Veterinary Parasitology 210 (2015) 141–144
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Study on the use of toltrazuril to eliminate Neospora caninum in congenitally infected lambs born from experimentally infected ewes S.S. Syed-Hussain a,b,∗ , L. Howe b , W.E. Pomroy b,∗ , D.M. West b , M. Hardcastle c , N.B. Williamson b a Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia b Institute of Veterinary Animal and Biomedical Sciences, Massey University, Palmerston North 4412, New Zealand c Gribbles Veterinary Pathology, PO Box 12049, Penrose, Auckland 1642, New Zealand
a r t i c l e
i n f o
Article history: Received 11 August 2014 Received in revised form 19 March 2015 Accepted 23 March 2015 Keywords: N. caninum Toltrazuril Treatment Congenital transmission Lambs Sheep
a b s t r a c t To determine if toltrazuril was effective in eliminating Neospora caninum infection from congenitally infected lambs. Twenty-eight ewes were allocated to 3 groups where animals in Groups A and B were inoculated with 1 × 107 N. caninum tachyzoites on Day 120 of gestation and Group C was maintained as a negative control group. Lambs born from ewes in Group A were treated with toltrazuril (20 mg/kg) on Days 0, 7, 14 and 21 after birth. Lambs in Groups B and C were untreated. All lambs in Groups A and B were seropositive at 12 weeks of age. At 12 weeks of age, no differences between lambs in Group A and Group B were observed in serological results (ELISA and western blot), presence of N. caninumrelated brain histopathological lesions or the number of organisms detected by qPCR. Group C remained negative for serology, detection of N. caninum DNA as well as histopathology throughout the study. Results indicate that N. caninum congenitally-infected lambs had a continuing infection with N. caninum despite being treated with toltrazuril. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Chemotherapy has the potential to be an effective control measure for neosporosis (Hasler et al., 2006) provided the drug used is effective. Toltrazuril, a symmetrical triazinone, is metabolized into ponazuril (toltrazuril sulfone). Both toltrazuril and ponazuril are effective against a broad spectrum of cyst-forming and non-cyst-forming
∗ Corresponding authors at: Institute of Veterinary Animal and Biomedical Sciences, Massey University, Palmerston North 4412, New Zealand. Tel.: +6463569099x85161; fax: +6463505699. E-mail addresses: [email protected] (S.S. Syed-Hussain), [email protected] (W.E. Pomroy). http://dx.doi.org/10.1016/j.vetpar.2015.03.019 0304-4017/© 2015 Elsevier B.V. All rights reserved.
apicomplexan protozoa (Haberkorn, 1996) including positive effects against N. caninum in vitro (Darius et al., 2004; Strohbusch et al., 2008) and in vivo, both in mice (Strohbusch et al., 2009) as well as in experimentally and naturally infected calves (Haerdi et al., 2006; Kritzner et al., 2002). However, in order for toltrazuril to reach its full effectiveness in clearing N. caninum infection, one study has shown that a supportive T cell-immune response is necessary (Ammann et al., 2004). Regardless, no chemotherapeutic approach has been shown to be safe and fully effective for the treatment of neosporosis in cattle (Dubey and Schares, 2011). One approach has been to treat neonates to eliminate the organism following congenital infection. The aim of this present study was to determine if toltrazuril was effective
142
S.S. Syed-Hussain et al. / Veterinary Parasitology 210 (2015) 141–144
in eliminating N. caninum infection from congenitally infected lambs.
using Minitab (Minitab Inc., PA, USA) and all statistical analyses were considered significant when p < 0.05.
