highest percentage was found at 28 dpi. All the Group D lambs were PCR negative (Table 2).

3.3.3. Lymph nodes Positive T. gondii PCR results were obtained in distal jejunal and pre scapular lymph nodes collected from all Group A lambs culled at 14, 28 and 42 dpi (88.9% of 72 PCRs were positive). Positive results were also observed in the lymph nodes of 8 out of the 12 lambs from the Group B (27.8% positive reactions). When lymph nodes from Groups C and D lambs were analyzed by PCR, only 1 pre-scapular

Table 2 Percentage (%) of positive samples to ITS1 PCR in the different samples studied. Group

dpi

Localization CNS

Lymph nodes

Heart

Skeletal muscles

Lung

Liver

Kidney

A

14 28 42

81.25 62.50 54.17

100.00 83.33 83.33

91.67 83.33 50.00

92.59 97.22 74.07

25.00 25.00 0.00

66.67 58.33 8.33

50.00 50.00 16.67

B

14 28 42

0.00 0.00 0.00

20.83 45.83 16.67

0.00 0.00 0.00

4.63 4.63 10.19

0.00 0.00 0.00

16.67 8.33 0.00

8.33 8.33 16.67

C

14 28 42

0.00 0.00 0.00

8.33 0.00 0.00

0.00 0.00 0.00

1.85 5.56 1.85

0.00 0.00 0.00

0.00 0.00 25.00

0.00 0.00 16.67

D

14 28 42

0.00 0.00 0.00

0.00 0.00 0.00

0.00 0.00 0.00

0.00 0.00 0.00

0.00 0.00 0.00

0.00 0.00 0.00

0.00 0.00 0.00

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lymph node sample collected from a Group C lamb was positive. 3.3.4. Liver, kidney and lung In liver samples collected from 8 out of 12 Group A lambs, T. gondii DNA was found, with positive PCR reactions decreasing from 66.67% at 14 dpi to less than 10% at 42 dpi. One out of the four liver samples collected from Group B lambs culled at 14 and 28 dpi tested T. gondii DNA positive, while at 42 dpi these were negative. In group C only samples from one of the lambs culled at 42 dpi tested positive. All hepatic samples from Group D lambs were T. gondii negative. Positive reactions were found in the kidney at the three dpi in groups A and B. However, while the frequency of positive results decreased towards the end of the experiment in group A, the opposite occurred in group B. In any cases, the frequency of T. gondii detection was always equal or, more commonly, lower in group B than in group A. Also one animal from Group C was PCR positive in its kidney at 42 dpi and all kidney samples of the 3 Group D lambs were negative. In the lung, positive amplification was found only in 2 out of 12 lambs from group A (1 at 14 dpi and 1 at 28 dpi). No lung samples from Groups B, C or D were positive. Focusing on the counts of positive and negative cases out of the total number of PCR reactions carried out (507, 48, 11 and 0 positives out of, respectively, 684, 684, 642 and 171 samples for groups A–D), statistical evidence of association between group and identification of parasite DNA by PCR was found (p < 0.0001). In particular, correspondence analysis revealed a strong direct bond between PCR negative cases and groups B (vaccinated and challenged), C (vaccinated and non-challenged) and D (non-vaccinated, non-challenged). 3.4. Genetic characterization of T. gondii using SAG3 and GRA6 nested PCR-RFLP Genotyping was carried out on selected tissue samples collected from Groups B and C lambs in order to genetically characterize the T. gondii strain identified, since these animals had been vaccinated with the type I strain (S48) and some were later challenged with the M4 strain, a type II strain. T. gondii genotype I (strain S48 present in the vaccine) and III (neither present in the vaccine or challenge) were identified in the Semitendinosus sample collected from a Group B lamb culled at 42 dpi where T. gondii DNA was amplified. Genotype III of T. gondii was identified in Trapezius and diaphragm samples from a Group C lamb culled at 28 dpi. Genotype II (M4 strain used for the challenge) was identified in distal jejunal and prescapular lymphnodes from Group B lambs culled at 14 and 28 dpi and Genotype I was present in the distal jejunal lymphode of one Group B lamb culled at 42 dpi. Genotype II was also typified in the kidney sample from one Group B lamb culled at 42 dpi. 3.5. Histopathology and IHC CNS. Glial foci (characterized by focal infiltrates of inflammatory and glial cells, Fig. 3a) and perivascular cuffs

