Kamran Ashraf

Alleviation of cyclic heat stress in broilers by dietary supplementation of mannan-oligosaccharide and Lactobacillus-based probiotic: Dynamics of cortisol, thyroid hormones, cholesterol, C-reactive protein, and humoral immunity

Heat stress (HS), one of the major problems of tropical and subtropical countries, adversely affects the production performance of poultry. Keeping this in view, the present study was designed to investigate some of the biological markers of HS in broilers as modulated by dietary supplementation of mannan-oligosaccharide (MOS) and a Lactobacillus-based probiotic (LBP), either alone or in combination. Two hundred fifty 1-d-old-chicks were randomly divided into 5 groups. From d 22, the birds were either kept at the thermoneutral zone (TN) or exposed to HS to the conclusion of study, d 42. Birds were fed either a corn-based basal diet (TN and HS groups) or the same diet supplemented with 0.5% MOS (HS-MOS group), 0.1% LBP (HS-LBP group), or their combination. Birds were immunized against Newcastle disease virus on d 4 (intraocular; live attenuated) and d 20 (drinking water; live attenuated) and infectious bursal disease virus on d 8 (intraocular; live intermediate strain) and d 24 (drinking water; live attenuated). Birds were killed on d 42 to collect serum for determination of cortisol, thyroid hormones, cholesterol, C-reactive protein, and postvaccinal antibody titers. Results revealed that dietary supplementations decreased (P < 0.05) the serum cortisol and cholesterol concentrations and increased (P < 0.05) thyroxine concentration compared with the HS group without affecting triiodothyronine concentration. The percentage of the C-reactive protein-positive birds was higher (P < 0.05) in the HS group compared with the TN group. Dietary supplementations improved humoral immunity against Newcastle disease virus and infectious bursal disease virus during HS. In conclusion, dietary supplementation of either MOS or LBP alone or in combination can reduce some of the detrimental effects of HS in broilers.

Single or combined effects of mannan-oligosaccharides and probiotic supplements on the total oxidants, total antioxidants, enzymatic antioxidants, liver enzymes, and serum trace minerals in cyclic heat-stressed broilers

This study was intended to explore the oxidative status of broilers under cyclic heat stress (HS) as modulated by supplementation of mannan-oligosaccharides (MOS) and a probiotic mixture (PM). Two hundred fifty 1-d-old chicks were randomly divided into 5 groups. From d 22, birds were either kept in a thermoneutral zone (TN; 26.7°C for 24 h/d) or subjected to HS (35 ± 1.1°C and 75 ± 5% RH for 8 h/d from 1000 to 1800 h) to the conclusion of the study on d 42. Birds were fed either a corn-based diet (TN and HS groups) or the same diet supplemented with 0.5% MOS (HS-MOS group), 0.1% PM (HS-PM group), or their combination as a symbiotic (SYN; HS-SYN group). On d 42, birds were killed by cervical dislocation to collect serum for the determination of total oxidants, total antioxidants, paraoxonase, arylesterase, ceruloplasmin, aspartate aminotransferase, alanine aminotransferase, and trace minerals. Heat stress increased (P < 0.05) total oxidants and total antioxidants and decreased (P < 0.05) paraoxonase and arylesterase, with no change in ceruloplasmin, aspartate aminotransferase, and alanine aminotransferase activities. Dietary supplementation decreased (P < 0.05) total oxidants and total antioxidants, with no effect on the activities of other enzymes. Heat stress did not influence serum copper, zinc, and manganese concentrations of birds when compared with those in the TN group. However, MOS increased (P < 0.05) concentrations of all the trace minerals, whereas SYN increased (P < 0.05) concentrations of only zinc and copper. It was concluded that MOS or PM supplementation, alone or as a SYN, may reduce some of the detrimental effects of HS, whereas MOS alone or as a SYN may improve the absorption of trace minerals.

Genetic evidence for hybridisation between Haemonchus contortus and Haemonchus placei in natural field populations and its implications for interspecies transmission of anthelmintic resistance.