2. Materials and methods
3. Results
Twenty-eight ewes were allocated to 3 groups where animals in Groups A (n = 11) and B (n = 12) were inoculated with 1 × 107 N. caninum tachyzoites on Day 120 of gestation and Group C (n = 5) was maintained as a negative control group. Lambs born from ewes in Group A (n = 12) were treated with toltrazuril (20 mg/kg) on Day 0, 7, 14 and 21 after birth. Lambs in Groups B (n = 10) and C (n = 7) were untreated. The ewes were approximately four years old, had been vaccinated for Toxoplasma gondii and Campylobacter fetus fetus and were free from N. caninum as indicated by negative serology using ELISA (Chekit* N. caninum Antibody Test Kit, IDEXX Laboratories, Australia) when sampled prior to removing them from their home farm. These ewes were the control group from Year 1 in a previous study on vertical transmission (Syed-Hussain et al., 2015). Blood samples were collected from ewes before inoculation and 4 weeks post-inoculation. In this study, ewes lambed outside in paddocks, thus obtaining pre-colostral blood from all new-born lambs was not feasible. Blood samples were taken from all lambs at 12 weeks of age and for Groups A and B also at 2 weeks of age. All lambs were euthanased at 12 weeks of age and brain and heart tissues were collected, then divided for storage at −20 ◦ C or fixed in 10% formalin. All sera collected were tested as previously described (Syed-Hussain et al., 2013) using a commercial ELISA test kit (Chekit* N. caninum Antibody Test Kit, IDEXX Laboratories, Australia). An S/P ratio of ≥11.8% was regarded as positive as described by Reichel et al. (2008). Sera from all ewes were also tested one month post-inoculation with a western blot as described previously (Syed-Hussain et al., 2013) to confirm their infection status and all sera from 12 week old lambs were similarly tested. Extraction of DNA from brain and heart samples stored at −20 ◦ C and the qPCR was as previously described (SyedHussain et al., 2015). Histological examination of the lambs’ brains stored in formalin was conducted for the detection of lesions consistent with N. caninum infection in the brain as previously described (Syed-Hussain et al., 2015). In brief, a total pathological score was calculated for each slide from sections involving the frontal lobe, temporal lobe and the cerebellum. One way ANOVA were performed to compare the antibody responses in animals in Groups A, B and C between ewes at pre-inoculation and 1 month post-inoculation, as well as all lambs at 2 and 12 weeks of age. All animals’ S/P values for ELISA were tested for normality and all were found to be normally distributed; one-way multiple comparison using Fisher’s method was performed. Brain lesion score values were not normally distributed and were analysed with the Mann–Whitney U test. A Chi-square method was conducted to determine if there was any association between treatment groups and numbers of lambs detected positive for N. caninum infection (ELISA, histology and total infected animals). All statistical analyses were performed
Twenty-one ewes from Groups A and B were seropositive by western blot after 4 weeks post inoculation. However, two ewes from Group A remained seronegative (S/P < 11.8% and western blot negative) and these ewes and their lambs were excluded from further analysis. The S/P values in the ELISA for the remaining inoculated ewes in Groups A and B ranged from 3% to 45% and 4% to 27% respectively, with 2 animals in both Groups A and B which were considered ELISA seronegative. None of the animals in Group C had antibodies detected by either western blot or