Post Feeding Research Articles

Effects of Blood Coagulate Removal Method on Aedes albopictus (Diptera: Culicidae) Life Table Characteristics and Vector Competence for Dengue Virus.

Commercially available blood can be used as an alternative to live animals to maintain mosquito colonies and deliver infectious bloodmeals during research studies. We analyzed the extent to which two methods for blood coagulate removal (defibrination or addition of sodium citrate) affected life table characteristics (i.e., fecundity, fertility, hatch rate, and adult survival) and vector competence (infection, dissemination, and transmission) of Aedes albopictus (Skuse) for dengue virus (DENV). Two types of bovine blood were tested at two extrinsic incubation temperatures (27 or 30°C) for DENV-infected and uninfected mosquitoes. Fully engorged mosquitoes were transferred to individual cages containing an oviposition cup and a substrate. Eggs (fecundity) and hatched larvae (fertility) were counted. At 14 and 21 d post feeding on a DENV-infected bloodmeal, 15 mosquitoes were sampled from each group, and vector competence was analyzed (bodies [infection], legs [dissemination], and saliva [transmission]). Differences in life table characteristics and vector competence were analyzed for mosquitoes fed blood processed using different methods for removal of coagulates. The method for removal of coagulates significantly impacted fecundity, fertility, and hatch time in the uninfected group, but not DENV-infected group. Infected mosquitoes showed significantly higher fecundity and faster hatch time than uninfected mosquitoes. We show no significant differences in infection or dissemination rates between groups; however, horizontal transmission rate was significantly higher in mosquitoes fed DENV-infected citrated compared with defibrinated blood. We expect the findings of this study to inform research using artificial blood delivery methods to assess vector competence.

The post feeding glycaemic and insulin response to copra meal in horses

Knowing the effect a feed ingredient has on post-feeding glycaemic and insulin responses is important when managing conditions like equine metabolic syndrome, laminitis and the polysaccharide storage myopathy form of tying up in horses. Feeds that cause minimal post-feeding disturbances to plasma glucose and insulin are desirable. This study was conducted to determine the post feeding glycaemic and insulin response in horses to copra meal (Copra Meal; 11% non-structural carbohydrate; NSC) and to compare this to the responses observed to low NSC pasture (Pasture; 7% NSC) and higher NSC extruded pellet (Pellet; 25.3% NSC) and sweetfeed (Sweetfeed; 33.7% NSC) rations.Copra Meal did not increase plasma glucose levels above those observed in horses grazing the low NSC Pasture while the Pellet and Sweetfeed rations caused significant post-feeding rises in plasma glucose. The Pellet and Sweetfeed rations also raised post feeding plasma insulin levels significantly compared to Pasture and Copra Meal. Copra Meal raised plasma insulin levels significantly higher than that observed in horses grazing Pasture from 15 to 60min post feeding, after which time there was no significant difference. The results of this study demonstrated that the NSC content of a feed ingredient will influence the post-feeding glycaemic and insulin response to that feed, with low NSC feeds giving significantly lower responses than higher NSC feeds. The insulin response to Copra Meal observed in this study was small both in magnitude and time in comparison to levels reported in the literature needed to induce laminitis and as such are unlikely to be physiologically significant. Copra Meal is a feed ingredient that could be considered for use in diets for horses needing to remain on a low NSC diet that will cause minimal disturbance to their post feeding glucose and insulin levels.

Temporal analysis of 16 STR loci in human blood drawn from two culicid mosquitoes cultured at different temperatures

