Interactions between equine lymphocytes and equine herpesvirus-1 (EHV-1)

Equine herpesvirus-1 (EHV-1) infects and causes contagious respiratory disease, epidemic abortion and sporadic neurological disease. In general, EHV-1 infection elicits the stimulation of both humoral and cellular immune responses in horses. As EHV-1 is a persistent virus, its infection leads to latent infection during the first two weeks with chronic reactivations. Virus neutralising antibodies combat EHV-1 infection by reducing nasal virus shedding, particularly in the nasopharynx, but they play insignificant roles in controlling systemic spread by cell-associated viraemia to the respiratory tract and lymphatic tissues. The cytotoxic T lymphocyte (CTL) response is the most crucial aspect of EHV-1 specific cell mediated immunity and its function is well described to protect against clinical disease, viraemia and nasopharyngeal virus shedding. EHV-1 immediate early gene products (gene 64) have been shown to be targets for EHV-1 specific CTL. Moreover, vaccination of ponies with the EHV-1 gene 64 protein induces cell mediated immune responses resulting in the reduction of clinical and virological disease. This important finding underlines the substantial role of viral gene 64 in cell mediated immunity and highlights its potential for improved vaccination strategies. Therefore, the overall aim of this study was to identify peptide sequences of CTL epitopes within EHV-1 immediate early protein’s Fragment D (encoded by gene 64) in ponies expressing the MHC class I A3/B2 allele. In order to achieve this, in vitro cellular techniques were established and T lymphocyte responses to EHV-1 were quantified as a prerequisite for identifying CTL target peptides. This first investigation aimed to measure activation of equine peripheral blood mononuclear cells (PBMC) with either phorbol 12-myristate 13-acetate (PMA) or infectious EHV-1 by assessing interferon gamma (IFNγ) synthesis detected by flow cytometry as a prelude to stimulating PBMC with gene 64 protein or peptides. The intra-assay and inter-assay variations of IFNγ synthesis were characterised and the coefficients of variation of both assays were 8.42% and 5.8%. The phenotype of medium-stimulated and PMA-ionomycin stimulated PBMC comprised principally T cells as defined by antibodies specific for equine leukocyte markers. Intracellular IFNγ expression after in vitro EHV-1 stimulation of primed PBMC was assessed and revealed a very low percentage of IFNγ secreting cells in the responding population. In order to induce cells to use for the preparation of equine T lymphocyte and CTL lines/clones, in vitro EHV-1 stimulation of PBMC in bulk culture was characterised. Phenotypic analyses and viability of EHV-1 stimulated PBMC showed that 5% NHS in culture medium at 7 days stimulation was more reliable to induce CD8+ cells compared with 10% FCS, indicating the preferred use of 5%NHS in culture media to generate and assay equine CTLs. The next aim was to develop EHV-1 specific T cell lines and clones from whole virus (EHV-1). Several attempts were made to generate these using mouse anti equine CD3 monoclonal antibody (mAb) (clone UCF6G), autologous rhuIL-2 and mitomycin C treated PBMC as APC, but these failed. Using two different approaches; 5-ethynyl-2’-deoxyuridine (EdU) and Cu (I)-catalysed cycloaddition ‘click’ reaction assay and tritiated thymidine [3H] uptake to show lymphocyte activation in response to anti-CD3 monoclonal antibody, it was demonstrated that the anti-CD3 mAb was able to activate cell proliferation and allowed cell division and population expansion. To further assist the establishment of CTL lines and clones, the peptides of the EHV-1 immediate early protein’s fragment D (amino acids 750-1143 in the IE protein) were investigated using an IFNγ ELISPOT assay. This approach was optimised and characterised using medium, mitogen and EHV-1 as the negative and positive controls. A library of 128 synthetic peptides was obtained comprising molecules of 14 amino acids in length, overlapping by 11 amino acids. All peptides were constructed into pool arrays linearly and overlapping peptides were not contained within the same pool. The optimal concentration of the peptide pool was determined and chosen as 0.048μg per well. This concentration was also used for screening the individual peptides. The results showed that several peptide pools produced high number of spot-forming-cells significantly greater than the threshold for positivity and most of individual peptides stimulated an IFNγ response from PBMC, suggesting that IFNγ synthesis is too insensitive to detect CD8+ IFNγ + responses. In summary, this study has demonstrated the potential of new and relevant in vitro techniques that can be applied to the investigation of CTL target peptide(s) of EHV-1 as presented by the A3/B2 allele in horses