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(Fig. 3b) were quantified in each of the CNS samples collected during the post mortem examination. All samples taken for histopathology were also treated by immunohistochemical, labelling of T. gondii antigen, for identification of structures compatible with parasite tissue cysts (Fig. 3c) or tachyzoites (Fig. 3d). Lesions were present in all groups, but they were more frequent in groups A and B. In both these groups lesions (glial foci and perivascular cuffs) were more frequent in the frontal and temporal cortex, hypothalamus and hippocampus (additional file 1). There was no clear difference in the distribution of the lesions within the CNS among the different time points studied (i.e. 14, 28 and 42 dpi) or groups, nor in the distribution of the IHC positive signal (data not shown). Results of the quantification of lesions in the CNS are shown in Table 3. Group A showed the highest number of glial foci, when compared with the rest of the groups, occurring at 14 dpi. From there, the number decreased in the subsequent time points. Perivascular cuffs showed an inverse pattern to glial foci in this group as they were scarce at 14 dpi but increased towards the end of the experiment. The number of animals from group A where tissue cysts were observed under IHC labelling was higher at 14 dpi than on other days, while tachyzoites were observed in the same number of animals (25%) at the three time points (Table 3). The number of tissue cysts was variable, between 1 and 5 per animal, and no more than in 2 areas, out of the 10 studied, were positive in each animal. Clear differences in the frequency of observed lesions were detected for Group B lambs in comparison to Group A lambs. Both glial foci and perivascular cuffs observed at 14 dpi were scarcer when compared with the quantity at the same time point in the Group A animals. Then, an increase in the number of both types of lesions was recorded at 28 dpi. Finally, at 42 dpi the number of both lesions returned the similar numbers as at 14 dpi. Also, less positive labelled structures were found in Group B, either tissue cysts or tachyzoites, observed using the T. gondii IHC, finding only a few tissue cysts at 14 and 28 dpi, as well as tachyzoites only observed in mesencephalon of one of the lambs culled at 28 dpi. The number of perivascular cuffs and glial foci quantified in the CNS samples collected from the Groups C and D lambs remained at low levels at 14, 28 and 42 dpi (Table 3). No positive labelled T. gondii-like structures were observed in any of the samples collected from the Groups C and D lambs. 3.5.1. Heart and skeletal muscle Small foci characterized by the infiltration of mononuclear cells were recorded in the heart and skeletal muscle samples collected from all the lambs regardless of the group. All the groups showed a tendency towards fewer foci of inflammation as the experiment progressed, except group C, where no clear tendency was observed. Positive labelling for parasite structures was found in lambs from Groups A (50% of studied animals) and B (8% of studied animals). This labelling was mostly of tissue cysts at 14 dpi in group A, and the only positive animal from group B, where no tachyzoites were observed in the muscles of any animal. Tachyzoites were found only in the heart sample from 1 animal from group A at 14 dpi. No positive labelling was

Please cite this article in press as: Katzer, F., et al., Immunization of lambs with the S48 strain of Toxoplasma gondii reduces tissue cyst burden following oral challenge with a complete strain of the parasite. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.07.003

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Fig. 3. Lesions found in the brain of challenged lambs and Toxoplasma gondii specific labelling by immunohistochemistry. (a) Brain. Glial foci characterized by the focal accumulation of glial cells, lymphocytes, macrophages and plasma cells. H/E; (b) brain. Perivascular cuff denoted by the accumulation of mononuclear inflammatory cells in the Virchow–Robin space. H/E; (c) brain. Tissue cysts positively labelled against toxoplasma antigens. IHC, (d) lymph node. Intracelular parasitophorous vacuole containing a well-deliniated tachyzoite-like structure. IHC.