ELISA. Table 1 shows the details of N. caninum detection by ELISA, western blot, PCR and histology in neonatal lambs (n = 10) that were stillborn or died within 3 weeks of birth as well as viable lambs (n = 28) that were euthanased at 12 weeks of age. No abortions occurred in any ewes. 3.1. Detection of N. caninum in non-viable lambs In Group A, one of two dead neonates was seropositive by western blot, had lesions consistent with N. caninum infection in the brain (score of 34) and N. caninum DNA was detected in the brain and heart. The other dead neonate had no signs of infection. At Week 3, one of the treated lambs died due to injury. It was serologically positive by ELISA (S/P = 22%), western blot, had lesions consistent with N. caninum infection in the brain (score of 33), a tissue cyst in the brain and N. caninum DNA was detected in the brain. In Group B, there was evidence of N. caninum infection by western blot, qPCR and histopathology in 5/7 of the neonates that died within 3 weeks of birth. In Group C one ewe died in late pregnancy with one foetus that tested negative for N. caninum infection. 3.2. Detection of N. caninum in viable lambs At 2 weeks of age, ELISA antibody responses for lambs in Groups A and B were not significantly different from each other (p > 0.05) with mean S/P values (proportion seropositive) of 11% (5/11) and 10% (5/10), respectively. At 12 weeks of age, ELISA antibody responses for lambs in Group B were significantly higher than those in Group A (p < 0.05) with mean S/P values (proportion seropositive) being 30% (9/11) and 21% (9/10), respectively. All lambs in Groups A and B were positive by western blot at 12 weeks of age including those that were seronegative by ELISA (Table 1). None of the lambs in Group C (control) were seropositive by ELISA or western blot at 12 weeks of age. At 12 weeks of age, N. caninum DNA was detected in the brain samples of 1/11 and 2/10 lambs in Group A and B, respectively, which quantitatively was equivalent to the DNA of less than 1 tachyzoite/mg of tissue in both lambs. No N. caninum DNA was detected in the brains of lambs from Group C (control). Histological lesions (Table 1) consisted of multifocal to coalescing, randomly arranged and variably sized areas
S.S. Syed-Hussain et al. / Veterinary Parasitology 210 (2015) 141–144
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Table 1 Positive detection of N. caninum using ELISA, western blot, qPCR and histological lesions consistent with N. caninum in neonates (died within 3 weeks of birth) and lambs at 12 weeks of age. Groups‡
ELISA (mean SP value)
Western blot
qPCR
Histology Number of positive lambs
Lesion score range of positive lambs
Total – infectedT
Neonates that died within 1 week of birth
A B C
1¶ /3 0/7 0/1
2/3 4/7 0/1
2/3 5/7 0/1
2/3 5/7 0/1
33–34 5–94 0
2/3 5/7 0/1
Lambs at 12 weeks of age
A B C
9/11a (21%b ) 9/10a (30%a ) 0/7b (−8%c )
11/11 10/10 0/7
1/11 2/10 0/7
8/11a 9/10a 0/7b
2–22a 1–30a 0b
11/11a 10/10a 0/7b
For lambs at 12 weeks of age means followed by the same letter within columns do not differ from each other (p < 0.05). ‡ Neonates/lambs born from: Group A (ewes-inoculated on Day 120 gestation, lambs treated), and B (ewes inoculated on Day 120 of gestation, lambs not treated) and Group C (un-inoculated ewes used as control, lambs untreated). T– animals are considered positive when detected positive by ELISA, western blot, PCR or by presence of histological lesions. § One ewe in Group C died at end of pregnancy with one foetus. ¶ This lamb in Group A died due to injury on Day 16 after the 3rd treatment with toltrazuril and had suckled.
