Sample pooling for real-time PCR detection and virulence determination of the footrot pathogen <Emphasis Type="Italic">Dichelobacter nodosus</Emphasis>

Dichelobacter nodosus is the principal cause of ovine footrot and strain virulence is an important factor in disease severity. Therefore, detection and virulence determination of D. nodosus is important for proper diagnosis of the disease. Today this is possible by real-time PCR analysis. Analysis of large numbers of samples is costly and laborious; therefore, pooling of individual samples is common in surveillance programs. However, pooling can reduce the sensitivity of the method. The aim of this study was to develop a pooling method for real-time PCR analysis that would allow sensitive detection and simultaneous virulence determination of D. nodosus. A total of 225 sheep from 17 flocks were sampled using ESwabs within the Swedish Footrot Control Program in 2014. Samples were first analysed individually and then in pools of five by real-time PCR assays targeting the 16S rRNA and aprV2/B2 genes of D. nodosus. Each pool consisted of four negative and one positive D. nodosus samples with varying amounts of the bacterium. In the individual analysis, 61 (27.1%) samples were positive in the 16S rRNA and the aprV2/B2 PCR assays and 164 (72.9%) samples were negative. All samples positive in the aprV2/B2 PCR-assay were of aprB2 variant. The pooled analysis showed that all 41 pools were also positive for D. nodosus 16S rRNA and the aprB2 variant. The diagnostic sensitivity for pooled and individual samples was therefore similar. Our method includes concentration of the bacteria before DNA-extraction. This may account for the maintenance of diagnostic sensitivity. Diagnostic sensitivity in the real-time PCR assays of the pooled samples were comparable to the sensitivity obtained for individually analysed samples. Even sub-clinical infections were able to be detected in the pooled PCR samples which is important for control of the disease. This method may therefore be implemented in footrot control programs where it can replace analysis of individual samples.     

A monoclonal antibody against a canine CD45 homologue: analysis of tissue distribution, biochemical properties and in vitro immunological activity

This report describes the characterisation of a monoclonal antibody (mAb), AB6, which recognises specifically a cluster of canine leukocyte surface molecules. The immunogen used for obtaining the AB6 mAb was a lysate of canine peripheral blood mononuclear cells (PBMC). This novel mAb belongs to the IgG2a isotype, and reacted in Western blot with four different canine leukocyte glycoproteins with apparent molecular weights of 180, 190, 205 and 220 kDa. The AB6 mAb recognised the majority of canine peripheral blood leukocytes as determined by flow cytometry (97%). It also exhibited a broad reactivity pattern against lymphoid and myeloid cells, inhibited the proliferation of mitogen-stimulated canine PBMC and did not recognise human PBMC and murine splenocytes. The biochemical properties, cell and tissue specificity, and in vitro biological activity of the AB6 mAb indicate that it recognises a canine CD45 homologue. The mAb could become a valuable diagnostic and research tool for the evaluation of immune functions in dogs.     

Integrated approach for improving small scale market oriented dairy systems in Pakistan: participatory rural appraisal and economic opportunity survey

Livestock production is an integral part of the rain-fed and irrigated agriculture system in Pakistan. Animal production is closely interlinked with the cropping systems and play a crucial role in the rural economy. Participatory rural appraisals and economic opportunity surveys were conducted in two ecological zones (irrigated and rain-fed) and two dairy production systems (peri-urban and mixed livestock). Major constraints in animal health, nutrition and reproduction were identified and interventions were suggested to overcome these difficulties. The economic opportunity survey revealed that maximum opportunity to enhance farmers’ income is to increase milk production per day per animal, which can be accomplished through coordinated improvements in nutrition, reproduction and genetics.     

Variable number of tandem aminoacid repeats in adhesion-related CDS products in Mycoplasma hyopneumoniae strains

The Mycoplasma hyopneumoniae genome contains at least 22 regions with a variable number of tandem nucleotide repeats (VNTRs) within coding DNA sequences (CDSs). In this work, the VNTR-containing CDSs were analysed in order to evaluate their degree of variation, possible correlations with antigenic properties, and their potential to be used as a basis for a strain typing PCR assay. We have analysed the VNTRs in five M. hyopneumoniae strains (J, 7448, 7422, PMS, and 232), based on published genomic sequences and on amplified and sequenced DNA segments. These VNTRs are distributed among 12 genes, most of which encode putative surface proteins, including known adhesins. The number of repeat units in any of the VNTRs is highly variable among the analysed strains, but they are, without exception, translationally in frame, and, therefore, code for a variable number of aminoacid repeats (VNTARs). These VNTARs determine putative structural, physicochemical and antigenic variations in the corresponding proteins, with potential implications for aspects associated to M. hyopneumoniae pathogenicity, such as cell adhesion and interactions with the host immune system. Considering that the characterized VNTARs are relatively stable, at least in vitro, and their sizes are strain-specific, we have developed a VNTR-based PCR assay for M. hyopneumoniae strain identification, useful for enzootic pneumonia (EP) diagnosis, strain typing, and distinction of circulating field isolates from vaccine strains in animals vaccinated against EP.     

