First report of Echinococcus ortleppi and genotype G6 of E. canadensis cluster from southern Punjab, Pakistan and a global overview on genetic structure and host adaptation of E. ortleppi.

Cystic echinococcosis (CE), caused by Echinococcus granulosus sensu lato, is a neglected tropical disease known mainly for its zoonotic nature. CE is endemic to Pakistan, however, the disease is not given due consideration and millions of people remain at health risk. This study was undertaken to assess the species and genotypes of E. granulosus sensu lato in sheep, buffaloes and cattle, brought to slaughterhouses of two major cities (Multan and Bahawalpur) of south Punjab, Pakistan. A total of 26 hydatid cyst specimens were characterized through complete cox1 mitochondrial gene (1609 bp) sequencing. Species and genotypes of E. granulosus sensu lato discovered in the southern Punjab consisted of E. granulosus sensu stricto (n =21), E. ortleppi (n=4) and genotype G6 of the E. canadensis cluster (n=1). Of E. granulosus s.s. isolates, the genotype G3 was predominantly involved in causing infections to the livestock of this region. Since all of these species are zoonotic, wide and effective surveillance studies are required to ascertain the risks to human population in Pakistan. Additionally, a global overview on cox1 phylogenetic structure of E. ortleppi was carried out. Despite widespread occurrence, the species is mostly limited to the southern hemisphere. The highest burden has been reported in South America (62.15%) and Africa (28.44%) and by far the most common host is cattle, accounting for >90% of cases.

Biotic Potential Induced by Different Host Plants in the Fall Armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae).

Fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is a polyphagous insect pest of many important crops. To evaluate the influence of host plants on the biology and survival of the Pakistani population of S. frugiperda, we examined life table parameters of S. frugiperda raised on maize, sorghum, wheat, and rice. The development rate was significantly higher on the maize crop than on the other three host plants. Different larval diets affected development time and fecundity. S. frugiperda attained the fastest larval development (16 days) on maize and the slowest development (32.74 days) on rice. Adult females from maize-fed larvae laid 1088 eggs/female, those from sorghum-fed larvae laid 591.6 eggs/female, those from wheat-fed larvae laid 435.6 eggs/female, and those from rice-fed larvae laid 49.6 eggs/female. Age stage-specific parameters also indicated the higher fecundity, higher life expectancy, and higher survival of S. frugiperda on maize plants than on the other three hosts. Larval diets had a significant varying effect on the finite and intrinsic increase rates, reflecting that maize was the most suitable diet. The findings of the present study are useful for predicting population dynamics especially in areas cultivating Poaceae crops, except maize, to develop sustainable integrated pest management strategies for this pest.

Genetic structure and phylogeography of Echinococcus granulosus sensu stricto genotypes G1 and G3 in Pakistan and other regions of the world based on nad5 gene.

Pakistan is a neglected endemic focus for Echinococcus granulosus sensu lato, a zoonotic parasite species complex with the ability to infect wide spectrum of hosts. Wide gaps exist in literature for etiological agents of cystic echinococcosis (CE) in Pakistan due to a very low number of studies on identifying the exact genotypes involved in epidemiological manifestation of this disease. Focusing on transmission patterns and epidemiological dynamics, this study aimed at investigating infective genotypes among the cattle population of south Punjab, Pakistan, employing a mitochondrial marker nad5 (680 bp). Nucleotide sequences retrieved from 28 hydatid cyst isolates displayed considerable intraspecific variation revealing the existence of G3 and G1 strains of Echinococcus granulosus sensu stricto. The G3 genotype emerged as the predominant cause (78.57%) of hydatidosis in cattle. Apart from this, to understand phylogeographical relations, homologous nucleotide sequences of the partial nad5 gene from six major regions of the world were employed in the population genetics analysis to have an insight into genetic variability and demographics of G3 genotype in particular. Diversification of G3 and its haplotypes in Pakistan (n = 11) and other regions of the world (India, Iran, Turkey, Italy and France) was demonstrated. It was further demonstrated that the South Asian population (Pakistan and India) was highly differentiated from the other regions. It could, therefore, be speculated that G3 is diverging and expanding its population with South Asia as the main focal point.