of gliosis, necrosis, non-suppurative perivascular cuffing and non-suppurative meningeal inflammatory infiltration. Most of the lambs in Group A (8/11) and B (9/10) were positive for brain lesions with no significant difference in the number of lambs with lesions or in the lesion scores (p > 0.05) between these 2 groups. This indicates that treatment with toltrazuril did not have an effect on reducing the severity of lesions seen in these lambs. A comparison was also made between neonates (Groups A and B combined) and lambs at 12 weeks of age in Groups A and B. No significant differences (p > 0.05) were seen in the number of neonates with brain histopathological lesions and lesion scores when compared with those of lambs in Groups A and B when killed at 12 weeks of age. No lesions were seen in any lambs in Group C. 4. Discussion The results of this study indicate that treatment of congenitally infected newborn lambs with toltrazuril at 20 mg/kg at weekly intervals did not effectively eliminate the N. caninum infection from these lambs. A small number of studies have been conducted investigating the pharmacokinetics of toltrazuril in a range of animals although none of these include its behaviour in sheep. In general the elimination is reasonably slow. In one study in young cattle the half-life of toltrazuril sulfone was 58 h with a Cmax at about 48 h (Dirikolu et al., 2009a,b). In this study the levels in the blood showed a steady decline and had reduced to about half this Cmax value by 6 days. Toltrazuril has also been shown to have a relatively slow elimination rate in pigs (Lim et al., 2010), llamas (Prado et al., 2011) and horses (Dirikolu et al., 2009a). To date there are no published pharmacological studies in sheep. Assuming a similar behaviour in sheep to that in cattle supports the use of repeated treatments after several days. The use of weekly treatments in this present study was arbitrary. The lack of a measurable response may reflect either that the drug will never be effective or the dose and/or frequency of treatment was inappropriate. Inoculation of sheep 30 days before birth allows sufficient time for at least some tissue cysts to be formed in
the lambs, presuming that foetal lambs are infected soon after inoculation (McAllister et al., 1996). To date there are no studies where toltrazuril treatment has been unequivocally directed against N. caninum bradyzoites within cysts although several studies have been conducted where it is not clear which stage of N. caninum is present at the time of treatment. Toltrazuril and its metabolites have been shown in a number of studies to be effective against tachzyoites (Kritzner et al., 2002; Strohbusch et al., 2008). Using a similar approach to that of the present study with a murine model, Strohbusch et al. (2009) demonstrated that the treatment of neonatal mice with toltrazuril reduced, but did not eliminate, the infection. Although not specifically stated, presumably both tachyzoites and bradyzoites would have been present in these treated neonatal mice. A related study treated pregnant mice with toltrazuril immediately after inoculation with N. caninum which significantly reduced foetal infection but given the timing involved would suggest the treatment was directed against the tachyzoites (Gottstein et al., 2005). In a study with the related protozoan T. gondii, treatment with toltrazuril was not totally effective in controlling infection in recently infected lambs (Kul et al., 2013) which presumably had bradyzoites of this organism in their tissues at the time of treatment. In this study, the most compelling evidence to indicate that lambs were infected is the presence of histopathological lesions, although N. caninum DNA was detected in a small number of animals. For ewes inoculated with Neospora at Day 120 of pregnancy (McAllister et al., 1996) histopathological lesions were a more consistent finding than the presence of organisms. In the present study it was expected that treated lambs would have fewer residual lesions and they would be more localised than were observed in the untreated animals. It is also noteworthy that there was no significant difference in the number of animals with histopathological lesions or in the lesion score between the neonates and lambs at 12 weeks of age although the mean score of lesions was lower in the latter. A possible explanation for this observation is that tachyzoite proliferation could still be occurring in the neonates but it is likely that only bradyzoites were present in lambs by 12
