Molecular detection of Anaplasma in apparently healthy Cholistan breed of cattle from the Bahawalpur district, Pakistan.

The present study was designed to report the prevalence of Anaplasma sp. in blood samples of Cholistan breed of cattle from Bahawalpur District and to determine the risk factors associated with the prevalence of this parasite. A total of 148 blood samples were randomly collected from apparently healthy cattle. On the sampling sites, data on the characteristics of the animals (species, gender, age) were collected through questionnaires. 47 blood samples (31.8% of total) produced the 577 base pairs DNA fragment specific for 16S rRNA gene of Anaplasma sp. by PCR amplification. Out of 47 Anaplasma sp. positive PCR products, 9 were found to be Anaplasma marginale by restriction with BssNa1 and 9 were confirmed to be Anaplasma phagocytophilum (A. phagocytophilum) as they amplified 550 bp fragment from the amplified MSP 2 gene of this species. Risk factor analysis indicated that the presence of parasite was not limited to a particular sex or age group of the infected animals. Comparison of hematological profile revealed that Anaplasma sp. positive cattle had significantly reduced levels of mean corpuscular volume (P=0.02) and eosinophils (P=0.02) than in parasite negative animals. While studied serum biochemical profile remain unaffected when compared between the two groups.

Molecular detection of Anaplasma in apparently healthy Cholistan breed of cattle from the Bahawalpur district, Pakistan

The present study was designed to report the prevalence of Anaplasma sp. in blood samples of Cholistan breed of cattle from Bahawalpur District and to determine the risk factors associated with the prevalence of this parasite. A total of 148 blood samples were randomly collected from apparently healthy cattle. On the sampling sites, data on the characteristics of the animals (species, gender, age) were collected through questionnaires. 47 blood samples (31.8% of total) produced the 577 base pairs DNA fragment specific for 16S rRNA gene of Anaplasma sp. by PCR amplification. Out of 47 Anaplasma sp. positive PCR products, 9 were found to be Anaplasma marginale by restriction with BssNa1 and 9 were confirmed to be Anaplasma phagocytophilum (A. phagocytophilum) as they amplified 550 bp fragment from the amplified MSP 2 gene of this species. Risk factor analysis indicated that the presence of parasite was not limited to a particular sex or age group of the infected animals. Comparison of hematological profile revealed that Anaplasma sp. positive cattle had significantly reduced levels of mean corpuscular volume (P=0.02) and eosinophils (P=0.02) than in parasite negative animals. While studied serum biochemical profile remain unaffected when compared between the two groups.

A report on the molecular detection and seasonal prevalence of Trypanosoma brucei in Dromedary Camels from Dera Ghazi Khan District in Southern Punjab (Pakistan).

The present study was designed for molecular detection of Trypanosoma brucei through PCR, by using kinetoplast DNA (kDNA) maxicircle primers, on seasonal basis and to demonstrate the effect of this parasite on complete blood count and selected parameters of serum biochemistry in camels from Southern Punjab (Pakistan). A total of 291 camel blood samples (61 male, 230 females) were collected from Dera Ghazi Khan District in Pakistan during March 2012 till February 2013 for Trypanosoma brucei detection by blood smear screening, micro hemato centrifugation and Polymerase chain reaction techniques. Twenty eight out of 291 blood samples (9.62%) produced a 164 bp DNA fragment specific for T. brucei. Only 6 blood samples (2.06%) were found parasite positive by microscopic examination and 13 (4.46%) were positive for microhematocrit centrifugation technique. Seasonal PCR based prevalence of trypanosomiasis was 6.9%, 13.7%, 9.7% and 8.1% during spring, summer, autumn and winter seasons respectively. T. brucei prevalence was not restricted to a particular age group or and gender of the studied animals (P > 0.05). A significant increase in WBC (P = 0.001), neutrophils (P = 0.004), ALT (P = 0.028) and decreased RBC (P < 0.000), hemoglobin (P < 0.000) and packed cell volume (P < 0.000) were detected in parasite positive as compared to the parasite negative blood samples. In conclusion, PCR is a more reliable and sensitive technique than conventional microscopic blood screening and microhematocrit centrifugation for the detection of T. brucei in camel blood. We recommend the use of PCR for the effective prophylactic detection of T. brucei in livestock in order to reduce economic losses.

