Muhammad Kasib Khan

Gastrointestinal helminthiasis: prevalence and associated determinants in domestic ruminants of district Toba Tek Singh, Punjab, Pakistan

The objective of the present study was to determine the prevalence and associated determinants (e.g., sex, age, on-farm management and husbandry) of gastrointestinal (GI) helminths in the domestic animals of district Toba Tek Singh, Punjab, Pakistan. For this purpose, 1,140 cattle, 1,140 buffaloes, 660 goats, 840 sheep, and 156 camels were randomly selected and their fecal samples were screened every other week for a year using a modified floatation technique. The samples positive for strongyle-type eggs had the parasite species identified using coproculture. It was found that the prevalence of GI helminths was significantly higher (P < 0.05) in sheep (44.17%; 371/840) than in other livestock. Sheep were followed in order by goats (40.15%; 265/660), buffaloes (39.82%; 454/1,140), and cattle (33.68%; 384/1,140). The important helminth species identified were Fasciola (F.) gigantica, Fasciola hepatica, Haemonchus contortus, Toxocara vitulorum, Trichostrongylus spp., Oesophagostomum spp., Ostertagia spp., Cooperia spp., Strongyloides spp., Moniezia spp., and Trichuris spp. The prevalence of GI helminths except F. hepatica and F. gigantica was significantly higher in grazing animals, females (P < 0.05) and young (P < 0.05) of all the host species when compared with stall-fed animals, males and adults, respectively. Using ponds and rivers/canals as drinking water were found to have significant influence (P < 0.05) on the prevalence of GI helminths. The results provide a baseline data for planning future research and control strategies against GI helminthes.

Bovine fasciolosis: prevalence, effects of treatment on productivity and cost benefit analysis in five districts of Punjab, Pakistan.

The present study reports the prevalence, effects of treatment and cost benefit analysis of bovine fasciolosis in five districts of Punjab Province viz Sargodha, Jhang, Muzaffargarh, Lodhran and Layyah. From each of the five districts, 80 animals were selected and fortnightly screened through standard coprological procedures for a period of one year for the presence of eggs of Fasciola species. Of 4800 faecal samples analyzed, 1222 (25.46%) were found positive for fasciolosis. The occurrence of Fasciola (F.) gigantica (22.40%) was higher (P<0.05) than F. hepatica (3.06%). Highest month-wise prevalence (P<0.05) of fasciolosis was found in winter (39.08%) followed in decreasing order by spring (29.50%), autumn (20.33%) and summer (12.92%). District-wise prevalence of fasciolosis was highest (P<0.05) in Sargodha (40.31%) and lowest in Layyah (11.77%) while other districts were having intermediate values of prevalence of fasciolosis. Species-wise prevalence of fasciolosis was found higher (P<0.05) in buffaloes (30.50%) as compared to cattle (20.42%). However, there were no age and sex-related differences (P>0.05) in prevalence of fasciolosis. A strong positive association of grazing (OR=1.81), mixed farming of small and large ruminants (OR=1.39), stagnant pond bathing (OR=2.24) and river/canal bathing (OR=2.06) was found with the prevalence of fasciolosis as compared to stall feeding, separate farming of small and large ruminants and rivers/canal/tap water bathing, respectively. Post-treatment average milk increase of 0.62 L per animal per day with 0.35% higher fat was observed in fasciolicide-treated animals with the cost benefit ratio of 3.9. The results provided significant data on the epidemiology of five districts of Punjab province which may be helpful for the planners and small holder dairy farmers for control of fasciolosis in the study districts.

Anthelmintic activity of Trianthema portulacastrum L. and Musa paradisiaca L. against gastrointestinal nematodes of sheep.