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weeks of age. These are located within cysts that protect them from being detected by the lamb’s immune response. It should be noted that the power to observe a difference was limited with the current experimental design. The usefulness of serological status as a measure of treatment efficacy is less clear. Interestingly treated lambs were all seropositive but showed significantly lower antibody levels compared to those that were not treated, suggesting higher antigenic stimulation in the non-treated lambs. This is similar to results in experimentally infected calves where one group was treated with toltrazuril (Kritzner et al., 2002). Conversely, in a different study by Haerdi et al. (2006) no difference was observed in antibody titres between treated and non-treated congenitally infected newborn calves at 12 weeks of age and by 6 months of age, treated calves had higher antibody titres suggesting the successful treatment had actually promoted a higher humoral response. In the current study, despite some of the lambs in Groups A and B being seronegative by ELISA at 12 weeks of age, all of the congenitally infected lambs were positive by western blot indicating antibodies against Neospora were still present. Testing with qPCR was of limited use in detecting N. caninum in this study and consequently of limited help to evaluate the efficacy of treatment. This particular qPCR assay (Okeoma et al., 2005) has shown a high degree of sensitivity and is able to detect the presence of N. caninum DNA from
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Study on the use of toltrazuril to eliminate Neospora caninum in congenitally infected lambs born from experimentally infected ewes S.S. Syed-Hussain a,b,∗ , L. Howe b , W.E. Pomroy b,∗ , D.M. West b , M. Hardcastle c , N.B. Williamson b a Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia b Institute of Veterinary Animal and Biomedical Sciences, Massey University, Palmerston North 4412, New Zealand c Gribbles Veterinary Pathology, PO Box 12049, Penrose, Auckland 1642, New Zealand
a r t i c l e
i n f o
Article history: Received 11 August 2014 Received in revised form 19 March 2015 Accepted 23 March 2015 Keywords: N. caninum Toltrazuril Treatment Congenital transmission Lambs Sheep
a b s t r a c t To determine if toltrazuril was effective in eliminating Neospora caninum infection from congenitally infected lambs. Twenty-eight ewes were allocated to 3 groups where animals in Groups A and B were inoculated with 1 × 107 N. caninum tachyzoites on Day 120 of gestation and Group C was maintained as a negative control group. Lambs born from ewes in Group A were treated with toltrazuril (20 mg/kg) on Days 0, 7, 14 and 21 after birth. Lambs in Groups B and C were untreated. All lambs in Groups A and B were seropositive at 12 weeks of age. At 12 weeks of age, no differences between lambs in Group A and Group B were observed in serological results (ELISA and western blot), presence of N. caninumrelated brain histopathological lesions or the number of organisms detected by qPCR. Group C remained negative for serology, detection of N. caninum DNA as well as histopathology throughout the study. Results indicate that N. caninum congenitally-infected lambs had a continuing infection with N. caninum despite being treated with toltrazuril. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Chemotherapy has the potential to be an effective control measure for neosporosis (Hasler et al., 2006) provided the drug used is effective. Toltrazuril, a symmetrical triazinone, is metabolized into ponazuril (toltrazuril sulfone). Both toltrazuril and ponazuril are effective against a broad spectrum of cyst-forming and non-cyst-forming
∗ Corresponding authors at: Institute of Veterinary Animal and Biomedical Sciences, Massey University, Palmerston North 4412, New Zealand. Tel.: +6463569099x85161; fax: +6463505699. E-mail addresses: [email protected] (S.S. Syed-Hussain), [email protected] (W.E. Pomroy). http://dx.doi.org/10.1016/j.vetpar.2015.03.019 0304-4017/© 2015 Elsevier B.V. All rights reserved.
apicomplexan protozoa (Haberkorn, 1996) including positive effects against N. caninum in vitro (Darius et al., 2004; Strohbusch et al., 2008) and in vivo, both in mice (Strohbusch et al., 2009) as well as in experimentally and naturally infected calves (Haerdi et al., 2006; Kritzner et al., 2002). However, in order for toltrazuril to reach its full effectiveness in clearing N. caninum infection, one study has shown that a supportive T cell-immune response is necessary (Ammann et al., 2004). Regardless, no chemotherapeutic approach has been shown to be safe and fully effective for the treatment of neosporosis in cattle (Dubey and Schares, 2011). One approach has been to treat neonates to eliminate the organism following congenital infection. The aim of this present study was to determine if toltrazuril was effective
142
S.S. Syed-Hussain et al. / Veterinary Parasitology 210 (2015) 141–144
in eliminating N. caninum infection from congenitally infected lambs.
using Minitab (Minitab Inc., PA, USA) and all statistical analyses were considered significant when p < 0.05.