found in any of the samples studied from Groups C or D. Tissue cysts compatible with Sarcocystis spp. were observed in the samples of heart and muscle from all studied animals. PCR analysis on these samples confirmed that lambs were infected with Sarcocystis tenella (data not shown).

studied animals), and only a few tachyzoites in one animal culled at 28 dpi. Positively labelled tachyzoites were only observed in 1 lymph node of one lamb from Group B and another lamb from Group C, both culled at 28 dpi. All the other lymph nodes samples were IHC negative.

3.5.2. Lymph nodes Both lymph nodes tested from each of Group A animals, showed mild thickening of the cortex with an increased number of reactive lymphoid follicles (mild follicular hyperplasia). No changes were observed in the lymph nodes from the other groups. Immunohistochemical labelling showed pseudo cysts or parasitophorous vacuoles of T. gondii only in the distal jejunal lymph node collected from one of the Group A lambs culled at 14 dpi. Single tachyzoites were labelled in several lymph node samples collected from lambs from Group A culled at 14 dpi (75% of

3.5.3. Liver, kidney and lung Small foci of non suppurative hepatitis were observed mainly in the tissue samples collected from Group A at 14 dpi and Group B at 14 and 28 dpi. Lambs from Groups C and D showed scarce small foci of non suppurative inflammation with no clear differences among the studied time-points. Similarly, a mild non purulent interstitial nephritis was observed in a number of animals from all the Groups. No evident lesions were found in the lungs. No positive T. gondii like structures were detected using IHC in the liver, kidney or lung samples.

Table 3 Average number of lesions (glial foci and perivascular cuffs) and percentage of animals with positive immunohistochemical labelling for toxoplasma antigen (tissue cysts and tachyzoites) in the CNS of each group. Group

A B C D a b

HE

IHC

14 dpi

28 dpi

42 dpi

14 dpi

28 dpi

42 dpi

13.5/11.5a 1.25/1.25 0.5/4 1/1

2.75/17.25 8/26.25 0.5/1 2/4

1.75/23 1.25/3.25 1/6 0/0

75/25b 25/0 0/0 0/0

50/25 25/25 0/0 0/0

50/25 0/0 0/0 0/0

Average number of glial foci/perivascular cuffs. % of animals within the group positive for immunohistochemically labelled tissue cysts/tachyzoites.

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4. Discussion This study shows that vaccination of lambs with the incomplete S48 strain of T. gondii reduces the parasite burden and formation of tissue cysts following an oral challenge with a virulent strain. These results show that it may be possible to use vaccination to reduce the risk of transmission of T. gondii to humans through consumption of undercooked meat. The effect of vaccination is relevant to public health, as insufficiently cooked T. gondii infected meat is a major source of infection for humans and sheep are the most frequently infected animal among commonly farmed livestock species (Kijlstra and Jongert, 2008). The results from our study are comparable to those of previous experiments in pigs with another incomplete (RH), self-limiting strain of T. gondii (Dubey et al., 1991; Kringel et al., 2004). Results from these combined studies show that vaccination with an incomplete strain of T. gondii (unable to form cysts) reduces, but does not completely prevent, the formation of tissue cysts following a further infection with a complete strain. Previous studies have found that vaccination of pigs with a subunit vaccine, based on crude rhoptry proteins, showed less protection than immunization with an incomplete strain (RH) against tissue cyst formation (Garcia et al., 2005). The use of live complete strains as a vaccination strategy seems to be more successful in preventing tissue cyst formation. Studies carried out in lambs and rats have shown that a complete strain of T. gondii with low virulence in mice (ME-49) is able to reduce significantly the formation of tissue cysts by a virulent strain in a subsequent infection (Freyre et al., 2008; Falcon and Freyre, 2009). Considering these results, it is tempting to hypothesize, that the lack of complete protection, with the incomplete strains could be related to the absence of a bradyzoite stage in the vaccination strain. Thus, the use of a complete strain of T. gondii for immunization would allow the host immune system to process and respond to a larger variety of antigens, especially those related to the bradyzoite and tissue cysts stages, before the infection with the virulent strain as other authors have suggested (Falcon and Freyre, 2009). Several field tests have shown that vaccination with the attenuated vaccine based on the S48 strain of T. gondii significantly reduces the occurrence of abortions in sheep exposed to a second infection during pregnancy (Wilkins et al., 1988; Buxton, 1993). Furthermore, it was found by bioassay in cats and mice that this vaccine did not persist in the tissues of lambs from day 10 after vaccination (Wilkins et al., 1988; Buxton, 1993; Buxton and Innes, 1995). However, genetic characterization of two PCR positive samples from one vaccinated and challenged animal, culled 6 wpi, showed the DNA amplified belonging to genotype I parasites, most probably from the S48 strain used for vaccination. The higher sensitivity of the current PCR technique used in this study, together with the fact that PCR amplification detects DNA, and does not determine the viability or stage of the parasite, may be a plausible explanation for this discrepancy as the bioassay in mice or cat only detects viable parasites. Furthermore, immunohistochemical labelling showed the presence of tachyzoite-like structures in the lymph nodes of animals belonging to both