AbstractFemale mosquitoes feed on human blood, which can be collected to analyze human short tandem repeat (STR) sequences; these are specific to each human individual. Analysis of STRs might help in identification of a person found near a crime scene. Aedes aegypti and Culex pipiens mosquitoes fed on human blood were cultured at 18°C or 40°C (median temperature for summer and winter time in Riyadh governorate, Saudi Arabia) for 3, 6, 12, 24, 48 and 72 h. In A. aegypti, human DNA concentration was reduced with time at both temperatures. At 18°C, we obtained full STR profiles up to 48 h post feeding on human blood while none of the 16 loci were obtained at 72 h. At 40°C, we missed six sites at 12 h after blood sucking, 12 at 24 h, and 15 at 48 h and 72 h. In C. pipiens cultured at 18°C, full profiles were developed up to 48 h following blood feeding while we could not amplify five sites at 72 h. At 40°C, mortality among females was 50% at 24 h and 100% at both 48 h and 72 h; however, we had full profiles in all samples including dead insects. This research addressed the possibility of using mosquitoes in forensic research by DNA genotyping by changing the mosquito culturing temperature and mosquito genus. Our findings proved that different types of mosquito change the temporal pattern of STR analysis and showed that the mosquito culturing temperature affects the integrity of DNA for STR analysis.

The effect of banana (Musa acuminata) peels hot-water extract on the immunity and resistance of giant freshwater prawn, Macrobrachium rosenbergii via dietary administration for a long term: Activity and gene transcription.

The non-specific immune parameters, disease resistance and immune genes expressions in Macrobrachium rosenbergii were evaluated at 120 days of post feeding the diets containing the extracts of banana, Musa acuminate, fruit's peel (banana peels extract, BPE) at 0, 1.0, 3.0 and 6.0 g kg(-1). Results showed that prawns fed with a diet containing BPE at the level of 1.0, 3.0 and 6.0 g kg(-1) for 120 days had a significantly higher survival rate (30.0%, 40.0% and 56.7%, respectively) than those fed with the control diet after challenge with Lactococcus garvieae for 144 h, and the respective relative survival percentages were 22.2%, 33.3%, and 51.9%, respectively. Dietary BPE supplementation at 3.0 and/or 6.0 g kg(-1) for 120 days showed a significant increase total haemocyte count (THC), granular cell (GC), superoxide dismutase (SOD) activity, phenoloxidase (PO) activity, transglutaminase (TG) activity, and phagocytic activity and clearance efficiency to L. garvieae infection, and meanwhile, the significant decrease in haemolymph clotting times and respiratory bursts (RBs) per haemocyte of prawns were revealed. Furthermore, the mRNA expressions of prophenoloxidase (proPO), lipopolysaccharide and β-1,3-glucan binding protein (LGBP), peroxinectin (PE), transglutaminase (TG), and crustin (CT) were significantly increased. We therefore recommend that BPE can be used as an immunomodulator for prawns through dietary administration at 6.0 g kg(-1) for a long term (over 120 days) to modify immune responses and genes expression following the enhanced resistance against pathogens.

The effects of cinnamaldehyde, monensin and quebracho condensed tannin on rumen fermentation, biohydrogenation and bacteria in continuous culture system

Abstract The objective of this experiment was to evaluate the effects of different feed additives (cinnamaldehyde, monensin, and quebracho condensed tannin extract) on fermentation, trans fatty acids (FA) formation and selected strains of rumen bacteria. Four continuous culture systems were used in 4 × 4 Latin square designs with 4 periods of 10 days each. Treatment diets were: control diet (44:56 forage to concentrate; CON), control plus cinnamaldehyde (CIN) at 400 mg/L, control plus monensin (MON) at 12 mg/L, and control with quebracho condensed tannin extract (QTAN) at 100 g/kg of diet (DM basis). Fermenters were fed treatment diets three times daily at 120 g/day and overflow (effluent) samples were collected from each fermenter on days 8, 9 and 10 of each period to estimate nutrients digestibility and FA composition. On day 10 of each period, three samples were collected from each fermenter at 3 and 6 h post morning feeding for volatile fatty acids (VFA), ammonia-N and bacterial analyses. Compared with the CON diet, feed additives had no effects (P > 0.05) on apparent dry matter (DM), neutral detergent fiber (NDF) and organic matter (OM) digestibility but apparent protein digestibility decreased (P 0.05) on the DNA abundance of Anaerovibrio lipolytica and Butyrivibrio SA. In conclusion, results demonstrate that the feed additives used in this study affected the fermentation and biohydrogenation process. Addition of feed additives reduced the formation of C18:0 but only MON and CIN increased VA formation. MON and CIN effects on VA formation may in part be explained by their effects on B. proteoclasticum.