2. Materials and methods
3. Results
Twenty-eight ewes were allocated to 3 groups where animals in Groups A (n = 11) and B (n = 12) were inoculated with 1 × 107 N. caninum tachyzoites on Day 120 of gestation and Group C (n = 5) was maintained as a negative control group. Lambs born from ewes in Group A (n = 12) were treated with toltrazuril (20 mg/kg) on Day 0, 7, 14 and 21 after birth. Lambs in Groups B (n = 10) and C (n = 7) were untreated. The ewes were approximately four years old, had been vaccinated for Toxoplasma gondii and Campylobacter fetus fetus and were free from N. caninum as indicated by negative serology using ELISA (Chekit* N. caninum Antibody Test Kit, IDEXX Laboratories, Australia) when sampled prior to removing them from their home farm. These ewes were the control group from Year 1 in a previous study on vertical transmission (Syed-Hussain et al., 2015). Blood samples were collected from ewes before inoculation and 4 weeks post-inoculation. In this study, ewes lambed outside in paddocks, thus obtaining pre-colostral blood from all new-born lambs was not feasible. Blood samples were taken from all lambs at 12 weeks of age and for Groups A and B also at 2 weeks of age. All lambs were euthanased at 12 weeks of age and brain and heart tissues were collected, then divided for storage at −20 ◦ C or fixed in 10% formalin. All sera collected were tested as previously described (Syed-Hussain et al., 2013) using a commercial ELISA test kit (Chekit* N. caninum Antibody Test Kit, IDEXX Laboratories, Australia). An S/P ratio of ≥11.8% was regarded as positive as described by Reichel et al. (2008). Sera from all ewes were also tested one month post-inoculation with a western blot as described previously (Syed-Hussain et al., 2013) to confirm their infection status and all sera from 12 week old lambs were similarly tested. Extraction of DNA from brain and heart samples stored at −20 ◦ C and the qPCR was as previously described (SyedHussain et al., 2015). Histological examination of the lambs’ brains stored in formalin was conducted for the detection of lesions consistent with N. caninum infection in the brain as previously described (Syed-Hussain et al., 2015). In brief, a total pathological score was calculated for each slide from sections involving the frontal lobe, temporal lobe and the cerebellum. One way ANOVA were performed to compare the antibody responses in animals in Groups A, B and C between ewes at pre-inoculation and 1 month post-inoculation, as well as all lambs at 2 and 12 weeks of age. All animals’ S/P values for ELISA were tested for normality and all were found to be normally distributed; one-way multiple comparison using Fisher’s method was performed. Brain lesion score values were not normally distributed and were analysed with the Mann–Whitney U test. A Chi-square method was conducted to determine if there was any association between treatment groups and numbers of lambs detected positive for N. caninum infection (ELISA, histology and total infected animals). All statistical analyses were performed