Effects of non-medicated and medicated urea molasses multinutrient blocks on dry matter intake, growth performance,body condition score and feed conversion ratio of saanen lactating does fed conventional diets

In this study, 24 Saanen lactating does raised by a smallholder in Kemahang, Tanah Merah, Kelantan were randomly assigned to four groups with six goats in each group. The trial included evaluation of four dietary treatments, that is, T1: control group fed on basal diet only, which consisted of 3 kg Napier grass (Pennisetum purpureum) and 1 kg commercial goat pellet. Animals in T2, T3 and T4 received equal amounts of basal diet with supplementation of urea molasses multi-nutrient block (UMMB), medicated urea molasses multi-nutrient block (MUMB) and commercial mineral block (CMB) respectively. The total dry matter intake (DMI) (kg/d) in T2 (1.28) and T3 (1.24) were significantly higher (p0.05) between treatments on average daily gain (ADG) and body measurements. Highest ADG (g/d) were recorded in T2 (53.57) followed by T3 (45.63), T4 (39.68) and T1 (37.70). Similar trend was also recorded in body condition score (BCS) but there were no significant differences (p>0.05) between treatments. At the end of the 90 days of feeding trial, both T2 and T3 showed acceptable BCS, that is, at 3.25 and 3.08 respectively, while low BCS were recorded in T1 (2.63) and T4 (2.71). There was significant difference (p<0.05) between treatments on feed conversion ratio (FCR) which were at 0.84, 0.95, 1.20 and 1.46 for T2, T3, T4 and T1 respectively. Both UMMB and MUMB were effective in enhancing appetite, DMI and ADG of the dairy goats, apart from minimising weight loss during lactation

Effects of non-medicated and medicated urea molasses multi-nutrient block supplements on nutrient intake and blood mineral profile of lactating Saanen goats

Poor nutrition is a major contributor to the performance of dairy goats on smallholder farms. Hence, strategic supplementation is required to overcome nutritional deficiencies. This paper reviews the effects of urea molasses multi-nutrient block (UMMB) and medicated urea molasses multi-nutrient block (MUMB) supplements in comparison with imported commercial mineral blocks (CMB) on nutrient intake and blood mineral profile of Saanen lactating goats. A 90-day feeding trial was conducted at Yusof Ecofarm, Tanah Merah, Kelantan, Malaysia. Twenty four (24) Saanen lactating goats were assigned to four dietary treatments with six (6) goats in each group based on completely randomized block design. Goats in T1 (control) were fed with basal diet only that comprise of roughages and goat pellet while T2, T3 and T4 received equal amount of basal diet and allowed for ad-libitum licking of UMMB, MUMB and CMB respectively. Total dry matter intake (DMI) (kg/d) were 1.14, 1.29, 1.25 and 1.16 in T1, T2, T3 and T4 respectively whereas the UMMB, MUMB and CMB intakes (g/d) were 86.8, 50.4 and 36.6 respectively. There were significant differences (p<0.05) between treatments on nutrient intake. Crude protein (CP), crude fiber (CF), ether extract (EE), acid detergent fiber (ADF) and neutral detergent fiber (NDF) were significantly higher (p<0.05) in T2 and T3 as compared to T1 and T4. The serum concentrations of calcium (Ca), ferum (Fe), copper (Cu) and zinc (Zn) were significantly higher (p<0.05) in T2 with the values of 33.60 ppm, 341.67 ppb, 270.00 ppb and 138.30 ppb respectively as compared to other treatments. Hence, the findings of this research revealed that both UMMB and MUMB were effective in improving nutrient intake and blood mineral profile of lactating Saanen goats

Effects of medicated and non-medicated multi-nutrient block supplementation on gastrointestinal parasite infestation and blood hematological parameters of lactating saanen goats