Improving rumen ecology and microbial population by dried rumen digesta in beef cattle.

Four Thai native beef cattle with initial body weight (BW) of 91.8 ± 4.75kg were randomly assigned according to a 4 × 4 Latin square design to receive four concentrates replacement levels of soybean meal (SBM) by dried rumen digesta (DRD) at 0, 33, 67, and 100% on dry matter (DM) basis. All cattle were fed rice straw ad libitum while additional concentrate was fed at 0.5% BW daily. The experiment was conducted for four periods of 21days. Rumen fluid was analyzed for predominant cellulolytic bacterial population by using real-time PCR technique. Increasing levels of DRD did not alter total feed intake, ruminal pH and temperature, and plasma urea nitrogen (P > 0.05). Protozoa and fungal population were not differed by DRD supplementation while population of bacteria at 4h post feeding was increased when SBM was replaced with DRD at 66 and 100% DM. Population of total bacteria and R. flavefaciens at 4h post feeding were significantly highest with inclusion of 100% of DRD in the ration. The experimental diets has no effect on excretion and absorption of purine derivatives (P > 0.05), while microbial crude protein and efficiency of microbial N synthesis were significantly increased with DRD inclusion in the diet and highest with 100% DRD replacement (P > 0.05). Replacement of SBM by DRD at 100% DM improved the rumen ecology and microbial population in beef cattle fed on rice straw.

Shotgun Proteomic Analysis of Protein Expression in Mosquito Ovaries Post Blood Meal

Female mosquitoes require a blood meal for oogenesis and receive a substantial iron load in this meal in the form of holo‐transferrin and hemoglobin. During the course of digestion and absorption in the 72 hours post blood feeding, the iron concentration in the gut decreases 10‐fold while that in the ovaries more than doubles. Eggs are laid with ~125 ng Fe each; the majority coming from the blood meal. Iron is mitogenic and iron deposition in the ovaries may induce changes in the endogenous protein expression profile over time. Proteomic analysis of extracts from developing ovaries and eggs revealed proteins that are expressed at time intervals of 24 and 72 hours post blood feeding. These analyses may provide further insight into the effect of a blood meal on mosquito iron metabolism and mosquito fecundity.

358 TGR5 Activation Is Neuroprotective in Hepatic Encephalopathy

Background and Aims: Infiltration of neutrophils in the liver contributes to the development of alcoholic liver diseases (ALD). However, the factors that regulate neutrophil infiltration and functions remain unclear. miR-223 is one of the most abundant miRNAs expressed in neutrophils, the purpose of this study is to investigate its role in the pathogenesis of ALD. Methods: The mouse model of chronic-plus-binge ethanol feeding was employed in miR223-/mice and their wild-type controls. Liver injury and inflammation were determined. Results: Real-time PCR analyses demonstrated the expression of miR-223 levels in neutrophils was about 24 fold and 15 fold higher than that in hepatocytes and Kupffer cells/macrophages, respectively. Chronic-plus-binge ethanol feeding markedly increased miR-223 expression in hepatic neutrophils. Compared with wild-type mice, miR223-/mice had higher levels of serum ALT and AST, and greater number of peripheral neutrophils post ethanol feeding. Immunostaining analyses revealed that the numbers of MPO positive neutrophils in the liver were elevated after chronic-plus-binge ethanol feeding with higher levels in miR223-/mice than in wild-type mice. The oxidative reaction as demonstrated by MDA and HNE staining in ethanol-fed miR223-/mice was stronger than ethanol-fed wild-type mice. In consistent with this notion, neutrophils from miR223-/mice produced higher levels of ROS compared with those from wild-type mice. In conclusion, miR223 protects against alcoholic liver injury by suppressing neutrophil infiltration and ROS production; miR223 could be a novel therapeutic target for the treatment of ALD.