Evaluation of anthelmintic effects of Trianthema (T.) portulacastrum L. (Aizoaceae) whole plant and Musa (M.) paradisiaca L. (Musaceae) leaves against prevalent gastrointestinal worms of sheep was done that may justify their traditional use in veterinary clinical medicine. In vitro anthelmintic activity of the crude aqueous methanolic extract (CAME) of both the plants was determined using mature female Haemonchus (H.) contortus and their eggs in adult motility assay (AMA) and egg hatch test (EHT), respectively. In vivo anthelmintic activity of crude powder (CP) and CAME in increasing doses (1.0-8.0 g kg(-1)) was determined in sheep naturally infected with mixed species of nematodes using fecal egg count reduction test (FECRT) and larval counts. The study design also included untreated as well as treated controls. Fecal egg count reduction and larval counts from coprocultures were performed pre- and post-treatments to assess the anthelmintic activity of the plants. CAME of T. portulacastrum and M. paradisiaca showed a strong in vitro anthelmintic activity and pronounced inhibitory effects on H. contortus egg hatching as observed through AMA and EHT, respectively. Both plants exhibited dose and time dependent anthelmintic effects on live worms as well as egg hatching. M. paradisiaca (LC(50)=2.13 μg mL(-1)) was found to be more potent than T. portulacastrum (LC(50)=2.41 μg mL(-1)) in EHT. However, in vivo, maximum reduction in eggs per gram (EPG) of faeces was recorded as 85.6% and 80.7% with CAME of T. portulacastrum and M. paradisiaca at 8.0 g kg(-1) on 15th day post-treatment, respectively as compared to that of Levamisole (7.5 mg kg(-1)) that caused 97.0% reduction in EPG. All the species of gastrointestinal nematodes (GINs), i.e. Haemonchus contortus, Trichostronglyus spp., Oesophagostomum columbianum and Trichuris ovis which were prevalent, found susceptible (P<0.01) to the different doses of CP and CAME of both plants. The data showed that both T. portulacastrum and M. paradisiaca possess strong anthelmintic activity in vitro and in vivo, thus, justifying their use in the traditional medicine system of Pakistan.

Prevalence and associated risk factors for bovine tick infestation in two districts of lower Punjab, Pakistan.

Bovine tick infestation is still a serious nuisance to livestock and the dairy industry of Pakistan. The current paper reports the prevalence and associated risk factors for bovine tick infestation in the districts Layyah and Muzaffargarh of lower Punjab, Pakistan. A questionnaire-based survey was conducted to identify and to quantify variation in the prevalence of bovine tick infestation with respect to host (age, species, sex, and breed) and environmental (geographical area and climate) determinants. Multiple stage cluster random sampling was used and 3500 cattle and buffaloes from the two districts were selected. Prevalence of bovine tick infestation was significantly higher (OR=1.95; p<0.05) in cattle (1076/1475; 72.9%) than in buffaloes (957/2025; 47.3%). Hyalomma anatolicum anatolicum was the major tick species (33.5%; 1173/3500), followed by Rhipicephalus sanguineus (13%; 456/3500). The highest monthly prevalence in both the districts was found in July. Ticks were not found in Layyah from November to March and in Muzaffargarh from December to March. The average number of ticks was proportional to the prevalence of infestation. Also, tick infestation in a 7cmx7cm dewlap of the animal was proportional to that of the rest of body. Prevalence of tick infestation was associated (p<0.05) with district, host species and breed. In cattle, prevalence of tick infestation was associated (p<0.05) with age and sex of host. The results of this study provide better understanding of disease epidemiology in the study districts, which will help for planning of control strategies.