In a 90-day feeding trial, twenty four (24) Saanen lactating does were assigned to four treatment group. The treatments were feeding: basal diet only as a control (T1), basal diet with urea molasses multi-nutrient block (UMMB) (T2), basal diet with medicated urea molasses multi-nutrient block (MUMB) (T3) and basal diet with commercial mineral block (CMB) (T4). There were significant differences (p0.05) between treatments on WBC, LYM, MON, GRA, RBC, HCT, MCV, MCH, PLT, MPV and PCT. However, significant effects (p<0.05) on HBG, MCHC, RDW and PDW were observed in T2 and T3. This research showed that UMMB and MUMB were effective in controlling parasite infestation in Saanen lactating dairy goats apart from improving their blood hematological parameters. Comparison with CMB showed that it is practical to be used for parasite control

Utilisation of oil palm fronds as ruminant feed and its effect on fatty acid metabolism

Inclusion of oil palm fronds (OPF) pellets (200 g kg-1 DM) in a complete animal feed has been found to increase the unsaturated fatty acid content in ruminants. However, given the low-fat content of OPF (21 g kg-1 DM), changes in ruminal fatty acid (FA) metabolism will only result in nutritionally relevant differences in animal tissues when OPF enhances conservation of polyunsaturated fatty acid (PUFA) from external sources. Additionally, given the low metabolisable energy value (4.9 to 6.5 MJ (ME) kg-1 DM) of OPF, supplementation of OPF with an energy-dense feed compound such as fat is of interest. Thus, this approach could also be used in combination with other dietary fat supplementation strategies to further manipulate fatty acid concentration of ruminant tissues and products for human consumption

The roles of soybean lecithin in aquafeed: a crucial need and update

Soybean lecithin is extensively used as the dietary supplementation of phospholipids in animal production. Soybean lecithin plays significant roles in aquafeed as growth promoter, feed enhancer, immunity modulator and antioxidant activity stimulator for aquaculture species. Besides, soybean lecithin is also reported to help aquaculture species being resilient to physical and chemical stressors. In this review, common sources, chemical structure and mode of action of lecithin, with highlight on soybean lecithin application in aquaculture over four-decadal studies published between 1983 and 2023, were evaluated and summarized. By far, soybean lecithin is best-known for its beneficial effects, availability yet cost-effective for aquafeed formulation. Findings from this review also demonstrate that although nutritional profile of long-chain polyunsaturated fatty acids and phosphatidylcholine from egg yolk and marine sources are superior to those from plant sources such as soybean, it is rather costly for sustainable application in aquafeed formulation. Moreover, commercially available products that incorporate soybean lecithin with other feed additives are promising to boost aquaculture production. Overall, effects of soybean lecithin supplementation are well-recognized on larval and juvenile of aquaculture species which having limited ability to biosynthesis phospholipids de novo, and correspondingly attribute to phospholipid, a primary component of soybean lecithin, that is essential for rapid growth during early stages development. In addition, soybean lecithin supplementation plays a distinguish role in stimulating maturation of gonadal development in the adults, especially for crustaceans

The effect of physical and biological pre-treatments of oil palm fronds on in vitro ruminal degradability

Physical pre-treatment of the oil palm frond (OPF) is known to loosen the lignocellulose while the biological pre-treatment is capable in degrading the lignin, making the substrates more accessible for rumen microbes. This study aimed at assessing the efficacy of physical, biological and combination of both pre-treatments of OPF on the in vitro ruminal degradability. Five different samples of OPF pre-treatments were used in this study; OPF was subjected to the physical pre-treatment (POPF), OPF to the biological pre-treatments using an enzyme extract of each Ganoderma lucidum (BGL) and Lentinula edodes (BLE), respectively. Another two samples were subjected to a combination of physical and biological pre-treatments of G. lucidum (CGL) and L. edodes (CLE) respectively. The control was non-treated OPF. Two fistulated Katjang goats consuming 440 g/kg OPF and 897 g/ kg commercial pellet daily on dry matter basis were used as rumen fluid donors. In vitro incubation was carried out at 39°C for 24 hours. Proportions of volatile fatty acid were measured at the end of incubation by gas chromatography. Results showed that concentrations of lignin following all pre-treatment methods were significantly lower (p<0.05) at 150(POPF), 90(BGL), 119(BLE), 100(CGL) and 120(CLE) g/ kg DM as compared to the FOPF (190 g/kg). After 24 hours of incubation, the cumulative gas of all treatment groups differed significantly from FOPF. Both BGL and CGL showed significantly higher propionate and butyrate concentrations as well as apparent rumen degradable carbohydrate with 6.57 mg and 6.54 mg, respectively as compared to the FOPF. It appeared that BGL and CGL resulted in higher lignin degradation that increased the in vitro rumen degradability. In conclusion, biological pre-treatment with enzyme extract of WRF, either alone or in combination are promising to improve the quality of OPF