Host-Plant Variations Affect the Biotic Potential, Survival, and Population Projection of Myzus persicae (Hemiptera: Aphididae).

The green peach aphid, Myzus persicae Sulzer (Hemiptera: Aphididae), a polyphagous insect pest is a major threat to a wide range of crops worldwide. Aiming to evaluate the life history traits of M. persicae, feeding on different host plants, we used five vegetables: cabbage, Brassica oleracea (Brassicaceae); chinese cabbage, B. rapa (Brassicaceae); chili pepper, Capsicum annum (Solanaceae); crown daisy, Chrysanthemum coronarium (Asteraceae); and eggplant, Solanum melongena (Solanaceae). TWOSEX-MSchart software was used for the statistical analysis about the age-stage, two-sex life table theory. The highest fecundity (69.65 individuals) rate of M. persicae, intrinsic rate of increase (r = 0.425 d-1), finite rate of increase (λ = 1.531 d-1), net reproductive rate (R0 = 69.65 offspring), and shortest mean generation time (T = 9.964 d) were recorded on the chili pepper plant. Whereas, lower fitness occurred on cabbage. The findings attained from population growth parameters indicate that chili pepper is the most susceptible plant, while cabbage is resistant to aphids. Population projection results also supported this statement, as the final total population size on cabbage was significantly lower than other plants. The reported information would be useful for devising integrated pest management programs, particularly those involving M. persicae. This information also suggests the adaptability of M. persicae causing economic damage to these vegetable cultivars.

Extracts of Euphorbia nivulia Buch.-Ham. showed both phytotoxic and insecticidal capacities against Lemna minor L. and Oxycarenus hyalinipennis Costa.

Many phytochemicals can affect the growth and development of plants and insects which can be used as biological control agents. In this study, different concentrations of crude, hexane, chloroform, butanol, and aqueous extracts of Euphorbia nivulia Buch.-Ham., an endemic plant of the Cholistan desert in South Punjab of Pakistan, were analysed for their chemical constituents. Their various concentrations were also tested for their phytotoxic and insecticidal potential against duckweed, Lemna minor L., and the dusky cotton bug, Oxycarenus hyalinipennis Costa. various polyphenols, i.e., quercetin, gallic acid, caffeic acid, syringic acid, coumaric acid, ferulic acid, and cinnamic acid were detected in different concentrations with different solvents during the phytochemical screening of E. nivulia. In the phytotoxicity test, except for 100 µg/mL of the butanol extract gave 4.5% growth regulation, no phytotoxic lethality could be found at 10 and 100 µg/mL of all the extracts. The highest concentration, 1000 µg/mL, of the chloroform, crude, and butanol extracts showed 100, 63.1, and 27.1% of growth inhibition in duckweed, respectively. In the insecticidal bioassay, the highest O. hyalinipennis mortalities (87 and 75%) were recorded at 15% concentration of the chloroform and butanol extracts of E. nivulia. In contrast, the lower concentrations of the E. nivulia extracts caused the lower mortalities. Altogether, these findings revealed that E. nivulia chloroform extracts showed significant phytotoxicity while all the extracts showed insecticidal potential. This potential can be, further, refined to be developed for bio-control agents.

Comprehensive study based on mtDNA signature (nad1) providing insights on Echinococcus granulosus s.s. genotypes from Pakistan and potential role of buffalo-dog cycle.