Incidence and molecular characterisation of lumpy skin disease virus in Zimbabwe using the P32 gene

Between January, 2013 and December, 2014, there was a lumpy skin disease (LSD) outbreak that affected cattle in different localities of Zimbabwe. The outbreak resulted in severe economic losses to the livestock industry. A retrospective study was conducted by examining stored veterinary records of the LSD outbreak at the Central Veterinary Laboratory (CVL) in Harare, Zimbabwe. Over the 2-year period, a total of 10,038 cases and 880 deaths (8.77 %) were recorded. LSD was reported from all regions of the country, with the highest incidence occurring in Mashonaland West (30.95 %) and Midlands province (14.59 %). The frequency of reported outbreaks was highest in March and April, with the lowest reported cases occurring in November. A total of 25 representative specimens (skin biopsies) were collected from nodular skin lesions of infected cattle, and after viral DNA isolation, the P32 gene was successfully amplified, by using PCR, in 88 % (22/25) of all assayed specimens. Out of the 22 samples that showed amplification, 16 (73 %) were selected for DNA sequencing, and from these, 13 sequences were submitted to GenBank and assigned accession numbers: KX033494, KX033495, KX033496, KX033497, KXO33498, KX033499, KX033500, KX033501, KX033502, KX033503, KX033504, KX033505 and KX033506. Phylogenetic analyses of the 13 sequences was done by using MEGA 7 and showed that the viruses formed two major clusters implying that at least two strains of LSDV are in circulation in Zimbabwe. This study provides the first report on the incidence and molecular characterisation of LSDV in Zimbabwe.     

Nutraceutic effect of free condensed tannins of <Emphasis Type="Italic">Lysiloma acapulcensis</Emphasis> (Kunth) benth on parasite infection and performance of Pelibuey sheep

Forty-five Pelibuey sheep were experimentally infested with nematodes to evaluate the effect of three free condensed tannin (FCT) levels of Lysiloma acapulcensis on fecal egg counts (FECs), packed cell volumes (PCV), ocular mucosa colors (OMC), average daily gain (ADG), and adult nematode count. Five treatments were used: 12.5, 25.0, and 37.5 mg of FCT kg^?1 of body weight (BW); sterile water (control); and ivermectine (0.22 mg kg^?1 of BW) as chemical group. The data were processed through repeated measurement analysis. Even though the three FCT doses decreased (P?<?0.05) the FEC, the highest reduction was obtained with 37.5 mg kg^?1 of BW. No differences were observed in PCV and OMC. Higher ADG (P?<?0.05) was observed with 37.5 mg kg^?1 of BW of FCT. The count of adult nematodes (females and males) in the higher dose of FCT was similar to chemical treatment. Dose of 37.5 mg kg^?1 of BW decreased the parasite infection and improved the lamb performance. Therefore, this dose could be used as a nutraceutic product in sheep production.     

Apparent metabolizable energy value of whole date palm (<Emphasis Type="Italic">Phoenix dactylifera</Emphasis> L.) and its possible use as a feedstuff for aged laying hens

This study was conducted to evaluate the feeding value of whole date palm (WDP) with and without enzyme supplementation on aged laying hen’s performance. Apparent metabolizable energy value of WDP was determined by the total collection method using ten adult leghorn cockerels. WDP was substituted with a corn-soybean meal basal diet at 40% level, and then the metabolizable energy of this experimental diet and basal diet was determined. After that, a total number of 256 Bovanz 95-week-old hens were randomly allocated into eight groups consisting of four replicates of eight birds each, based on a 4 × 2 factorial arrangement of the treatments. Eight iso-caloric and iso-nitrogenous experimental diets including one corn-soybean meal-based control diet and two, three, and four corn-soybean meal-based diets included 70, 140, and 210 g/kg ground WDP, respectively. Each of the diets supplemented with two levels of an enzyme (0.0 and 0.07 g/kg Natozim Plus). There was no significant difference in feed intake, feed conversion ratio, egg production, egg mass, eggshell thickness, and Haugh unit among the treatments (P > 0.05). However, yolk color score significantly decreased as dietary WDP level increased. The serum biochemical metabolites were not significantly affected by WDP and enzyme supplementation (P > 0.05). There were no significant differences in the relative weight of different organs except for abdominal fat. Our findings show that using WDP up to 21% of the diet was more economic and had no adverse effect on productive performance and serum metabolites of laying hens. However, WDP had an adverse effect on yolk color which can be ameliorated by carotenoid supplementation.     