Palm date meal as a non-traditional ingredient for feeding aquatic animals: A review

Protein ingredients in aquafeed are one of the most important factors responsible for the development and sustainability of aquaculture. Currently, because of high costs and fluctuating production, some animal and plant protein sources are unable to satiate the increasing demand from the fish feed manufacturers. Aquatic animals’ nutritional requirements have been accorded particular focus with less costly feedstuff in aqua feed given extra weightage. There has been increasing attention in recent years on finding methods to recycle the animal and plant by-products for feed preparation. Due to its vital amino acid content as well as high protein composition, palm date meal (PDM) which is a renewable and sustainable resource is expected to be a viable raw material option for replacing protein ingredients (e.g., fish meal and soybean meal) or as a supplement in fish feed. PDM is an agro-industry by-product which left from dates as waste in several countries. This article reviewed the current research including the source, derivatives, and the potential of PDM as a possible alternative to the conventional plant and animal protein sources. Also, the added value of using PDM waste in aquafeed to reduce the feed cost, enhance this e growth rate of fish, improve the health and well-being of fish, and subsequently sustain the aquaculture industry. Therefore, this review paper will illuminate the possibility of PDM as a promising feed source and also the present knowledge and future perspectives about the application of PDM in aquaculture

Interactions between equine lymphocytes and equine herpesvirus-1 (EHV-1)