359 Thrombospondin-1 Is Associated With the Neurological Complications Associated With Acute Liver Failure

Background and Aims: Infiltration of neutrophils in the liver contributes to the development of alcoholic liver diseases (ALD). However, the factors that regulate neutrophil infiltration and functions remain unclear. miR-223 is one of the most abundant miRNAs expressed in neutrophils, the purpose of this study is to investigate its role in the pathogenesis of ALD. Methods: The mouse model of chronic-plus-binge ethanol feeding was employed in miR223-/mice and their wild-type controls. Liver injury and inflammation were determined. Results: Real-time PCR analyses demonstrated the expression of miR-223 levels in neutrophils was about 24 fold and 15 fold higher than that in hepatocytes and Kupffer cells/macrophages, respectively. Chronic-plus-binge ethanol feeding markedly increased miR-223 expression in hepatic neutrophils. Compared with wild-type mice, miR223-/mice had higher levels of serum ALT and AST, and greater number of peripheral neutrophils post ethanol feeding. Immunostaining analyses revealed that the numbers of MPO positive neutrophils in the liver were elevated after chronic-plus-binge ethanol feeding with higher levels in miR223-/mice than in wild-type mice. The oxidative reaction as demonstrated by MDA and HNE staining in ethanol-fed miR223-/mice was stronger than ethanol-fed wild-type mice. In consistent with this notion, neutrophils from miR223-/mice produced higher levels of ROS compared with those from wild-type mice. In conclusion, miR223 protects against alcoholic liver injury by suppressing neutrophil infiltration and ROS production; miR223 could be a novel therapeutic target for the treatment of ALD.

Dynamic expression of miRNAs across immature and adult stages of the malaria mosquito Anopheles stephensi

BackgroundMicroRNAs are small non-coding RNAs that are involved in various biological processes including insect development. Anopheles stephensi serves as primary vector of malaria parasite in Asia and exhibits holometabolous life cycle that involves four different stages of development. Regulation and role of mosquito miRNAs during various stages of mosquito development remain largely unknown.MethodsHigh throughput small RNA sequencing was employed for identification and profiling of miRNAs across immature and adult stages of malaria vector, which were further validated using Northern hybridization and real time PCR. Target prediction and pathway analysis was carried out to understand the role of regulated miRNAs in insect development. Degradome sequencing was employed to identify cleaved targets of some regulated miRNAs. Loss of function strategy was employed for miR-989 to understand its probable role in female reproductive process.ResultsSmall RNA sequencing and data analysis revealed 111 and 14 known and novel miRNAs respectively across all stages of Anopheles stephensi. Nine miRNAs showed gender specific regulation across different stages of mosquito development. Analysis of miRNAs revealed regulation of 24 and 26 miRNAs across different stages of male and female mosquito development respectively. mRNA targets and significant pathways targeted by regulated miRNAs were identified for each stage of mosquito development. Degradome sequencing revealed twenty nine cleaved targets of insect miRNAs. MicroRNA-989 showed significant up-regulation in the adult female as compared to adult male mosquito. Knockdown of miR-989 expression in adult female using miRNA specific antagomir affected targets playing roles in protein binding, proteolysis and nucleic acid binding in ovary tissue of female mosquito post blood feeding.ConclusionsThis is the first comprehensive effort to understand regulation of Anopheles stephensi miRNAs across developmental stages of male and female mosquito. Preliminary role of regulated miRNAs in mosquito development was revealed by target prediction and pathway analysis. MicroRNA-989 emerged to have important roles in adult female mosquitoes showing significant up-regulation which was further studied using miR-989 specific antagomir. This study provides insights into mosquito development and reproductive process and has implications for effective control of mosquito population required for reducing spread of mosquito-borne infectious diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0772-y) contains supplementary material, which is available to authorized users.