Genetic hybridisation between parasitic nematode species has potentially important consequences. It could lead to the introgression of genes between species including those involved in pathogenicity, host specificity, transmission and drug resistance. It could also complicate diagnosis and control. However, there are few compelling examples of its occurrence in parasites in the field. Haemonchus contortus and Haemonchus placei are two closely related parasitic nematode species that predominantly infect small ruminants and cattle, respectively. They are capable of experimental hybridisation when adult worms of each species are transplanted into the same individual host. Given that co-infection occurs in both small ruminants and cattle, there is potential for hybridisation in the field. However, this has not been definitively demonstrated and its extent is unknown. We investigated the occurrence of co-infection and interspecies hybridisation in H. contortus and H. placei in field populations from small ruminants from Pakistan and southern India using a number of independent genetic markers. Haemonchus contortus and H. placei co-infections were common in Pakistan but not in southern India where H. placei appeared to be absent in small ruminant hosts. In the former region, a number of worms were identified that were heterozygous for fixed, species-specific rDNA internal transcribed spacer 2 (ITS-2) single nucleotide polymorphisms. Genotyping of these ITS-2 heterozygotes with an additional four nuclear markers conclusively demonstrated them to be F1 interspecies hybrids. Mitochondrial NADH dehydrogenase subunit 4 haplotype analysis demonstrated that four of the hybrid worms had a H. placei maternal parent and one had a H. contortus maternal parent showing that hybridisation could occur in either direction. Interestingly, one of these hybrids contained an H. contortus isotype-1 β-tubulin benzimidazole resistance allele, suggesting there is a potential for interspecies introgression of drug resistance loci. We believe this is the first definitive genetic evidence of hybridisation between H. contortus and H. placei in the field and represents the most comprehensive genetic evidence of F1 hybrids between any human or livestock parasitic nematode species to date. Further, it suggests that interspecies transmission of anthelmintic resistance mutations warrants further investigation.

Neospora caninum prevalence in dogs raised under different living conditions.

Neospora caninum is an important cause of abortion in dairy cattle worldwide. Dogs are important in the epidemiology of N. caninum because they act as definitive hosts shedding oocysts in the environment. Vertical transmission of the parasite is well recognized as an important aspect of the epidemiology of the parasite but the importance of horizontal transmission has been less studied. A N. caninum competitive ELISA was used to examine serum samples from 600 dogs that were raised under 4 different living conditions. Samples from 138 dogs living on 24 dairies with a prevalence (0-70%) of anti-N. caninum antibodies in the cattle, 294 pet dogs without neurological signs, 76 from pet dogs exhibiting neurological signs, and 92 stray dogs were examined. The overall seroprevalence of N. caninum was 23.5% (95% CI = ± 2.99) in the 600 dogs. Significant (P < 0.05) differences were observed between the 4 different populations of dogs. The number of N. caninum positive samples were: 51 (36.9%, 95% CI = ± 3.09) of 138 dogs from dairies, 31 (10.5%, 95% CI = ± 6.38) of 294 pet dogs without neurological signs, disorders, 22 (28.9%, 95% CI = ± 6.70) of 76 pet dogs with neurological signs, and 37 (40.2%, 95% CI = ± 2.83) of 92 stray dogs. Seropositivity to N. caninum in dogs from dairies was associated with a high prevalence of N. caninum antibodies in the cattle. At the 3 dairies where no dogs were present, the seroprevalence to N. caninum in the cattle was significantly lower (P < 0.05) than in the 21 dairies where dogs were present. Seroprevalence was significantly higher (P < 0.05) in male dogs (97 of 366; 26.5%, 95% CI = ± 3.40) than in female dogs (44 of 234; 18.8%, 95% CI = ± 5.65). Seroprevalence in dogs increased with age suggesting postnatal exposure to N. caninum infection however, this increase was not significant (P > 0.05). The prevalence of N. caninum antibodies was not significantly (P>0.05) different in dogs based on breed. These findings suggest a relationship between N. caninum infection of dogs from dairies and cattle on these dairies. However, further research is required to determine what is the most important way dogs acquire infection and how to prevent dogs from shedding oocysts.

Molecular evidence shows that the liver fluke Fasciola gigantica is the predominant Fasciola species in ruminants from Pakistan

Fascioliasis is an important disease affecting livestock, with great costs to producers worldwide. It has also become a serious issue for human populations in some endemic areas as an emerging zoonotic infection. There are two Fasciola species of liver fluke responsible for this disease, which occur worldwide, Fasciola hepatica and Fasciola gigantica. Identifying these two species on the basis of adult or egg morphology requires specialist knowledge due to the similarity of characters, and may misidentify putative intermediate or hybrid forms. In this study we sequenced the internal transcribed spacer 2 (ITS-2) rDNA of liver flukes collected from multiple species of hosts from seven localities in the Punjab and Baluchistan provinces of Pakistan, to determine the distribution of these two species. All 46 flukes processed in this study, collected from seven sites, showed the rDNA ITS-2 genotype corresponding to F. gigantica, contradicting previous reports, based on adult and egg morphology, that both species are present in Pakistan, with F. hepatica being the more common.