Pakistan has long been considered neglected endemic region for Echinococcus granulosus. Limited surveillance studies have failed to epidemiologically draw complete picture on geographical presence and etiological agents of cystic echinococcosis. Amidst such lacunae, current study explored main transmission routes of this disease through molecular characterization of hydatid cyst isolates obtained from sheep (n = 35), goats (n = 26), cattle (n = 30) and buffalo (n = 30) from the four provinces of Pakistan. Two strains of E. granulosus sensu stricto, G1/G3, and their haplotypes were observed to be cycling in sympatry in the domestic ungulate populations. G3 genotype had higher prevalence (66.94%) in the hosts compared to G1 genotype (33.06%) which was not surprising, considering the large buffalo population in Pakistan. Haplotypic analysis revealed presence of 9 different haplotypes configured in a double clustered network with two centrally positioned haplotypes referred to as G3 (PKH1) and G1 (PKH6). Population demographics and genetic variability indices suggested expanding parasitic population in multiple host spectrum. Elucidating local transmission patterns of E. granulosus sensu stricto, buffalo-dog cycle emerged as one of the dominant causes of G3 dispersal in contrary to other global studies. Adaptability of G3 to environmental conditions of Pakistan and high affinity for buffaloes emphasize on heterogeneous nature of this strain in contrast to G1. However, more studies involving larger datasets and mitochondrial sequences could confirm this hypothetically formulated inference.

Immunomodulatory and ameliorative effects of Lactobacillus and Saccharomyces based probiotics on pathological effects of eimeriasis in broilers.

Eimeria infection is very important in broilers and causes heavy economic losses in extensive farming system due to reduced weight gains, high mortality and poor feed conversion ratio (FCR). Under the circumstances, there is a dire need to devise effective control strategies to avoid/counteract this infectious threat. This study was conducted to assess the immunomodulatory and ameliorative effects of Lactobacillus and Saccharomyces based probiotics against Eimeria infection in broilers. The results showed statistically higher (P < 0.05) lymphoproliferative responses in experimental groups treated either with Lactobacillus or Saccharomyces based probiotics, as compared to control group. Further higher antibody titers (geomean titers) were also recorded in chickens of experimental groups treated with probiotics as compared to those of control group. The probiotic treated groups also revealed significantly improved (P < 0.05) FCRs as compared to control group. In challenge experiment, significantly lower (P < 0.05) oocyst counts were recorded in broilers treated with probiotics, when compared with control group. Further, experimental groups also revealed significantly higher (P < 0.05) daily weight gains and protection rates as compared to control. The data regarding the lesion scoring showed that chickens treated with probiotics had higher values of percent protection against intestinal and caecal lesion, when compared with those of control group. In conclusion, supplementation of probiotics proved very useful to enhance the immunological and performance potentials of broilers which subsequently provided protection against Eimeria infection. Further studies on the physico-chemical properties along with commercial feasibility and cost benefit analysis of these probiotic species are needed for wise selection to get maximum profit from broiler industry.

Evaluation of immunostimulatory and immunotherapeutic effects of tropical mushroom (Lentinus edodes) against eimeriasis in chicken.

Mushrooms (Lentinus edodes) were processed for hot water (HWE), methanolic (ME), and polysaccharide (PSE) extracts. Polysaccharides were isolated through ion exchange (DEAE cellulose) and size exclusion (Sephadex G-100) chromatography. Monosaccharides including maltose (0.282%), glucose (0.113%), and mannose (0.451%) were identified, qualitatively and quantitatively, from the isolated polysaccharides through high-performance liquid chromatography. The whole study was divided into two experiments. Experiment 1 was meant for the evaluation of HWE and ME; whereas, experiment 2 was meant for the evaluation of PSE for immunostimulatory and immunotherapeutic activities. The cellular and humoral immune responses were demonstrated through lymphoproliferative response to phytohemagglutinin-P (PHA-P) and anti-body response to sheep red blood cells (SRBCs), respectively. The immunotherapeutic effects of these extracts were demonstrated against eimeriasis in terms of lesion scoring, oocysts per gram of droppings, and percent protection. Cell-mediated immune responses observed at 24, 48, and 72 h post-PHA-P injection were significantly higher (P < 0.05) in chickens administered with any of the three extracts (PSE, ME, and HWE), when compared with the controls. Humoral immune response in terms of anti-SRBCs anti-body titers was also observed higher in chickens administered with mushroom extracts. In the challenge experiment, significantly higher (P < 0.05) OPG and lesion scores were observed in controls as compared to the groups administered with mushroom extracts (HWE, ME, and PSE). Significantly higher (P < 0.05) percent protection against eimeriasis was observed in all groups administered with different extracts of L. edodes as compared to controls. In conclusion, L. edodes extracts showed immunostimulatory potential which persisted against eimeriasis in chicken.