Twenty-one ewes from Groups A and B were seropositive by western blot after 4 weeks post inoculation. However, two ewes from Group A remained seronegative (S/P < 11.8% and western blot negative) and these ewes and their lambs were excluded from further analysis. The S/P values in the ELISA for the remaining inoculated ewes in Groups A and B ranged from 3% to 45% and 4% to 27% respectively, with 2 animals in both Groups A and B which were considered ELISA seronegative. None of the animals in Group C had antibodies detected by either western blot or ELISA. Table 1 shows the details of N. caninum detection by ELISA, western blot, PCR and histology in neonatal lambs (n = 10) that were stillborn or died within 3 weeks of birth as well as viable lambs (n = 28) that were euthanased at 12 weeks of age. No abortions occurred in any ewes. 3.1. Detection of N. caninum in non-viable lambs In Group A, one of two dead neonates was seropositive by western blot, had lesions consistent with N. caninum infection in the brain (score of 34) and N. caninum DNA was detected in the brain and heart. The other dead neonate had no signs of infection. At Week 3, one of the treated lambs died due to injury. It was serologically positive by ELISA (S/P = 22%), western blot, had lesions consistent with N. caninum infection in the brain (score of 33), a tissue cyst in the brain and N. caninum DNA was detected in the brain. In Group B, there was evidence of N. caninum infection by western blot, qPCR and histopathology in 5/7 of the neonates that died within 3 weeks of birth. In Group C one ewe died in late pregnancy with one foetus that tested negative for N. caninum infection. 3.2. Detection of N. caninum in viable lambs At 2 weeks of age, ELISA antibody responses for lambs in Groups A and B were not significantly different from each other (p > 0.05) with mean S/P values (proportion seropositive) of 11% (5/11) and 10% (5/10), respectively. At 12 weeks of age, ELISA antibody responses for lambs in Group B were significantly higher than those in Group A (p < 0.05) with mean S/P values (proportion seropositive) being 30% (9/11) and 21% (9/10), respectively. All lambs in Groups A and B were positive by western blot at 12 weeks of age including those that were seronegative by ELISA (Table 1). None of the lambs in Group C (control) were seropositive by ELISA or western blot at 12 weeks of age. At 12 weeks of age, N. caninum DNA was detected in the brain samples of 1/11 and 2/10 lambs in Group A and B, respectively, which quantitatively was equivalent to the DNA of less than 1 tachyzoite/mg of tissue in both lambs. No N. caninum DNA was detected in the brains of lambs from Group C (control). Histological lesions (Table 1) consisted of multifocal to coalescing, randomly arranged and variably sized areas
S.S. Syed-Hussain et al. / Veterinary Parasitology 210 (2015) 141–144
143
Table 1 Positive detection of N. caninum using ELISA, western blot, qPCR and histological lesions consistent with N. caninum in neonates (died within 3 weeks of birth) and lambs at 12 weeks of age. Groups‡
ELISA (mean SP value)
Western blot
qPCR
Histology Number of positive lambs
Lesion score range of positive lambs
Total – infectedT
Neonates that died within 1 week of birth
A B C
1¶ /3 0/7 0/1
2/3 4/7 0/1
2/3 5/7 0/1
2/3 5/7 0/1
33–34 5–94 0
2/3 5/7 0/1
Lambs at 12 weeks of age
A B C
9/11a (21%b ) 9/10a (30%a ) 0/7b (−8%c )
11/11 10/10 0/7
1/11 2/10 0/7
8/11a 9/10a 0/7b
2–22a 1–30a 0b
11/11a 10/10a 0/7b
For lambs at 12 weeks of age means followed by the same letter within columns do not differ from each other (p < 0.05). ‡ Neonates/lambs born from: Group A (ewes-inoculated on Day 120 gestation, lambs treated), and B (ewes inoculated on Day 120 of gestation, lambs not treated) and Group C (un-inoculated ewes used as control, lambs untreated). T– animals are considered positive when detected positive by ELISA, western blot, PCR or by presence of histological lesions. § One ewe in Group C died at end of pregnancy with one foetus. ¶ This lamb in Group A died due to injury on Day 16 after the 3rd treatment with toltrazuril and had suckled.