Equine herpesvirus-1 (EHV-1) infects and causes contagious respiratory disease, epidemic abortion and sporadic neurological disease. In general, EHV-1 infection elicits the stimulation of both humoral and cellular immune responses in horses. As EHV-1 is a persistent virus, its infection leads to latent infection during the first two weeks with chronic reactivations. Virus neutralising antibodies combat EHV-1 infection by reducing nasal virus shedding, particularly in the nasopharynx, but they play insignificant roles in controlling systemic spread by cell-associated viraemia to the respiratory tract and lymphatic tissues. The cytotoxic T lymphocyte (CTL) response is the most crucial aspect of EHV-1 specific cell mediated immunity and its function is well described to protect against clinical disease, viraemia and nasopharyngeal virus shedding. EHV-1 immediate early gene products (gene 64) have been shown to be targets for EHV-1 specific CTL. Moreover, vaccination of ponies with the EHV-1 gene 64 protein induces cell mediated immune responses resulting in the reduction of clinical and virological disease. This important finding underlines the substantial role of viral gene 64 in cell mediated immunity and highlights its potential for improved vaccination strategies. Therefore, the overall aim of this study was to identify peptide sequences of CTL epitopes within EHV-1 immediate early protein’s Fragment D (encoded by gene 64) in ponies expressing the MHC class I A3/B2 allele. In order to achieve this, in vitro cellular techniques were established and T lymphocyte responses to EHV-1 were quantified as a prerequisite for identifying CTL target peptides. This first investigation aimed to measure activation of equine peripheral blood mononuclear cells (PBMC) with either phorbol 12-myristate 13-acetate (PMA) or infectious EHV-1 by assessing interferon gamma (IFNγ) synthesis detected by flow cytometry as a prelude to stimulating PBMC with gene 64 protein or peptides. The intra-assay and inter-assay variations of IFNγ synthesis were characterised and the coefficients of variation of both assays were 8.42% and 5.8%. The phenotype of medium-stimulated and PMA-ionomycin stimulated PBMC comprised principally T cells as defined by antibodies specific for equine leukocyte markers. Intracellular IFNγ expression after in vitro EHV-1 stimulation of primed PBMC was assessed and revealed a very low percentage of IFNγ secreting cells in the responding population. In order to induce cells to use for the preparation of equine T lymphocyte and CTL lines/clones, in vitro EHV-1 stimulation of PBMC in bulk culture was characterised. Phenotypic analyses and viability of EHV-1 stimulated PBMC showed that 5% NHS in culture medium at 7 days stimulation was more reliable to induce CD8+ cells compared with 10% FCS, indicating the preferred use of 5%NHS in culture media to generate and assay equine CTLs. The next aim was to develop EHV-1 specific T cell lines and clones from whole virus (EHV-1). Several attempts were made to generate these using mouse anti equine CD3 monoclonal antibody (mAb) (clone UCF6G), autologous rhuIL-2 and mitomycin C treated PBMC as APC, but these failed. Using two different approaches; 5-ethynyl-2’-deoxyuridine (EdU) and Cu (I)-catalysed cycloaddition ‘click’ reaction assay and tritiated thymidine [3H] uptake to show lymphocyte activation in response to anti-CD3 monoclonal antibody, it was demonstrated that the anti-CD3 mAb was able to activate cell proliferation and allowed cell division and population expansion. To further assist the establishment of CTL lines and clones, the peptides of the EHV-1 immediate early protein’s fragment D (amino acids 750-1143 in the IE protein) were investigated using an IFNγ ELISPOT assay. This approach was optimised and characterised using medium, mitogen and EHV-1 as the negative and positive controls. A library of 128 synthetic peptides was obtained comprising molecules of 14 amino acids in length, overlapping by 11 amino acids. All peptides were constructed into pool arrays linearly and overlapping peptides were not contained within the same pool. The optimal concentration of the peptide pool was determined and chosen as 0.048μg per well. This concentration was also used for screening the individual peptides. The results showed that several peptide pools produced high number of spot-forming-cells significantly greater than the threshold for positivity and most of individual peptides stimulated an IFNγ response from PBMC, suggesting that IFNγ synthesis is too insensitive to detect CD8+ IFNγ + responses. In summary, this study has demonstrated the potential of new and relevant in vitro techniques that can be applied to the investigation of CTL target peptide(s) of EHV-1 as presented by the A3/B2 allele in horses

Effect of pretreated oil palm frond in enhancing the ruminal degradability, growth performance and meat quality of goats