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vaccinated groups (challenged and unchallenged), suggesting that the frequent detection by PCR may be due to the presence of tachyzoites still multiplying at different localizations in the host. The fact that S48 parasites remain in the organism longer than previously described should not constitute any risk of parasite transmission from vaccinated animals to humans, as tachyzoites, the probable stage of the S48 parasite present in the organism, are not infectious by oral route. Further studies on pigs, carried out with a similar experimental design, and testing through bioassay for infectivity of muscle from vaccinated-non challenged piglets have shown them to be non infectious (Burrells et al., in preparation). Parasite DNA was amplified in five out of six of the vaccinated unchallenged animals, but the only positive sample with enough amplified DNA to perform genotyping was identified as genotype III, which was neither used in the challenge or vaccine inocula. Genotype III T. gondii has been recently identified in samples from wild animals in Great Britain (Burrells et al., 2013), which shows that this genotype is circulating and it is plausible that the experimental animals were exposed to it potentially through contaminated fodder. Another possibility, is that some animals were infected before the beginning of the study, despite the seronegative status prior to vaccination. It has been previously described that ELISA values for serological antibodies can drop after five weeks post-infection in pigs inoculated with genotype III parasites (Pardini et al., 2012). Histological analysis was most informative in samples from the brain, as there were many mild, non-specific lesions in the muscles of animals from all groups, most probably produced by Sarcocystis spp. infection, which is very frequent among sheep. Lesions in the brain (i.e. glial foci and perivascular cuffs) were detected earlier (14 dpi) in the non vaccinated challenged lambs, and the perivascular cuffs were clearly more frequent in this group at 42 dpi when compared to vaccinated challenged group. Similar findings have been described in mice, where fewer lesions and tissue cysts were described in the brains of vaccinated mice when compared to non-vaccinated (Parmley et al., 2002). As these authors suggested, the most probable interpretation is that vaccination reduces the dissemination, so fewer parasites reach the brain or muscles. The parasite burden, determined by the number of PCR positive results in the brain and other tissues, observed in the current study was clearly higher in the non vaccinated lambs, supporting this hypothesis. As the lambs in this study have been immunized with tachyzoites of the S48 strain, which could not develop tissue cysts, it is likely that the immune response in the vaccinated lambs was able to reduce the number of circulating tachyzoites. The fact that all the challenged lambs, both vaccinated and non vaccinated, reach similar ELISA OD values for specific antibodies suggests that their immune system might have been exposed to a similar dose of parasites. Thus, it is tempting to hypothesize that the immunization with S48 strain did not interfere with the colonization of the enterocytes and globet cells by the sporozoites. Instead, the immune response would attack the parasite in a subsequent stage, as an intracellular tachyzoite, while it disseminates through the host.