Nutrient digestibility and rumen fermentation of dried cassava peels and brewers dried grains based diets by West African dwarf (WAD) sheep

In the tropics, supplementing concentrates with grasses, especially during the dry season could improve protein to energy ratio in ruminants thereby improving optimum production. Also, supplementation with a protein source enhances the production capacity of ruminants. Therefore, this study was designed to determine the nutrient digestibility and rumen fermentation kinetics of Panicum maximum supplemented with brewers dried grains and dried cassava peels based diets by West African Dwarf (WAD) lambs. Nine lambs of about 9 months in age and bodyweight ranging from 10.5kg-16.5kg were randomly divided into 3 groups and assigned to the following diets: Diet 1-100% Panicum maximum, Diet 2 - 81% DCP + 13.50% PKC + 2% Oyster shell + 2% Salt + 1.50% Premix and Diet 3 - 81% BDG + 13.50% PKC + 2% Oyster shell + 2% Salt and 1.50 Premix. Result showed that animals on diet 1recorded the least significant (p< 0.05) dry matter digestibility (55.00%) to those on diets 2 and diet 3. Animals on diet 3 recorded the best crude protein digestibility (66.88%) which was significantly different (p<0.05) from those on diets 1 and 2 (53.04% and 56.79%). Animals on diet 1 recorded the highest significant (p<.0.05) amount of ammonia nitrogen at O and 4hours post feeding. The same trend seems to occur for volatile fatty acids (acetate, propionate and butyrate) at O hour post feeding. However, at 4 hour post feeding, animals on diet 3 recorded a slight numerical advantage over other animals. It can therefore be deduced from this study that diet 3 was most preferred as evident from the chemical composition of the feed and nutrient digestibility of the lambs.Keywords: Dried cassava peels, brewer’s dried grains, digestibility, fermentation kinetics and lambs.

Milk Production and Income over Feed Costs in Dairy Cows Fed Medium-roasted Soybean Meal and Corn Dried Distiller's Grains with Solubles.

The aims of this study were to determine the effects of feeding medium-roasted soybean meal (SBM) and corn dried distiller’s grains with solubles (CDDGS) in dairy cows on milk production and income over feed costs. A randomized complete block design experiment was conducted with 24 crossbred multiparous Holstein Friesian dairy cows in early- and mid-lactation. Four dietary treatments were as follows: basal diet without feed substitute (Control), 7.17% dry matter (DM) roasted SBM replaced for concentrate (R-SBM), 11.50% DM CDDGS replaced for concentrate (DDGS), and 3.58% DM roasted SBM plus 5.75% DM CDDGS replaced for concentrate (SB-DG). The roasted SBM was produced using a medium-heated treatment at 100°C for 180 min. Dry matter intake was not affected by feeding high rumen undegradable protein (RUP) sources, but the replacement of roasted SBM and CDDGS for concentrate significantly improved (p<0.001) RUP intake (0.90, 0.86, and 0.88 kg/d corresponding to R-SBM, DDGS, and SB-DG) compared to the control (0.61 kg/d). Feeding roasted SBM and CDDGS alone or in combination had no significant effect on milk composition of dairy cows (p>0.05), whereas milk yield was significantly increased by 3.08 kg/d in the SB-DG group relative to the control group (p<0.01). Net income was meaningfully increased (p<0.05) from 4th week post feeding, the SB-DG group reached the greatest net income ($3.48/head/d) while the control group had the lowest value ($2.60/head/d). In conclusion, the use of CDDGS alone or in combination with medium-roasted SBM as substitute for concentrate in lactating dairy cattle diet led to improved milk production and net income over feed costs without affecting total dry matter intake and milk composition, while feeding medium-roasted SBM seemed to show intermediate values in almost parameters.

Dietary supplement of banana (Musa acuminata) peels hot-water extract to enhance the growth, anti-hypothermal stress, immunity and disease resistance of the giant freshwater prawn, Macrobrachium rosenbergii