The effect of infection with mixed Eimeria species on hematology and immune responses following Newcastle disease and infectious bursal disease booster vaccination in broilers

Background: Coccidiosis is an important parasitic disease of chickens, causing high mortality and morbidity. This morbidity is believed to be correlated with altered population dynamics of blood cells and immunocompromisation. Objectives: This study investigated the effects of mixed Eimeria species (viz., tenella, maxima, acervulina and necatrix) infection on hematology and immune responses following Newcastle disease (ND) and infectious bursal disease (IBD) booster vaccination in broilers. Animals and methods: One-day-old broiler chicks (Hubbard; n = 200) were divided into two equal groups A and B. On day 16, group A was infected orally with Eimeria species (7 × 104 sporulated oocysts), whereas group B served as control. Both groups were analyzed for hematological parameters on post-infection days 6–8. Sera from both groups were analyzed for antibody titers against ND and IBD vaccines. On day 8 post-infection, lymphoid organs were also examined. Results: Significantly lower (P < 0.05) levels of plasma proteins, globular volume, hemoglobin concentration, packed cell volume, total erythrocytes, mean corpuscular volume and mean corpuscular hemoglobin concentration were found in infected chickens compared with non-infected control chickens. In addition, the infected group exhibited significantly increased (P < 0.05) numbers of different leukocytes. Infected chickens also showed significantly lower antibody titers against ND and IBD with decreased relative organ weights of all lymphoid organs except spleen. Conclusion and recommendations: Mixed species of Eimeria adversely affected the hematology and immune efficiency of broilers. Thus, inexpensive immune potentiators and hemotonics along with appropriate anti-coccidial medications are suggested to avoid the complications and subsequent economic losses.

Response of maternally isolated rock pigeons (Columba livia domestica) to different dietary concentrations of mannan-oligosaccharide

Prebiotics are being used as an alternative to antibiotic growth promoters in poultry industry with a variable degree of success. This trial was conducted to investigate the effect of dietary mannan-oligosaccharide (MOS) supplementation on growth performance, selected colonic bacterial population, and immune response in maternally-isolated rock pigeons. The pigeons (n = 36) were randomly distributed in 4 treatment groups (n = 9 per group) with 3 replicates or pens (n = 3) in each group. Birds were fed either a corn/wheat-based basal diet (control group/CON) or the same diet supplemented with 0.1%, 0.2%, or 0.5% MOS for 35 d. On d 35, birds were killed to collect visceral organs, colonic contents, and serum. Colonic contents were used to enumerate selected microbiota and serum was used to detect antibody titer against the Newcastle disease virus. Cell-mediated immunity was determined by measuring the skin thickness following 2,4-dinitrochlorobenzene challenge. Results showed that supplementation did not affect the BW of birds. During wk 4, feed intake was significantly higher in the 0.2% (187.9 ± 0.86) and the 0.5% (186.4 ± 0.86) MOS-supplemented groups compared with the CON group (180.7 ± 0.86). Gizzard weights (with and without digesta) were significantly higher in the MOS 0.1% (10.67 ± 0.33 and 8.22 ± 0.26) and the MOS 0.2% (9.91 ± 0.33 and 7.94 ± 0.26) groups compared with the CON group (7.56 ± 0.33 and 6.25 ± 0.26). Small intestinal weight was significantly higher in the MOS 0.2% group (14.71 ± 0.56) compared with the CON group (9.56 ± 0.56). Lengths of small intestine (MOS 0.1% = 92.56 ± 1.69, MOS 0.2% = 90.79 ± 1.69, MOS 0.5% = 90.57 ± 1.69) in all the MOS-fed groups and large intestine in the 0.1% (3.50 ± 0.02) and the 0.5% (3.47 ± 0.02) MOS-fed groups were significantly higher than the CON group (small intestine = 77.63 ± 1.69, large intestine = 2.41 ± 0.02). Weights of heart, liver, pancreas, and large intestine remained unaffected. Feeding of MOS did not influence colonic population of Escherichia coli, Clostridia, and Bifidobacteria. Lactobacilli count was significantly higher in the 0.2% MOS-fed group (9.77 ± 0.12) compared with the CON group (9.19 ± 0.16). Mannan-oligosaccharide did not affect the immune response of the birds as antibody titer against the New Castle disease virus and the cell-mediated immunity remained similar in all the treatment groups. In conclusion, dietary MOS supplementation influenced only the colonic lactobacilli population without any apparent effects on the production performance in growing pigeons.

