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Serofpidemiology, Zoonotic Potential And Chemotherapy Of Neosporosis In Dogs And Cattle

By: Muhammad Mudasser Nazir | Prof. Dr. Azhar Maqbool.
Contributor(s): Dr. Muhammad Lateef | Prof. Dr.
Material type: materialTypeLabelBookPublisher: 2010Subject(s): Department of Parasitology | Phd. ThesisDDC classification: 1575,T Dissertation note: The aim of current study was to demonstrate the most important features of Neospora caninum infection in Pakistan. In the present study, I examined the prevalence of N. caninum in 7 districts of the country and to accessed the efficacy of various drugs against the parasite in cell culture. For the achievement of this purpose, the core objectives were, To have an overview on the overall seroprevalence of neosporosis throughout the country by means of cELISA in aborting, at risk and clinically healthy cows. To check the correlation of Iscom ELISA and cELISA, and determination of prevalence of N. caninum by means of Iscom ELISA on milk samples. To identify the transmission of disease towards human. To determine the efficacy of various drugs against N. caninum. In phase 1, overall seroprevalence of N. caninum in dairy cattle (detected by means of cELISA, VMRD, Inc., Pullman, WA, USA) was found to be 43.4% with a significant difference (P < 0.05) of seropositivity among all 18 herds (n = 5 aborting herds, n = 13 non-aborting/clinically healthy herds) selected from 7 districts of Pakistan. The seropositivity of cattle to N. caninum antibodies was significantly higher in aborting animals (52.7%) as compared to non-aborting cows ( 41.5%), indicating a significant difference between aborting and non-aborting cattle. In case of pregnant and non-pregnant animals, similar findings were recorded in our study. A significantly higher rate of seroprevalence was observed in pregnant dams (59.8%) than non-pregnant cattle (35.2%). Overall, higher serological prevalence was evaluated during the summer season (61.1%) in all areas followed by autumn (46.9%), spring (34.9%) and least seropositivity was observed in winter season (26.6%). The difference in seropositivity was significantly different among all age groups, greater in animals older than 2 years of age. Furthermore, the prevalence was statistically significant (P < 0.05) among cattle of different breeds. Seroprevalence in cases of crossbred animals were higher followed by exotic and indigenous breeds. Phase 2, describes the seroprevalence of N. caninum in clinically healthy dairy cows. A selection of 760 animals from 13 dairy herds located in Punjab and Sindh Province, Pakistan to demonstrate the presence or absence of the Neospora caninum infection in commercial dairy cattle. The serostatus of the cows towards N. caninum was detected by cELISA (VMRD, Pullman, WA). Out of 760 animals, (43.2%) were seropositive to N. caninum. A significant difference of positivity was recorded among all 13 dairy herds. Age wise prevalence though not statistically significant among all age groups, was greater in animals over 2 years of age and least in heifers. Variation was also observed in samples from cattle of different breeds. A significantly higher prevalence was observed in crossbred animals than in purebred and nondescript cattle. Seasonal prevalence was higher during summer season than rest of the seasons. The seroprevalence of N. caninum in pregnant cows was significantly greater than in non-pregnant animals. Second experiment of this phase describes the seroprevalence of 240 animals from 5 herds with a high rate of abortion, the percentage of seropositivity observed in these herds was 43.8%, slightly higher than the clinically healthy and non-aborting cattle. No significant difference was observed among all sample locations in this experiment. However, significant difference of positivity was recorded among different breeds of cattle. Age wise prevalence, though not statistically significant (P > 0.05), was greater in animals older than 2 years of age. The assessment of milk samples from lactating cows were also determined for Neospora caninum antibodies by means of Iscom ELISA (SANOVIR® Sanova Biotech AB, Uppasala, Sweden) and showed a good level of agreement (r² = 0.9959) between the two tests (cELISA and Iscom ELISA). Although, the cELISA (VMRD, Inc., Pullman, USA) expressed a higher seropositivity and sensitivity than Iscom ELISA (Sanova Biotech AB, Uppasala, Sweden). Therefore, both of the ELISA tests (cELISA