In the present study, Macrobrachium rosenbergii were fed with diets containing extracts of banana, Musa acuminate, fruit's peel (banana peels extract, BPE) at 0, 1.0, 3.0 and 6.0 g kg−1. The non-specific immune parameters, disease resistance and anti-hypothermal stress were evaluated at 2, 4, 8, 16 and 32 days of post feeding. Also, we demonstrated the percent weight gain (PWG), percent length gain (PLG), feeding efficiency (FE), and survival rate of giant freshwater prawn at 30, 60, 90, and 120 days of post feeding. The PWG, PLG, FE and survival rate of prawns fed at 0, 1.0, 3.0 and 6.0 g kg−1 BPE-containing diets after 120 days were 69.5%, 75.4%, 77.8% and 83.3%; 21.8%, 23.6%, 27.8% and 33.9%; 0.60, 0.72, 0.75 and 0.90; and 55.4%, 62.2%, 62.3% and 75.3%, respectively. After 32 days of post feeding, a significant increase in total haemocyte count (THC), different haemocyte count (DHC), respiratory bursts (RBs), superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, phenoloxidase (PO) activity and transglutaminase (TG) activity, and meanwhile, a decreased haemolymph coagulation time was observed. Furthermore, phagocytic activity and clearance efficiency of prawns against Lactococcus garvieae infection were significantly increased. Prawns challenged with L. garvieae after 32 days of feeding at 1.0, 3.0 and 6.0 g kg−1 had a significantly higher survival rate (33.3%, 40.0% and 56.7%) than those fed with the control diet. Subsequently, hypothermal (14 °C) stress was 43.4%, 50.0% and 50.0%, respectively. Altogether, we therefore recommend the dietary BPE administration at 6.0 g kg−1 promotes growth, anti-hypothermal stress, and enhance immunity and resistance against L. garvieae in M. rosenbergii.

Clinical features and management of equine post operative ileus: Survey of diplomates of the European Colleges of Equine Internal Medicine (ECEIM) and Veterinary Surgeons (ECVS).

There is a need for an improved understanding of equine post operative ileus (POI), in terms of both clinical definition and optimal management. Although the pharmacological strategies that are used to treat POI continue to evolve, little is known about the supplementary strategies used to prevent and manage this condition. To report the current strategies used to diagnose, prevent and manage POI following emergency abdominal surgeries. Cross-sectional survey. An electronic survey invitation was sent by email to 306 European college diplomates (European Colleges of Equine Internal Medicine, ECEIM n = 120, and Veterinary Surgeons, ECVS n = 186). The response rate was 33% (100 of 306). The median reported estimated incidence of POI was 10-20%. The presence of reflux on nasogastric intubation was the main criterion used to define POI. Lesions involving the small intestine were thought to be the leading risk factors for developing POI. Anti-inflammatory drugs, antimicrobial drugs and i.v. fluids were the primary preventative strategies when managing cases at high risk for POI. Flunixin meglumine and lidocaine were the drugs most commonly used to treat horses with POI. Supplementary preventative and treatment strategies for POI included control of endotoxaemia, fluid therapy, early ambulation and judicious timing of post operative feeding. Appreciation of the potential risk factors associated with the development of POI appeared to have an impact on the choice of management strategies that are implemented. The majority of ECEIM and ECVS Diplomates in the survey used flunixin meglumine and lidocaine, often in combination, to treat horses with POI, which is likely to reflect the contributory role of inflammation in its pathophysiology. Various supplementary strategies were used to prevent and manage POI risk factors intraoperatively and post operatively.

A slot blot immunoassay for quantitative detection of Plasmodium falciparum circumsporozoite protein in mosquito midgut oocyst.

There is still a need for sensitive and reproducible immunoassays for quantitative detection of malarial antigens in preclinical and clinical phases of vaccine development and in epidemiology and surveillance studies, particularly in the vector host. Here we report the results of sensitivity and reproducibility studies for a research-grade, quantitative enhanced chemiluminescent-based slot blot assay (ECL-SB) for detection of both recombinant Plasmodium falciparum circumsporozoite protein (rPfCSP) and native PfCSP from Oocysts (Pf Oocyst) developing in the midguts of Anopheles stephensi mosquitoes. The ECL-SB detects as little as 1.25 pg of rPfCSP (linear range of quantitation 2.5–20 pg; R2 = 0.9505). We also find the earliest detectable expression of native PfCSP in Pf Oocyst by ECL-SB occurs on day 7 post feeding with infected blood meal. The ECL-SB was able to detect approximately as few as 0.5 day 8 Pf Oocysts (linear quantitation range 1–4, R2 = 0.9795) and determined that one Pf Oocyst expressed approximately 2.0 pg (0.5–3 pg) of native PfCSP, suggesting a similar range of detection for recombinant and native forms of Pf CSP. The ECL-SB is highly reproducible; the Coefficient of Variation (CV) for inter-assay variability for rPf CSP and native PfCSP were 1.74% and 1.32%, respectively. The CVs for intra-assay variability performed on three days for rPf CSP were 2.41%, 0.82% and 2% and for native Pf CSP 1.52%, 0.57%, and 1.86%, respectively. In addition, the ECL-SB was comparable to microscopy in determining the P. falciparum prevalence in mosquito populations that distinctly contained either high and low midgut Pf Oocyst burden. In whole mosquito samples, estimations of positivity for P. falciparum in the high and low burden groups were 83.3% and 23.3% by ECL-SB and 85.7% and 27.6% by microscopy. Based on its performance characteristics, ECL-SB could be valuable in vaccine development and to measure the parasite prevalence in mosquitoes and transmission-blocking interventions in endemic areas.