Please cite this article in press as: Katzer, F., et al., Immunization of lambs with the S48 strain of Toxoplasma gondii reduces tissue cyst burden following oral challenge with a complete strain of the parasite. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.07.003

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We report in this study that parasite burden can be effectively reduced in lambs, orally challenged with a complete strain T. gondii by prior vaccination with S48 strain tachyzoites. Although vaccination did not completely prevent tissue cyst formation following challenge, as a number of tissue samples from vaccinated animals carried T. gondii DNA, it still remains to be elucidated whether these samples would be infective or not, as the presence of parasite DNA does not necessarily provide evidence for the presence of viable parasite. Further studies, including infectivity assessment by bioassay are currently underway to address this issue. This study shows that immunization of lambs with the live incomplete S-48 strain of T. gondii is an effective and feasible strategy to reduce parasite burden in meat and thus, reduce the risk to public health. 5. Conclusion Vaccination of lambs with the incomplete strain S48 of T. gondii does not prevent infection following challenge but significantly reduces T. gondii burden in the tissues. Acknowledgements B1 and NED obtained from BRC Toxoplasma, Limoges, France. FK, LI, FC, PMB, YP, BH were supported by the Scottish Government, Rural and Environmental Sciences and Analytical Services (RESAS). This project was partially funded by the European Union Seventh Framework Network of Animal Disease Infectiology Research Facilities (NADIR; reference number FP7-228394). JB was supported by CSIC through the JAE-Doc program, financed in part by European Social Fund (ESF); AB, by the Moredun Foundation and GC by the Instituto Nacional de Tecnología Agropecuaria (INTA), Argentina. References Bartley, P.M., Wright, S.E., Zimmer, I.A., Roy, S., Kitchener, A.C., Meredith, A., Innes, E.A., Katzer, F., 2013. Detection of Neospora caninum in wild carnivorans in Great Britain. Vet. Parasitol. 192 (1–3), 279–283, http://dx.doi.org/10.1016/j.vetpar.2012.10.001. Benavides, J., Maley, S., Pang, Y., Palarea, J., Eaton, S., Katzer, F., Innes, E.A., Buxton, D., Chianini, F., 2011. Development of lesions and tissue distribution of parasite in lambs orally infected with sporulated oocysts of Toxoplasma gondii. Vet. Parasitol. 179 (1–3), 209–215, http://dx.doi.org/10.1016/j.vetpar.2011.03.001. Burrells, A., Bartley, P.M., Zimmer, I.A., Roy, S., Kitchener, A.C., Meredith, A., Wright, S.E., Innes, E.A., Katzer, F., 2013. Evidence of the three main clonal Toxoplasma gondii lineages from wild mammalian carnivores in the UK. Parasitology, 1–9, http://dx.doi.org/10.1017/S0031182013001169. Burrells, A., Benavides, J., Canton, G., Garcia, J.L., Bartley, P.M., Nath, M., Thomson, J., Chianini, F., Innes, E.A., Katzer, F. Vaccination of pigs with the S48 strain of Toxoplasma gondii – safer meat for human consumption, in preparation. Buxton, D., 1993. Toxoplasmosis: the first commercial vaccine. Parasitol. Today 9, 335–337. Buxton, D., Blewett, D.A., Trees, A.J., McColgan, C., Finlayson, J., 1988. Further studies in the use of monensin in the control of experimental ovine toxoplasmosis. J. Comp. Pathol. 98 (2), 225–236. Buxton, D., Innes, E.A., 1995. A commercial vaccine for ovine toxoplasmosis. Parasitology 110 (Suppl.), S11–S16. Buxton, D., Maley, S.W., Thomson, K.M., Trees, A.J., Innes, E.A., 1997. Experimental infection of non-pregnant and pregnant sheep with Neospora caninum. J. Comp. Pathol. 117 (1), 1–16.

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