and Iscom ELISA) for the detection of N. caninum antibodies in dairy cattle can perform better in lactating animals. The Iscom ELISA has some advantages over cELISA as it's easy to collect milk samples than serum samples, moreover Iscom ELISA is cheaper and easy to use but has low sensitivity than cELISA and cannot be used in dry animals. The percentage of positivity detected through Iscom ELISA on individual milk samples were 61.4% and 76.6% by cELISA. In phase 3, an epidemiological study was conducted to determine seroprevalence of N. caninum in dogs of different breeds and age groups. The serum samples of dogs were analyzed by cELISA (VMRD, Pullman, USA) showed a seropositivity of 23.5%. There was no significant difference of seropositivity among various sample locations, highest prevalence was observed in Muzaffar Garh (31.9%), followed by Gujranwala (27.9%), Lahore (25.1%), Hafizabad (20.2%) and least prevalence was recorded in district Okara (14.6%). A significant difference in prevalence of N. caninum antibodies between male (26.1%) and female (18.8%) dogs were recorded. The difference in seroprevalence was not significant among all age groups. The samples with no age record showed a highest prevalence (29.5%) and least seropositivity was observed in adult dogs of 3-6 years of age (18.7%). During Summer season, highest positivity to N. caninum was (31.0%) recorded while the lowest prevalence (16.0%) was observed in Winter season. Phase 4, describes the seroprevalence and transmission of N. caninum in humans. A selection of 52 serum samples from humans was analyzed for the presence of N. caninum antibodies. The serostatus of the humans towards N. caninum antibodies (IgG) was determined by using commercially available antigen coated IFAT slides (VMRD, Inc., Pullman, Washington USA) and human conjugate. Overall very low prevalence (1.9%) of N. caninum antibodies was reported in this study. Only one case was found to be positive, these findings indicate that no strong evidence of N. caninum infection in humans. In phase 5, in vitro drug trials was conducted to access the best efficacy of three commercially available drugs. We found that among three anticoccidial drugs i.e Clindamycin, Diclazuril and Sulfadiazine, Diclazuril has best inhibitory effect against N. caninum tachyzoites in cell culture followed by Clindamycin and sulfadiazine.
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The aim of current study was to demonstrate the most important features of Neospora caninum infection in Pakistan. In the present study, I examined the prevalence of N. caninum in 7 districts of the country and to accessed the efficacy of various drugs against the parasite in cell culture.
For the achievement of this purpose, the core objectives were,
To have an overview on the overall seroprevalence of neosporosis throughout the country by means of cELISA in aborting, at risk and clinically healthy cows.
To check the correlation of Iscom ELISA and cELISA, and determination of prevalence of N. caninum by means of Iscom ELISA on milk samples.
To identify the transmission of disease towards human.
To determine the efficacy of various drugs against N. caninum.
In phase 1, overall seroprevalence of N. caninum in dairy cattle (detected by means of cELISA, VMRD, Inc., Pullman, WA, USA) was found to be 43.4% with a significant difference (P < 0.05) of seropositivity among all 18 herds (n = 5 aborting herds, n = 13 non-aborting/clinically healthy herds) selected from 7 districts of Pakistan. The seropositivity of cattle to N. caninum antibodies was significantly higher in aborting animals (52.7%) as compared to non-aborting cows ( 41.5%), indicating a significant difference between aborting and non-aborting cattle. In case of pregnant and non-pregnant animals, similar findings were recorded in our study. A significantly higher rate of seroprevalence was observed in pregnant dams (59.8%) than non-pregnant cattle (35.2%). Overall, higher serological prevalence was evaluated during the summer season (61.1%) in all areas followed by autumn (46.9%), spring (34.9%) and least seropositivity was observed in winter season (26.6%).
The difference in seropositivity was significantly different among all age groups, greater in animals older than 2 years of age. Furthermore, the prevalence was statistically significant (P < 0.05) among cattle of different breeds. Seroprevalence in cases of crossbred animals were higher followed by exotic and indigenous breeds.