Rapid and sensitive detection of Bartonella bacilliformis in experimentally infected sand flies by loop-mediated isothermal amplification (LAMP) of the Pap31 gene.

BackgroundCarrion' disease, caused by Bartonella bacilliformis, remains truly neglected due to its focal geographical nature. A wide spectrum of clinical manifestations, including asymptomatic bacteremia, and lack of a sensitive diagnostic test can potentially lead to a spread of the disease into non-endemic regions where competent sand fly vectors may be present. A reliable test capable of detecting B. bacilliformis is urgently needed. Our objective is to develop a loop-mediated isothermal amplification (LAMP) assay targeting the pap31 gene to detect B. bacilliformis. Methods and FindingsThe sensitivity of the LAMP was evaluated in comparison to qPCR using plasmid DNA containing the target gene and genomic DNA in the absence and presence of human or sand fly DNA. The detection limit of LAMP was 1 to 10 copies/µL, depending on the sample metrics. No cross-reaction was observed when testing against a panel of various closely related bacteria. The utility of the LAMP was further compared to qPCR by the examination of 74 Lutzomyia longipalpis sand flies artificially fed on blood spiked with B. bacilliformis and harvested at days (D) 1, 3, 5, 7 and 9 post feeding. Only 86% of sand flies at D1 and 63% of flies at D3 were positive by qPCR. LAMP was able to detect B. bacilliformis in all those flies confirmed positive by qPCR. However, none of the flies after D3 were positive by either LAMP or qPCR. In addition to demonstrating the sensitivity of the LAMP assay, these results suggest that B. bacilliformis cannot propagate in artificially fed L. longipalpis.ConclusionsThe LAMP assay is as sensitive as qPCR for the detection of B. bacilliformis and could be useful to support diagnosis of patients in low-resource settings and also to identify B. bacilliformis in the sand fly vector.

Pathology of gout induced by high protein diets in growing broilers

The inestigation was carried out on the gross and histo-pathological changes that occur due to different protein diet in broilers. A total of 100, day-old broiler chicks were randomly divided into 5 groups having 20 chicks each. The chicks were fed with diets having 10 per cent (group A), 15 per cent (group B), 20 per cent (group C), 30 per cent (group D) and 40 per cent (group E) crude protein for 28 days. Post mortem examination of birds showed that relative weight of kidney was increased in birds of group E on day 28 post feeding. Grossly deposition of urate crystals in visceral organs, petechial haemorrhages and enlargement of kidney were observed. The salient histopathological changes included tophi formation, severe degenerative and necrotic changes in kidney and mild fatty changes and necrosis of hepatocytes in liver. The histopathological changes in heart, spleen and bursa of fabricius in the birds of group E on day 14 were similar to the naturally gout affected birds.