Anthelmintic Drugs: Their Efficacy and Cost-Effectiveness in Different Parity Cattle

Abstract Gastrointestinal nematodes are responsible for economic losses in bovines and are characterized by reduced milk production, decreased working efficiency, and even death. In our study, the effect of different anthelmintic treatments on nematode control in different parity cattle (Friesian crossbreds) at calving and their effect on milk yield were evaluated. The economics of anthelmintics and farm benefits in terms of increased milk production after deworming was also calculated. We screened cattle of first and second parity for nematodes. Animals were randomly selected in each group. In first parity animals, there were 23 positive cattle found, which were divided into 3 different groups, while in second parity animals there were 20 positive cattle which were also divided into 3 groups. For treatment of gastrointestinal nematodes, we used albendazole (velbazine) at 10 mg/kg body weight and levamisole (Nilverm®) at 7.5 mg/kg. In this study, both drugs were found effective in controlling nematode infections in cattle. Percentage reduction of eggs per gram (EPG) by albendazole was 48.20, 85.34, and 93.90% and 51.54, 81.43, 91.74% on day 7, 14, and 21 in first and second parity animals, respectively. Percentage reduction of EPG by levamisole was 44.45, 76.92, and 88.03% and 46.60, 73.78, 85.43% on day 7, 14, and 21 in first and second parity animals, respectively. The average increase in milk production in albendazole-treated groups was 0.39 and 0.92 L per day while increases in levamisole treated groups were 0.27 and 0.55 L per day in first and second parity cattle, respectively. After treatment, albendazole increased the milk fat by 0.07 and 0.1% while levamisole decreased by 0.02 and 0.05% in first and second parity cattle, respectively. It is concluded that anthelmintic treatments of recently calved cattle have a significant effect on milk production due to the nematode control. Milk production increased significantly in second parity cattle following anthelmintic treatment as compared to first parity animals. Levamisole had a negative effect on fat concentration in cattle while albendazole-treated cattle showed a positive effect. Albendazole has been found more efficient in reducing EPG of helminths in both parity animals as compared to levamisole-treated animals while the cost–benefit ratio of levamisole was greater than albendazole.

Emergence and the spread of the F200Y benzimidazole resistance mutation in Haemonchus contortus and Haemonchus placei from buffalo and cattle

Benzimidazoles have been intensively used in the livestock sector, particularly in small ruminants for over 40 years. This has been led to the widespread emergence of resistance in a number of small ruminant parasite species, in particular Haemonchus contortus. In many counties benzimidazole resistance in the small ruminants H. contortus has become severely compromising its control; but there is a little information on benzimidazole resistance in H. contortus infecting buffalo and cattle. Resistance to benzimidazoles have also been reported in the large ruminant parasite, Haemonchus placei, but again there is relatively little information on its prevalence. Hence it is extremely important to understand how resistance-conferring mutations emerge and spread in both parasites in the buffalo and cattle host in order to develop the approaches for the recognition of the problem at an early stage of its development. The present study suggests that the F200Y (TAC) mutation is common in H. contortus, being detected in 5/7 populations at frequencies between 7 to 57%. Furthermore, 6/10 H. placei populations contained the F200Y (TAC) mutation, albeit at low frequencies of between 0.4 to 5%. The phylogenetic analysis suggests that the F200Y (TAC) mutation in H. contortus has emerged on multiple occasions in the region, with at least three independent emergence of resistance alleles across the populations. In contrast, the F200Y (TAC) resistance-conferring mutation in H. placei is only seen on a single haplotype. A high level of haplotype frequency of the susceptible alleles in the region, suggests that the unique resistance conferring-mutation has spread from a single emergence; likely by anthropogenic animal movement. Overall, these results provide the first clear genetic evidence for the spread of benzimidazoles resistance-conferring mutations to multiple different locations from a single emergence in H. placei; while supporting previous small ruminant-based observations of multiple emergence of resistance mutations in H. contortus.

Population genetics of benzimidazole-resistant Haemonchus contortus and Haemonchus placei from buffalo and cattle: implications for the emergence and spread of resistance mutations

The population genetics of nematode parasites are poorly understood with practical reference to the selection and spread of anthelmintic resistance mutations. Haemonchus species are important to study the nematode population genetics due to their clinical importance in ruminant livestock, and the availability of genomic resources. In the present study, it has been examined that Haemonchus contortus and Haemonchus placei populations from three buffalo and nine cattle hosts. Seventy-three individual adult worms of H. contortus and 148 of H. placei were analysed using a panel of seven microsatellite markers. The number of alleles per locus in H. contortus and H. placei indicated that all populations were polymorphic for the microsatellites used in the present study. Genetic diversity parameters included high levels of allelic richness and heterozygosity, indicating effective population sizes, high mutation rates and high transmission frequencies in the area. Genetic structure parameters revealed low genetic differentiation between and high levels of genetic variation within H. contortus and H. placei populations. Population dynamic analyses showed an absence of heterozygosity excess in both species, suggesting that there was no deviation from genetic drift equilibrium. Our results provide a proof of concept for better understanding of the consequences of specific control strategies, climatic change or management strategies on the population genetics of anthelmintic resistance alleles in Haemonchus spp. infecting co-managed buffalo and cattle.