An assessment of benzimidazole resistance against caprine nematodes in Central India

Current status of resistance to benzimidazole (BZ) group of anthelmintic drugs against caprine nematodes in Central India at Amanala goat farm, Jabalpur, Madhya Pradesh (M. P.), was systematically investigated using faecal egg count reduction (FECR) test and egg hatch test (EHT). Besides, allele-specific PCR (AS-PCR) was deployed to ascertain the susceptible genotype (alleles) especially of the Haemonchus contortus. Randomly selected 30 goats, irrespective of age and sex, were divided into three groups of 10 each, to serve as treated and untreated controls. It was ensured that the animals were not administered with an anthelmintic drug for the past 3 months prior to undertaking the study, and faecal egg counts were estimated. FECR test evidenced fenbendazole resistance by partial elimination (24.90%) copro-egg counts in the treated group of animals vis-à-vis controls with a lower confidence interval of ?26%. Further, EHT revealed ED-50 value of 0.335 μg of thiabendazole/ml, confirming benzimidazole resistance in the animals of that farm. AS-PCR showed that 62% of H. contortus larvae were homozygous resistant (rr), 24% heterozygous (rS) and 14% homozygous susceptible (SS). The genotypic frequencies of three genotypes (rr, rS and SS) were significantly (P < 0.01) different. The prevalence of benzimidazole resistance allele (r) was also significantly (P < 0.01) higher (74%) as compared to susceptible allele (S) (26%). The resistance to benzimidazole has been discussed while emphasizing improved managemental practices designed to reduce exposure of the goat population to parasites, minimize frequency of anthelmintic use at optimum dose and rotational use of different chemical groups of medicines with different mode of action, so as to overcome and combat the upcoming problem in the field.     

Current status of cattle production system in Nyagatare District-Rwanda

A study was conducted to characterize the cattle production systems in Nyagatare District, Eastern Province of Rwanda using pre-tested questionnaires, interviews with key informants as well as focus group discussions in a period of 2 months. The respondents were selected by multi-stage sampling at sector and cell levels. Based on the procedure of Krejcie and Morgan (Educational and Psychological Measurement 30:607–610, 1970) to determine the overall sample size, the result indicated that the majority (98.3%) of farms were privately owned by large families of five to seven members, and most farmers (53.9%) had only primary education. Most respondents (52.6%) were in the age bracket of 41–50 years and were mainly (48.3%) located within 3 km from trading centers. The farm size averaged 6.5 ± 0.8 ha and most farms (64.7%) were fenced except in Rukomo Sector (50%) where zero grazing prevailed. Natural pastures (savanna grass land) were the main feed resource; tethering (9%) and communal grazing had diminished. Napier grass was the main planted forage (93.2%), followed by Chloris guyana (3.1%) and Brachiara (1.2%). Leguminous forages were rarely (2.5%) reported. Vita-mineral and salt block supplements, hay, and crop residues were the predominant supplementary feed stuffs used except in Karangazi and Rwemiyaga Sectors where only vita-mineral block predominated. However, maize and rice brans were reported to be the main feed stuffs used in supplementary feeding of lactating cows. Most farmers (89.7%) reported shortage of water as most of the farmers trekked their cattle to the nearest valley dams (59.2%), rivers (21.1%), and a few 6% had water on farms. Indigenous cattle were predominant (67.03%) followed by cross-breeds (28.37%) and exotics (4.6%) while all farmers kept small ruminants. Natural breeding predominated (74.9%) and most farms (60.6%) had animal houses most of which were temporary (52.8%). The reported mean age at first calving (AFC) was highest (40.2 ± .33) for Ankole and the lowest (29.1 ± .50) months for exotic cattle. Calving interval was shorter in local breeds than 65.7 ± 3.0 in exotic. The mean dairy milk yield was lowest for Ankole cattle 2.4 ± .08 as compared to the exotics (10.42 ± .36) and their crosses (7.2 ± .34). The main challenges were diseases, shortage of water, feeds, and inadequate extension services. Same observation was reported by Okello (African Journal of Range and Forage Science 22(3), 2005) in Uganda.     

L-carnitine significantly decreased aging of rat adipose tissue-derived mesenchymal stem cells

Mesenchymal stem cells are undifferentiated cells that have the ability to divide continuously and tissue regeneration potential during the transplantation. Aging and loss of cell survival, is one of the main problems in cell therapy. Since the production of free radicals in the aging process is effective, the use of antioxidant compounds can help in scavenging free radicals and prevent the aging of cells. The aim of this study is evaluate the effects of L-carnitine (LC) on proliferation and aging of rat adipose tissue-derived mesenchymal stem cells (rADSC). rADSCs were isolated from inguinal region of 5 male Rattus rats. Oil red-O, alizarin red-S and toluidine blue staining were performed to evaluate the adipogenic, osteogenic and chondrogenic differentiation of rADSCs, respectively. Flow cytometric analysis was done for investigating the cell surface markers. The methyl thiazol tetrazolium (MTT) method was used to determine the cell proliferation of rADSCs following exposure to different concentrations of LC. rADSCs aging was evaluated by beta-galactosidase staining. The results showed significant proliferation of rADSCs 48 h after treatment with concentrations of 0.2 mM LC. In addition, in the presence of 0.2 mM LC, rADSCs appeared to be growing faster than control group and 0.2 mM LC supplementation could significantly decrease the population doubling time and aging of rADSCs. It seems that LC would be a good antioxidant to improve lifespan of rADSCs due to the decrease in aging.