Phase 2, describes the seroprevalence of N. caninum in clinically healthy dairy cows. A selection of 760 animals from 13 dairy herds located in Punjab and Sindh Province, Pakistan to demonstrate the presence or absence of the Neospora caninum infection in commercial dairy cattle. The serostatus of the cows towards N. caninum was detected by cELISA (VMRD, Pullman, WA). Out of 760 animals, (43.2%) were seropositive to N. caninum. A significant difference of positivity was recorded among all 13 dairy herds. Age wise prevalence though not statistically significant among all age groups, was greater in animals over 2 years of age and least in heifers. Variation was also observed in samples from cattle of different breeds. A significantly higher prevalence was observed in crossbred animals than in purebred and nondescript cattle. Seasonal prevalence was higher during summer season than rest of the seasons. The seroprevalence of N. caninum in pregnant cows was significantly greater than in non-pregnant animals.
Second experiment of this phase describes the seroprevalence of 240 animals from 5 herds with a high rate of abortion, the percentage of seropositivity observed in these herds was 43.8%, slightly higher than the clinically healthy and non-aborting cattle. No significant difference was observed among all sample locations in this experiment. However, significant difference of positivity was recorded among different breeds of cattle. Age wise prevalence, though not statistically significant (P > 0.05), was greater in animals older than 2 years of age.
The assessment of milk samples from lactating cows were also determined for Neospora caninum antibodies by means of Iscom ELISA (SANOVIR® Sanova Biotech AB, Uppasala, Sweden) and showed a good level of agreement (r² = 0.9959) between the two tests (cELISA and Iscom ELISA). Although, the cELISA (VMRD, Inc., Pullman, USA) expressed a higher seropositivity and sensitivity than Iscom ELISA (Sanova Biotech AB, Uppasala, Sweden). Therefore, both of the ELISA tests (cELISA and Iscom ELISA) for the detection of N. caninum antibodies in dairy cattle can perform better in lactating animals. The Iscom ELISA has some advantages over cELISA as it's easy to collect milk samples than serum samples, moreover Iscom ELISA is cheaper and easy to use but has low sensitivity than cELISA and cannot be used in dry animals. The percentage of positivity detected through Iscom ELISA on individual milk samples were 61.4% and 76.6% by cELISA.
In phase 3, an epidemiological study was conducted to determine seroprevalence of N. caninum in dogs of different breeds and age groups. The serum samples of dogs were analyzed by cELISA (VMRD, Pullman, USA) showed a seropositivity of 23.5%. There was no significant difference of seropositivity among various sample locations, highest prevalence was observed in Muzaffar Garh (31.9%), followed by Gujranwala (27.9%), Lahore (25.1%), Hafizabad (20.2%) and least prevalence was recorded in district Okara (14.6%). A significant difference in prevalence of N. caninum antibodies between male (26.1%) and female (18.8%) dogs were recorded. The difference in seroprevalence was not significant among all age groups. The samples with no age record showed a highest prevalence (29.5%) and least seropositivity was observed in adult dogs of 3-6 years of age (18.7%). During Summer season, highest positivity to N. caninum was (31.0%) recorded while the lowest prevalence (16.0%) was observed in Winter season.
Phase 4, describes the seroprevalence and transmission of N. caninum in humans. A selection of 52 serum samples from humans was analyzed for the presence of N. caninum antibodies. The serostatus of the humans towards N. caninum antibodies (IgG) was determined by using commercially available antigen coated IFAT slides (VMRD, Inc., Pullman, Washington USA) and human conjugate. Overall very low prevalence (1.9%) of N. caninum antibodies was reported in this study. Only one case was found to be positive, these findings indicate that no strong evidence of N. caninum infection in humans.
In phase 5, in vitro drug trials was conducted to access the best efficacy of three commercially available drugs. We found that among three anticoccidial drugs i.e Clindamycin, Diclazuril and Sulfadiazine, Diclazuril has best inhibitory effect against N. caninum tachyzoites in cell culture followed by Clindamycin and sulfadiazine.

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