The global burden of fasciolosis in domestic animals with an outlook on the contribution of new approaches for diagnosis and control

Fasciolosis is an economically important disease for livestock, as well as being zoonotic. Recent figures on the prevalence of this disease have caused alarm concerning its potential for an increased prevalence in the future. The prevalence of fascioliosis has been documented from different regions of the world, helping us identify areas where future research needs to be focused. This manuscript is a review of the current status of the disease, the pathogenic species involved, diagnostic techniques (with new modifications and comparative specificity, sensitivity, and rapidity of these tests), chemotherapy, and vaccination. This also encompasses inaccurate reports on vaccination and drug development as well as the latest technologies to find promising candidates for drugs and vaccines. Drugs with lower efficacy have been used on some farms which lead to exacerbation of the clinical disease, presumably due to the development of drug resistance. Future studies should be focused on (1) the use of the most reliable diagnostic tests for periodic monitoring of the disease, (2) insights of the ecobiology and transmission dynamics of the snail intermediate host and the best possible methods of their control, (3) in vitro and in vivo testing of chemotherapeutic compounds using sensitive methods, and (4) the identification of novel drug and vaccine candidates using modern molecular markers. This approach may help increase the reliability of chemotherapeutic agents and control nuisance, ultimately reducing the economic losses attributable to the livestock industry around the world.

Prevalence of Helminth Infections in Dairy Animals of Nestle Milk Collection Areas of Punjab (Pakistan)

Abstract The objective of the current research project was to document the prevalent helminths of dairy animals of Nestle milk collection areas of Punjab (Pakistan). For this purpose, seven high milk-producing areas of Punjab province including Farooqa, Kot Adu, Dunya Pur, Layyah, Mor Mandi, Shorkot and Jalapur were selected. The animals were randomly selected and screened for parasitic eggs through standard coprological examination procedures. The helminth species found prevalent in the study areas included; Ascaris vitulorum, Fasciola hepatica, Haemonchus contortus, Bunostomum phlebotomum,Ostertagia circumcinta, Oesophagostomum radiatum, and Trichostrongylus spp. The possible determinants associated with the prevalence of these parasites were also studied in this project. The results of this study provided a basic epidemiological data for planning a wide scaled helminth control program in the above-mentioned high producing areas of Pakistan.

Molecular epidemiology of Theileria annulata and identification of 18S rRNA gene and ITS regions sequences variants in apparently healthy buffaloes and cattle in Pakistan

A molecular epidemiological survey was conducted to determine the prevalence of piroplasms in buffaloes and cattle from Sheikhupura and Okara districts of Punjab, Pakistan using reverse line blot (RLB) hybridization assay. The genetic diversity within 18S rRNA gene and ITS regions sequences of various obtained Theileria species (spp.) was also investigated. Briefly, 102 blood samples from buffaloes and cattle in the study districts were collected on blood collection cards and brought to the laboratory. DNA was extracted; the V4 hypervariable region of 18S rRNA was amplified and analyzed using RLB. Out of total samples analyzed, 61 (59.8%) were hybridized with Babesia/Theileria (B/T) genus-specific probe. Only one species of piroplasm was detected in buffaloes and cattle in study districts, i.e. Theileria (T.) annulata. Six samples only hybridized with B/T genus-specific and Theileria genus-specific probes but not with any species-specific probe indicating the presence of novel species or variants. The sequences of 18S rRNA gene and ITS regions of these six samples revealed the presence of T. annulata variants as confirmed through sequence identity estimation and phylogenetic analyses. Meanwhile, an unexpected sequence variation was observed within the 18S rRNA gene and ITS regions sequences of T. annulata identified in the present study. This is the first report on the simultaneous detection of species of piroplasms infecting buffaloes and cattle in Pakistan and molecular characterization of T. annulata 18S rRNA gene and ITS regions. The present study may address the new insights into the epidemiology of theileriosis which will help researches in designing control strategies and developing various molecular diagnostic tools at national level.

Identification and characterization of a novel 34 kDa merozoite protein in Babesia orientalis.