A report on the molecular detection and seasonal Trypanosoma brucei in Dromedary Camels from Dera Ghazi Khan District in Southern Punjab (Pakistan)

The present study was designed for molecular detection of Trypanosoma brucei through PCR, by using kinetoplast DNA (kDNA) maxicircle primers, on seasonal basis and to demonstrate the effect of this parasite on complete blood count and selected parameters of serum biochemistry in camels from Southern Punjab (Pakistan). A total of 291 camel blood samples (61 male, 230 females) were collected from Dera Ghazi Khan District in Pakistan during March 2012 till February 2013 for Trypanosoma brucei detection by blood smear screening, micro hemato centrifugation and Polymerase chain reaction techniques. Twenty eight out of 291 blood samples (9.62%) produced a 164 bp DNA fragment specific for T. brucei . Only 6 blood samples (2.06%) were found parasite positive by microscopic examination and 13 (4.46%) were positive for microhematocrit centrifugation technique. Seasonal PCR based prevalence of trypanosomiasis was 6.9%, 13.7%, 9.7% and 8.1% during spring, summer, autumn and winter seasons respectively. T. brucei prevalence was not restricted to a particular age group or and gender of the studied animals (P > 0.05). A significant increase in WBC (P = 0.001), neutrophils (P = 0.004), ALT (P = 0.028) and decreased RBC (P < 0.000), hemoglobin (P < 0.000) and packed cell volume (P < 0.000) were detected in parasite positive as compared to the parasite negative blood samples. In conclusion, PCR is a more reliable and sensitive technique than conventional microscopic blood screening and microhematocrit centrifugation for the detection of T. brucei in camel blood. We recommend the use of PCR for the effective prophylactic detection of T. brucei in livestock in order to reduce economic losses.

Prevalence of Anaplasma phagocytophilum in horses from Southern Punjab (Pakistan).

The present study was designed to optimize a PCR-RFLP protocol for the molecular detection of Anaplasma sp. and to compare its prevalence in blood samples of equines from Southern Punjab (Pakistan) and to find out the risk factors involved in the spread of anaplasmosis. A total of 210 blood samples were collected from equines from 2 sampling sites (Dera Ghazi Khan and Khanewal districts). Data on the animals' characteristics (age, species and gender) were collected through survey. PCR amplified the 577bp product specific for 16S rRNA gene of Anaplasma spp. in 9 blood samples (4.3% of total), [Dera Ghazi Khan (N = 3) and Khanewal (N = 6)]. These Anaplasma spp. positive blood samples were used for PCR amplification using A. phagocytophilum specific primers and parasite was detected in all of them. Also it was revealed that the characteristics of the animals i.e. age, gender, species had no significant association with the presence of Anaplasma sp. Hematological parameters remained unaffected while lymphocyte count was significantly lowered in A. phagocytophilum positive samples.

PCR based detection of Theileria lestoquardi in apparently healthy sheep and goats from two districts in Khyber Pukhtoon Khwa (Pakistan).

The present study was carried out to determine the prevalence of Theileria lestoquardi from two districts of Khyber Pukhtoon Khwa (Kohat and Peshawar) in Pakistan and also to report the risk factors associated with the spread of ovine theileriosis. A total of 165 blood samples were collected from sheep (N = 44) and goats (N = 121) from randomly selected herds. Data on the characteristics of animals and the herds were collected through questionnaires. Five (3%) out of total 165 samples produced 730 base pairs DNA fragment, through PCR amplification of 18S SSU rRNA gene, specific for T. lestoquardi. All positive samples were from district Kohat while samples from Peshawar were found negative for this parasite. Statistical analysis indicated a significant association (P = 0.005) between sampling site and prevalence of T. lestoquardi. It was observed that presence of tick on the ruminant (P = 0.0007) and the dogs associated with the herd (P = 0.001) were highly significant risk factor for the spread of ovine theileriosis. It was also observed that mixed herds (containing both sheep and goats) were more prone to the parasite. We have concluded that PCR is a sensitive and reliable diagnostic tool for detection of T. lestoquardi in blood samples of small ruminants and can be used for the prophylactic screening and treatment of this blood parasite in order to increase the live stock production in Pakistan.

A comparison of two different techniques for the detection of blood parasite, Theileria annulata, in cattle from two districts in Khyber Pukhtoon Khwa Province (Pakistan).

The present study was carried out to determine the prevalence of Theileria annulata in large ruminants from two districts, Peshawar and Kohat, in Khyber Pukhtoon Khwa (Pakistan). Blood samples were collected from 95 cattle. Data on the characteristics of animals and herds were collected through questionnaires. No significant risk factors were found associated with the spread of tropical theileriosis in the study area. Two different parasite detection techniques, PCR amplification and screening of Giemsa stained slides, were compared and it was found that PCR amplification is a more sensitive tool (33.7% parasite detection), as compared to smear scanning (5.2% parasite detection) for the detection of Theileria annulata. 32 out of 95 animals, from both districts, produced the 721-bp fragment specific for Theileria annulata.