Shiitake Culinary-Medicinal Mushroom, Lentinus edodes (Agaricomycetes): A Species with Antioxidant, Immunomodulatory, and Hepatoprotective Activities in Hypercholesterolemic Rats.

Lentinus edodes is a culinary-medicinal mushroom that has an established history of use in Asian therapies. The mushroom offers well-documented beneficial health effects such as antihypercholesterolemic, antitumor, and antibacterial activities. In this study, dried powder of L. edodes fruiting bodies was used to evaluate immunomodulatory, hepatoprotective, and antioxidant effects in hypercholesterolemic rats. Albino rats (n = 24) were divided into 3 groups: the control (CON) group, the hypercholesterolemia-only group (HCG), and the L. edodes group (LEG). Hypercholesterolemia was induced in rats in the HCG and LEG by feeding cholesterol and cholic acid in a chow maintenance diet (CMD) for 24 days. The CON group was fed the CMD throughout the experiment. The HCG continued on the high-cholesterol diet without any L. edodes supplement. The LEG was fed the high-cholesterol diet supplemented with L. edodes for an additional 42 days. Various biological health biomarkers, such as total antioxidant capacity, total oxidant status, arylesterase, paraoxonase activity, and liver enzymes in serum were studied to evaluate antioxidant and hepatoprotective responses. Cell-mediated immunity was evaluated in each group through a delayed type of hypersensitivity reaction. The total oxidant status decreased significantly (P ≤ 0.05) after administration of L. edodes in the diet. The cell-mediated immune response significantly increased (P ≤ 0.05) in the LEG. The significant decrease in liver enzymes supports the hepatoprotective effect of L. edodes. In conclusion, the results show the immunomodulatory, hepatoprotective, and antioxidant activities of L. edodes supplementation in hypercholesterolemic rats.

Immunomodulating and Antiprotozoal Effects of Different Extracts of the Oyster Culinary-Medicinal Mushroom Pleurotus ostreatus (Higher Basidiomycetes) Against Coccidiosis in Broiler.

The culinary-medicinal oyster mushroom Pleurotus ostreatus, procured from local sources, was processed for hot water and methanolic extraction. Extracts obtained were subjected to proximate analysis to determine the amount of crude protein, crude fiber, ash, ether, and nitrogen-free extracts. These extracts were evaluated for immunomodulating and antiprotozoal effects against coccidiosis in a broiler. Cellular immune investigation revealed significantly higher (P < 0.05) lymphoproliferative response to phytohemagglutinin-P in groups administered P. ostreatus extracts compared with controls. Humoral immune investigation revealed higher immunoglobulin (total Ig, IgG, and IgM) titers against sheep red blood cells in treated groups compared with controls. However, nonsignificant (P > 0.05) findings were observed in investigations of lymphoid organs. Antiprotozoal studies revealed a significantly higher (P < 0.05) percentage of protection against coccidiosis in groups administered P. ostreatus extracts when compared with controls. Moreover, lesion scoring and oocysts per gram of droppings observed in the control group were significantly higher (P < 0.05) compared with those in groups administered hot water and methanolic extracts of P. ostreatus. Results concluded that hot water and methanolic extracts of P. ostreatus had strong immune-enhancing activities. Further, these extracts also had excellent antiprotozoal activities against coccidiosis in a broiler.