of gliosis, necrosis, non-suppurative perivascular cuffing and non-suppurative meningeal inflammatory infiltration. Most of the lambs in Group A (8/11) and B (9/10) were positive for brain lesions with no significant difference in the number of lambs with lesions or in the lesion scores (p > 0.05) between these 2 groups. This indicates that treatment with toltrazuril did not have an effect on reducing the severity of lesions seen in these lambs. A comparison was also made between neonates (Groups A and B combined) and lambs at 12 weeks of age in Groups A and B. No significant differences (p > 0.05) were seen in the number of neonates with brain histopathological lesions and lesion scores when compared with those of lambs in Groups A and B when killed at 12 weeks of age. No lesions were seen in any lambs in Group C. 4. Discussion The results of this study indicate that treatment of congenitally infected newborn lambs with toltrazuril at 20 mg/kg at weekly intervals did not effectively eliminate the N. caninum infection from these lambs. A small number of studies have been conducted investigating the pharmacokinetics of toltrazuril in a range of animals although none of these include its behaviour in sheep. In general the elimination is reasonably slow. In one study in young cattle the half-life of toltrazuril sulfone was 58 h with a Cmax at about 48 h (Dirikolu et al., 2009a,b). In this study the levels in the blood showed a steady decline and had reduced to about half this Cmax value by 6 days. Toltrazuril has also been shown to have a relatively slow elimination rate in pigs (Lim et al., 2010), llamas (Prado et al., 2011) and horses (Dirikolu et al., 2009a). To date there are no published pharmacological studies in sheep. Assuming a similar behaviour in sheep to that in cattle supports the use of repeated treatments after several days. The use of weekly treatments in this present study was arbitrary. The lack of a measurable response may reflect either that the drug will never be effective or the dose and/or frequency of treatment was inappropriate. Inoculation of sheep 30 days before birth allows sufficient time for at least some tissue cysts to be formed in
the lambs, presuming that foetal lambs are infected soon after inoculation (McAllister et al., 1996). To date there are no studies where toltrazuril treatment has been unequivocally directed against N. caninum bradyzoites within cysts although several studies have been conducted where it is not clear which stage of N. caninum is present at the time of treatment. Toltrazuril and its metabolites have been shown in a number of studies to be effective against tachzyoites (Kritzner et al., 2002; Strohbusch et al., 2008). Using a similar approach to that of the present study with a murine model, Strohbusch et al. (2009) demonstrated that the treatment of neonatal mice with toltrazuril reduced, but did not eliminate, the infection. Although not specifically stated, presumably both tachyzoites and bradyzoites would have been present in these treated neonatal mice. A related study treated pregnant mice with toltrazuril immediately after inoculation with N. caninum which significantly reduced foetal infection but given the timing involved would suggest the treatment was directed against the tachyzoites (Gottstein et al., 2005). In a study with the related protozoan T. gondii, treatment with toltrazuril was not totally effective in controlling infection in recently infected lambs (Kul et al., 2013) which presumably had bradyzoites of this organism in their tissues at the time of treatment. In this study, the most compelling evidence to indicate that lambs were infected is the presence of histopathological lesions, although N. caninum DNA was detected in a small number of animals. For ewes inoculated with Neospora at Day 120 of pregnancy (McAllister et al., 1996) histopathological lesions were a more consistent finding than the presence of organisms. In the present study it was expected that treated lambs would have fewer residual lesions and they would be more localised than were observed in the untreated animals. It is also noteworthy that there was no significant difference in the number of animals with histopathological lesions or in the lesion score between the neonates and lambs at 12 weeks of age although the mean score of lesions was lower in the latter. A possible explanation for this observation is that tachyzoite proliferation could still be occurring in the neonates but it is likely that only bradyzoites were present in lambs by 12
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weeks of age. These are located within cysts that protect them from being detected by the lamb’s immune response. It should be noted that the power to observe a difference was limited with the current experimental design. The usefulness of serological status as a measure of treatment efficacy is less clear. Interestingly treated lambs were all seropositive but showed significantly lower antibody levels compared to those that were not treated, suggesting higher antigenic stimulation in the non-treated lambs. This is similar to results in experimentally infected calves where one group was treated with toltrazuril (Kritzner et al., 2002). Conversely, in a different study by Haerdi et al. (2006) no difference was observed in antibody titres between treated and non-treated congenitally infected newborn calves at 12 weeks of age and by 6 months of age, treated calves had higher antibody titres suggesting the successful treatment had actually promoted a higher humoral response. In the current study, despite some of the lambs in Groups A and B being seronegative by ELISA at 12 weeks of age, all of the congenitally infected lambs were positive by western blot indicating antibodies against Neospora were still present. Testing with qPCR was of limited use in detecting N. caninum in this study and consequently of limited help to evaluate the efficacy of treatment. This particular qPCR assay (Okeoma et al., 2005) has shown a high degree of sensitivity and is able to detect the presence of N. caninum DNA from