A novel Babesia orientalis 34 kDa protein (designated BoP34) was obtained by immunoscreening of a cDNA expression library using B. orientalis infected water buffalo serum. The complete nucleotide sequence of the BoP34 was 1088 bp, which contained one open reading frame (ORF), two untranslated regions (UTRs) and a poly (A) tail. The length of ORF was 933 bp, encoding a polypeptide of 310 aa with a predicted size of 34 kDa. BLAST analysis showed that the nucleotide sequence of BoP34 had 71% similarity with that of the Babesia bovis gene XM_001611335, which encodes a nuclear movement family protein. This suggested that BoP34 is a homologous of the movement family protein. Structural analysis of the BoP34 protein indicated a CS domain which may interact with the ATPase domain of the heat shock protein 90. A truncated version of BoP34 was cloned into the expression vector pET-32a and subsequently expressed and purified from the Escherichia coli Rosetta™ (DE3) pLysS stain as a Trx-fusion (designated rBoP34-T). Antibodies in the serum of a B. orientalis-infected water buffalo were able to recognize this protein in immune-bloting analysis. Rabbit antibodies raised against rBoP34-T could detecte native BoP34 (34 kDa) in B. orientalis-infected water buffalo erythrocytes. These results suggested that BoP34 might be a good diagnostic antigen for the specific detection of anti-B. orientalis antibody in water buffalo. Further research is required to explore the biological function and diagnostic potential of this molecule.

Prevalence and associated risk factors of haemoparasites, and their effects on hematological profile in domesticated chickens in District Layyah, Punjab, Pakistan

Several haemoparasites commonly infect avian species, including chicken that significantly decline productivity and ultimately lead to high mortality rate. The current study was designed to assess the prevalence and associated risk factors of haemoparasites in domesticated chickens in and around District Layyah, Punjab Province, Pakistan. For this purpose, blood samples from a total of 384 backyard chickens were collected from wing vein using sterile insulin syringe. The parasites were identified from Giemsa stained thin blood smears based on morphological features using standard keys. Results demonstrated that a total of 265 backyard chickens (69%) were infected with haemoparasites in District Layyah. According to genus-wise distribution, 31.5%, 24.4% and 13% prevalence of genera Plasmodium/Haemoproteus, Leucocytozoon and mixed species were recorded, respectively. Among associated risk factors, the prevalence was relatively higher in females, chicks, naked neck breeds, scavengers feeding patterns and chickens reared at fully open coops type. The information given in the study could be of much importance in planning of an effective haemoparasites control program at District and Provincial level.

Immunological and Therapeutic Evaluation of Wheat (Triticum aestivum) Derived Betaglucans against Coccidiosis in Chicken

This study was carried out to determine the immunomodulatory and therapeutic effects of wheat derived beta-glucans (purified) against avian coccidiosis. Briefly, beta-glucans from wheat bran were extracted and purified using standard procedures. A total of 250 broiler chicks (day-old) were reared at experimental station, Department of Parasitology, University of Agriculture, Faisalabad for this study. At 7 th day of their age, birds were subdivided into five equal groups (n=50). Groups A, B and C were orally administered with graded doses of purified beta-glucans (50, 100 and 150 mg/kg of body weight) for three consecutive days; whereas, positive control (group D) was administered with vitamin-E at the dose rate of 87mg/kg; while negative control (group E) was served with phosphate buffered saline (PBS solution). At 14 th day post administration of graded doses, 30 chickens from each group were randomly selected and used to monitor cellular and humoral immune responses, while remaining birds (n=20) in each group were challenged with mixed species of genus Eimeria for therapeutic evaluation. Overall, chickens in group A administered with 50 mg/kg b.wt. purified beta-glucans showed significantly higher immune responses in terms of enhanced humoral and cell mediated immunity as compared to those of other beta-glucans administered and E groups. While, the immune responses showed by group A were comparable with group D. After challenge with Eimeria, the groups A and D also showed maximum weight gains with low oocyst counts and maximum percent protection against lesions in both caecum and intestine. On the other hand, a minimum daily weight gain with increased number of oocysts in chickens was observed in control group (PBS) during therapeutic evaluation. These findings suggest that the beta-glucans derived from wheat may have immunomodulatory and therapeutic effects against Eimeria infection in chickens.