The alternative source of ruminant feed is waste from agricultural byproducts such as oil palm fronds (OPF). With an annual production of 13 million tons and low cost, the OPF is of interest in Malaysia as it is abundant. However, OPF contains high neutral detergent fibre with 20% lignin, which can be major constraints to OPF use as livestock feed. It is in line with poor digestibility of OPF in cattle (400 g/kg DM). Consequently, the energy content of OPF ranges between 4.9 and 5.6 MJ of metabolisable energy (ME)/kg DM. There is a need to treat OPF to improve its nutritional value and digestibility to be used as animal feed. Biological pretreatment with enzyme extract from white rot fungi (WRF) is considered a promising technique because of its preferential degradation of lignin with minimum dry matter loss. To make biological pretreatment more effective, a combination of biological with other pretreatments might be necessary. To date, some physical pretreatments have been developed to upgrade the OPF, which include pressing using traditional sugarcane machine. Physical pretreatment may increase particle size, which allows the increase of rumination duration, chewing activity, and ruminal pH, which could offer a favourable environment for rumen microorganisms to thrive. Therefore, the current study was conducted to assess the effect of physical and biological pretreatments of OPF using enzyme extract from WRF on nutritive value and lignocellulosic composition. The study was also investigated to evaluate the potential of physical and biological pretreatments of OPF in enhancing the in vitro rumen degradability, growth and health performances, as well as meat quality of goats. Five different samples of OPF pretreatments were used in this study. The petiole of OPF was subjected to the physical pretreatment (POPF) by pressing using a conventional sugarcane pressing machine and the OPF, which consisted of petiole, leaflet and rachis were biologically pretreated using an enzyme extract of each Ganoderma lucidum (G. lucidum; BGL) and Lentinula edodes (L. edodes; BLE), respectively. Another two samples were subjected to a combination of physical and biological pretreatments of G. lucidum (CGL) and L. edodes (CLE), respectively. The control was non-treated OPF (FOPF). The results revealed that biological pretreatment with enzyme extract of G. lucidum and L. edodes either alone or combination with physical pretreatment reduced crude fibre and increased crude protein. The BGL, CGL, BLE and CLE reduced crude fibre by 15%, 16.6%, 14% and CLE were increased by 58.4%, 58.8%, 48.5% and 54.85% respectively All pretreatments decreased hemicellulose contents significantly as compared to control, but no significant differences were observed among pretreatments. Similarly, the lignin contents (% DM) of all pretreated OPFs showed a significant decrease as compared with the non-treated OPF, but treatments with BGL (10.00) and CGL (10.00) showed the lowest lignin content of OPF (p<0.05). However the physical, biological and combined pretreatments did not change the cellulose content of the OPF (p<0.05). The reduction of lignin content of OPF may indicate the cellulose and hemicellulose are converted into simple fermentable sugars such as glucose and xylose. Physical and biological pretreatments may help to reduce the crystallinity of the cellulose fibre in the OPF while also reducing the size of the materials. For in vitro study, two fistulated Katjang goats consuming OPF and commercial pellet daily were used as rumen fluid donors. In vitro incubation was carried out at 39°C for 24 hours. Both BGL and CGL showed significantly higher propionate and butyrate concentrations as well as apparent rumen degradable carbohydrate (ARDC) with 6.57mg and 6.54mg, respectively as compared to the FOPF. It appeared that BGL and CGL resulted in higher lignin degradation that increased the in vitro rumen degradability, indicating that rumen microbes' access to cellulose was greatly improved. However, comparing these two white rot fungi, G. lucidum seemed more promising for improving the in vitro rumen degradability. Biological pretreatment with enzyme extract of G. lucidum combined with physical pretreatment improved the nutritional values of OPF by decreasing the lignin contents, consequently improving the ruminal degradability along with high total gas production, high VFA and high ARDC. Thus, the biological pretreatment with enzyme extract of G.lucidum was selected to pretreat the OPF for feeding trial study in goats. Twenty, 5-month old male, cross-bred Boer goats with the average initial body weight 21.8±0.5 kg were assigned to treatments in a completely randomised block design of 120-day feeding trial. Five treatment diets containing 20% of OPF with different OPF pretreatments, 50% Napier grass and 30% goat concentrates were used in this study. The T1 consisted of nontreated OPF, T2 contained physically pretreated OPF, T3 with biological pretreated OPF with enzyme extract from WRF, T4 with OPF undergone combination of physical and biological pretreatments and the control (CD) consisted of the basal diet without OPF. During the feeding trial, feed intake and growth performance of goats were measured. After 120-day of the feeding trial, a digestibility trial was carried out on all goats involving total faecal collection. The animals were then slaughtered for carcass traits and meat quality evaluations. The results showed that the intakes of DM, crude protein (CP) and ash were significantly higher in group T3 and T4. However, all pretreatment methods had no adverse effect on body weight (BW) gain. The digestibility of DM, CP, acid detergent fibre and neutral detergent fibre nd 13%, respectively in T4 group as compared with control group. Haematological profile was improved based on reference value in all groups. Total faecal egg respectively. Besides, hot and cold carcass weights, dressing percentage and meat quality measurements were not affected by the pretreatments. Nevertheless, biological pretreatment of OPF has the potential to promote the accumulation of mono- and polyunsaturated fatty acids in the meat. In conclusion, this study has demonstrated that physical and biological pretreatment of OPF either alone or in combination improved the quality of OPF by decreasing the lignin contents, consequently improving the in vitro ruminal degradability along with high total gas production, high VFA and high ARDC. Biological pretreatment of OPF also increased feed intake and some characteristics, chemical composition and meat quality