Effect of feeding feed blocks containing different levels of urea calcium sulphate mixture on feed intake, digestibility and rumen fermentation in Thai native beef cattle fed on rice straw

Abstract Four Thai native beef cattle were randomly assigned according to a 4 × 4 Latin square design to evaluate the effect of urea calcium sulphate mixture (U-cas) levels in feed block on feed intake, apparent digestibility of nutrients and rumen fermentation fed on rice straw based diet. The four dietary treatments were different levels of U-cas supplementation in feed block at 0, 120, 150 and 180 g/kg dry matter (DM), respectively. The present results revealed that rice straw intake increased with the increasing levels of U-cas inclusion in the feed block. The highest total intakes of DM and energy (ME, MJ/d) were in cattle receiving feed block containing U-cas at 180 g/kg DM, and followed by 150, 120 and 0 g/kg DM, respectively. Apparent digestibility of nutrients enhanced the increasing levels of U-cas supplementation in feed blocks, except acid detergent fiber. Rumen pH and temperature were not affected by U-cas levels inclusion in feed block. At 4 h post feeding, the concentration of ruminal ammonia nitrogen decreased with increasing levels of U-cas in feed blocks (P

A novel approach to systematically compare behavioural patterns between and within groups of horses

This study piloted a multiple aspect approach to assess differences or changes in equine behaviour, using physiological (heart rates – HR, salivary cortisol – SC) and behavioural measures in various routine (feed-time, riding, turning out) and test situations (novel situation tests NS: 2× reactivity and 2× handling, Cross-over design). In order to evaluate if this approach could identify different behavioural patterns, two distinct groups of riding school horses (selected by experienced equine professionals) were observed repeatedly over 6 weeks: 6 excitable/nervous horses (EX: Age: mean=14±3.4, 8–18yrs, BW 677±48kg, Body Condition [BC]=4.6±0.4) and 6 calm, quiet horses (CA: Age: mean=16±2.5, 12–18yrs, BW 645±35, BC 5.1±0.4).EX horses had consistently lower SC levels than CA horses (p<0.01), when coming in from the paddock in the morning. Therefore, there was a significant increase of SC after feeding for EX horses (+43%, p<0.05) but not for Group CA (only 16%). After feeding SC showed no difference between the two groups. Calm horses had significantly higher HRs (44±1.2bpm) while waiting for feed and post feeding (p<0.01) than EX horses (38±0.7; p<0.05). Behavioural measures showed significantly higher agitation scores for EX horses during feeding, walking to (p<0.01) and release into paddock (p<0.05). When ridden, EX horses showed a greater amount of threatening behaviour towards other horses (p<0.05) and CA horses showed a significantly greater reluctance to move forward (p<0.05). All mean agitation scores were significantly higher for Group EX (EX 2.3, CA 1.8, s.e.m. 0.3; p<0.01) and apart from feed time these showed good correlations with behaviour observations. In the NS reactivity test CA horses had a significantly higher HR variability than EX horses (CA: 64±10.5, EX: 41±3.5, p<0.05). There was no correlation between HRs and SC but in the NS handling test there was a correlation between SC and refusals to cross a bridge (p<0.01; rs[12]=0.70) as well as SC and agitation score (p=0.07; rs[12]=0.54). Horses which displayed a greater level of investigation scored lower in agitation behaviours (p<0.01, rs[12]=−0.68). The methodology applied, clearly and consistently highlighted differences between horse groups, although the physiological measures did not always follow assumptions. Within groups individual horses reacted differently in standardised test and routine situations, highlighting that multiple testing will increase the chances of observing subtle changes in behaviour or perceived stress.

Differential peptidomics highlights adipokinetic hormone as key player in regulating digestion in anautogenous flesh fly, Sarcophaga crassipalpis

Females of anautogenous flesh flies, Sarcophaga crassipalpis, need a protein meal in order to produce their first batch of eggs. This protein meal elicits an increase in midgut proteolytic activity that is under neuropeptidergic regulation. Time series of decapitation and rescue experiments of liver fed flies evidenced the need of a peptide factor released by corpora cardiaca (CC) within 4h post protein feeding in order to assure complete protein digestion. Q-Exactive quantitative differential peptidomics analysis on CC of sugar fed flies and flies 5h post protein feeding respectively, showed a unique consistent decrease in the stored amount of adipokinetic hormone (AKH) ranging between 16% up to 63%. Injection of AKH into liver fed decapitated flies as well as sugar fed intact flies resulted in dose dependent enhanced midgut proteolytic activity up to the level of intact protein fed flies. This suggests a key role of AKH in food depended reproduction.